1. The Landscape

Language-learning methdologies centering around Stephen Krashen’s input hypothesis have been slowly on the rise over the past several years—terms like “comprehensible input”, “mass immersion”, etc. come to mind. ~5-10 years ago, this method didn’t have so many people talking about it on the internet. At the time, I would point people mainly to 2 people/memeplexes: LingQ and Refold/MIA/AJATT. These are still good places to start if you’re unfamiliar with the topic. Before you commit to a language learning method—especially a method one where your effort is not transparently commensurate with reward—you’re going to need to be confident and motivated. So I recommend you go ahead and check out some of the videos I’ll link in these first 2 sections and go from there, seeing what vibes with you.

Refold, MIA, and AJATT are methods for learning languages—mainly Japanese, which, due to being one of the most difficult languages for English speakers, yet also the most enticing for nerds, it represents the vanguard of the language learning community and technologies made to aid you in your quest. A lot of the resources you’ll find and that I’ll link will be about Japanese, but it all basically applies to any other language. AJATT/MIA/Refold basically refer to the same thing just changing names and somewhat evolving and changing ownership over the course of a few years, to put it simply. It’s had a cult following on the internet for quite some time, and the core idea is just consuming as much Japanese media as possible, and your brain will automatically acquire the language. Everything else like brushing up on grammar is effectively a supplement to facilitate this process.

The Youtube channel Matt vs Japan is one of the OGs at actually disseminating the core ideas of AJATT (All Japanese All The Time). He then went on to make MIA (Mass Immersion Approach) which later got nuked for personal reasons, and basically became Refold (which is somehow monetized? I haven’t kept up with it, but it’s not necessary. What matters are the free resources they’ve laid out in their methodology, the online community surrounding it, and the resources therein. A mere methodology for consuming content fundamentally can’t really be monetized.

Over the years, many other people have started Youtube channels giving progress reports with these methodologies, and sometimes educating the youth on the method of watching anime all day and making Anki cards. I highly suggest you stick to this memeplex and not bother with any other content because there’s a high probability of it being misinformed and/or slop.

All that being said, any of these videos/youtube channels may or may not lay out every single bit of nuance behind comprehensible input. Especially as it pertains to you and your needs, preferences, strengths and weaknesses, etc. so I recommend you watch many of them critically, and plan your method for learning your target language.

Sentence Mining & Building Vocabulary

For your immersion, sentence mining or some way of tracking words and building your vocabulary, such as with a pre-made Anki deck, is the second most important thing after comprehensible input. (Allocating a disproportionate amount of your time on this component could even take you relatively far—but there are optimal ratios). Many tools exist for this. When you consume with a piece of media in your target language (reading, watching, or listening) you’ll have some kind of tool that records which words you know/don’t know, and, ideally, also recording which words you’re in the process of learning. If you’re unfamiliar with SRS I wrote a little bit about it in the post on Studying & Spaced Repetition, although that was specifically for non-language learning purposes (meanwhile language learning is probably the most common usecase of SRS).

When you sentence mine to create targeted sentence cards, you find an i+1 sentence when consuming media, meaning it contains a single unknown word. Note that the best pre-made decks also follow this format, gradually starting from bare-bones vocabulary and only using words used prior, gradually increasing the repertoire. After a certain point though, the word frequency lists these decks are based on lacks catering to your own domain specificity and probably becomes boring. (But, rest assured… there are decks with 10,000+ cards, there’s 3 volumes of Remembering the Kanji, etc. but don’t bother, lol).

Like consuming scientific content? You’ll learn scientific words. I doubt “hydrogen” or “neuron” is particularly high on the word frequency list, yet it might be something you’d benefit from learning ASAP due to the content you like to consume. You get the idea. There do exist non-general frequency lists and decks made out of them, such as one based on anime. (You can also compute frequency lists yourself out of a data set if you were so inclined, and you could also systematically find i+1 sentences out of them, provided you’re using something which tracks your vocabulary, which is what I discuss below. I’m getting ahead of myself.)

Once you reach the mark of 500~2000 known words (especially if we’re talking Kanji/Hanzi), you understand a significant amount of material and can start easily mining 5-10+ sentences daily.

(From: Forever a student: Chinese character frequency list - News articles)

Matt vs Japan - Sentence Cards vs Vocab Cards: In-Depth Comparison discusses what your flashcards will be looking like; it’s hotly debated which exact format is optimal. To some extent, it comes down to preference.

Anyways - for actually actually implementing sentence mining, you have 2 options:

1. Migaku. There are other software solutions I used to recommend but think lacks features at this point, LingQ (a good explanatory video), or, as a free alternative, Learning with Texts. All great pieces of software; I got quite proficient in German with LingQ, but I would use Migaku if I had to start over due to the fact you can use it on basically any website and use it in real-time on videos (Youtube, Netflix, or mpv/asbplayer + texthooker for any local file) without needing to import anything or whatever.

2. Pop-up dictionary like JMDict/Yomitan, + mpv + scripts for sentence-mining and importing to Anki. You might want to add Language Reactor to the mix too. This is much less versatile than tools like Migaku in almost every way, which aim to immediately create a setup like this, but it is free and open-source. If that is appealing to you, I recommend https://anacreondjt.gitlab.io/, https://animecards.site/, and https://tatsumoto-ren.github.io/ as a good place to start when creating your setup. Having your cards exist in the Anki ecosystem (i.e., your local files, where you can batch edit/analyze things, etc.) is quite nice though; interoperability between Anki and ‘Migaku Legacy’ is possible, however! It’s not something I’ve used personally though.

Anyways: no, you don’t actually need to use Anki or a structured SRS system if you immerse enough, especially if you have a word tracking tool to reduce cognitive strain, which is why I said that word tracking is essentially rather than SRS per se. When reading with a tool like pop-up-dictionary, especially if it has tracking like Migaku/LingQ, you’re still taking advantage of the testing effect by inevitably scratching your head trying to comprehend it. I still highly recommend supplemental SRS if you’re learning a difficult language, especially Japanese/Chinese. It’s very high reward:effort, especially if you have to memorize certain things, rather than just see what sticks in what you immerse with. That being said, Steve Kaufmann himself doesn’t use the SRS functionality in LingQ, and neither did I when using it for German, instead relying purely on input on LingQ for vocabulary retention. Was this gonna slide for something way more difficult like Chinese? This is much less likely. 5-10 words is quite fair for Japanese/Chinese.

In summary: as stated in the tl;dr, my method is basically just something like:

• 80% of your time you can allocate to the language should be spent doing active immersion with Migaku. Start with videos with subtitles in your target language, then move on to reading and audio-only after they become comprehensible (especially if they work better with your daily routine or the type of material you’d simply like to consume). Ideally get versatility in all 3, but technically if you only care about one of those 3 domains, nothing is stopping you.
• ~15% of your time reviewing SRS. Before you rely solely on sentence mining i+1 sentences for acquiring new words (if ever), you must get a pre-made flashcard deck of ~500-2k cards. Ankiweb has a ton of these, and I’ve linked some below.
• ~5% of your time learning grammar points or otherwise “studying” the language.
• Passive immersion/passive listening throughout the day (during times where you can partially put your attention to the language, such as while at the gym, while chopping wood, while carrying water, etc.) if possible is also quite valuable - even at the beginning stages.

Grammar + Other Resources

You still need a good book/online resource that you can consult every now and then for learning grammar, etc. as a supplement to your immersion. I think that—especially if you’re on the higher end of free time you can dedicate to learning a language—not allocating at least a little time to grammar is slightly ridiculous. It would be a wast eof time expecting to learn everything related to grammar only via immersion, especially if the language is a difficult one. Even just 5-30 minutes a day will go a long way and fill in the gaps for you.

• Start with the Refold Detailed Roadmap. I haven’t read this in forever but this should equip you with just about everything you need to know (terminology, priors, a more nuanced explanation behind the methodology) to approach other resources.
• Refold Community Dashboard - Notion has links to resources for a ton of languages and their respective sub-pages(though for some reason it’s kinda hard to navigate. For instance I have no idea how to get to the one for Mandarin, yet it links back to this page in directory structure at the top??)
• https://github.com/kelciour/mpv-scripts
• https://github.com/Ajatt-Tools

Japanese

• The best Anki Deck is probably ‘JP1K decks’ like this.

Get the version where the front of the card has the isolated target word though.

• Tae Kim is probably the best grammar guide out there. It’s short and to the point; very well-known in the community. Avoid books like Genki.

Traverse these links for finding useful stuff. For instance, sites like https://kitsunekko.net should you need to download subtitles. There’s all kinds of stuff you might want along your journey.

• Refold Japanese - Google Docs
• https://animecards.site/
• https://anacreondjt.gitlab.io/
• https://tatsumoto-ren.github.io/ is an excellent resource; I recommend looking at its many FAQ articles

(Mandarin) Chinese

• Best introductory Anki Deck is probably either what comes with Migaku, or Refold Mandarin 1k.

• Mnemonic-based methods, i.e. the “Marilyn Method” make a very good case for themselves. HanziHero is probably the best implementation of this. Mandarin Blueprint’s method is also quite good, but their course is really expensive; I wouldn’t really recommend it unless you’re literally just rich.

  • I may implement this in the future. HanziHero is really cool in theory, but it lacks features: personally I found myself basically never using their mnemonics, especially due to the fact you can’t even customize them. So if you don’t vibe with them, too bad. You can’t choose ‘actors’ or ’locations’ that are personally meaningful to you (which Mandarin Blueprint does allow for). r- has to be Robinhood instead of Rick Owens, etc.. Additionally: they sometimes give components meanings that don’t really correspond with their literal etymology, or even contexts in which they’re being used. E.g., calling 艮 ‘silver’ (when it actually means “tough” and depicts a person looking back) or 行 ‘sandal’ (which does relate to locomotion, but it depicts a street intersection, not a sandal). To me, this is inexcusable; it feels like you’re bastardizing your understanding of the words’ real origins. The pictographic/ideographic origin of Chinese characters and the way the components interact to create characters is undoubtedly one of the most—probably the most—fascinating aspect of the language. (This is why I think it’s probably a shame that simplified Chinese was invented instead of sticking with traditional, but that’s a whole other take). Why lie to yourself about what characters are depicting? Can your memory not rest easy and place its trust in thousands of years of linguistic development? Hubris!

• Refold Mandarin - Refold Mandarin Resources. This here is beautiful.

• Refold Mandarin - Google Docs

• https://www.dong-chinese.com/. Has a good dictionary, and Youtube videos sorted by difficulty.

• https://www.vidioma.com/ Youtube videos sorted by difficulty

• https://hanzicraft.com for dictionary + character decomposition.

• https://rtega.be/chmn/index.php?subpage=41 dictionary + etymology + (sometimes) mnemonics.

Ancient Languages

You can’t immerse yourself in a language that’s dead, now can you? Well, for Latin, there’s probably actually a decent ammount of audio out there considering it’s somewhat popular, and it has the excellent book Lingua Latina per se Illustrata (and sites that provide notes), which follows a ground-up approach to learning. (This method of pure-input slightly above your current level has recently been popularized with Dreaming Spanish - although it’s not text-only).

But if the language you want to learn is really dead, then you’re probably not aversive to doing it the old fashioned way anyways, and that’s just the “grammar-translation” method. There’s not exactly much of a purpose in training yourself to speak a language with no native speakers, or be “fluent” in it, per se. Work through textbooks, learning grammar and understanding more and more sentences, until you can read what you want. Our is an era of AI though; I imagine it will be able to generate a plethora of resources and translate things and even create audio/video not too long from now. Stuff like this video is something you might find interesting.

Ancient Greek

The 3 most distinguished dialects would probably be: Homeric, Attic (Plato, Aristotle) and Koine (Septuagint), but there’s also Ionic, Doric, and a few others. But if there’s one dialect to own the title “Ancient Greek”, it’d be Attic - most textbooks are about it, and it has enough similarities with the other dialects and with modern Greek, such that jumping from one to another wouldn’t be too difficult or anything.

• Lingua Gracae per se Illustrata is a WIP project that replicates LLPSI.
• Athenaze
• Hansen & Quinn, Greek: An Intensive Course + Answer key, notes

If you know Latin, maybe you could also get by with reading some material right off the bat, say in a biglotic text, since something like 10% of Latin words are of Greek origin, they have similar morphologies, and similar grammars. See Sihler, Andrew L. New Comparative Grammar of Greek and Latin or something like that.

Palī

It’s probably a good idea to learn Sanskrit first, which is a treasure anyways, and is very standardized: the Aṣṭādhyāyī being a spectacle of scholarly rigour, to a degree that took contemporary linguistics took a long time to even approach ints comprehensiveness.

• https://palistudies.blogspot.com/p/resources.html
• A Course in the Pali Language
• https://www.buddha-vacana.org/ or https://www.tititudorancea.com/z/tipitaka_english.htm for Pali-English biglottic texts.
• Johansson’s Pali Texts Explained to the Beginner has an appendix comparing Sanskrit and Pali.

Color Vision

You can read the Wikipedia page on color blindness for a primer on what we’re about to get into.

(This is not an accurate ’test’ per se for those with color blindness, and it is only a simulation. In reality it’s actually a bit difficult to simulate what the result of color blindness looks like, as it’s not like you just edit out a color channel from the image)

Essentially, in dichromacy, a gene for one of the opsins is absent. In anomalous trichromacy, all 3 opsins are present, but one of them has (a significant amount of) genetic mutations (a “hybrid gene”; an “L-M chimera”) that alters its spectral sensitivity (this is what those glasses excel at):

(From: The molecular basis of variation in human color vision)

Color blindness is more common in males (5-8% of men vs. <1% in females) because OPN1LW/OPN1MW are found on the X chromosome (OPN1SW is on chromosome 7). Females, of course, have 2 X chromosomes, thus if one lacks the gene for an opsin, this would also make her a carrier of color blindness.

On the flip side, this is also somewhat the basis for theories about human tetrachromacy. Indeed, it’s probably only possible in females, such as when an additional, different OPN1LW/OPN1MW is somehow expressed.

This genetic basis for this is actually rather common (~12%), but true ‘functional tetrachromacy’ is a rarity, and something that hasn’t been studied too extensively. In The dimensionality of color vision in carriers of anomalous trichromacy, they showed that 1 woman out of 24 with deuteranomaly (who thus are assumed to be retinal/non-functional tetrachromats) exhibited true tetrachromacy. The artist Concetta Antico is a living tetrachromat who’s gotten her fair share fo media attention. Apparently she has access to ~100 million colors, as opposed to ~1-10 million (conflicting sources…).

The Graphium Sarpedon, the bluebottle butterfly, has 15 different photoreceptors: (From: The More, the Better? A Butterfly with 15 Kinds of Light Sensors in Its Eye)

The mantis shrimp is famous for having 12 different photoreceptors, but unfortunately: Despite the impressive range of wavelengths that mantis shrimp have the ability to see, they do not have the ability to discriminate wavelengths less than 25 nm apart. It is suggested that not discriminating between closely positioned wavelengths allows these organisms to make determinations of its surroundings with little processing delay. Having little delay in evaluating surroundings is important for mantis shrimp, since they are territorial and frequently in combat. (Wikipedia)

(From: A different form of color vision in mantis shrimp (2014): Theoretical approaches have predicted that between four and seven photoreceptor types are all that is needed to accurately encode the colors of the visible spectrum.)

Avenues for Modification

Consider what occured in evolution: in humans and some other primates, OPN1LW branched off from OPN1MW as we escaped from the nocturnal bottleneck.

(Source)

Or more subtle changes, like for instance, in Spectral-tuning mechanisms of marine mammal rhodopsins and correlations with foraging depth Fasick et al. describes how bottleneck dolphin rhodopsin blue-shifted during evolution (since it dives deep in the big blue) from an λmax of ~500 nm to 488 nm via a substitution of 3 amino acids.

Molecular determinants of human red/green color discrimination: The human red and green color vision pigments are identical at all but 15 of their 364 amino acids, and yet their absorption maxima differ by 31 nm… the spectral difference between these 2 pigments is shown to be determined by 7 and only 7 amino acid residues. For example, one of the residues is the Ser180Ala SNP (rs1557157655) on OPN1LW where it has a ~4-7 nm longer λmax. This SNP actually accounts for small differences in the general human population.

And to reiterate: as mentioned in the beginning, anomalous opsins are somewhere in between, with a few of these 7 residues differing. And, again: that’s part of what we see in human retinal tetrachromacy, where a 4th, anomalous cone that differs from its sister on the other X chromosome is added. Therefore, let’s design a whole gamut! Because now for the fun stuff.

So back in 2009, The Neitz Lab cured color blindness(?) in squirrel monkeys, whose males (and some females) are natural dichromats:

• Gene therapy in colour | Nature

• Gene therapy for red-green colour blindness in adult primates (Manusco et al. 2009)

  • As you can see in Fig. 3, it took an entire 20 weeks after the gene therapy for a sudden onset of color discrimination, which persisted at similar levels from 24 weeks onwards, after 2 years.
    • 

• Curing color blindness–mice and nonhuman primates (Neitz & Neitz 2014)

Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment (2007)

Thing is, these are animals. We don’t actually have proof they’re perceiving red/green just like a trichromatic would. Perhaps it’s merely an enhanced sensitivity within the deuteranope spectrum somehow?

The Neitz Lab did their best. For instance, a hallmark of dichromatic vision is the neutral point, in the middle of the visible spectrum where the sensitivity of both cones overlaps and light appears achromatic. In deuteranopes this is ~500 nm, and ~495 for protanopes. Manusco et al. writes: As shown in Fig. 3b and c, both monkeys’ measured thresholds for these additional hues were similar to their thresholds for DW = 490 nm, demonstrating they now lacked a spectral neutral point and have become truly trichromatic. Furthermore, treated monkeys were able to discriminate blue-green (DW = 490 nm) when it was tested against a red-violet background (DW = −499 nm), instead of the grey background, indicating that the monkeys’ newly-acquired “green” and “red” percepts were distinct from one another.

Some concerns are further discussed in Is adding a new class of cones to the retina sufficient to cure color-blindness? (2015): we simulate how replacing the pigment of some cones is expected to influence the outcomes on the behavioral test used so far. The simulations show that this test does not provide conclusive evidence that the animals acquired the ability to make new chromatic distinctions. In our view, it is therefore premature to claim that human color-blindness can be cured through gene therapy. They also reference how, importantly, unlike other aspects of vision, color deprivation in early life does not preclude the proper development of color vision later in adulthood (e.g.: Striking absence of long-lasting effects of early color deprivation on monkey vision (1990))

Ultimately? Unless we figure out how to talk to monkeys about the nature of mind, we need better studies to give us more concrete inductive evidence, or we need to just try it in humans in order for us to really know how your vision changes and what the world looks like.

Furthermore, let us consider the physiology of cone cells more closely:

(These are rod cells, but cone cells are the exact same mechanism) (From: Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling)

So the thing is, cone cells (unlike rod cells) aren’t continually replaced or anything during one’s lifetime, and each cell only expresses a single opsin gene; we can’t just replace the expression of a certain percentage of cone cells with another. As shown in the Neitz Lab study, the AAV encoding human L-opsin was targeted to M cones, leading to a coexpression of both types on the same cone cell (the resulting neural signaling being something rather difficult to wrap your head around?)

Perhaps even more interesting, there are currently human clinical trials underway for the treatment of achromatopsia—monochromacy, amongst other things. There are ~6 genes that can cause achromatopsia. Unlike monochromacy proper/cone monochromacy, achromatopsia is an abberation in the downstream phototransduction cascade. All 3 opsins are still present; the cells are dysfunctional.

Nonetheless, there are still some things we can learn from these cases, like the question of how the perception of color is acquired in the visual cortex, and things of that sort. Where are these people though? I do not know.

• Cortical Visual Mapping following Ocular Gene Augmentation Therapy for Achromatopsia (2021)

• A demonstration of cone function plasticity after gene therapy in achromatopsia (2022)

• Seeing color following gene augmentation therapy in achromatopsia (2023) In humans.

  • We suggest that treated CNGA3-achromatopsia patients can perceive a stimulus’s color attribute, although in a manner that is different and very limited compared with sighted individuals.

Gene Therapy for Color Blindness (2017)

And indeed, before there were human trials, the prerequisite treatment in animal models has also been demonstrated to be successful: AAV-Mediated Cone Rescue in a Naturally Occurring Mouse Model of CNGA3-Achromatopsia (2012), etc.

Beyond the Visible Spectrum

What about UV or infrared vision? What about X-ray vision, or maybe radio wave vision?

In cases of aphakia, such as with cataracts surgery (which Claude Monet had done in one eye! It gave his vision a bluish tint) prior to the invention of UV-blocking intraocular lenses in the 1980s, ultraviolet light (down to ~300 nm) is in fact visible—it’s the lens of the eye that filters out wavelenghts under ~400 nm. And to all 3 cones no less, leading to those with aphakia seeing UV as slightly bluish (since the S-cone is a bit more sensitive, as you may assume) white.

UV-sensitive tetrachromats, such as certain birds, do indeed have a cone specialized for detecting light in the UV range:

Perhaps the sequence for one of these SWS1 genes similar in homology to human OPN1SW that peaks in the UV range could be investigated for our purposes. cf. Evolution of color vision in primates - Evolutionary pathway of SWS1 - Wikipedia. Or, we could simply modify the existing amino acid sequence of human OPN1SW in order to shift its λmax further towards UV, as described in the previous section.

But the thing about UV light is that it’s not exactly healthy to your cells. It goes without saying neither is X-ray or gamma ray vision. The thing about animals with UV vision not facing negative repurcussions—it’s theorized they simply don’t live long enough to face the repurcussions of it.

As for infrared, as Wikipedia explains, there is no limit necessarily, just an exponential drop in sensitivity after 700 nm. Powerful NIR lasers are visible to the naked eye up to about 1050 nm, but that’s not exactly an everyday scenario.

Reason being, we’re limited by chemistry. A photon’s energy is proportional to its frequency: E = hν. Frequency is inversely related to wavelength, such that hν = hc/λ. And when we get to the infrared range, the energy is too low for 11-cis-retinal to be photoisomerized into all-trans retinal (this is an integral step of the phototransduction cascade, if the photon part didn’t make that obvious.)

(From: The quest of vision: retina’s daily challenges)

But interestingly, as Retina, Retinol, Retinal and the Natural History of Vitamin A as a Light Sensor explains: Although retinal is the universal chromophore for all vitamin A-based light sensors, the exact isomer of retinal can be different between species. For example, aquatic animals are known to shift absorption maxima of visual pigments by using the A1 (11-cis retinal) or A2 (11-cis-3,4-dehydroretinal) chromophore. There are also examples of terrestrial vertebrates using vitamin A2-based visual pigments, which belong to the most red-shifted visual pigments (e.g., absorption maximum of 625 nm). A2 pigments absorb longer wavelengths of light compared to the A1 version because of the extension of the conjugated chain of the chromophore.

Maybe just maybe, if amino acid structure of opsins is not the bottleneck for making the deepest red cone possible, we could benefit from squeezing out some red sensitivity by making the switch to vitamin A2 (which in humans is produced from al-trans-retinol via CYP27C1), and also making sure we support the metabolism of it. But… it’s pretty minute difference:

(From: Vitamin A1/A2 chromophore exchange: Its role in spectral tuning and visual plasticity)

Replacement of the chromophore has been done in vitro at least: Chromophore switch from 11-cis-dehydroretinal (A2) to 11-cis-retinal (A1) decreases dark noise in salamander red rods

Eye Color

WIP section, because honestly I’m not sure by what means the pigmentation of the iris would actually be meaningfully altered after initially being created, since I don’t believe there’s any sort of continuous renewal process.

This one’s just for fun, especially since it should be pretty easy as far as gene editing goes, with no complicated biochemical mechanism we’re altering here—just flipping some SNPs. That does require CRISPR in this case, rather than AAV. And, as we’ll discuss in the next section, I think it’s possible this may even be doable with eye drops.

Well, Wikipedia has a number of references for the matter. OCA2 and HERC2 are the main genes. But there are hundreds of SNPs. And of course a wide array of genes for other pigments.

In albinism, the iris is actually able to become red (not a good look—especially in more severe cases where the pupil is also completely red—but to each their own) or in less severe cases, violet, which when the iris is decorated with eye-catching lacunae, and a contrasting (read: pigmented) limbal ring, I find to be quite attractive—at least in the, like, 3 different people you see when you look up “albino purple eyes”. So maybe it’s something you could shoot for. The actress Elizabeth Taylor was famous for haing deep blue eyes that looked violet in certain lighting conditions (and also having distichiasis, double eyelashes.)

Gene Therapy (Do-It-Yourself?)

Is retinal gene therapy via eyedrops possible? For our purposes I’m skeptical, but who knows. Fistly, we ought to only bring our gene vector into the retina and nowhere else. Don’t ask me what would even happen if cones started to be expressed in areas they shouldn’t (my guess is that that’s highly unlikely), or if they would even survive or integrate with anything, etc.. With eyedrops, the drug obviously first enters the cornea, conjunctiva, lens, iris, etc. with little of it reaching the postior segment (Recent Perspectives in Ocular Drug Delivery (2015) - PMC) owing to the series of barriers before reaching the cone cells in the retina.

There was indeed a case where HSV-1 was delivered via eyedrops and successfully cured a boy’s blindness related to dystrophic epidermolysis bullosa, where type VII collagen formation is disrupted—which exists in epithelial regions of the eye—not the retina (and, you know, only the retina).

Even intravitreal injection could be too diffusive in that regard. But take the case of treating retinal vein occlusion: the central retinal vein is located in the most postior segment of the eye, and anti-VEGF drugs are injected intravitreally (whereby it diffuses evenly to the entire retina) there, in order to counteract neovascularization/edema/etc. in certain conditions like macular degeneration. However: The Neitz lab in Efficiency of gene therapy via intravitreal injection in primate cones (2022) and Poster Session II: Intravitreal gene therapy in primate reaches extrafoveal cones (2023) showed that intravitreal AAV can indeed reach the retina and the fovea. And reaching the fovea/macula lutea is definitely important for our purposes: essentially it is the region of the eye necessary for detailed, photopic vision, and it is where cone cells are most densely concentrated (especially M- and L-cones, as done in this study), with no rods present, as opposed to the high density of rod cells in the periphery of the retina:

The more invasive and complex subretinal injection is how Manusco et al. delivered their AAV into squirrel monkeys (via 3 separate injections, along with a cocktail of other drugs so that things went smoothly), and it’s how other retinal gene therapies (For example, Luxturna®/Voretigene neparvovec, which obtained FDA approval in 2017) are being performed: Subretinal Injection Techniques for Retinal Disease: A Review, Gene therapy in retinal diseases: A review (2021).

Now performing intravitreal injection of a genetically engineered virus into your own eyeballs is not something I recommend you do at home. But, I will note that in theory, a great deal of genetic engineering (say injecting something intramuscularly instead) is literally a thing you can do at home. I’ll link a few things that explain the process better than I can:

• DIY Human CRISPR Guide = The Odin

The YouTube channel The Thought Emporium has a series Whose Gene is it Anyway where he constructs an exact plan/shopping list for knocking in/out certain genes in plants or in vitro cells. You may have seen his video I Genetically Engineered MYSELF to Fix Lactose Intolerance where he makes his microvilli express E. coli lactase, via AAV, delivered orally in microcrystalline cellulose pills. A single dose made him fully lactose tolerant for 12 months before the benefits started to even slightly fade.

Why this and similar potential therapies aren’t well-known and more ubiquitously used is beyond me and it’s proof the general public is way behind science. It’s like when I realized jetpacks aren’t science fiction one time when I was a kid. What’s even less obvious to some is that genetic engineering isn’t even exactly cutting-edge technology. It was achieved as early as the 1970s (like a number of other great things). Such a profoundly beneficial technology (~2-3% of human diseases are single-gene disorders—not bad) has gone under the radar probably due to bullshit biology reasons, since I guess things are easier said than done. But once you realize how easily things are in fact said, it’s hard to see why we’re not seeing larger mass adoption.

As far as I know, price (and legality, but that’s what Honduras is for) is the main hang-up when it comes to visiting gene therapy clinics. How much it is just mark up and some initial intellectual labor? Probably most of it. Some of these treatments such as Hemgenix®/Etranacogene dezaparvovec (intravenous AAV delivery of Factor IX), while sometimes covered by medical insurance (look how they did Shkreli?) can cost illions.

Remember that some of this is theoretically able to be done DIY, obtaining plasmids and the like simply by ordering them online. You just might have to find a cartel opthamologist.

You can watch the 4-episode TV series Unnatural Selection to get a better portrait of the ‘genetic biohacker’ spirit I’m talking about. Amongst other people, they follow Aaron Traywick (who was unfortunately later found dead in a sensory deprivation tank in 2018 with ketamine in his system). His views on gene editing erred on the anarchistic side; something that could have led us all into a very interesting timeline. He had grandiose (but one must wonder if they weren’t realistic, and benevolent) aspirations on curing an array of diseases such as HIV and AIDS via gene editing. They also cover some members of the crew at The Odin (Jo Zayner, David Ishee, et al.) who share a similar sentiment. David Ishee is one of the few people/biohackers so far to undergo (self-administered!) gene therapy for ‘designer’ purposes while keeping the public in the loop: increasing follistatin levels. This is a pretty well-studied therapy in the longevity field (that includes in humans, for muscular dystrophy and the like) compared to the millions of other things one could do, so I see the rationale. He successfully did it in mice first, and then on himself, but ultimately with questionable results (at least in terms of the effect on his physique, IIRC?) for reasons unknown. Bryan Johnson recently joined the ranks, and before that there were people like Liz Parrish, who has received gene therapy for increasing follistatin, klotho, and telomerase. Or Brian Hanley, who gave himself an extra copy of GHRH as proof-of-concept for the treatment of AIDS—however that works.

Pharmacopoeia

I’ll proceed to infodump all the relevant substances in the sphere. Read the wikipedia articles, some studies, and spend a bit of time reading anecdotes, and you’ll essentially be pretty caught up with the ‘canon’.

I’ve put personal ratings next to most of the noots I’ve personally taken, but bear in mind subjectivity yields ± a star or 2; no brain/organism is exactly alike and I won’t bother trying to be context-invariant. It’s also on a bit of a relative scale: by all means, I can’t wait for the day all the five-star things I’ve listed pale in comparison to certain future therapies.

I’ve also left out a fair bit of ‘supplements’; I want to focus on compounds with more brain-selective MOAs rather than improving general metabolism/health with cognition being downstream (not that that isn’t perfectly legitimate) as well as whatever pharmeceuticals such as antidepressants where incurring a net benefit is rather far from universal (the lack of side effects is a requirement for being a proper nootropic)

9-Me-BC

Controversy around potential toxicity.

ABT-089

• α4β2 partial agonist.

  • Weak α6β2β3 partial agonist, and even weaker α7 partial agonist / α3β4 antagonist (which further helps with nicotine cessation)

• Some studies on ADHD were shown to be ineffective. Stopped at phase 2. Still may be promising for other cognitive benefits, like Improved visual working memory in nonhuman primates (delayed matching test) etc. It also significantly increases vivid/lucid dreaming.

• Administration of the nicotinic acetylcholine receptor agonists ABT-089 and ABT-107 attenuates the reinstatement of nicotine-seeking behavior in rats

• See also other studies on α4β2 partial agonists, like Ispronicline (TC1734, AZD-3480):

  • Effects of TC-1734 (AZD3480), a selective neuronal nicotinic receptor agonist, on cognitive performance and the EEG of young healthy male volunteers
    • Substantial cognitive benefit. U-shaped increase in performance with the word recall task, linear increase in digit vigilance (speed), picture recognition (sensitivity index), power of attention (speed), and quality of episodic memory.
    • Pretty much linear increase in alpha ‘centroid’ and alpha peaks. Decrease in absolute delta and theta power.

• [ABT-089: Pharmacological Properties of a Neuronal Nicotinic Acetylcholine Receptor Agonist for the Potential Treatment of Cognitive Disorders]

• ABT-089, but not ABT-107, ameliorates nicotine withdrawal-induced cognitive deficits in C57BL6/J mice

• Central nicotinic receptor agonists ABT-418, ABT-089, and (–)-nicotine reduce distractibility in adult monkeys

• Selectivity of ABT-089 for α4β2* and α6β2* nicotinic acetylcholine receptors in brain

  • ABT-089 had partial agonist activity (7-23% of nicotine) and high selectivity for α4α5β2 nAChR as evidenced by loss of activity in thalamus of α5-/- mice. ABT-089 stimulated [3H]-dopamine release (57%) exceeded the activity at α4β2 nAChR, that could be explained by the activity at α6β2* nAChR*

• NEURONAL NICOTINIC RECEPTOR AGONISTS FOR THE TREATMENT OF ATTENTION-DEFICIT/HYPERACTIVITY DISORDER: FOCUS ON COGNITION

  • ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride]: II. A novel cholinergic channel modulator with effects on cognitive performance in rats and monkeys
    • These relatively modest effects of ABT-089 under standard conditions can be increased dramatically when the monkeys perform the task in the presence of a visual distractor stimulus introduced during delay intervals. Under these conditions, ABT-089 completely reinstated normal performance: Central nicotinic receptor agonists ABT-418, ABT-089, and (-)-nicotine reduce distractibility in adult monkeys Methylphenidate is also active in the distractor model, although its effects are not as impressive as those obtained with ABT-089:
    • Administered acutely, ABT-089 only marginally improved the spatial discrimination water maze performance of septal-lesioned rats. However, more robust improvement (45% error reduction on the last training day) was observed when ABT-089 was administered continuously
      • Continuous infusion of (-)-nicotine produced comparable improvement in the spatial discrimination water maze performance of septal-lesioned rats, but a 40-fold higher dose of (-)-nicotine was required
    • ABT-089 was efficacious at 1.3 – 4.0 μmol/kg/day (in rats) but not at a higher dose of 13 μmol/kg/day, resulting in a U-shaped dose response curve
  • ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties
    • ABT-089 has efficacy comparable to nicotine in evoking ACh release from rat hippocampal synaptosomes:
    • ABT-089 is only about 70% as efficacious and 25-fold less potent than nicotine in inducing release of dopamine from striatal slices
    • In contrast to nicotine and ABT-418, which activate dopaminergic neurons in ventral tegmental area (VTA) slices, ABT-089 was inactive in this assay at concentrations up to 10 μM
  • Agonists at neuronal nAChRs can act postsynaptically to improve cognitive function but can also increase the release of a number of neurotransmitters involved in cognitive function The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory
  • Was as efficacious as nicotine and slightly more potent than nicotine in inducing ACh release from prefrontal cortex in rats after local application: [Differential cholinergic “footprints” evoked by nicotine- and the a4β2-selective partial agonist ABT-089 in prefrontal cortex]

• ABT-089, a neuronal nicotinic receptor partial agonist, for the treatment of attention-deficit/hyperactivity disorder in adults: results of a pilot study

  • Improved spatial working memory, numeric working memory, and selective attention (reducing commission errors).

• Double Dissociation of Nicotinic α7 and α4/β2 Sub-receptor Agonists for Enhancing Learning and Attentional Filtering of Distraction

  • The alpha-7 nAChR agonist PHA-543613 selectively enhanced the learning speed of feature values but did not modulate how salient distracting information was filtered from ongoing choice processes. In contrast, the selective alpha-4/beta-2 nAChR agonist ABT-089 did not affect learning speed but reduced distractibility.
  • Prefrontal α7 and α4β2 receptors show a layer-specific expression profile with stronger α4β2 expression in thalamic recipient layer VI and α7 more prominent expression in layer V, which is rich in striatal projection neurons: Layer-specific modulation of the prefrontal cortex by nicotinic acetylcholine receptors
    • 
      • In LII/III, only interneurons were activated. (and yet I see both nAChRs on the pyramidal spines?). Together, these results suggest that in the PFC nAChR activation results in inhibition of LII/III pyramidal neurons.*

• Half life of 1.7 hours, but apparently 24+ hour cognition enhancement is seen for multiple of the α4β2 partial agonists:

  • Long-lasting cognitive improvement with nicotinic receptor agonists: mechanisms of pharmacokinetic–pharmacodynamic discordance

Dose: ~2mg intranasal may be the best. It is orally bioavailable, but there are multiple anecdotes of similar oral doses not yielding comparable effects, instead needing ~8-20mg.

Random α4β2 notes

• Estradiol is a PAM. Zinc potentiates α4-containing somehow.
• Selective a4b2 Nicotinic Acetylcholine Receptor Agonists Target Epigenetic Mechanisms in Cortical GABAergic Neurons
• Stimulation is associated with Growth Hormone secretion.
  • Association of a Nicotinic Receptor Mutation with Reduced Height and Blunted Physostigmine-Stimulated Growth Hormone Release
    • People with the inactive CHRNA4 mutation Ser248Phe are an average of 10 cm (4 inches) shorter than average and predisposed to obesity
• Stimulation of dopamine release by nicotinic acetylcholine receptor ligands in rat brain slices correlates with the profile of high, but not low, sensitivity α4β2 subunit combination (i.e. stimulating α4₃β2₂)

Structure

• Can have 3 or 2 of each: α4₂β2₃$ has high sensitivity to Nicotine, lower sensitivity to acetylcholine, and a low Ca2+ permeability relative to $α4₃β2₂$ (which has another binding site)
  • So what does that even tell us about the net effect of nicotine administration? No idea really.
• Two distinct allosteric binding sites at α4β2 nicotinic acetylcholine receptors revealed by NS206 and NS9283 give unique insights to binding activity-associated linkage at Cys-loop receptors

Subtypes

• 

• Additional Acetylcholine (ACh) Binding Site at α4/α4 Interface of (α4β2)2α4 Nicotinic Receptor Influences Agonist Sensitivity in other words, just $α4₃β2₂$.

  • Notice the isoform-specific β2-β2 vs α4-α4 interfaces:
    • Agonist occupation of the α4(+)-α4(-) interface leads to channel gating. α4-α4 contains a Zn2+ site that either inhibits or potentiates depending on its concentration, whereas the β2-α4 homology is only an inhibitory site.
      • Previously: Non-Agonist-Binding Subunit Interfaces Confer Distinct Functional Signatures to the Alternate Stoichiometries of the α4β2 Nicotinic Receptor: An α4–α4 Interface Is Required for Zn2+ Potentiation
        • Zn2+ inhibition is voltage-dependent on (α4)2(β2)3 but not on (α4)3(β2)2.

Desensitization

• Regulation of α4β2 Nicotinic Receptor Desensitization by Calcium and Protein Kinase C
  • After prolonged nicotine treatment, α4β2 nAChRs accumulate in a “deep” desensitized state, from which recovery is very slow. We suggest that PKC-dependent phosphorylation of α4 subunits changes the rates governing the transitions from “deep” to “shallow” desensitized conformations and effectively increases the overall rate of recovery from desensitization. Long-lasting dephosphorylation may underlie the “permanent” inactivation of α4β2 receptors observed after chronic Nicotine treatment
  • PKC enhanced rate of recovery: α4β2 receptors containing a mutant α4 subunit that lacks a consensus PKC phosphorylation site exhibited little recovery from desensitization.
• Recovery from desensitization of neuronal nicotinic acetylcholine receptors of rat chromaffin cells is modulated by intracellular calcium through distinct second messengers
  • Desensitization of nAChRs, evoked by 2 sec focal application of nicotine, which largely raised [Ca2+]i, was not affected by intracellular application of agents that activate or depress protein kinase C (PKC) or A (PKA) or inhibit phosphatase 1, 2 A and B.

Expression

• Located on MSNs. Not sure where else it’s mostly expressed besides the stritum. Both post- and pre-synaptic.
• Higher availability of α4β2 nicotinic receptors (nAChRs) in dorsal ACC is linked to more efficient interference control
• Finds itself on GABAergic neurons in the VTA.

α4β2α5

• The α4β2α5 nicotinic cholinergic receptor in rat brain is resistant to up‐regulation by nicotine in vivo #Read

  • Suggests a regulatory role for α5 nAChR?
  • Subunit Composition and Pharmacology of Two Classes of Striatal Presynaptic Nicotinic Acetylcholine Receptors Mediating Dopamine Release in Mice
    • α5 nAChR knockout: diminished nicotine-sitmulated dopamine release
    • The β2 subunit is an absolute requirement for both classes. In contrast, deletion of β4 or α7 subunits had no effect
  • Virtually all of the α5-containing nAChRs in the rat hippocampus, striatum, cerebral cortex, and thalamus are α4β2α5 nAChRs.
  • The α5 subunit is associated in ~37% of the nAChRs in the hippocampus, ~24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus.
    • The fact that this resistance to up-regulation was seen in four different brain regions, suggests that it is the presence of the a5 subunit, rather than factors such as specific brain region or cell type that confers this property on a4b2a5 receptors in vivo.
    • Density is not even increased by chronic administration of nicotine.
  • Nicotine-induced increases in nAChRs in brain or neuronal cell lines are not accompanied by changes in subunit mRNAs, nor is de-novo protein synthesis of nAChR subunits required (Penget al.1994; Wanget al.1998).
    • Therefore, studies of the mechanisms underlying nicotine-induced up-regulation of nAChRs have focused on post-translational changes, including:
      • Increased assembly of the subunits intoreceptors (Wanget al.1998; Nashmiet al.2003)
        • The much higher level of nicotine-induced up-regulation in most transfected cells may result from the constant driving force of their constitutively active promoter leading to over-expression of nAChR subunits. Thus, the large nicotine-induced increase in nAChRs seen in most transfected celllines may reflect, to a large extent, enhanced assembly and/orincreased maturation of nascent nAChR oligomers formedfrom excess subunits and, to a lesser extent, decreased receptor degradation.
      • Decreased degradation of the receptors (Peng et al.1994;Wang et al.1998)
        • In brain, where a large excess of subunits is less likely to be the norm, this may be a more important mechanism.
          • α4β2 is perhaps rapidly regraded, while nicotine slows this, leading to upregulation (demonstrated in Peng et al.1994; Kuryatov et al.2005), and α5 may slow this down.
      • Increased maturation of nascent receptors (Kuryatovet al.2005;Salletteet al.2005)
      • Conversion of receptors from alow affinity to a high affinity conformation (Buisson andBertrand 2001; Vallejoet al.2005).

• α5 lacks a Y190 residue found in all other a subunits (Karlin and Akabas 1995), and without this residue it probably cannot contribute to an agonist binding site.

  • In fact, although α5 has the two cysteine residues at approximately positions 192 and 193 that are common to all nAChR α subunits, it has highest sequence homology with the b3 subunit (Boulter et al. 1990). Thus, the a5 and b3 subunits may represent a branch point at which these two classes of subunits diverged.
    • Interestingly, the presence of β3 in α6β2β3 provides resistance to downregulation by nicotine.

• Ca2+ Permeability of the (α4)3(β2)2 Stoichiometry Greatly Exceeds That of (α4)2(β2)3 Human Acetylcholine Receptors

  • Increased Ca2+ conductance several fold vs. regular α4β2.
    • α4, α5, and β3 subunits all have a homologous glutamate in M2 that contributes to high Ca2+ permeability, whereas β2 has a lysine at this position.
  • We show that Ca2+ permeability is determined by charged amino acids at the extracellular end of the M2 transmembrane domain

Agmatine

Rating: ★★. Helps misophonia, probably from antagonizing eNMDAR (due to being a polyamine site antagonist). eNOS giving you better pumps while antagonizing iNOS and nNOS is really unique as well. I can attest to it being somehow vaguely therapeutic for depression. It’s not too bad of a general supplement, but it’s not all good either (CB1 agonism, nAChR antagonism, PPAR, β-oxidation, etc.)

• https://men-elite.com/2020/05/22/agmatine-an-absolutely-amazing-amino-acid-for-your-whole-body/

• Oxidative stress-induced nNOS deactivation: Agmatine enhances the NADPH oxidase activity of neuronal NO synthase and leads to oxidative inactivation of the enzyme I mean, I wonder if it’s a net negative in terms of NOS activity?

• Agmatine acts as an antagonist of neuronal nicotinic receptors.

• The molecular and metabolic influence of long term agmatine consumption (Nissim et al., 2014)

ALCAR

I personally never bothered with it due to the anti-thyroid/pro-FAO and pro-cortisol effects of carnitine.

• Upregulates D1 without acute dopamine increase or D2 upregulation; ALCAR upregulating D1 increases PKA→CREB, which seems meh at first but apparently ALCAR does NOT activate ΔFosB or CDK5:

  • Repeated acetyl-l-carnitine administration increases phospho-Thr34 DARPP-32 levels and antagonizes cocaine-induced increase in Cdk5 and phospho-Thr75 DARPP-32 levels in rat striatum

• Effects of Long-term Acetyl-L-carnitine Administration in Rats: I. Increased Dopamine Output in Mesocorticolimbic Areas and Protection toward Acute Stress Exposure

Amphetamine

Rating: ★★★. I’m scared it destroys your brain and that I would become addicted, but with the few times in my life I’ve tried it, it made studying advanced topics as effortless as reading the morning paper and yet more engaging than they’ve ever been (this is not news to anyone who knows the slightest thing about adderall) so one simply cannot dock too many points. In fact I think stacking it with (afaik yet-to-exist) compounds that abolish neurotoxicity (such was the dream with antioxidants like Deferoxamine, SkQ1, and Selegiline) is a possible route to ’nootropic escape velocity’. (Related is the study of MDMA by QRI, et al. which is a whole different monster)

• https://www.astralcodexten.com/p/know-your-amphetamines
• Sensitization/upregulation of dopamine via microdosing stimulants - a viable strategy? is this real?
• Leo’s Biohacking Protocol To Make His Adderall Prescription More Effective
  • The rationale behind how amphetamines are prescribed is suboptimal and (more) unsustainable. Instant release and low doses (<10 mg, but even just a few mg is efficacious) is preferable.
• Cycle off at least weekly, adding in GDNF and PGC-1α activators to repair oxidation, and σ1 agonists.

Dextroamphetamine

Stick with this. More dopaminergic and mental without the peripheral side effects.

Methamphetamine

Long-Term Treatment with Low Doses of Methamphetamine Promotes Neuronal Differentiation and Strengthens Long-Term Potentiation of Glutamatergic Synapses onto Dentate Granule Neurons

The neuroprotective potential of low-dose methamphetamine in preclinical models of stroke and traumatic brain injury HED = 0.08 mg/kg = ~5.5mg.

Ashwagandha

Like most herbals, it does way too many things. I wouldn’t fuck with it.

ASP2905

KCHN3 inhibitor, which is concentrated in the frontal lobe. This potentiates the recurrent excitation of delay neurons via preventing K+ efflux out of the dendritic spine in the PFC, a central component of working memory (cf. Amy Arnsten’s research on the dlPFC, guanfacine, etc.):

• 

• ASP2905, a specific inhibitor of the potassium channel Kv12.2 encoded by the Kcnh3 gene, is psychoactive in mice

  • Inhibited meth-induced hyperlocomotion, but did not affect spontaneous locomotion.
  • Treats certain symptoms of Schizophrenia

• The KCNH3 inhibitor ASP2905 shows potential in the treatment of attention deficit-hyperactivity disorder (Takahashi, 2018)

  • KCNH3 overexpression in mice is associated with cognitive deficits, and knockout mice exhibit enhanced performance in attention.
  • Increased efflux of Dopamine and Acetylcholine in the Medial Prefrontal Cortex.
    • 
  • 

Anecdote: methylphendate-like headspace.

ASP4345

D1 PAM.

Atomoxetine

• NET inhibitor. The thing about this is that NET is responsible for Dopamine reuptake in the Prefrontal Cortex (where DAT expression is minimal) rather than striatum/NAcc like traditional stims.
• NMDA antagonist (to some extent). Possible μ antagonist? Yeah, I think it has some off-target effects. Liver toxic. How fun. See reboxetine instead.
• Altered gene expression in the prefrontal cortex of young rats induced by the ADHD drug atomoxetine (Lempp et al.)

Bacopa

• AChEi, ChAT activation, monoamine potentiation, lowers blood pressure.

Brilliant Blue G

P2x7 antagonist.

• Completely suppresses Amyloid β-induced neuronal death, while not affecting APP cleavage, via inhibiting the NLRP inflammasome.
• Brilliant Blue G improves cognition in an animal model of Alzheimer’s disease and inhibits amyloid-β-induced loss of filopodia and dendrite spines in hippocampal neurons (2014)
  • Amyloid β-induced loss of filopodia and spine density in cultured hippocampal neurons was prevented… BBG prevents the learning and memory impairment and cognitive deficits induced by the toxicity of soluble Aβ, and improves the development of dendritic spines in hippocampal neurons in an Alzheimer’s model mouse.

P2X7

• ATP receptor; ionotropic. Found in microglia, macrophage, retina, endometrium.

  • Requires higher ATP levels than other P2X, but response is potentiated by reducing concentration of divalent cations, as they block it.
    • Some P2X receptors rapidly desensitize ATP, while ones like P2X2 remain open as long as ATP is bound.
  • Technically, ADP and AMP are weak agonists.
  • The ionotropic ATP receptor subunits P2X1–6 receptors play important roles in synaptic transmission, yet the P2X7 receptor has been reported as absent from neurons in the normal adult brain. R

• Usually a dimer but can form heteromers with a few other P2X subunits.

• Activation leads to recruitment of Pannexin to form Panx1, a Ca2+-gated ATP export channel.

• K+ efflux→PAF→NEK7 (NIMA-related kinase), binding to and activating NLRP3.

  • Nek7 is an essential mediator of NLRP3 activation downstream of potassium efflux
  • So how the hell does intracellular potassium of all things disinhibit this binding of Hek7?

• A2A is the entry point from which cumulative sleep deprivation (ie aging/time passing) upregulates P2X7. P2X] also upregulates A2A, so it’s a positive feedback loop. ATP release from extended P2X7 activation also activates P2X7, more positive feedback loop. This whole area is a ripe target.

• P2X7 receptors induce degranulation in human mast cells

• Maternal P2X7 receptor inhibition prevents autism-like phenotype in male mouse offspring through the NLRP3-IL-1β pathway

  • Activation of the purinergic P2X7 receptors is necessary and sufficient to convert maternal immune activation (MIA) to Autism-like features in male offspring mice.

• Soluble P2X7 receptor is elevated in the plasma of COVID-19 patients and correlates with disease severity - biorxiv

• Hyperactivation of P2X7 receptors as a culprit of COVID-19 neuropathology

• P2X7 Receptors Amplify CNS Damage in Neurodegenerative Diseases

Extracellular ATP

• The Elusive P2X7 Macropore
  • Extracellular ATP causes reversible permeabilization of mammalian cell plasma membranes due to P2X7R-dependent formation of a large conductance pore (the ‘macropore’).
  • Cholesterol in Lipid Rafts inhibits P2X7R-associated permeability increases. Palmitoylation opens it.
  • Regulation of P2X Purinergic Receptor Signaling by Cholesterol
    • Defects in the C-terminal inhibited plasma expression of the receptor, probably leading to ubiquination.
    • The activation of Acid Sphingomyelinase in microglia cells was shown to be necessary for P2X7 receptor-dependent microvesicle shedding and the release of IL-1β: Acid sphingomyelinase activity triggers microparticle release from glial cells
      • This causes a positive feedback mechanism as Ceramide and other sphingolipid is then able to displace cholesterol.
      • This is not directyl related to obesity: The ATP-P2X7 signaling axis is dispensable for obesity-associated inflammasome activation in adipose tissue
    • In macrophages, the synthesis of Leukotrienes through activation of PLA2 and mobilization of arachidonic acid is required for the P2X7-mediated reduction in the parasitic load of infected cells.
• Inhibition of connexin 43 hemichannel-mediated ATP release attenuates early inflammation during the foreign body response

Neurogenesis

• As a ‘pattern recognition receptor’, its function is perhaps recognizing malformed/damaged neurons in response to extracellular ATP activity on Microglia. This is how cohesive connections are assessed during Neurogenesis. In highly neuroinflammatory states (like Alzheimer’s?) this process “feed forward”.
  • https://en.wikipedia.org/wiki/Pattern_recognition_receptor also includes TLR, and some other things (CLR, RLR, NLR/NLRP).
• The role of P2X7 receptors in a rodent PCP-induced schizophrenia model
  • P2X7 knockout in mice promotes defects in cognition and social activity. IT Doubles surface expression of NR2A and NR2B.
• Impaired interleukin-1beta and c-Fos expression in the hippocampus is associated with a spatial memory deficit in P2X(7) receptor-deficient mice

• immature neurons fire much more quickly and are more disinhibited and have a much higher p2x7 receptor expression than should mature neurons. Increased p2x7 activity should disinhibit young neurons and that would be responsible for increased plasticity in young neurons but it also contributes to cell death

• Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons

Bromantane

Rating: ★★★. Never noticed much besides an increase in verbal fluency, especially since the benefits are long-lasting.

• https://old.reddit.com/r/NooTopics/comments/11n3m3g/an_update_to_the_literature_of_bromantane/, https://old.reddit.com/r/NooTopics/comments/ufdwng/bromantane_spray_research_dump_shipping_prices/, https://old.reddit.com/r/NooTopics/comments/sfisay/a_breakdown_on_bromantane_nasal_spray/, https://old.reddit.com/r/NooTopics/comments/pyyuwz/finally_elucidating_the_mysterious_bromantane/

Its primary action is upregulation of Tyrosine Hydroxylase, AADC, and increasing GDNF (possibly via HDAC1 inhibition, extrabolating from the other adamantane-containing drugs like amantadine and memantine). PDE10 inhibitor. GAT-3 inhibitor in high doses. Possible inhibitor of Kir2.1.

• Mechanisms of Action of Ladasten: Activation of Gene Expression for Neurotrophins and Mitogen-Activated Kinases
  • This paper suggests the increase in striatum BDNF and following ERK1/2 expression underlies Bromantane’s long-term dopaminergic effects
• Time Course of Histone Deacetylase 1 and Acetylated H3 and H4 Histones in the Brain of Rats Treated with Ladasten
• Effects of ladasten on dopaminergic neurotransmission and hippocampal synaptic plasticity in rats

Bupropion

Some bad some good.

• Atypical antidepressant. NET/DAT (in high doses) inhibitor and non-competitive antagonist of α4β2, α3β4, and α1β1γδ.
  • Ki-63μΜ Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus
• 
  • Compare to dl-Methylphenidate’s DAT/NET IC50 of 20 and 51 respectively (.35 vs .39 ratio). The difference is in the metabolites as you can see.
• 
• Associated with a high risk of release of mediators from mast cells. I don’t think it activates H1 though.
• Psychopharmacology of bupropion in normal volunteers.
  • α3β4 nAChR antagonism isn’t cognitively impairing.
• [Pharmacokinetic and pharmacodynamic of bupropion: integrative overview of relevant clinical and forensic aspects]
  • Dwoskin (2006) can exacerbate depression and increase suicidal thoughts.
  • Should be administered early in the morning as it exacerbates insomnia.
  • Vivid dreams, hallucinations, unusual thoughts/behavior, confusion, tremors, agitation, anxiety, swollen glands, joint pain, increased blood pressure
• Inhibits CYP2D6 (58 mM IC50?)
• [Seizures after overdoses of bupropion intake]
  • Pronounced increase in catecholamines is the main factor, I believe. QT prolongation can be seen.
• [Alcohol significantly lowers the seizure threshold in mice when co-administered with bupropion hydrochloride]
  • In mice, 116mg/kg (660 HED) without, or 89.4 for ethanol/bupropion.
• Bupropion increases striatal vesicular monoamine transport
  • Rapidly, reversibly, and dose-dependently increased vesicular DA uptake; an effect also associated with VMAT2 protein redistribution.

• Molecular interaction of bupropion with nicotinic acetylcholine receptors
  • Where in the brain it binds is important: binds to presynaptic α4β2 on GABAergic neurons in the Ventral Tegmental Area and α3β4 nAChR in the habenulo-intrerpeduncular pathway.
  • Bnding affinity for α4 is rougly equal to α1.
• Recent Developments in Novel Antidepressants Targeting α4β2-Nicotinic Acetylcholine Receptors
• Acute effect of the anti-addiction drug bupropion on extracellular dopamine concentrations in the human striatum: An 11C raclopride PET study
  • Bupropion , 100 mg, had no effect alone but abolished the sedation and auditory vigilance impairment produced by alcohol when co-administered.

Caffeine/Coffee (Coffeine)

Rating: ★★★★. Pure Wakefulness is capable of eliciting divine euphoria and connection to the universe, and I thrive on the noradrenergic drive it gives, BUT I think the long-term effects on the brain are questionable. I am currently taking a break until further notice.

• Antagonizes the IP3 receptor, competitive antagonist of the ionotropic Glycine Receptor, and voltage-independent activator of the ryanodine receptors.
• Antagonizing Adenosine A1-D1 heterodimers increases release of dopamine in the dorsal Striatum and Nucleus Accumbens core (not shell).
• Adenosine is a vasodilator?
• Moderate inhibition of Acetylcholinesterase R R
• Increases BDNF in the Hippocampus
• Activatives Histaminergic neurons?
• Suppressess TRPA1
• Prevents or even reverses various forms of Fibrosis in the liver. R
• Reduces atherogenesis R
• Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain
• Caffeine inhibits exercise-induced increase in tryptophan hydroxylase expression in dorsal and median raphe of Sprague-Dawley rats Red Ginseng also does this. Idk mechanism
• Enhances Estrogen metabolism/detox R
  • By competing for the Cytochrome P450 oxidase system, can inhibit clearance of Estrogen from the Liver? R
• Caffeine does not entrain the circadian clock but improves daytime alertness in blind patients with non-24-hour rhythms
• Caffeine reduces low-frequency delta activity in the human sleep EEG
  • Delta waves
• Ergogenic effects of caffeine are mediated by myokines - via being an Ca2+ ionophore andor AMPK activator, enhancing secretion of Myokines.
  • Increases IL-6 in skeletal Muscle and blood but not the liver of mice. But, the IL-6 in skeletal muscle alleviates rodent model NAFLD.
• [Caffeine enhances acetylcholine release in the hippocampus in vivo by a selective interaction with adenosine A1 receptors.]
• Regular Caffeine Intake Delays REM Sleep Promotion and Attenuates Sleep Quality in Healthy Men
  • 20 male coffee consumers who reported 478 +- 102.8 mg/day: they were given 3x150 mg for 10 days, or 3x150 mg caffeine for 8 days then placebo (for the last 2 days I suppose)
  • 
• The effect of caffeine on working memory load-related brain activation in middle-aged males
  • Load-related encoding activation was greater in the DLPFC, and lower in the thalamus.
• [Adenosine A2A, but not A1, receptors mediate the arousal effect of caffeine]
  • Increased wakefulness in wild-type and A1 knockout, but not in A2A knockout.

• Chronic Caffeine Alters the Density of Adenosine, Adrenergic, Cholinergic, GABA, and Serotonin Receptors and Calcium Channels in Mouse Brain
  • Saw no A2A upregulation!
    • In platelets, neutrophils, etc. it is upregulated, but that’s different from the brain. What’s more significant is other studies on the brain.
      • [A1 and A2A adenosine receptors and A1 mRNA in mouse brain: effect of long-term caffeine treatment (1997)]
        • Upregulated A2A in the striatum
  • 5-HT1 and 5-HT2: +26-30%
  • mAChR & nAChR: +40-50%
  • Benzodiazepine-binding site: +65%, and slightly decreased affinity.
  • Density of cortical nitrendipine-binding sites in Calcium Channels: +18%.

Cerebrolysin/Cortexin

As a vegan I haven’t exactly bothered into looking into it too much, but there’s a lot of info and anecdotes on the NooTopics discord server. (This goes for most things).

It’s pretty infamous for a certain Longecity thread where he was convinced prions were giving him strange autoimmune reactions. There have been absolutely zero reports of this in the literature. The presence of prions is impossible: prions are ≥25 kDa, whereas peptides are filtered such that they don’t contain peptides >5 kDa. This single user did quite a number on cerebrolysin’s whole reputation; something truly surreal.

Citicoline

Rating: ★★. I’m super sensitive to this stuff and get existential depression from taking any dose worth its salt. It does increase concentration before that kicks in though.

One of the better choline sources.

• Intravenously injected CDP-choline increases blood pressure and reverses hypotension in haemorrhagic shock: effect is mediated by central cholinergic activation (increases Vasopressin)

• Influence of citicoline on citalopram-induced antidepressant activity in depressive-like symptoms in male mice

  • ONLY citicoline at the 5 mg/kg dose had shifted its status from being noneffective to become significantly effective in the co-administered group ~30mg HED.

Choline

Made primarily in the liver via phosphatidylethanolamine methylation.

• https://chrismasterjohnphd.com/blog/2010/12/04/meeting-choline-requirement-eggs-organs/

  • The Sweet Truth About Liver and Egg Yolks — Choline Matters More to Fatty Liver Than Sugar, Alcohol, or Fat
    • Lab rats are choline deficient. It is capable of completely protecting lab rats against NAFLD induced by sugar+fat+alcohol.
      • So, pregnant rats fed 3x their normal amount had their progeny see a lifelong 30% increase in visuospatial and auditory memory.
    • Effect of different dietary fats on choline requirement of rats (1957)
      • Saturated fats require more choline than PUFAs to prevent liver steatosis.
    • This is rather remarkable, because much lower concentrations of sucrose started spontaneously producing fatty liver disease in lab rats in the late 1970s and early 1980s once the American Institute of Nutrition set standards for purified rodent diets that relied exclusively on isolated vitamins and minerals rather than whole-food supplements like cod liver oil, yeast, and wheat germ.

• Choline Redistribution during Adaptation to Choline Deprivation

  • Deprivation is achieved by feeding PEMT-/- mice a choline-deficient diet. Lethal due to liver failure. Apparently lacking MDR2 (multiple drug-resistant protein 2) allows hepatic choline recycling!
    • The mice lacking PEMT still initiated choline redistribution.
  • Normal levels of choline-containing metabolites were maintained in the brains of choline-deficient Mdr2–/–/Pemt–/– mice for 90 days despite continued choline consumption via oxidation. Choline oxidase activity had not been previously detected in the brain. Plasma levels of choline were also maintained for 90 days, whereas plasma phosphatidylcholine levels decreased by >60%.

• By weight, the more common forms:

  • PC increases choline stores and is ~13%.
  • Alpha-GPC is more for enhancing its release in the brain, and is 40% choline by weight.
  • ‘Lecithin’ is 10-20% PC, thus like 1-2% choline.
  • Phosphatidylcholine = 15%.
  • Citicoline is ~18.5% by weight.
  • Betaine is worth something like 25% its does in choline?
    • CMJ recommends to have it be ≤50% of choline intake if you choose to supplement with it.

• I think supplementation-induced depression is possibly from excessive methylation via conversion into Trimethylglycine and onwards to DMG → sarcosine. Longecity. Another explanation is maybe overencoding of negative experience due to excessive brain ACh R

• Water-soluble choline bompounds (PChol/GPC) can enter the portal circulation of the liver intact. Lipid-soluble (PC/SPM (sphingomyelin) are either hyrdrolysed by phospholipases or enter the lymph incorporated into chylomicrons. R

Requirements

• CMJ - Meeting the Choline Requirement — Eggs, Organs, and the Wheat Paradox
  • Bear in mind only methionine can actually be used to make choline, while B6/8/12 and TMG merely spare it.
  • PEMT ctually creates homocysteine in the process of creating choline! So, if your PEMT is running smoothly, you can make your own choline, but you still need more betaine and B vitamins to neutralize the homocysteine that’s generated in the process. If your PEMT engine is working like this, however… well, then, you’ve got another problem: If PEMT isn’t using up your methionine to make choline, the methionine is just going to go further on down that pathway shown above and make more homocysteine anyway! Thus, although having a PEMT gene that Uncle Buck would surely envy might lower our choline requirement, consuming lots of methionine won’t help us at all. In fact, extra methionine just gives us more homocysteine and thereby increases our need for choline, betaine, folate, B12, and B6.
• The Chris Masterjohn calculator says I need 1124 mg Choline daily. WTF!
  • The calculator uses SNPs in the folate transporter (SLC19a1, rs1051266, G80A), the enzyme that helps make the precursor to methylfolate (MTHFD1, rs2236225, G1958A), and the enzyme that helps make methylfolate itself (MTHFR, rs1801131, A1298C and rs1801133, C677T) to develop a “methylfolate score.” This is used to calculate how much choline you should aim for.
  • It then uses a polymorphism in the enzyme that helps make phosphatidylcholine, a specific form of choline that protects against fatty liver disease, promotes gall bladder health, and facilitates healthy digestion of dietary fats and absorption of fat-soluble vitamins (PEMT, rs7946, 5465G>A). This is used to tell you the likelihood that missing your choline requirement will hurt these functions in your body.

Clausenamide

• The anti-dementia drug candidate, (−)-clausenamide, improves memory impairment through its multi-target effect (2016)
  • Mild elevation of intracellular Ca2+ concentrations -> neuron survival + outgrowth & antagonism of neural apoptosis induced by growht factor deprivation.
    • Intracellular Ca2+ levels are mainly regulated by the following factors: (1) extracellular Ca2+ influx into the cytoplasm, (2) Ca2+ release from intracellular stores, (3) Ca2+ clearance via either outflow from the cytoplasm to the extracellular space or accumulation into internal Ca2+ stores, and (4) cytoplasmic Ca2+ buffering.
  • Modulation of the cholinergic system
    • In vitro promotion of ChAT in frontal cortex neurons -> suppored cholinergic neuron survival + neurite outgrowth & synaptosomal ACh release.
    • In vivo: Increased ChAT in the neocortex, hippicampus, and sitratum: [Effects of (-), (+)clausenamide on anisodine-induced acetylcholine decrease and associated memory deficits in the mouse brain (1998)]. reversibly inhibited ACE with much lower potency than inhibitors like galantamine.
  • Upregulation of synaptic plasticity
    • There are two forms of LTP in the hippocampus: N-Methyl-D-aspartate-(NMDA-) or VGCC-dependent LTP. Clau induced the latter. LTP is not inhibited by nimodipine, indicating VGCC (potentiation) is only necessary for induction but not maintenaince.
    • Did not bind to NMDAR, indicating it is NMDAR independent: [Effects of (-), (+) clausenamide on central N-methyl-D-asparate receptors in rodents (1997)].
    • APV, an NMDAR blocker, had no effect on clau-induced LTP: [Two forms of long-term potentiation induced by different compounds (Xu et al 2007)]
    • However, chronic administration of clau promoted the expression of synaptic NMDA receptors:
      • (1) increasing NMDA receptor density in synaptic membranes,
      • (2) increasing NMDA receptor affinity to their endogenous ligands as indicated by the (-)-clau-increased Bmax values of NMDA receptors in the synaptic membrane, and
      • (3) ameliorating oxidative stress-induced synaptic membrane fluidity, which facilitates NMDA receptor turnover in synaptic membrane.
    • Increased mossy fiber sprouting and expression of GAP43
  • Activation of cellular and molecular signaling pathways involved in learning and memory.
  • Inhibits Tau hyperphorsphorylation and Amyloid β-induced intracellular Ca2+ overload.
    • APP mice are generated by overexpression of the mutant APP gene and are characterized by senile plaque overload and related apoptosis in the central nervous system.
    • There’s a Chinese study out there where it improved symptoms in human Alzheimer’s patients - 400mg/week, once a week.
    • Anti-apoptotic in five regards:
      • (1) low potassium in cerebellar granule cells, (2) growth factor deprivation in cortical neurons,
      • (3) 6-OHDA in high BAXα-expressing PC12 cells,
        • Inhibited BAX-α-induced cytochrome C release, possibly by increasing glutathione content?
      • (4) ischemia/reperfusion in rats, and (5) Aβ1-40 infusion- and natural aging in rat brain
      • Inhibits expression of p53, c-Myc, etc.
      • PKC-MEK negatively regulates GSK-3β (-> Tau hyperphosphorylation.) Akt and mitogen-activated protein kinase enhance C-type lectin-like receptor 2-mediated platelet activation by inhibition of glycogen synthase kinase 3α/β (CLEC-2 and GPVI (potentiated by AKT/MAPK:) activate platelets through Src, Syk, and PLCγ-2)
      • And Clau surely increases PKC, which was shown in Xu 2005. Clau-induced microtubule protection was at least in part mediated by PP1, responsible for tau dephosphorylation.
  • They go relatively in-depth regarding its chemistry. Its nootropic effects are chirality-dependent. Clau was more potent than piracetam (5-10mg/kg vs. 500mg/kg respectively) for improving performance in memory-impaired animals.
• Previously, Recent advances in the study of (–)clausenamide: chemistry, biological activities and mechanism of action (Oct 2014)
  • NGF & BDNF induce small elevations of Ca2+ in neurons
  • Shown to be a potassium channel antagonist (preventing efflux) and inducing membrane depolarization.
• (−)Clausenamide facilitates synaptic transmission at hippocampal Schaffer collateral-CA1 synapses (2012)
  • Calcium release from endoplasmic reticulum is mediated by two main types of receptors: RyRs and IP3-Rs. Ryanodine blockage of RyR suppressed synaptic facilitation, whereas IP3 blockage showed no effect, suggesting even PLC might not even be involved.
  • CAMKII dependent.
  • PKA inhibitor: no effect.
  • So it’s turning out to be a slightly exotic cascade, I guess. VGCC -> RyR -> CAMKII -> CREB -> Egr1, BDNF, etc. - CAMKII -> p-ERK peaked 5 and 30 minutes in hippocampus and cortex respectively. p-nCREB 9 minutes after.
• Study on the Nootropic Mechanism of (-)Clausenamide - Influence on the Formation of Synapses in Mouse Brain (Jiang & Zhang (1998/2006)
  • Increased cortical thickness by 10% in 4 weeks.
    • Gintrux links this for demonstrating cortical thickness and intelligence: Cognitive ability changes and dynamics of cortical thickness development in healthy children and adolescents (2013)
    • Could be due to intracellular Ca2+ influencing neuronal cytoarchitecture.
  • 29% growth of synapse density in a region of the CA3.
• ()-Clausenamide Potentiates Synaptic Transmission in the Dentate Gyrus of Rats (Xu et al 2005)
• Physiological signature of a novel potentiator of AMPA receptor signalling (Szulc et al., 2018) not even chinese
  • We have synthesized a novel small molecule based on the pyrrolidinone–containing core structure of Clausenamide.: BRS-015
  • Overall quite similar to clausenamide; no NMDAR effects, chirality, etc. They hypothesize AMPA potentiation is via CAMKII -> γ-8.
  • Potentiated inward currents evoked by local application of l–glutamate onto CA3. It facilitated the induction of mossy fibre LTP, but the magnitude of potentiation was smaller than that observed in untreated slices.
  • Asymmetrical synapses between large mossy fibre terminals and thorny excrescences in CA3 pyramidal neurons contain an average number of AMPA receptors exceeding 4 times the number reported for C/A synapses.
    • [Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain]
    • Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus (1998)
  • C/A (not CA) synapses can be void of AMPAR, while mossy fibre synapses have smaller variability.
  • High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus
    • Higher GluR1 expression at A/C (associational/commissural synapses) synapses, compared to mossy fibre synapses which have subtypes more equally.
      • Well that’s a whole other rabbithole.
• [Activation of ERK1/2-CREB pathway during potentiating synaptic transmission of (-)clausenamide in rat dentate gyrus. (2012)]
• Increases Choline Acetyltransferase.
• Gintrux: The cortex begins to thin after the age of five or six as part of the normal aging process. This study is the first to show the association between cortical thickness and development in full scale IQ, and has potentially wide-ranging implications for the pedagogical world and for judicial cases in which the defendant’s IQ score could play a role in determining the severity of the sentence
  • Could this be a correlate for the cut-off point for development intelligence in early life?

Cordyceps Militaris

Rating: ★★. Noticeable increase in endurance, but I think its long-term effects on D2, adenosine, and lovastatin content are questinoable.

D21

aka TAT-D21.

• The heteromer flips the relationship between dopamine concentration and which receptor gets stimulated: D1 gets active in low concentrations and D2 in high cincentrations, contrary to norm. Though I’ve also heard that it essentially just nullifies dopaminergic signaling, and that splitting the dimer essentially ‘differentiates’ D1 and D2 and allows much more flexible LTP and LTD that is seen in adolescence; D21 increases with age.

• D21 is much more prevalent (~250% d21/d1 ratio compared to control) in those with Depression. Naturally then, the peptide has standard antidepressant effects. ==Induces calcium release by being Gq-coupled== somehow. R. Many studies will measure D1 or D2 individually, and they do no such thing. Only co-expressive cells.

  • Probably driven by D1: certain in vitro thingies resulted in PIP turnover. Possibly from high concentrations activating another receptor. R

• Differential receptor crosstalk in DRD1-DRD2 heterodimer upon phasic and tonic dopamine signals (May 2021) & Visualization of differential GPCR crosstalk in DRD1-DRD2 heterodimer upon different dopamine levels (June 2022) looks like a refined version if biorxiv is for drafting or something

  • High dopamine levels preferentially make the heteromer inhibit D1 signalling, whilst low dopamine levels make the heteromer inhibit D2 signalling:Upon treatment of 10 nM dopamine, D21 heterodimer induces a similar level of cAMP to D1 only (possibly because D2 activity in the heterodimer is inhibited at this nanomolar DA level) In contrast, at 10 µM dopamine, the cAMP level by D21 was significantly lower than the one by D1.

• Intranasal Delivery of a Peptide with Antidepressant-Like Effect (Brown & Liu 2014)

  • It doesn’t split the peptide or anything, or change protein levels, (idk, maybe Cogmetics’ does?) but simply prevents the interaction between D1 and D2 at the dimer.
  • Lowered locomotor activity. Thus it is not a stimulant. However it is anti-immobility - they swam more. Yeah idfk.
  • Elevates BDNF in the Prefrontal Cortex, and decreased levels are seen in major depressive disorder.
    • However, Dopamine D1-D2 receptor heteromer signaling pathway in the brain: emerging physiological relevance (Hasbi et al. 2011) suggests that D21 activation could result in transcription of BDNF in cultured neurons derived from the ventral striatum.
      • There is “requisite” D1/D2 synergism for certain reward processes:
        • Striatal Fos expression is indicative of dopamine D1/D2 synergism and receptor supersensitivity.
        • Concurrent activation of dopamine D1 and D2 receptors is required to evoke neural and behavioral phenotypes of cocaine sensitization !!

• There are also D1-D3 and D2-D5 heterodimers, as well as D1-NMDAR and D5-GABA-A heterodimers.

  • Direct interactions between NMDA and D1 receptors: a tale of tails (Lee & Liu 2004)
  • Regulation of dopamine D1 receptor function by physical interaction with the NMDA receptors (Lee & Liu et al. 2004)

• Δ-Tetrahydrocannabinol Increases Dopamine D1-D2 Receptor Heteromer and Elicits Phenotypic Reprogramming in Adult Primate Striatal Neurons BUT CBD attenuated most of this.

• Daily THC and withdrawal increase dopamine D1-D2 receptor heteromer to mediate anhedonia and anxiogenic-like behavior through a dynorphin and kappa opioid receptor mechanism

• Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer

• Differential receptor crosstalk in DRD1-DRD2 heterodimer upon phasic and tonic dopamine signals

• Dopamine D1-D2 receptor heteromer expression in key brain regions of rat and higher species: Upregulation in rat striatum after cocaine administration (Hasbi et al. 2020)

  • Bam says the Orbitofrontal Cortex is possibly receiving translocated d21, from areas where the difference in mRNA and presence is the inverse, such as the Olfactory Tubercle.

Calcium

• Perhaps it’s been known well that D1 activates phospholipase C? D2 agonists potently enhance AA release, initiated by increasing intracellular Ca2+ or stimulating constitutive purinergic receptors - which trigger Ca2+. Naturally, all this stimulates phospholipase A2. R.
• Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth
  • CAMK IIα increases BDNF expression.
• Rats have a lower prescence and importance of the heteromer in their brains than humans. The further up in intelligence you go across species, the more prevalent. It implies that the heteromer counteracts brain power/force in some way, and it does play the role of a negative dopamine regulator (it lowers dopamine transmission in general by lowering dopa release). In theory, society is like a Swiss army knife in making the d1/d2 heteromer appear. (wtf? lol stoner.)
• Dopamine D1 and D2 Receptor Co-activation Generates a Novel Phospholipase C-mediated Calcium Signal (Lee et al. 2004)
  • Coexpression barely changes adenylyl cyclase activity in these cells compared to D1 alone. PKA, PKC, or PI3K inhibition did not change calcium levels, but PLC inhibiton shut it down >90% (and blocked Gq-coupled P2Y receptors along the way).
  • Gi/o is not directly involevd in the calcium signal. Gi and Gq crosstalk does not appear to underlie, since neither couple to Gq to begin with. Good reasoning?
  • Virtually all Medium Spiny Neurons that express D1 also technically express D2.
  • Coexpression did not affect the ligand binding pocket of either receptor.
  • In the rat frontal cortex, interneurons only expressed D2, unlike pyramidal neurons.
  • Heteromers of the CCR2 and CCR5 chemokine receptors and heteromers of the μ- and δ-opioid receptor may couple to G proteins distinct from those associated with homogeneous populations of their constituent receptors, but interestingly the cellular responses is not different from that of the individual receptors.
• Dopamine receptor-mediated Ca(2+) signaling in striatal medium spiny neurons (Tang et al. 2004)
  • ~40% MSN elicit robust repetitive Ca2+ oscillations following application of dopamine - PLC-dependent.

Deferoxamine

Rating: ★. Didn’t notice anything. See: Scattered Notes: Deferoxamine

Dihexa

High affinity to hepatocyte growth factor and potentiates c-Met, the protein it encodes.

Pretty controversial. It’s supposedly 7x more potent than BDNF for improvint alzheimer’s-like cognitive impairment. This sounds awesome to a lot of people but it really dosen’t help.

• Dihexa is not bdnf
  • Bam says Dihexa mainly works in the sensory systems, but notes it’s weird how the cortical systems increase autistic symptoms from being given different sensory information?

Donepezil

• Developed under the ‘cholinergic hypothesis’ of Alzheimer’s, i.e. over-activation of ACE, which isn’t always the truth let alone the whole story.
• Acetylcholinesterase inhibitor and σ1 agonist (14.6 nM).
  • Non-competitive inhibitor, unlike galantamine
• Inhibits voltage-activated Sodium Channel currents and delays Kir currents and fast transient potassium currents.
• [Mechanisms of alpha7-nicotinic receptor up-regulation and sensitization to donepezil induced by chronic donepezil treatment.]
  • Upregulates α7 nAChR in the hippocampus. This was prevented by coadministration with PI3K inhibitor.
• Donepezil impairs memory in healthy older subjects: behavioural, EEG and simultaneous EEG/fMRI biomarkers
• Donepezil modulates nicotinic receptors of substantia nigra dopaminergic neurones
• Modulates α4β2, I think.
  • Depressed Nicotine currents that were in vitro “induced by brief puffer pulses, through a glass micropipette positioned above the slice”.

D-Serine

Obsoleted by Neboglamine. Sarcosine is also a safer option.

• Chronic d-serine supplementation impairs insulin secretion

• Antibiotic, and not in a good way; apparently it kills important microbes involved in sugar metabolism.

• Metabolism by DAAO (highly expressed in the kidney) produces pyruvate, ammonium, and hydrogen peroxide. Not fun.

Erythropoietin

• https://www.leoandlongevity.com/blog/categories/epo-for-the-brain

There’s demonstrated proof that engaging in cognitive work increases the long term potentiation or the “connections between neurons” through a variety of mechanisms and models that test this are called “environmental enrichment” within the literature. Increasing the load or demand that we place on our neurons when we learn strengthens the connections between neurons as well as neuronal morphology through induction of functional hypoxia.

• Introducing neuroepo:
  • Nasal administration of the neuroprotective candidate NeuroEPO to healthy volunteers: a randomized, parallel, open-label safety study n=25 and 20% them got headaches and 20% saw increase in liver enzymes. They call that ‘well tolerated’.
  • The Effect of Neuroepo on Cognition in Parkinson’s Disease Patients Is Mediated by Electroencephalogram Source Activity
• Erythropoietin: a candidate compound for neuroprotection in schizophrenia showed 5,000 IU/kg in mice injected ip.
• Emerging biological roles for erythropoietin in the nervous system
  • 
    • JAK2-STAT is found on the C-terminus of Erythropoietin Receptor (and βCR)
• Erythropoietin Enhances Hippocampal Response during Memory Retrieval in Humans
  • In healthy humans, 40,000 IU rhEPO IV increased the hippocampal response during memory retrieval 1 week later!
• Erythropoietin enhances hippocampal long-term potentiation and memory (in young healthy mice)
  • This selective improvement was maintained for an EPO treatment-free period of another 3 weeks
  • It exerts hematopoieis-independt effets on the NS. Maybe this was obvious but I assumed it was all by proxy of something to do with hematology at least.
    • Derivatves like CEPO (carbamoylated EPO) exert similar neuroprotective properites, but not erythropoietic effects. Epobis counts too I’m sure.
  • Made some interesting electrical changes in CA1 slices, especially quite a huge drop in short-term depression fEPSP slope.
  • Decreased amplitude and frequency of spontaneous EPSC in CA1 and increased SIPSC frequency - 50%?
  • Did not reveal differences in epression of synaptic proteins or postsynaptic receptor proteins and their subunits.
  • Evoked excitatory postsynaptic current (EPSC) amplitudes in EPO-treated neurons were reduced to about 60% of control in these autapic neurons… why did they choose them..? No diference in NMDA/AMPA ratio.
  • Synaptic boutons per neuron increased, but reduces the amount of primed vesicles? EPO is likely to reduce the number of active synapses without altering total synapse number
  • Hematocrit seems irrelevant. In our study, the hematocrit was already back to control levels when we still observed a significant effect on
• Expression of constitutively active erythropoietin receptor in pyramidal neurons of cortex and hippocampus boosts higher cognitive functions in mice
  • Constitutively active = no need for agonist.
• Effects of recombinant human erythropoietin on cognition and neural activity in remitted patients with mood disorders and first-degree relatives of patients with psychiatric disorders: a study protocol for a randomized controlled trial
• Differential effects of erythropoietin on neural and cognitive measures of executive function 3 and 7 days post-administration
• Erythropoietin improves operant conditioning and stability of cognitive performance in mice
  • It is not produced solely by the kidneys, but is also produced in astrocytes. In line with renal production, hypoxia enhances it here, too.
    • Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes: they did not directly stimulate it, rather they increased mRNA level.
  • They did the 5CSRTT (Five Choice Serial Reaction Time Task). It was consistenyl superior in like every measure.
    • α7 nAChR knockout exhibits defecit in 5-CSRTT. R
• Acute and chronic elevation of erythropoietin in the brain improves exercise performance in mice without inducing erythropoiesis
  • The increase in maximal exercise performance is independent of changes in total hemoglobin mass, whole blood volume, and cardiovascular parameters. Also greater self-esteem and mood/euphoria.
  • Tg21 transgenicity overexpresses rhEPO independent of oxygen.
    • Effects of erythropoietin administration on cerebral metabolism and exercise capacity in men
      • 3 days of high-dose NO effect in healthy subjects on exercise capacity or cognition.
• Recombinant erythropoietin improves cognitive function in chronic haemodialysis patients
  • Adjusting for control, their WAIS IQ increased by about 6 points.
• Prolonged astrocyte-derived erythropoietin expression attenuates neuronal damage under hypothermic conditions
• Regulation of muscle and metabolic physiology by hypothalamic erythropoietin independently of its peripheral action
  • While blood EPO raises with aging and obesity, hypothalamic EPO decreased.
  • Aged mice were chronically treated with EPO in the hypothalamic ventricle, showing an increase in lean mass, while body weight and fat mass decreased as a result of a moderate reduction of food intake. Both muscle and metabolic functions were improved.
• Intranasal erythropoietin therapy in nervous system disorders

Neurogenesis

• One of the most promisingly yet somehow inconspicuously framed studies on cognitive enhancement I’ve seen Introducing the brain erythropoietin circle to explain adaptive brain hardware upgrade and improved performance
  • rhEPO (recombinant human) treatment of young rodents or EPO receptor (EPOR) overexpression in Pyramidal Neuro]s caused remarkable and enduring cognitive improvement, together with enhanced LTP. The ‘brain hardware upgrade’, underlying these observations, includes an EPO induced ~20% increase in Pyramidal Neurons and Oligodendrocytes in CA hippocampi in the absence of elevated DNA synthesis.
  • Reduces Microglia numbers and dampens their activity as prerequisites for undisturbed EPO-driven differentiation of pre-existing local neuronal precursors.
  • It’s extremely potent and thus very sparse and hard to measure its relative expression.
• Erythropoietin protects primary hippocampal neurons increasing the expression of brain-derived neurotrophic factor
• Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus (2016)
  • Similar group to the study below.
  • BrdU labels mitotic brain cells I guess.
    • At week 4 we found a reduction in BrdU+ cells in DG/SVZ, likely as a consequence of temporary depletion of progenitors or negative feedback on neurogenesis.
    • Increased apoptosis, potentially because of abnormal crowding of maturing neurons.
  • Healthy young mice (starting at 4 weeks old or 11 weeks old) 3-week EPO administration i.p. (5000IU/kg = HED ~27k) every other day.
  • Increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under ’enduring cognitive challenge’, they were preserved. This is concomitant with an increased hippocampal volume. in the 11->14 week mice.
    • They of course both increased, in similar amounts, because new axons need to be myelinated.
    • OPC = olgodendrocyte precursor cells. Can differentiate into them without dividing.
  • this EPO effect involves stimulation of precursor differentiation rather than proliferation or anti-apoptosis
    • Decreased number and diameter of ’neurospheres’, culture of clustered NSCs. Inhibitory of their proliferation.
  • Pax6 and doublecortin-positive cells (i.e. immature) were reduced after 3 days compared to control. This may imply that the increase in neurons was due to accelerated differentiation of neural progenitors.
  • They used leucine incorporation as a marker of protein synthesis sans proliferation. And it actually increased ~20%.
• Functional hypoxia drives neuroplasticity and neurogenesis via brain erythropoietin (2020)
  • High-ose EPO amplifies auto-/paracine EPO/EPOR signaling prompting emergence of new CA1 neurons and enhanced dendritic spine densities.
• Matrix metalloproteinase 2 (MMP-2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration
  • EPO activates endothelial cells, which promote endogenous neuroblast migration by secreting MMPs that degrade extracellular matrix (ECM) components.
• Regeneration in the nervous system with erythropoietin
  • Relies on WISP1 signaling to foster stem cell survival and to block FOXO activity. Relies on mTOR for induction of plasticity/differentiation/etc.
  • In contrast to its mTOR activation I suppose, in some conditions it increases AMPK and auotphagy activity.
  • EPO increases WNT signaling expression.
    • +Wnt3 expression: Survival, neuron-like differentiation and functionality of mesenchymal stem cells in neurotoxic environment: the critical role of erythropoietin
      • NGF levels remained the same, contrary to hypothesis. LIF (type of IL-6) was upregulated in hypoxia and normoxia.
  • Expression is increased by cytokines like TNF-α, IL-1β and IL-6. Stimulated by Selenium depletion, xenon, and microglial inhibitors.
    • Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes
      • Stimulatory effect of IGF-1/2 on EPO was not affected by oxygen concentration, obviously unlike its normal induction from hypoxia.

Epobis

• Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects
• A new agonist of the erythropoietin receptor, Epobis, induces neurite outgrowth and promotes neuronal survival

Galantamine

I don’t believe it has tolerance, but it and other rapidly irreversible AChEis upregulate AChE with long-term use: Acetylcholinesterase and its inhibition in Alzheimer disease

Ginkgo Bilboa

Rating: ★★. I don’t really notice anything (Swanson brand. Maybe I’ll try Ginkgold one day).

• Ginkgo biloba extract (EGb761) influences monoaminergic neurotransmission via inhibition of NE uptake, but not MAO activity after chronic treatment
  • Inhibits NET, SERT, DAT, and in high concentrations, MAO. After 14 days, only NET is significantly decreased.
• Terpene Trilactones from Ginkgo biloba Are Antagonists of Cortical Glycine and GABAA Receptors (2003)
  • Contains Bilobalide, a Glycine Receptor antagonist.
  • Role of glycine receptors and glycine release for the neuroprotective activity of bilobalide (2008)
• GABA-A NAM. Apparently it upregulates GABA-A after 1-2 weeks.
• Very weak Acetylcholinesterase inhibitor. Inhibits NOS. Blocks Aldosterone.
• Strongly potentiates α1 Glycine receptors.
• Upregulates GABA-A5 or some shit?
• In rats it may inhibit MAO-B but apparently this has not been demonstrated in humans.
• Blood thinner via PAF (platelet-activating factor) inhibition.
• Contains Amentoflavone.
• A single amino acid determines the toxicity of Ginkgo biloba extracts
  • their lack of toxicity in humans is not in doubt. These extracts are, however, highly toxic to insects.
  • Insect GABA receptors contain Ala at their 2’ position in the pore. Substitution with Val, which is the equivalent residue in vertebrate GABA-A receptor α-subunits, decreases ginkgolide potency by up to 10,000-fold.
• Ginkgo biloba extract promotes osteogenic differentiation of human bone marrow mesenchymal stem cells in a pathway involving Wnt/β-catenin signaling
• Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide
  • Inhibits PDH, SDH, SUCLG, CS
• Stabilization of mitochondrial function by Ginkgo biloba extract (EGb 761)
• https://raypeatforum.com/community/threads/ginkgo-biloba-is-anti-serotonin-and-pro-dopamine.34051/post-520338
  • Ginkgo biloba normalises stress-elevated alterations in brain catecholamines, serotonin and plasma corticosterone levels
  • The Ginkgo biloba extract EGb 761® and its main constituent flavonoids and ginkgolides increase extracellular dopamine levels in the rat prefrontal cortex
• Polysomnographic effects of adjuvant ginkgo biloba therapy in patients with major depression medicated with trimipramine
• Good for bones:
  • Ginkgo Biloba improves bone formation during fracture healing: an experimental study in rats
  • Effects of the extract of Ginkgo biloba on the differentiation of bone marrow mesenchymal stem cells in vitro
  • Ginkgo biloba extract promotes osteogenic differentiation of human bone marrow mesenchymal stem cells in a pathway involving Wnt/β-catenin signaling
  • Potential antiosteoporotic effect of ginkgo biloba extract via regulation of SIRT1-NF-kB signaling pathway
• Gingkgolic acid is a toxin. Extracts aim to achieve a minimum concentration of this. Life Extension may be the lowest according to Odder_Sea.
• Action Mechanism of Ginkgo biloba Leaf Extract Intervened by Exercise Therapy in Treatment of Benign Prostate Hyperplasia
  • I don’t get this experimental model, tbh. They applied T and E2 to these rats.
  • Ginkgo alone had no effect on T, 5α-reductase, and dihydrotestosterone (DHT), but suppressed androgen receptor (AR), aromatase, E2 and estrogen receptor (ER), and the proliferating cell nuclear antigen (PCNA); Ex alone significantly reduced T, aromatase, E2, ER, AR, and PCNA, but highly raised DHT. While Ginkgo + Ex androgenically downregulated T, aromatase, E2, and ER, but upregulated DHT, AR, and PCNA, implying Ginkgo + Ex tended to worsen BPH.
• Ginkgo biloba and cerebral bleeding: a case report and critical review
  • We report the case of a young woman who made chronic use of G. biloba and suffered from cerebral bleeding without any structural abnormalities.

The anecdotes: improves focus and (long-term) memory.

Guanfacine

Rating: ★★. It’s not for me—but I can see it being very helpful for some. When you read about people being prescribed Intuitiv (guanfacine XR), etc. they’ll describe it as removing an element of anxiety, and that removing sudden drives to action makes it easier to focus. But I’m on the other side of the coin; having that ‘push’ is totally how I’ve learned to work.

• Scientific rationale for the use of α2A-adrenoceptor agonists in treating neuroinflammatory cognitive disorders (Arnsten et al. 2023)
  • α2A agonists deactive macrophages; should reduce the release of kynurenine and GCP-II.
  • 
• Guanfacine’s mechanism of action in treating prefrontal cortical disorders: Successful translation across species (Arnsten 2007)
  • In classic circuits, such as in the visual cortex PKA (and therefore increased PDE4 activity) enhances plasticity. Well welcome to opposite world.
  • 
    • Notice the omission of RER-mediated signaling in ‘classical’ circuits. Why though?

• Effects of acute and sub-chronic administrations of guanfacine on catecholaminergic transmissions in the orbitofrontal cortex
  • Sub-chronic (7 days iirc) systemic administration:
    • Reduced norephinephrine release in OFC, LC, and thalamic reticulra nucleus.
    • Reduced GABA release in Medial Dorsal Nucleus
    • Enhanced AMPA-induced glutamate, NE, and DA release in OFC.
  • Obviously subchronic is the name of the game, but checking what the acute effects are could be relevant for learning how long benefits take to kick in.
  • **Attenuation of direct noradrenergic LC-OFC transmission at the resting stage and enhancement of direct co-releasing catecholaminergic LC-OFC transmission via GABAergic disinhibition in the intermediate LC-OFC pathway. **
  • Inhibiting HCN (via (guanfacine) inhibiting cAMP or blocking HCN directly) (which colocalizes with α2A) activates limbic-frontal cortex network connectivity, enhancing signal-to-noise ratio and improving focus on a particular stimulus:
    • Alpha2A-adrenoceptors strengthen working memory networks by inhibiting cAMP-HCN channel signaling in prefrontal cortex (Wang, Arnsten et al. 2007)
      • Commentary: Molecules to remember
      • Guanfacine, But Not Clonidine, Improves Planning and Working Memory Performance in Humans
        • The 29 μg/kg dose of guanfacine improved spatial working memory and planning… had no effect on attentional set-shifting.
          • An attentional set is formed when a subject learns that a set of rules can be applied to complex stimuli in order to differentiate relevant from irrelevant cues. Two stages within the AST protocol measure aspects of cognitive flexibility: reversal and the extra-dimensional shift. At the reversal stage, the previously negative stimuli within one dimension (medium in this example) is now positive. At the extra-dimensional shift stage, when the irrelevant dimension (odor in this example, perhaps as opposed to visual) becomes the relevant dimension. R
            • *The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents..
        • Clonidine is nonselective for α2 Adrenergic Receptor.
      • α2A blockade profoundly impairs spatial Working Memory. R (using yohimbine)
      • Recent studies indicate that elevated cAMP signaling in PFC impairs behavioral measures of WM. In contrast to LTM obviously.
• Chronic Administrations of Guanfacine on Mesocortical Catecholaminergic and Thalamocortical Glutamatergic Transmissions
  • chronic guanfacine administration did not affect intrathalamic GABAergic transmission, but it phasically enhanced thalamocortical glutamatergic transmission
  • Acutely: postsynapic LC→OFC/VTA efferents reduces noradrenergic neurotransmission.
  • Proposed downregulation of α2A in the LC

• Chronic stimulation of alpha-2A-adrenoceptors with guanfacine protects rodent prefrontal cortex dendritic spines and cognition from the effects of chronic stress (Hains, Arnsten et al. 2015)
• Guanfacine as an alpha-2-agonist inducer of growth hormone secretion—a comparison with clonidine
• Role of Alpha2a-Adrenergic Heteroreceptors in Stress-Induced Reinstatement of Cocaine Associated Behaviors: Implications for the Pharmacological Treatment of Stress-Driven Relapse of Drug Use
  • (Along with α2A of course), guanfacine also activates excitatory Gi-coupled heteroreceptors in the bed nucleus of the stria terminalis (BNST). I think α2A-x heteroreceptors, whatever they are
    • Okay so basically α2A can act as a typical autoreceptor, but it can also act as a ‘heteroreceptor’. mGluR3 also doies this.
    • A key brain region in driving stress-induced relapse.

dlPFC

https://beta.nootropicswiki.org/article/dlpfc-modulation

• Stress signalling pathways that impair prefrontal cortex structure and function (Arnsten 2009)

  • So the bad thing about Ca2+ in the dlPFC microcircuits is that it opens SK channels.
  • 
  • 
  • 

• Neuromodulation of Thought: Flexibilities and Vulnerabilities in Prefrontal Cortical Network Synapses (Arnsten et al. 2012)

  • Dynamic network connectivity = neuromodulators rapidly and flexibly altering efficacy of synaptic connections (in the dlPFC III recurrent excitatory microcircuits, of course) without changing the architecture.
    • Dynamic Network Connectivity: A new form of neuroplasticity (Arnsten et al. 2010)
      • neuromodulation plays an essential role in shaping the contents of our “mental sketch pad”, thus coordinating arousal state with cognitive state.
        • Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey
          • Depletion of catecholamines from the dlPFC was as detrimental as ablating the dlPFC itself!
    • Genetic insults to DNC proteins are commonly linked to Schizophrenia.
      • Impairment of NMDA for sure
  • Gives us a walkthrough on Goldman-Rakic’s findings from start to finish.
    • dlPFC neurons can represent visual space in the absence of sensory stimulation, the foundation of abstract thought—beautifully put
      • this basic representational operation is the building block of more complex dlPFC operations such as behavioral inhibition and cognitive control The prefrontal landscape: implications of functional architecture for understanding human mentation and the central executive (Goldman-Rakic 1996)
    • Intrinsic circuit organization of the major layers and sublayers of the dorsolateral prefrontal cortex in the rhesus monkey Layer V also exhibits all this stuff, but to a lesser extent.
    • It is likely that most Response cells reside in layer V, as they are selectively influenced by D2
    • Layer V neurons bear the majority of mRNAs encoding the five distinct dopamine receptor subtypes in the primate prefrontal cortex
      • Thus, levels of D1 and D2 mRNAs are noticeably stronger in the striatum than in the cortex, whereas D4 and D5 expression is clearly higher in the cortex. D3 transcripts are equivalent
  • Ramón y Cajal: In mice the basal dendrites [of pyramidal cells] are short and have few branches, in man they [the basal dendrites] are numerous, long and highly branched……as one ascends the animal scale the psychic cell becomes larger and more complex; it’s natural to attribute this progressive morphological complexity, in part at least, to its progressive functional state
  • Patricia Goldman-Rakic A Remembrance (Arnsten 2003) and similar work of hers in realation to Goldman-Rakic. Arnsten has many
    • Delay cells are usually spatially tuned, firing across the delay period for the neuron’s preferred direction, but decreasing firing for all other nonpreferred directions (the preferred direction for this Delay cell is indicated by a red asterisk). The microcircuits underlying Delay cell firing reside in deep layer III (and possibly in layer V as well) and are described in detail in the text. In contrast to Delay cells, Response cells are often inhibited during the delay period and instead fire leading up to, during, and/or after the motor response, initiating action and/or providing feedback. These neurons are thought to reside in layer V.
  • Unlike other areas in the cortex where LTP/CREB induces rapid changes in dendritic morphology, layer III dlPFC neurons remain long and thin, even in very old monkeys.
    • This geometric shape facilitates the rapid gating of synapses via ion channel opening, likely by limiting the spine’s volume and extending the distance that the signal must travel.
      • E.g. something histological: The spine neck filters membrane potentials
  • even mild pressure can impair the ability to find insightful solutions to problems A brain mechanism for facilitation of insight by positive affect
    • *positive mood enhances insight, at least in part, by modulating attention and cognitive control mechanisms via ACC, perhaps enhancing sensitivity to detect non-prepotent solution candidates. *
  • There is also endogenous catecholamine production in the dlPFC of some primate species, including humans

• A dopamine gradient controls access to distributed working memory in the large-scale monkey cortex this is some 200 IQ shit:
  • Depletion of dopamine from the prefrontal cortex and complete ablation of the prefrontal cortex cause similar working memory deficits
  • Too little or too much presynaptic PFC D1 stimulation disrupting delay period activity:

    • [A novel dopamine D1 receptor agonist excites delay-dependent working memory-related neuronal firing in primate dorsolateral prefrontal cortex]

    • Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory (Vijayraghavan, Arnsten, et al 2007)

      • delay cell firing. Low levels of D1 receptor stimulation are associated with noisy neuronal representations of visual space.
        • (There are additional dimensions to this if you want an accurate picture of working memory and arousal, but it’s a decent approximate model)
        • As for left vs. right while still being optimal, it’s something like creativity vs. focus:
      • D1 agonists had suppressive effects on firing of PFC neurons engaged in working memory tasks: moderate levels of suppression preferentially reduced firing to NPDs, leading to an enhancement in spatial tuning, whereas at higher levels of D1R stimulation, physiological suppression became overwhelming, leading to losses in spatial information capacity and detuning of spatial memory-related information
        • A well-studied phenomenon: Supranormal Stimulation of D1 Dopamine Receptors in the Rodent Prefrontal Cortex Impairs Spatial Working Memory Performance (Zahrt 1997)
          • Let us go deeper: Noise stress impairs prefrontal cortical cognitive function in monkeys: evidence for a hyperdopaminergic mechanism
• Dorsolateral Prefrontal Contributions to Human Working Memory
  • DlPFC damage was associated with deficits in the manipulation of verbal and spatial knowledge, with left dlPFC necessary for manipulating information in working memory and right dlPFC critical for manipulating information in a broader range of reasoning contexts.
  • Monitoring operations are thought to support the active retention of information in WM and computational mechanisms for manipulating items are recruited for updating/selecting between these representations:

Human Studies

• Guanfacine extended release in children and adolescents with attention-deficit/hyperactivity disorder: a placebo-controlled trial
  • Even though the half life is pretty long, it rapidly peaks in plasma—hence XR.
• Guanfacine and clonidine, alpha 2-agonists, improve paired associates learning, but not delayed matching to sample, in humans .1 or 2mg. increased subjective feelings of sedation.
• Effect on Primary Sleep Disorders When Children With ADHD Are Administered Guanfacine Extended Release aged 6-12
  • REM, NREM, and SWS were reduced in proportion to the overall sleep reduction.
• Central effects of guanfacine and clonidine during wakefulness and sleep in healthy subjects
  • 
  • Both drugs (1 or 2mg for guan) reduced systolic blood pressure without significantly altering diastolic blood pressure, pulse rate and objective performance parameters.
  • Guanfacine 1.0 mg did not alter REM sleep and 2.0 mg of guanfacine had less effect than both doses of clonidine in this respect.
  • guanfacine’s action on REM sleep began 5 h after the dose
  • Guanfacine’s peak effects were 4-6 hours after ingestion (observations were limited to 6 hours). 26% suppression.
• [Efficacy of guanfacine extended release assessed during the morning, afternoon, and evening using a modified Conners’ Parent Rating Scale-revised: Short Form.]
  • Response is consistent throughout the day regardless of time of administration.
• Lack of effects of guanfacine on executive and memory functions in healthy male volunteers 1 or 2 mg.
  • This study found no improvement of prefrontal memory or executive functions after guanfacine. Negative effects on blood pressure and poorer effects on digit span backward and slower reaction time indicate a mild sedative effect of guanfacine at these doses, possibly via mechanisms of autoreceptor down-regulation.
• Methylphenidate, Guanfacine, and Combined Treatment Effects on Electroencephalography Correlates of Spatial Working Memory in Attention-Deficit/Hyperactivity Disorder age 7-14.
  • Spatial working memory task, measuing ACC and primary visual cortex.
  • Both monotherapies had limited effects on EEG measures, with guanfacine further showing detrimental effects o performance.
• The Effects of Guanfacine on Working Memory Performance in Patients With Localization-Related Epilepsy and Healthy Controls (2008)

IDRA-21

Obsoleted by TAK-653, and arguably retroactively obsoleted by (pi)racetam. Read this for some ampakine lore.

Insulin (Intranasal)

Rating: ★★. More stable postprandial energy levels, and appetite suppression is alright when cutting. But it’s not very prominent to a young lad such as myself.

• Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase
• Broadening the Defintion of Brain Insulin Resistance in Aging and Alzheimer’s Disease
  • Alzheimer’s brains displayed region-specific reductions in insulin concentrations together with elevations in Insulin Receptor densities Rng
  • The brain even has localized insulin production.
• Insulin promotes glycogen storage and cell proliferation in primary human astrocytes
• Role of Insulin in Neurotrauma and Neurodegeneration: A Review
• Hyperinsulinemia Induces Insulin Resistance in Dorsal Root Ganglion Neurons
  • Chronic treatment (this means prolonged! Cells were in insulin soup for 24 h.) resulted in increased basal AKT phosphorylation. More importantly, acute insulin stimulation after chronic insulin treatment resulted in blunted phosphorylation of Akt, S6K, and GSK-3β.
    • In obese rats, insulin-stimulated Akt1 activity is decreased in muscle and adipose tissue but increased in liver and vice versa for Akt2
    • AKT phosphorylation is reduced in Adipocytes and skeletal Muscle of type 2 diabetic patients or from chronic insulin treatment inducing IR.
  • Interestingly, when the cells were treated with PI3K pathway inhibitor, but not MAPK pathway inhibitor, chronic insulin treatment did not block acute insulin treatment-induced Akt phosphorylation
  • Hyperinsulinemia increases Drp1, used for Mitochondrial Fission
• Insulin promotes rapid delivery of N-methyl-D- aspartate receptors to the cell surface by exocytosis (2001) (Xenopus oocytes)
  • Increases open time probability and NR1 surface expression.
• Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner (CA1)
• Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current (CA1)
• Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward
• Brain insulin receptors and spatial memory. Correlated changes in gene expression, tyrosine phosphorylation, and signaling molecules in the hippocampus of water maze trained rats
  • Insulin receptor mRNA is raised during memory tasks. How’s that for LTP?

Intranasal

• Insulin increases excitation and reward through activation of Nachar receptors on somatostatin interneurons which promote synchronicity in the brain and disinhibition of dopamine expressing cells.
• Intranasal insulin protects against substantia nigra dopaminergic neuronal loss and alleviates motor deficits induced by 6-OHDA in rats
• Intranasal insulin administration may be highly effective in improving cognitive function in mice with cognitive dysfunction by reversing brain insulin resistance
• Intranasal insulin improves memory in humans: superiority of insulin aspart
• Intranasal Insulin Improves Age-Related Cognitive Deficits and Reverses Electrophysiological Correlates of Brain Aging Intranasal does not alter bodily insulin receptors. It just delivers it to the brain.
• Intranasal insulin administration appears safe, does not affect systemic glucose control, and may provide acute improvements of cognitive function in patients with type 2 DM R
• Awake intranasal insulin delivery modifies protein complexes and alters memory, anxiety, and olfactory behaviors
• Effect of intranasally administered insulin on cerebral blood flow and perfusion; a randomized experiment in young and older adults
  • Young group was mean age 22 and not diabetic or anything.
• Central insulin administration improves whole-body insulin sensitivity via hypothalamus and parasympathetic outputs in men (2014)
  • Nondiabetic participants. 10 of which ~21.8 BMI: + 41 $\pm$ 8% increase in peripheral insulin sensitivity. 5 33.2 BMI obese patients saw… -0.7 $\pm$ 18% change.
    • This change was associated with hypothalamic activity.
  • Began with 6.25 mU/kg insulin bolus followed by 0.25mU/kg/min iv. Then, 160 U insulin intranasal.
    • Same as this group’s later study: Hypothalamic and Striatal Insulin Action Suppresses Endogenous Glucose Production and May Stimulate Glucose Uptake During Hyperinsulinemia in Lean but Not in Overweight Men (2017)
  • 
• Dose-Dependent Effects of Intranasal Insulin on Resting-State Brain Activity
  • 
  • Nine healthy young men. 0, 40, 80, or 160. After application of 160 IU as a nasal spray, ~0.1 enter the bloodstream, as shown in previous studies.
  • No differences for LH, FSH, Test, ACTH, cortisol, TSH.
• Intranasal Insulin Administration Dose-Dependently Modulates Verbal Memory and Plasma β-Amyloid in Memory-Impaired Older Adults
  • Plasma insulin and glucose levels were unaffected by treatment.
  • 20 IU was the most efficacious dose.
  • Promotes breakdown of Amyloid β
  • CSF Amyloid β levels are reliably lower in Alzheimer’s! Evaluation of CSF-tau and CSF-Abeta42 as diagnostic markers for Alzheimer disease in clinical practice Mixed results for plasma, though. In this study, it was usually increased.
  • Intranasal insulin improves memory in humans reduced plasma Cortisol. This may facilitate Aβ clearance via Insulin Degrading Enzyme.
• It is reasonable to speculate that in the long term, chronic intranasal administration may produce similar adverse effects centrally. Long-duration trials that include a late-stage test of acute brain insulin resistance could address this concern. Even such trials may not go far enough: the extent to which dose timing and frequency impact central insulin resistance is also a mystery. This despite the fact that, for instance, insulin sensitivity of peripheral tissues exhibits a clear circadian pattern in healthy humans (Boden et al., 1996). Additionally, the impact of dose frequency is poorly understood. With these thoughts in mind, future trials investigating both timing and frequency may help to develop an optimal dosing “schedule” that effectively balances efficacy and safety. R
• Nasal insulin changes peripheral insulin sensitivity simultaneously with altered activity in homeostatic and reward-related human brain regions
• Effects of intranasal insulin on hepatic fat accumulation and energy metabolism in humans
  • Overweight, elderly T2D group and lean healthy participants (control). Hepatic insulin sensitivity as not changed. In control, hepatocellular lipids decreased by 35% and hepatic ATP concentration increased by 18% after 3h.
• Intranasal Insulin Therapy for Alzheimer Disease and Amnestic Mild Cognitive Impairment
  • Benefits persisted 2 months after for the 20 IU group.
• Insulin given intranasally induces hypoglycaemia in normal and diabetic subjects (1982) Hard to read graphs…
• 40IU QID: Intranasal insulin did not demonstrate statistically significant improvements on overall mood, aspects of emotional processing, neurocognitive function, or self-reported quality of life patient reported outcomes. R

Istradefylline

Rating: ★★★★. I thrive on the kind of ‘impulsivity’ it gives you (i.e. even on the level of micro-behaviors like googling 3 things that show up in my head to find the answer for something instead of lazily paying attention to one laconic query), but it’s too bad it’ll reliably give you insomnia if you take too much.

• A2A antagonist. Wikipedia mentions how this eventually leads to D2 disinhibition.
  • Disrupting A2A-CB1 heterodimers will actually upregulate CB1.
• Weak CYP3A4 inducer (in vitro)? Metabolized by CYP1A1, CYP3A4, CYP3A5.
• Istradefylline, an adenosine A2a receptor antagonist, inhibits the CD4+ T-cell hypersecretion of IL-17A and IL-8 in humans
• Doesn’t even affect sleep: Istradefylline improves daytime sleepiness in patients with Parkinson’s disease: An open-label, 3-month study
• JC Denton says it will have an additive effect on striatal dopamine and partially restoring normal frontal DA (I guess that depends on parkinson’s/excess GDNF as in the study) whereas it’s guanfacine that will really balance out striatal and frontal.
• Lack of tolerance to motor stimulant effects of a selective adenosine A(2A) receptor antagonist
  • No change in A1, A2A, D1, D2 or their mRNA.
• [Istradefylline is recommended for morning use: a report of 4 cases.]
• Role of adenosine A2A receptors in hot and cold cognition: Effects of single-dose istradefylline in healthy volunteers (20mg)
  • Subjects on istradefylline interpreted social situations more positively (Social Information Preference)
  • Effected risk adjustment loss on the cambridge gamble task (which is interesting, I suppose. It activates OFC)
  • … we did not detect any other effects across a variety of hot and cold cognitive domains
  • Habitual coffee drinkers display a distinct pattern of brain functional connectivity
    • Higher stress levels, and associated with anxiety in males. Decreased functional connectivity in a network encompassing subcortical and posterior brain regions associated wtih somatosensory, motor, and emotional processing.
  • Learning and cognitive flexibility: frontostriatal function and monoaminergic modulation
  • Dopamine and Cognitive Control in Prefrontal Cortex
    • Let’s get back into this paradigm: sensory gating, maintaining and manipulating WM contents, and relaying motor commands.
  • How does adenosine control neuronal dysfunction and neurodegeneration? (Cunha 2016) (aaand it’s 77 pages of manuscript)
    • A2AR switch off A1R and CB1 receptors, bolster glutamate release and NMDA receptors to assist increasing synaptic plasticity in the ‘activated’ synapse; the parallel engagement of the astrocytic syncytium releases adenosine further inhibiting neighboring synapses, thus sharpening the encoded plastic change.
    • A1R are a hurdle for damage initiation, but they desensitize upon prolonged activation. However, if the insult is near-threshold and/or of short-duration, A1R trigger preconditioning, which may limit the spread of damage. Brain insults also up-regulate A2AR, probably to bolster adaptive changes, but this heightens brain damage since A2AR blockade affords neuroprotection in models of epilepsy, depression, Alzheimer’s, or Parkinson’s disease
  • There is also evidence, from a double-blind controlled trial in previously caffeine-naïve human volunteers, that acute caffeine can impact memory consolidation according to an Inverted-U function: Post-study caffeine administration enhances memory consolidation in humans
    • Pretty sure I heard fucking Huberman talk about this. Go figure it was published in Nature, his favorite. (In 2014)
  • Influences mental tracking: ADORA2A genotype modulates interoceptive and exteroceptive processing in a fronto-insular network (rs5751876) (no result for me)
    • The first thing to ask is if this is ‘moar A2A’, or less. Not sure. They do consume less caffeine, probably because of heightened sensitivity. So more??
    • TC was previously shown to be related to anxiety-related phenotypes.
    • TT risk genotype was associated with increased connectivity between the insula and the prefrontal cortex. The strength in connectivity correlated with interoceptive accuracy.
    • Carriers of the TT risk genotype furthermore displayed a reduced activity or deactivation in the striatum during executive control and decreased insula-striatal connectivity during the resting-state.
  • Working memory deficits in transgenic rats overexpressing human adenosine A2A receptors in the brain

Adenosine A2A

• Gs-protein coupled. As opposed to Adenosine A1, it is found in sleep-promoting neurons. It antagonizes the Histaminergic system.
• Modulation of adenosine A2A receptor function by interacting proteins. New targets for Huntington’s disease (2012) - a 133-page dissertation.
• An adenosine A2a agonist increases sleep and induces Fos in ventrolateral preoptic neurons
  • A2A antagonist blocks the sleep induced by PGD2 (which activates sleep-promoting neurons in ventrolateral preoptic area)
• Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1
  • Reduces Histamine and increases GABA, and promotes sleep.
  • PKA→GAT-1 and GABA uptake via reducing PKC.
• Downregulates M1 Macrophage phenotype.
• Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys
• Accumbal adenosine A2A receptor inactivation biases for large and costly rewards in the effort- but not delay-based decision making
  • “Go big or go home” mindset?
  • Nucleus Accumbens is important for cost-benefit decision making.
• Striatopallidal adenosine A2A receptor modulation of goal-directed behavior: Homeostatic control with cognitive flexibility
  • Striatum projects MSNs to the globus pallidus.
  • Striatopallidal A2AR exert an overall “break” control of a variety of cognitive processes. - antagonism enhances goal-directed behavior and cognitive flexibility.
  • 
• A2AR indirectly increases GABA via dopamine/D2 activation or via increasing Glutamate release Effect of adenosine A2A receptor stimulation on GABA release from the striatum of young and aged rats in vivo
  • In contrast, A2AR antagonists inhibit Glutamate release: Modulation of glutamate release and excitotoxicity by adenosine A2A receptors.
• Adenosine A2A Receptor Antagonists Affects NMDA Glutamate Receptor Function. Potential to Address Neurodegeneration in Alzheimer’s Disease
• Adenosine A2A receptors in neuronal outgrowth a target for nerve regeneration?
  • Neurite outgrowth depends on the receptor repertoire present at each moment: A2A activation promotes TrkB-induced dendritic branching, and PI3k/MAPK/PLC-induced axonal elongation!
• An Adenosine A2A Receptor Antagonist Improves Multiple Symptoms of Repeated Quinpirole-Induced Psychosis
  • Quinpirole: D2 and D3 agonist. Induces OCD-like behaviors. May have CB1 effects as well.
• Increases during aging, it seems:
  • Age-related shift in LTD is dependent on neuronal adenosine A2A receptors interplay with mGluR5 and NMDA receptors
    • Targeted neurogenesis pathway-based gene analysis identifies ADORA2A associated with hippocampal volume in mild cognitive impairment and Alzheimer’s disease
      • rs9608282 (no data for me): larger hippocampal volume (synaptic loss???). Upstream in the coding sequence, merely implying it could alter A2AR expression.
    • There is compelling evidence from animal models of a cortical and hippocampal upsurge of A2AR in glutamatergic synapses upon aging and AD (then proceeds to give 7 references)
    • A2A -> PKA/cAMP/CREB -> glutamate release & Ca2+ influx -> and then… hippocampus-dependent cognitive deficits?
      • The caffeine-binding adenosine A2A receptor induces age-like HPA-axis dysfunction by targeting glucocorticoid receptor function
        • A2AR overexpression -> loss of plasmatic corticosterone circvadian oscillation, and reduction of Glucocorticoid receptor levels in the hippocampus.
• Adenosine A2A Receptors Control Glutamatergic Synaptic Plasticity in Fast Spiking Interneurons of the Prefrontal Cortex
  • A2AR are enriched at mPFC (layer V) synapses.
  • At the network level, extracellularly induced LTP of population spikes was reduced by A2AR blockade. The interneuron-specificity of A2AR in controlling glutamatergic synapse LTP may ensure that during periods of high synaptic activity, a proper excitation/inhibition balance is maintained within the mPFC.
• Impact of in vivo chronic blockade of adenosine A2A receptors on the BDNF-mediated facilitation of LTP
  • Most of BDNF synaptic actions, namely on LTP, require activation of A2A.
    • … how? For one thing, is TrkB even expressed outside the basal ganglia? Or, why would synaptic effects of BDNF rely on adenosinergic efferents?
    • results showed that chronic blockade of A2AR in male Wistar rats inhibits the facilitatory action of BDNF upon LTP on hippocampal CA1 area and decreases both mRNA and protein levels of the TrkB-FL receptor in hippocampus

Heteromers

• G protein-coupled receptor heteromers are key players in substance use disorder.
• A2A homodimers as well.
• There A2A-D1 heterodimers along with A2A-D2 A2A-D2 Heteromers on Striatal Astrocytes: Biochemical and Biophysical Evidence
• A2A-CD73: Makes sense. Synergistic of course.
  • Ecto-5’-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions
    • Confirmed with proximity ligation assays
    • Moreover, CD73 KO mice displayed increased working memory performance and a blunted amphetamine-induced sensitization, mimicking the phenotype of global or forebrain-A2AR KO mice, as well as upon pharmacological A2AR blockade.
• A2A-mGluR5: synergistic.
• A2A-D3: adenosine antagonizes affinity and signaling of D3.
• A2A-oxytocin! Heterodimer of A2A and Oxytocin Receptors Regulating Glutamate Release in Adult Striatal Astrocytes

CB1

• A2A-D2-CB1 trimers as well.

• A2A-CB1 and A2A-CB2. They are antagonistic; A2A antagonism increases CB2 signaling.

• Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

  • Essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons
  • Co-stimulation of both receptors leads to strongly reduced downstream signaling (I mean why does it have any magnitude?)
  • DMR: dynamic mass redistribution - essentially a means to measure GPCR activation from light by a biosensor
    • Label-free is a benefit
  • Possibly facilitates Gαq signaling - just like D21?
    • Gq inhibition abrogate A2A and CB1 evoked changes in DMR.
    • Experiments with disrupting peptides on certain amino acids turned the heteromer into their respective classical signaling pathways.
  • Lots of stuff on Huntington’s disease and how expression changes. For instance, functional A2A-CB1 heteromers are expressed in Huntington’s disease in advanced but not late disease stages.

• A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation

  • Cocaine+A2A antagonism synergizes in reducing the firing rate of cholinergic interneurons: Medium Spiny Neuron M1 AChR inhibition → disinhibition of of Cav1.3, triggering postsynaptic Endocannabinoid release and a reduction of glutamatergic transmission via activating presynaptic CB1:
    • Indeed: in the presence of pirenzepine, a M1 receptor inhibitor, the electrophysiological effects of cocaine plus A2AR antagonists modulation on glutamatergic transmission were fully occluded.
      • Ironic how it’s dependent on Cav1.3 channels. Maybe it comes down to localization? It’s conflicting with the study they cite; it should follow that M1 inhibition would reduce VGCC activity.
    • Cites: Dopaminergic control of corticostriatal long-term synaptic depression in medium spiny neurons is mediated by cholinergic interneurons (2006)
      • Induction of LTD: dependent on D2 on the postsynaptic membrane.
      • These aspiny cholinergic Interneurons are autonomous pacemakers, leading to tonic ACh, thus tonic activation of striatal mAChR.
      • Lowering M1 AChR tone promotes LTD induction by disinhibiting critical intraspine Cav1.3 Ca2+ channels.
    • Previously: [The distinct role of medium spiny neurons and cholinergic interneurons in the D2/A2A receptor interaction in the striatum: implications for Parkinson’s disease]
    • VTA CB1 can increase dopamine firing rate: Independent presynaptic and postsynaptic mechanisms regulate endocannabinoid signaling at multiple synapses in the ventral tegmental area
    • Wtf at this point? Phasic dopamine release evoked by abused substances requires cannabinoid receptor activation

• Endocannabinoid signaling mediates psychomotor activation by adenosine A2A antagonists

  • eCB-mediated inhibition of indirect MSN increases movement. The theory is classical that direct MSN activation is pro-locomotion and iMSN = inhibiting movement.

• Adenosine–cannabinoid receptor interactions. Implications for striatal function (2010)

• Interaction of A2A adenosine and D2 dopamine receptors modulates corticostriatal glutamatergic transmission (2007)

• Dopamine Modulation of State-Dependent Endocannabinoid Release and Long-Term Depression in the Striatum (2005)

• Adenosine A2a blockade prevents synergy between μ-opiate and cannabinoid CB1 receptors and eliminates heroin-seeking behavior in addicted rats

• Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation

• Potentiation of cannabinoid signaling in microglia by adenosine A2A receptor antagonists

  • Microglial A2A and CB2 (heteromers also) are upregulated in AD
  • Blockade of A2ARi reduces amyloid burden and improves cognition in AD.
    • A1 adenosine receptors accumulate in neurodegenerative structures in Alzheimer disease and mediate both amyloid precursor protein processing and tau phosphorylation and translocation (2003)
      • High degree of colocalization for A1R and betaA4 amyloid in senile plaques and for A1R and tau in neurons with tau deposition, but without tangles, was seen.
      • A1->PKC->sAPP, A1->p21 & ERK->p->Tau.
      • adenosine A2A receptors, located mainly in striatal neurons in controls, appeared in glial cells in the hippocampus and cerebral cortex of patients
        • Indeed, idk if it’s usually expressed there: Adenosine A2 receptors: selective localization in the human basal ganglia and alterations with disease (1992)
          • Exclusively restricted to the Basal Ganglia, specifically: caudate nucleus, putamen, nucleus accumbens, olfactory tubercle and the lateral segment of the globus pallidus.
            • Not exactly, since it exists peripherally? Novel Players in the Aging Synapse: Impact on Cognition: A2A are particularly expressed in the lungs, spleen, thymus, heart, blood vessels, muscle, and brain… whereas in the neocortex and hippocampus, they are present at residual levels.
              • Plus this might be with AD patients anyways? Would need to check reference
          • Parkinson’s: density of adenosine A2 binding sites was comparable to that seen in control cases.
          • Huntington’s chorea: Denstiy was dramatically decreased

• Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys (2014)

  • Presynaptic: SCH-442416; Postsynaptic: KW-6002.
    • SCH antagonized while KW-6002 potentiated reinforcing effects of THC!
  • Previously: Reinforcing and neurochemical effects of cannabinoid CB1 receptor agonists, but not cocaine, are altered by an adenosine A2A receptor antagonist (2011)
    • Antagonist caused downward shifts of THC and anandamide dose-response curves (i.e. a decrease in effectiveness)
    • MSX-3 neither promoted reinstatement of extinguished drug-seeking behavior nor altered reinstatement of drug-seeking behavior by non-contingent priming injections of THC.

BDNF

• Transactivated with Trk Receptors.
• Adenosine A2A receptor activation is determinant for BDNF actions upon GABA and glutamate release from rat hippocampal synaptosomes
  • Both the inhibitory actions of BDNF on GABA release as well as the facilitatory action of the neurotrophin on glutamate release are dependent on the activation of adenosine A2AR by endogenous Adenosine. However, these actions could not be further enhanced by exogenous activation of A2AR.
• Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity
  • A2A agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts… Lipid raft integrity was also required for the effects of BDNF on hippocampal long-term potentiation at CA1 synapses.
  • Mimicked by Forskolin and blocked by PKA inhibitors… removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes.
• Activation of adenosine A2A receptors induces TrkB translocation and increases BDNF-mediated phospho-TrkB localization in lipid raft: implications for neuromodulation
• Adenosine A2a receptors modulate TrkB receptor-dependent respiratory plasticity in neonatal rats

Isoxazole-9

https://old.reddit.com/r/NootropicsFrontline/comments/muak0e/the_role_of_isoxazole9_on_invitro_invivo/

• Effects of Isx-9 and stress on adult hippocampal neurogenesis: Experimental considerations and future perspectives
  • It is also noteworthy that Isx-9-induced activation of endogenous neuronal gene programs is able to inhibit differentiation of neural progenitors into other cell types, even in the presence of strong gliogenic signals
• Isoxazole-9 reduces enhanced fear responses and retrieval in ethanol-dependent male rats
• Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells
  • Possibly toxic!!!
• Isoxazole 9 (ISX9), a small molecule targeting Axin, activates Wnt/β‐catenin signalling and promotes hair regrowth (Jan 2023)
• Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule
• ISX-9 potentiates CaMKIIδ-mediated BMAL1 activation to enhance circadian amplitude
• Anecdote: Permanently improved memory but also my limbs get numb/cold easier. It’s too fucking schizo to even talk about
• Anecdote: I megadosed intranasal multiple times. It feels so weird on my ears and brain but makes music way better and makes my brain so much more functionally awake. Weird side effects but super powerful with intranasal, sharpens mind a lot. What else activates wnt/beta-catenin as much as isx-9 then? The effects are phenomenal and the top 3 most powerful nootropics i have taken.
  • GSK3α/β is a negative regulator of β-catenin, these studies imply that reducing activity of the Wnt pathway increases the vulnerability to depression, mania, psychosis, and autistic behavior. Wnt/β-catenin signaling in brain development and mental disorders: keeping TCF7L2 in mind

J147

Inhibitor of ATP synthase (sounds fucked up). Upregulates NGF, Egr, and AMPK.

Whitens skin via suppressing α-MSH induced melanogenesis.

Kanna

Rating: ★★★★. Puts a smile on my face. But it often just doesn’t work, though that could be a failure to dose properly or poor quality extract.

• Inhibits Acetylcholinesterase.
  • VP doesn’t consider it much of an SSRI, I guess due to the dose required.
• Upregulates VMAT2, making it a monoamine-releasing agent (MRA) - this also includes histamine and GABA.
  • VP says the VMAT2 upregulation might only be on serotonergic neurons.,
• Possible cannabinoid agonist.
• Increases C-Reactive Protein?
• Contains the alkaloids: - (Δ7-)mesembrenone: SSRI and PDE4 inhibitor.
  • Mesembrine: SSRI, PDE4 inhibitor, MRA, VMAT2 upregulator.
    • ~500μg pure is probably the highest safe does according to VP.
    • MAO-Ai is basically negligible.
  • Mesembrenol
  • Tortuosamine
• 
• High-mesembrine Sceletium extract (Trimesemine™) is a monoamine releasing agent, rather than only a selective serotonin reuptake inhibitor - in vitro
  • Only mild AChEi.
  • 1 mg/ml of pure mesembrine achieved only a E30% AChE in hibition, while the complete alkaloid extract from Sceletium tortuosum inhibited AChE in a dose-dependent manner, with the highest concentration assessed (1 mg/ml) resulting in 87% inhibition, which suggests that the non-mesembrine portion of Sceletium may be responsible for the plant’s inhibitory effects on AChE activity. Somewhat surprisingly, a whole extract with low mesembrine content was reported to only inhibit acetylcholinester-ase activity by 7% (Harvey et al., 2011).
• Cannabinoid CB1 receptor binding and acetylcholinesteraseinhibitory activity of Sceletium tortuosum L.
• Modulation of glucocorticoid, mineralocorticoid and androgen production in H295 cells by Trimesemine™, a mesembrine-rich Sceletium extract

Lion’s Mane

Rating: ★★. I’d be lying if I said I didn’t enjoy the headspace and weird dreams (enough to take it for like a year straight under the premise, which may have been successful for all I know) but I think it’s mostly overhyped/marketed trash (at least for healthy people) at this point.

Downregulates D2, 5-AR inhibitor, and κ-opioid agonist.

L-Histidine

Rating: ★★★. Inconsistent, but it can be pretty fun sometimes.

• Tadaho Nakamura has interesting articles on histamine and cognition:

  • [Oral histidine intake improves working memory through the activation of histaminergic nervous system in mice.]

• Role of dietary histidine in the prevention of obesity and metabolic syndrome

  • Obese middle-aged chinese women received 2g 2x/day for 12 weeks. Average fat loss in the histidine group was 2.71 kg and TNF-α, IL-6 decreased.

LSD

Rating: ★★★★★

• Agonizes 5-HT2A, 5-HT2B, and 5-HT2C, partial agonist of 5-HT1A.
  • David Nichols in a presentation said it’s a fairly weak agonist on 5-HT2A— something like 30%. This train of thought is why Ray Peat calls LSD a ‘serotonin antagonist’.
• Histamine H2 antagonist (as are other hallucinogens).
• Agonizes AMPAR and κ-Opioid Receptor, and antagonizes Acetylcholine -> mGluR2 antagonism -> PI3K-AKT-mTOR pathway -> hippocampal synaptogenesis
• Defining the histamine H2-receptor in brain: the interaction with LSD
• Increased thalamic resting‐state connectivity as a core driver of LSD‐induced hallucinations
  • LSD‐induced functional connectivity measures between the thalamus and the right fusiform gyrus and insula correlated significantly with subjective auditory and visual drug effects
    • An important model suggested that hallucinogens disrupt thalamic gating of external and internal signals, leading to increased passage of information across the cortex: Serotonin research: contributions to understanding psychoses
• Hallucinogens in Mental Health: Preclinical and Clinical Studies on LSD, Psilocybin, MDMA, and Ketamine
• Psyhcedelics Promote Structural and Functional Neuroplasticity

L-Theanine

Say hello to the laughing stock (?). You might enjoy adding it to coffee/stimulants to attenuate certain side effects but I don’t think it’s exactly anything pro-cognitive.

L-theanine and Caffeine Improve Sustained Attention, Impulsivity and Cognition in Children with Attention Deficit Hyperactivity Disorders by Decreasing Mind Wandering (OR29-04-19)

Memantine

• NMDAR antagonist. D2 agonist, 5-HT3 antagonist, σ1 agonism but probably needs high doses

• Increases acetylcholine in the Nucleus Accumbens and Ventral Tegmental Area R

• Apparently nanostructured memantine selectively binds to eNMDAR not synaptic.

• Cognitive Enhancer Effects of Low Memantine Doses Are Facilitated by an Alpha7 Nicotinic Acetylcholine Receptor Agonist in Scopolamine-Induced Amnesia in Rats

  • 1.1 mg. This is what veganpermanently does (though he’s also done 5-25mg…)
  • Memantine is an α7 nAChR antagonist.
    • Ki 1 mM. Prolonged use upregulates it?

• Cognitive Enhancer Effects of Low Memantine Doses Are Facilitated by an Alpha7 Nicotinic Acetylcholine Receptor Agonist in Scopolamine-Induced Amnesia in Rats

• Novel Neuroprotective Mechanisms of Memantine: Increase in Neurotrophic Factor Release from Astroglia and Anti-Inflammation by Preventing Microglial Over-Activation

  • GDNF upregulaton was associated with histone hyperacetylation.

• Low-dose memantine attenuated morphine addictive behavior through its anti-inflammation and neurotrophic effects in rats

• How is it even used for dementia?

• Efficacy of Memantine as Adjunct Therapy for Autism Spectrum Disorder in Children Aged <14 Years

• Cancels/overpowers psychedelics via SLC7A11 (System Xc-) activation?

Modafinil

Rating: ★★★★★. It’s one of the most effective nootropics I’ve ever taken. See the post on Modafinil

NAC

More of a therapeutic (HPPD, tinnitus, OCD) than a nootropic for most people. Don’t expect it to be pro-cognitive.

Neboglamine

Rating: ★★★★. A scarily lucid potentiation of executive function and ‘motivation’ that feels purely rational and nothing to do with dopamine or anything. So many people get into nootropics for ‘getting things done’ and doing the things the better part of themselves wants to do without geting sidetracked on unimportant things, and I find negoblamine to be oddly effective at this.

• The only known NMDAR glycine site PAM, enhancing the binding of D-Serine.
  • According to sirsadalot, oral D-Serine causing molecular-level oxidative stress could be due to it ending up in extrasynaptic regions it doesn’t normally exist in.
• NR3 agonist, which unfortunately is anti-cognitive. Nonameuser thinks it inhibits PFC function.
• CR 2249: a new putative memory enhancer. Behavioural studies on learning and memory in rats and mice (1996)
• Cognition Enhancing Profile of CR 2249, a New NMDA-Glycine Site Modulator
• Antipsychotic-like effects of the N-methyl-D-aspartate receptor modulator neboglamine: an immunohistochemical and behavioural study in the rat.
• Novel NMDA Receptor Modulators: An Update
• Characterization of a novel putative cognition enhancer mediating facilitation of glycine effect on strychnine-resistant sites coupled to NMDA receptor complex

• See also apimostinel (NRX-1074) (sold on TeamLTR) and rapastinel (GLYX-13), modulators that bind to unique domains independent of the glycine site.
  • Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects
• There is also NYX-2925 (aka AGN-241751) and NYX-458.
• (It’s not an antagonist but apimostinel could be? There’s a biphasic dose response.) GLYX-13, a NMDA receptor glycine-site functional partial agonist, induces antidepressant-like effects without ketamine-like side effects
• Weak selective Noradrenaline reuptake inhibitor at high doses (104.5μM/L, 10x that of being a glycine site PAM)
  • Also has an NRI metabolite.

Nicotine

Rating: ★★

• Out of all the nAChR subtypes, its highest binding affinity is for α4β2, representing >90% of its binding in brain tissue, supposedly the primary mediatory of its addictiveness, since they release dopamine.
  • Another mechanism of its addiction is α4 nAChR upregulating inhibitory GABAergic neurons.
  • α4β2 Nicotinic Acetylcholine Receptors on Dopaminergic Neurons Mediate Nicotine Reward and Anxiety Relief
    • Mice lacking α4 nAChRs have elevated levels of basal anxiety.
  • For instance, GTS-21, an α7 agonist, does not activate the ’nicotine cue’ R
• Nicotine reverses hypofrontality in animal models of addiction and schizophrenia
  • The interesting part of the study is how it affects Somatostatin interneurons.
  • α5 nAChR: rs16969968 (Asp398Asn) (A/A (Me: AG)). Associated with nicotine dependence but lower risk of cocaine dependence. Results in loss of function.
    • Reduced resting-state functional connectivity, and circuits involving the PFC are altered: A genetically modulated, intrinsic cingulate circuit supports human nicotine addiction
      • Specifically, α4β2α5 nAChR function, which somehow ‘incorporates’ itself into heteromers like α4β2.
      • Previously: Association of nicotine addiction and nicotine’s actions with separate cingulate cortex functional circuits: Resting-state dACC-striatum functional connectivity may serve as a circuit-level biomarker for nicotine addiction
      • Molecular genetics of successful smoking cessation: convergent genome-wide association study results
        • The supplementary pdf has like a good 100 SNPs. Would like to see this on Nebula… it’s a lot of cool stuff; a lot of actin proteins. Even ADAM12 (one I have is rs7920207 AC)
  • Layer VI is one of only a handfyl of brain regions that express α5.
  • Deletion α5 in mice leads to PFC-linked behavioral deficits, altered cholinergic excitation and aberrant neuronal morphology in the PFC: Nicotinic acetylcholine receptors in attention circuitry: the role of layer VI neurons of prefrontal cortex
    • 
    • More smoking genes in the α3-α5-β4 cluster: Genome-wide meta-analyses identify multiple loci associated with smoking behavior
      • Strongest association was the α3 nAChR rs1051730 (A (Me: AG))
      • rs1329650 (G (Me: –))
      • rs1028936 (A (Me: AC))
      • rs3733829 (G (Me: AG))
        • EGLN2
      • Smoking cessation: rs3025343 (G (Me: AG))
      • BDNF: rs6265 (A (Me: TC (So AG?)))
        • Implcated in introversion, quicker mental decline in Alzheimer’s, impaired motor skills learning. It may be protective against depression though R
  • α5 nAChR is implicated in schizophrenia: ts. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased Somatostatin interneuron inhibitory drive over layer II/III pyramidal neurons - this resembles hypofrontality, and was reversed by chronic nicotine.
• Sustained phosphorylation of tyrosine hydroxylase at serine 40: a novel mechanism for maintenance of catecholamine synthesis
  • Upregulates striatal Tyrosine Hydroxylase for a few days after administration, probably downstream of GDNF (itself downstream of FGF2)
• Releases Kisspeptin
• Nicotinic modulation of neuronal networks: from receptors to cognition
• Stimulates presynaptic α7 nAChR in glutamatergic neurons, enhancing projections to DA neurons.
• Nicotine Effects and the Endogenous Opioid System
  • The endogenous opioid system participates in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception is mediated by α4β2 and α7 nAChRs, while NIC-induced HPA axis activation is mediated by α4β2, not α7, suggesting that the effects of NIC on the endogenous opioid system are mediated by α7, not α4β2.
• Activation and Desensitization of Nicotinic α7-type Acetylcholine Receptors by Benzylidene Anabaseines and Nicotine
• [Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes]
  • Nicotine decreases α4β2 responsiveness much faster than α3β2 nAChR.
• Inhibits 3α-HSD, boosting synthesis of DHT, etc. R
• 3 mg/kg/day for 6 weeks reduced 43% body weight gain and 65% blood insulin level, but had no effect on blood glucose level,… demonstrated that nicotine enhancement enhanced insulin sensitivity. R
• Chronic use downregulates NCAM
• Increased expression of VMAT2 in dopaminergic neurons during nicotine withdrawal
• Nicotine reduces discrimination between threat and safety in the hippocampus, nucleus accumbens and amygdala
  • Overactivation of the VTA.

Tolerance

• Chronic Nicotine Cell Specifically Upregulates Functional α4 Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path
  • Chronic nicotine does not change α4 receptor levels in dopaminergic neurons of VTA or Pars Compacta. Instead, upregulated, functional α4 receptors localize to the GABAergic neurons (Medium Spiny Neurons or interneurons??) of the VTA and Pars Reticula.
• Acute Nicotine-Induced Tachyphylaxis Is Differentially Manifest in the Limbic System
  • … whereas profound tolerance was observed in many cortical regions after the second of two paired nicotine injections at either 0.1 or 0.3 mg/kg, subcortical limbic structures showed only a weak trend for tolerance.
• Temporal change in human nicotinic acetylcholine receptor after smoking cessation: 5IA SPECT study
  • The binding potential of nAChRs in the brains of smokers decreased by 33.5% +/- 10.5% after 4 h of smoking cessation, increased by 25.7% +/- 9.2% after 10 d of smoking cessation, and decreased to the level of nonsmokers after 21 d of smoking cessation.
• β2-Nicotinic Acetylcholine Receptor Availability during Acute and Prolonged Abstinence from Tobacco Smoking
  • 
    • The acute decrease of availability in the Thalamus is quite weird. Not sure how that happens.
  • Nicotine induced increases in nAChR, termed “upregulation”, are conferred by a specific microdomain that is in the β2-subunit 34 and thus this upregulation is confined to nAChR that contain the β2-subunit

Noopept

Rating: ★★. Crazy laser focus. So crazy that I swear it bleeds into negatively affecting other cognitive faculties like short-term memory. It just felt weird sometimes. There are plenty of weird ass anecdotes out there, like it giving long-lasting 2D vision. So proceed at your own risk. There are better compounds out there for most of the things it does.

• AKA (N-Phenylacetyl-L-prolylglycine ethyl ester) Converted into phenylacetic acid, prolylglycine, and Cycloprolylglycine, a naturally-produced neurodipeptide which I think is how noopept exerts its effects.
• I believe it is much more glutamatergic than the other racetams; its vasodilatory effects can cause headaches which seem to be not uncommon. It can be used to kill NMDA antagonist trips (like Ketamine).
  • The drug action is based on the Antioxidant effect, the antiinflammatory action, and the ability to inhibit the neurotoxicity of excess calcium and glutamate, and to improve the blood rheology.
• Piracetam (400mg/kg (=~4.6g HED)) increased Alpha waves/Beta waves EEG activity in the left frontal cortex, and Alpha waves activity in both the right cortex and Hippocampus. Noopept (0.2mg/kg (=~2.27g HED)) increased Alpha waves/Beta waves activity… in all brain areas. The effect of Noopept in the alpha/beta1 ranges was replaced by increased beta2 activity after the eighth injection, while no effects were observed after the ninth one. R
• AMPA agonist: Studying specific effects of nootropic drugs on glutamate receptors in the rat brain

• Effect of noopept and afobazole on the development of neurosis of learned helplessness in rats Noopept will make you a non-fearful rat.

• Novel nootropic dipeptide Noopept increases inhibitory synaptic transmission in CA1 pyramidal cells (2010)

  • It was suggested that Noopept mediates its effect due to the activation of inhibitory Interneurons terminating on CA1 pyramidal cells. Results of current clamp recording of inhibitory interneurons residing in stratum radiatum confirmed this suggestion.

• Nootropic dipeptide noopept enhances inhibitory synaptic transmission in the hippocampus (2010)

  • 1mM significantly increased the frequency of spike-dependant spontaneous IPSCs whereas spike-independent mIPSCs remained unchanged, as well as the kinetic parameters (rise and decay time). It increasess the firing of GABA Interneurons.
    • I wouldn’t jump to the conclusion that this “inhibits memory” or something. I think it just makes you more selective, right? Or, it’s more like it enhances recall at the expense or encoding? However:
      • The original novel nootropic and neuroprotective agent noopept (2002)
        • In contrast to piracetam facilitating only the early stages of the memory process, noopept positively influences the Memory consolidation and retrieval steps as well… produces an additional selective anxiolytic action. The pronounced neuroprotective effect of noopept was demonstrated both in vivo (in cases of various forms of brain ischemia) and in vitro (on various neuronal models).

• Noopept improves the spatial memory and stimulates prefibrillar beta-amyloid antibody production in mice

• Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept

  • Increase in HIF-1α DNA-binding activity. Indeed, this may be its main MOA.
    • Its effects on HIF suggest that it shouldn’t be used continuously. -Peat. Interesting, since It is known that activation of the HIF system is now regarded as one of the main mechanisms of neuroprotection during hypoxia, cerebral ischemia, and neurodegenerative diseases. Relation to CO2??
  • Enhances Antioxidant activity.
  • Represses the stress-induced pSAK/JNK and pERK1.

NSI-189

Rating: ★★. It felt pretty awesome but I think it gave me chronic fatigue. Perhaps a shift from beta wave to alpha wave dominance increasing propensity for dissociation into more sedated states.

• Induces statistically-significant increases volume of the right Amygdala, which is reduced in size in major depressive disorder. The golden question is how bad this is, considering meditation atrophies it and all. R
• Enhancement of Mitochondrial Function by the Neurogenic Molecule NSI-189 Accompanies Reversal of Peripheral Neuropathy and Memory Impairment in a Rat Model of Type 2 Diabetes
  • This is a seriously underrated/potentially underappreciated mechanism. Apparently few compounds have such a comprehensive enhancement
• Elevated the expression of protein subunits of complexes III and V and activities of respiratory complexes I and IV in the cortex. Ameliorated loss of AMPK in sensory ganglia.
• An Early Valuation Of Neuralstem’s NSI-189 For MDD
• NSI‐189, a small molecule with neurogenic properties, exerts behavioral, and neurostructural benefits in stroke rats
  • Increased MAP2 expression. BDNF and SCF upregulation.

Its mechanism is pretty much unknown but it’s speculated to be adrenergic somehow.

• https://www.longecity.org/forum/topic/91594-nsi-189-mechanism-of-action/

  • JAK-STAT pathway inhibitor?. Lol. 5-HT1A and D2/D3, most likely agonism.
  • Some IC50s are supposedly: 14.2 DAT, 1.1 NET, 2.1 5-HT3, 11.1 5-HT7, 15.7 μ, 12.7 δ1.

• The neurogenic compound, NSI-189 phosphate: a novel multi-domain treatment capable of pro-cognitive and antidepressant effects

  • Works on the SCF complex (Skp, Cullin, F-box)

• Allostasist says NET inhibitor and 5-HT3 antagonist. And other people suggest possible TrkB downregulation.

• DAT: 14.2 IC50

• NET: 1.1

• SERT: >30

• 5-HT3: 2.1

• 5-HT7: 11.1

• μ: 15.7

• δ: 12.7

• https://docs.google.com/document/d/1jMJ84rKR8lWHVkkEnsZsWTBvonVVArjzw7WxOWDtSG0/edit every anecdote you could ever ask for. Wtf.

Octreotide

Potent somatostatin mimetic.

• Octreotide administration, under particular temporal conditions, enhances the responses of growth hormone to growth hormone-releasing hormone in normal subjects

Somatostatin

• Somatostatin Interneurons can express GABA but they also express SST. SST opposes the actions of GABA and promotes the inhibition of Parvalbumin interneurons which impart inhibition onto pyramidal cells. SST increases the efficiency of glutamate and the “fidelity” of neurotransmission.
  • Somatostatin enhances visual processing and perception by suppressing excitatory inputs to parvalbumin-positive interneurons in V1
• SST increases both muscarinic and nicotinic acetylcholine currents.
• He claims glutamate neurotoxicity is due to the starvation of glutamate neurons of glu and other excitatory AAs like I guess NMDA for example.
• Decline in SST = increased synorphin signalling/opioidergic tone hypothesis of brain aging.
  • Opioids activate SST interneurons, suppressing parvalbumin interneuron activity on pyramidal neurons, disinhibiting pyramidal cell activity leading to reward: Morphine coordinates SST and PV interneurons in the prelimbic cortex to disinhibit pyramidal neurons and enhance reward
  • In disorders such as autism, PV interneurons far outnumber those of SST leading to overinhibition.
• Decline in SST = increased D21 formation though GABA B, increasing D2, reducing NMDA via cholinergic deafferentation, and increasing mTOR signalling.
• Somatostatin dysfunction is the root cause of all neurodegenerative diseases and mental disorders including depression
  • Reduced brain somatostatin in mood disorders: a common pathophysiological substrate and drug target?
• Hypothalamic inflammation via endotoxins, ceramides, SFA, is a lead cause of diabetes. This is all these pathologies are ultimately mediated by a decline in insulin sensitivity and SST interneurons
• Somatostatin inhibitory interneurons are one of three populations of gabaergic interneurons within the brain that send gamma (GABA?) currents to pyramidal cells The foundations for optimal human health are grounded upon somatostatin interneurons especially within the hypothalamus
• The only GABAergic interneurons that express α7 nAChR, meaning it mediates the rewarding effects of cholinergic signaling.
• Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes
  • Agonists also inhibited cAMP formation dose-dependently, i.e. reduced Forskolin-induced endozepine release.
• The role of neuropeptide somatostatin in the brain and its application in treating neurological disorders
  • 

2-Oxazolidinones

Discussed pretty extensively on /r/researchchimicals

Cyclazodone

TAAR1 agonist

N-Methyl-Cyclazodone

Possible serotonergic effects.

Fenozolone

Pemoline

Probably the only sustainable one in terms of safety, but also the most difficult to obrain. Unknown MOA but likely has dopaminergic actions and is a possible AADC inhibitor (Pemoline and urinary excretion of catecholamines and indoleamines in children with attention deficit disorder)

It faded into obscurity after cases of liver failure in kids being prescribed pemoline for ADHD. But it would seem hepatotoxicity is overblown.

Pitolisant

• Histamine H3 partial inverse agonist, which enhances memory retrieval:
  • Central Histamine Boosts Perirhinal Cortex Activity and Restores Forgotten Object Memories
  • New procognitive enhancers acting at the histamine H3 and AMPA receptors reverse natural forgetting in mice: comparisons with donepezil and memantine in the object recognition task Drugs are called S3809 and S47445 respectively.
  • Histamine H3 receptor density is negatively correlated with neural activity related to working memory in humans
• Pitolisant, an inverse agonist of the histamine H3 receptor: an alternative stimulant for narcolepsy-cataplexy in teenagers with refractory sleepiness
• ‘Non-inferior’ to Modafinil at treating narcolepsy.
• Blocks hERG (Kv 11.2)
• Pitolisant-supported bridging during drug holidays to deal with tolerance to modafinil in patients with narcolepsy reverses Modafinil tolerance!

Phenidates

• cf. https://psychonautwiki.org/wiki/Substituted_phenidates

Methylphenidate

• Methylphenidate preferentially increases catecholamine neurotransmission within the prefrontal cortex at low doses that enhance cognitive function
• Methylphenidate Exposure Induces Dopamine Neuron Loss and Activation of Microglia in the Basal Ganglia of Mice
• Methylphenidate and μ opioid receptor interactions: A pharmacological target for prevention of stimulant abuse
  • supra-therapeutic doses of MPH produce rewarding effects (surrogate measure for addiction in humans) in a conditioned place preference paradigm and upregulate μ-Opioid Receptor activity in the Striatum and nucleus accumbens, brain regions associated with reward circuitry
    • This requires activation of D1, but not D2.

Dexmethylphenidate (Focalin)

Probably the most sustainable of the DRIs prescribed for ADHD.

IPPH (Isopropylphenidate)

Has a much greater affinity for DAT than NET.

Pinealon

https://unyieldingvigor.com/resources/b/pinealon-glu-asp-arg & https://old.reddit.com/r/NooTopics/comments/12lvvjq/pinealon_improves_cognition_and_performance_in/

https://penchant.bio/products/pinealon-spray

Pregnenolone

Rating: ★★. Good stuff, but I’m too young to get much out of it.

“A couple of times I saw men who were not quite suicidal, but extremely depressed, talking about quitting their job and just giving up, and they both happened to be sitting in a dark corner of the room with a glass of wine, wanting to retreat, even within the room as well as from life in general. And, thinking about the old bayonet studies and such, I put a pinch of pregnenolone in their wine; and within about 15 minutes, in both cases, they were grinning and talking about projects and went back to work and were just as happy as they could ever be.”

• Progesterone Pregnenolone & DHEA - Three Youth-Associated Hormones (Ray Peat)

• Increases dopamine release/response to Morphine in rat nucleus accumbens R

• Anti-Cancer.

• Improves joint mobility in arthritis, tissue elasticity in lungs, and eyesight.

• Enhances memory and mood (calms emotions, increased resilience) skin (improves circulation), normalizes aldosterone, and lowers estrogen.

• The most potent inhibitor of the stress signal: Ray Peat Forum

• Activates TRPM1/3 channels.

• Interferes with TLR4 activation.

• CB1 negative allosteric modulator.

  • Pregnenolone Can Protect the Brain from Cannabis Intoxication

• Excitatory neurosteroids DHEA(-S) and PGL at physiological concentrations participate in the inhibition of cortical neuronal degeneration elicited by staurosporine and glutamate, whereas the most potent positive modulator of GABA-A receptor - Allopregnanolone - has no effect. R

• FSH-induced aromatase activity in porcine granulosa cells: non-competitive inhibition by non-aromatizable androgens

  • Pregnenolone and Progesterone were inhibitory, but Testosterone was stimulatory.

• Pregnenolone itself doesn’t do much in the nervous system, but pregnenolone sulfate does exhibit cognitive/memory enhancing, antidepressant, anxiogenic, and proconvulsant effects.
• Potent negative allosteric modulator of GABA-A; weak positive allosteric modulator of NMDAR.
  • To a lesser extent, acts as a negative allosteric modulator of AMPAR, Kainate Receptor and Glycine receptors.
  • Subtype-dependence of N-methyl-d-aspartate receptor modulation by pregnenolone sulfate
    • Shown previously how P-S potentiates A>B and inhibits C$\approx$D: Inhibition of the NMDA response by pregnenolone sulphate reveals subtype selective modulation of NMDA receptors by sulphated steroids
    • the structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit is a key determinant for the PS effects
    • Acts at 2 distinct binding sites.
      • 
        • Interesting stuff in A: it initially inhibits the current, and then when it unbinds,
    • 20-oxo-5β-Pregnanolone-S (3α5βS) inhibits the responses.
    • responses induced by NR1A/NR2A and -NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR2C and NR2D.
• Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders
  • Acute obesogenic diet administration in mice impaired recognition memory due to defective production of pregnenolone in the hypothalamus.
  • POMC neurons have a far superior neuroSteroidogenesis capacity than AgRP neurons.
  • loss of Stard1 in POMC neurons impaired recognition memory performance in the face of normal metabolic and energy balance status. These findings indicate that inadequate neurosteroid production by POMC neurons is fundamentally implicated in cognition, but it is dispensable for energy homeostasis control
• The neurosteroid pregnenolone promotes degradation of key proteins in the innate immune signaling to suppress inflammation
  • Promotes ubiquination of TLR2/TLR4 adaptor protein TIRAP and TLR2 in Macrophages and microglia, and suppressed TLR2/TLR4-mediated IL-6 and TNF-α secretion.
• Brain distribution and behavioral effects of progesterone and pregnenolone after intranasal or intravenous administration
• Nanomolar Concentrations of Pregnenolone Sulfate Enhance Striatal Dopamine Overflow In Vivo
• Stabilizes Microtubules and stimulates assembly. RPF
  • Pregnenolone binds to microtubule-associated protein 2 and stimulates microtubule assembly Binds to MAP2. (Pregnenolone sulfate does not)

LTP

• Pregnenolone sulfate enhances long‐term potentiation in CA1 in rat hippocampus slices through the modulation of N‐methyl‐D‐aspartate receptors
  • Neuroactive steroid pregnenolone sulphate inhibits long-term potentiation via activation of alpha2-adrenoreceptors at excitatory synapses in rat medial prefrontal cortex.
    • PREGS inhibited induction of LTP in the Medial Prefrontal Cortex and had no influence on NMDA
• PREGS induces LTP in the hippocampal dentate gyrus of adult rats via the tyrosine phosphorylation of NR2B coupled to ERK/CREB [corrected] signaling
  • Total p-ERK levels increased but not levels of ERK. This is thanks to the calcium influx in NMDAR, and this is important for plasticity:
  • Which number on tyrosine NR2B? Not sure
• May have interactions with nACh receptors as well.
• Steroid pregnenolone sulfate enhances NMDA-receptor-independent long-term potentiation at hippocampal CA1 synapses: role for L-type calcium channels and sigma-receptors

Supplementation

Peat has said that literally every source on the market today is unsafe for one reason or another.

• 70-80% oral bioavailability.

• Men/women age 30 produce ~30-50mg/day. A dose of 300mg acts for a week. Dosing actually improves the body’s ability to produce its own pregnenolone. But high doses such as >100 inhibit androgens/DHT?

• We produce 5% as much in old age than we do in our youth - as is the case with DHEA and progesterone.

• 30-50 for DHEA+progesterone pthway, while 100-150 is more like mainly in the way of progesterone.

Intranasal

• Intranasal pregnenolone increases acetylcholine in frontal cortex, hippocampus, and amygdala-Preferentially in the hemisphere ipsilateral to the injected nostril (Fazari et al. 2019)
  • The second caveat is extremely interesting. Similarly, Previous studies have shown that the unilateral IN administration of l-DOPA increased extracellular dopamine only in the ipsilateral neostriatum of male Wistar rats, excluding the systemic transport of the drug from the nose to the central nervous system (De Souza Silva et al., 1997; Silva et al., 1997).
  • *Nuwayhid and Werling (2003) demonstrated that PREG *inhibits the NMDA-stimulated dopamine release in the striatum via Sigma Receptors with involvement of the coupled-Protein Kinase Cβ pathway.
    • Steroids modulate N-methyl-D-aspartate-stimulated 3H dopamine release from rat striatum via sigma receptors
      • I mean sure, everything probably expresses a little bit of anything. But it’s literally major a dopaminergic input - how much do its outputs matter? I’d say this study seems very significant but at the end of the day
      • 3H-dopamine lacks a double bond on the benzene. Are the consequences for this massive or what?
• Brain distribution and behavioral effects of progesterone and pregnenolone after intranasal or intravenous administration (Ducharme et al., 2011)
  • ~23% entered the blood. Brain levels were about two fold lower after intranasal administration.
    • From 10 to 60 minutes, Brain: 35 ± 2.6 -> 45 ± 18
    • Serum 3.7 -> 7.6
  • (Inj/g = percent of amount administered.) All this really asserts is preg’s affinity for brain regions.
    • 200pg was the goldilocks zone for memory enhancement, while 1fg or 200ng were without effect. They weighed 0.025 - 0.03kg, so that’s 0.00015pg/kg. HED = 0.0000229995pg/kg = 20 fucking femtograms of pregnenolone. I probably get more than that by just happening to be inhaling when I open the jar.
• Promnestic effects of intranasally applied pregnenolone in rats
  • 0.187 or 0.373 mg/kg = 0.056 or 0.112 mg = HED 2.1 or 4.2 mg.
  • 
    • C depicts how much time they spent in previous reward platforms as opposed to the new ones - this is a measure of relearning or whatever.

PRL-8-53

Rating: ★★★. 5mg sublingually definitely enhances both working and long-term memory (I’ll never forget how one time I photographically recalled the image of a wikipedia article I had been reading earlier because I wanted to read the rest of it) but it also feels kind of dirty. I don’t feel comfortable taking compounds with unknown MOAs.

Hypermnesic, spasmolytic. Only two studies done on it, in ‘74 and ‘78.

Derived from benzoic acid and Phenylmethylamine.

• Increased learning of words by 87.5-105% in poor performers, and 7.9-14% in high performers - but that’s just due to a ceiling effect. Was #1 by a decent margin in the /r/nootropics survey for memory.

• Displays Cholinergic properties (probably ACh uptake/synthesis). potentiates Dopamine and partially inhibits Serotonin signaling, and maybe synthesis.

  • I’ve heard people say it doesn’t really work without choline. Especially when stacked with TAK-653, for whatever reason.
  • It may inhibit the reuptake of dopamine, which depending on the mechanism such as DAT, may not be good long term. Also may be a direct agonist, though no stimulant-like symptoms have been shown even up to 200mg/kg.
  • Possibly a M2 AChR agonist.
  • Sirsadalot suspects it’s a DAAO inhibitor

• Possibly a HDAC inhibitor. Very contentious. https://old.reddit.com/r/Nootropics/comments/5bqrpm/i_figured_out_prl853s_mechanism_of_action/

Dose: ~5-10mg or so sublingually. Caustic to the teeth though. Can also do 1-5mg intanasally in water; easily dissolves.

Psychoplastogens

Iso-DMT, Tabernanthalog, etc.

• Identification of Psychoplastogenic N,N‑Dimethylaminoisotryptamine (isoDMT) Analogues through Structure−Activity Relationship Studies

• Evaluation of Isotryptamine Derivatives at 5-HT2 Serotonin Receptors

• The Subjective Effects of Psychedelics May Not Be Necessary for Their Enduring Therapeutic Effects

  • 6-MeO-iso produces no head twitch response. 6-MeO-Iso is an even more potent psychoplastogen than 5-MeO.

• Psychoplastogens: A Promising Class of Plasticity-Promoting Neurotherapeutics

• Psychedelics promote neuroplasticity through the activation of intracellular 5-HT2A receptors (Sheekey science show video)

  • Psychedelics and Other Psychoplastogens for Treating Mental Illness
    • Psychoplastogens are charactezied by, after single treatment, sustained effects on structural plasticity, notably in the PFC! Which means more excitatory projections to decrease depressive behavionr, addiction, etc.
      • Pretty sure it’s from Gαq → Ca2+ → TrkB
    • Brain-Derived Neurotrophic Factor Signaling and Subgenual Anterior Cingulate Cortex Dysfunction in Major Depressive Disorder
      • BDNF was not reduced in the aCC, but TrkB was, and thus various markers that are downstrean/dependent on BDNF, like SST, NPY, COST, Glutamate Decarboxylase 2, etc. (they note that GAD1 isn’t BDNF-dependent)
  • Positive correlation between lipophilicity (thus able to pass the membrane, which serotonin is not capable of - notice the methylation on the amine with DMT/Psilocin, which serotonin does not have) and the psychoplastogenic effect, with DMT being the strongest.
    • Clearly linked is a (mostly) inverse correlation between β-arrestin 2 Emax and Gq Emax and lipophilicity.
    • The ability to pass through the membrane is significant due to the presence of intracellular 5-HT2A in cortical neurons.
      • They gave the cells SERT, applied serotonin, and the dendritic outgrowth was similar to that of DMT.

• A role for the serotonin 2A receptor in the expansion and functioning of human transmodal cortex (Luppi et al. Sep 2023)

Racetams

Many would argue their AMPA PAM properties have been obsoleted by TAK-653. But they still have other actions (and some racetams have nothing to do with AMPA)

Aniracetam

Activates 5-HT2A, D2, D3.

• The aniracetam metabolite 2-pyrrolidinone induces a long-term enhancement in AMPA receptor responses via a CaMKII pathway
  • Enhances GluR1, GluR2, GluR3

Coluracetam

• Increases HACU. Mild AMPA PAM.

Fasoracetam

• Upregulates GABA-B.

Oxiracetam

Rating: ★★★. My 3rd favorite racetam. Feels really clean and was relatively consistent.

• Increases HACU and LTP.
  • Oxiracetam and aniracetam increase acetylcholine release from the rat hippocampus in vivo
• Oxiracetam and Zinc Ameliorates Autism-Like Symptoms in Propionic Acid Model of Rats

Phenylpiracetam

Rating: ★★★★★. Higher doses with no tolerance feel very dopaminergic. Eugeroic too; amazing preworkout if timed correctly.

Apparently it was originally invented to be taken at ~100mg daily and that its stimulating properties (which are amazing and sustainable) are treated as a temporary side effect (indeed, unfortunately, it only lasts 1-3 days before a break of ~1-2 weeks is necessary), but I refuse to believe you don’t just slap a phenyl group on perfectly good piracetam and act like dopaminergic properties were an accident.

• Affects the Casein Kinase enzymes. It could possibly exacerbate milk allergies due to this - very interesting.

• Decreases iNOS, increases eNOS

• Decreases the number of nAChRs and NMDARs???

• Agonizes α4β2 nAChR with IC50=5.86μm.

• The effects of scopolamine and the nootropic drug phenotropil on rat brain neurotransmitter receptors during testing of the conditioned passive avoidance task

  • 100mg/kg (HED>1g: madness)
  • I’ll have to look and see if all the effects measured were all after taking scopolamine.

• Possible actions on DAT: S-phenylpiracetam, a selective DAT inhibitor, reduces body weight gain without influencing locomotor activity .

• https://www.freepatentsonline.com/EP2891491A1.pdf (Rats) (10mg HED = ~113mg)

• Neuroprotective and anti-inflammatory activity of DAT inhibitor R-phenylpiracetam in experimental models of inflammation in male mice

Methylphenylpiracetam

σ1 PAM. Nothing like regular phenylpiracetam.

Phenylpiracetam Hydrazide (RGPU-95)

Piracetam

Rating: ★★★★. Inconsistent, but the way it increases fluidity of thought is fantastic. I had an amazing honeymoon period with it at the very least. Everyone should try a cycle of piracetam.

• Decreases the destabilizing effects Amyloid β, which causes lipid disorganization within cell membranes. Other racetams do this as well.
  • Increases (synthesis of) cytochrome B5
  • Inhibits stress-induced Prolactin increase
• Reduces Erythrocyte adhesion to vascular endothelium, hinders vasospasm, and facilitates microcirculation. R
• Influences Membrane fluidity in the whole body; protecting the cell against Hypoxia.
• Increases brain O2 consumption, in connection to ATP metabolism.
• Aldosterone receptors are involved in the mediation of the memory-enhancing effects of piracetam
  • Adrenalectomy blocks memory-enhancing effects of piracetam. This is abolished with administration of corticosterone or aldosterone so long as aldosterone receptors (Type I mineralcorticoid receptor) are not blocked.
• Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors (Ahmed & Oswald 2010)
  • GluR2/GluR3.
  • Along with Aniracetam, Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity.
  • Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators.
  • However, the binding sites for piracetam and Aniracetam differ considerably.

• Increases Dopamine in cerebral cortex and striatum, and Serotonin in the cortex, while reducing serotonin in the Striatum, Brain Stem and Hypothalamus. R
• Increases Noradrenaline by increasing Locus Coeruleus firing. R
• Increases Noradrenaline and Serotonin turnover in Hippocampus.

• Acetylcholinesterase inhibitor, in contrast to some other racetams that increase its synthesis.
• Increases hippocampal acetylcholine, and in aged mice, increases population of mAChR in frontal cortex by up to 40%.
  • Carbachol-induced accumulation of Inositol Monophosphates was elevated, suggesting piracetam can normalize functional deficits associated with aging
• Profound effects of combining choline and piracetam on memory enhancement and cholinergic function in aged rats
  • 200mg/kg increased Choline content in Hippocampus by 88%, and decreased Acetylcholine levels by 19%.
  • Choline administration raised choline content about 50% in the Striatum and Cerebral Cortex, and 6-10% increase in ACh levels. None in hippocampus.
    • The combination of choline and piracetam did not potentiate these effects seen with either drug alone, and in some cases, were worse.
  • Rats given 100mg/kg of piracetam+choline exhibited retention scores several times better than piracetam alone. 200mg/kg of choline or piracetam alone was still inferior.

Glutamate

• Enhances efficacy of AMPA-induced calcium influx and maximal density of AMPARs in synaptic membranes, due to the recruitment of a subset of AMPA receptors which normally don’t contribute to synaptic transmission.
  • Normalized the age-related elevated affinity of L-glutamate for NMDAR
• Binds to AMPARs with much lower affinity than ampakines or Aniracetam, but it can bind to multiple sites on the AMPA receptor, potentiating what binds to it, including piracetam itself.
• Larger doses can potentiate potassium-induced release of glutamate from hippocampal nerves.
• Significantly increases NMDAR density by 20% after 14 days of treatment.
• Allosteric modulator of certain CNS glutaminergic receptors. I wanna know how this prevents overactivation, because it does.

Pramiracetam

• More efficacious than coluracetam at increasing HACU. Not proven to be an AMPA PAM.

• Inhibits prolyl endopeptidase (aka prolyl oligopeptidase): Prolyl Oligopeptidase Enhances α-Synuclein Dimerization via Direct Protein-Protein Interaction

• There are multiple reports on Longecity/Reddit of this racetam causing long-term damage to cognition/working memory due to nitric oxide synthesis may result in glutamate excitotoxicity! 300mg/kg in rats (so >3g in humans…) increased NOS by approximately 20%, which is quite dangerous apparently.

  • Systemic administration of pramiracetam increases nitric oxide synthase activity in the cerebral cortex of the rat

RAP-103

Long-lasting inhibitor of CCR5 (also CCR2/CCR8). Downstream inhibition of GSK-3β reactivates myelinating cells in the brain, improving LTP/cognition. This also enhances opioid analgesia and reduces anxiety.

Rapamycin

The basic premise as a nootropic is intranasal adminitration to facilitate synaptic pruning.

Was originally discovered in the soil of Easter Island in 1975 from Streptomyces tsukubensis. Wtf. And that molecule: a macrolide.

• It inhibits Candida and other fungal infections, but it supresses the immune system by inhibiting activation of T Cells and B cells via reducing their sensitivity to IL-2.

  • Although, these things seem to have increased serum concentrations following treatment.

• Treatment with TOR-I results in a decrease in testosterone level, and an opposite increase in LH. Moreover, spermatogenesis seems to be disrupted by TOR-I and FSH levels are increased. R

• Independently increases PSD-95, NR2B expression, and glutamate recycling

• High levels in the periphery promote Beta Cell rest (somehow less sensitive to glucose)

• [Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system]

  • Many beneficial effects are via inhibiting mTORC1, and mTORC2 for the negative side effects.

• Chronic inhibition of mTOR by rapamycin modulates cognitive and non-cognitive components of behavior throughout lifespan in mice

  • Decreases mTORC1, but not mTORC2.

• Chronic Rapamycin Treatment Causes Glucose Intolerance and Hyperlipidemia by Upregulating Hepatic Gluconeogenesis and Impairing Lipid Deposition in Adipose Tissue

• Fasting and rapamycin: diabetes versus benevolent glucose intolerance

  • This mirrors the effect of fasting and very low calorie diets, which improve insulin sensitivity and reverse type 2 diabetes, but also can cause a form of glucose intolerance known as benevolent pseudo-diabetes.

• Rapamycin: one drug, many effects

• Induces Erythropoietin resistanec in kidney transplant patients - levels increase.

• Intranasal produces 25x+ brain concentrations with 1% the systemic exposure.

• Effects of Rapamycin on Insulin Brain Endothelial Cell Binding and Blood-Brain Barrier Transport

  • Acute did not affect BBB binding but chronic insigifnificantly slowed BBB transport. It slightly raised serum concentration of insulin and:

• Rapamycin for longevity: opinion article this is pretty informal

  • It can be regarded not as an “immunosuppressant” so much as reducing hyperimmunity. That’s what it’s used for in organ transplants
  • Whether this is by insulin or nutrients, overactivation of (mTOR->)S6K inhibits insulin signaling,
    • [Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1]
      • Amino acids mediate mTOR activation via class 3 PI3K (aka hVps34), as opposed to class 1. Infusion of Amino Acids leads to S6K1 activation and inhibition of insulin-induced class 1, leading to insulin resistance.
      • IRS-1 downregulatory phosphorylation is always by means of JNK??
        • A central role for JNK in obesity and insulin resistance
          • Absence of JNK results in decreased adiposity, significanty improved insulin ressistance and recepto signalling capacity.
  • Balancing Akt with S6K implications for both metabolic diseases and tumorigenesis
  • Ketogenic diets can cause symptoms of starvation psuedodiabetes (SPD)

• Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

Disease

• One thing to keep in mind about it potentiating Amyloid β in some pathologies is that the stuff may be protective!
• Intranasal rapamycin ameliorates Alzheimer-like cognitive decline in a mouse model of Down syndrome
  • Preventing systemic alterations avoids imunosuppresant effects in ther periphery. But the brain?
• Rapamycin Activates Mitophagy and Alleviates Cognitive and Synaptic Plasticity Deficits in a Mouse Model of Alzheimer’s Disease Get access Arrow
  • Effectively alleviates defecits of plasticity in APP models, promoting lysosome-mitophagosome fusion. Impedes Cytochrome C-mediated apoptosis, etc.
• Restoration of aberrant mTOR signaling by intranasal rapamycin reduces oxidative damage: Focus on HNE-modified proteins in a mouse model of down syndrome
  • Oxidation of Arginase-1 and PP2A play a part in brain damage associated with synaptic transmission failure and NFTs.
  • InRapa reduced ARG-1 protein-bound HNE and rescues its activitiy.
• mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer’s Disease
  • Restores eNOS-dependent cerebrovascular function funcions in AD models. (Since mTOR inhibits eNOS)
• Intranasal administration dring any point in the lifespan, besides late stage alzheimers, where it increases the toxicity of Amyloid β, will suppress its formation and disease progression and increase memory/cognition/mood.
• Therapeutic effects of rapamycin on MPTP-induced Parkinsonism in mice
  • IGF-1 inhibition mitigates MPTP-induced neurological dysfunction.

Roxadustat

Rating: ★★★. Increase in both physical and mental endurance. It gives an enjoyable headspace, though it’s all pretty subtle. 100 mg taken 4x/week (workout days).

- See also FG-4497.

• Elevated endogenous EPO by 2x
• Possible blood pressure increase - was not found in metanalyses and only in a small minority in clinical trial.
• Erythropoiesis gives a performance advantage due to additional oxygen capacity but too much over time can potentially become problematic if it leads to polycythemia. There should be low risk with roxadustat as it was shown to be safe in healthy men, but if you already have high rbc, hemoglobin, hematocrit or are on anabolic steroids for example(in which case you shouldnt use it anyway), the risk would be increased.
• The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells
• Roxadustat: Not just for anemia
  • In particular, it has the potential to increase the risk of pulmonary hypertension and vascular calcification, and aggravate inflammatory infections.
    • However, this is just based off a single case study of a diseased 73 year old woman, lol.
    • [Evaluation of the Carcinogenic Potential of Roxadustat (FG-4592), a Small Molecule Inhibitor of Hypoxia-Inducible Factor Prolyl Hydroxylase in CD-1 Mice and Sprague Dawley Rats]
• Roxadustat prevents Ang II hypertension by targeting angiotensin receptors and eNOS
  • HIF-1α inhibits AT1 and activates AT2 and eNOS.
• FG-4592 Improves Depressive-Like Behaviors through HIF-1-Mediated Neurogenesis and Synapse Plasticity in Rats
  • HIF-1α → CREB, BDNF, EPO, VEGF obviously, but also PSD-95 and Homer-1.
• Hypoxia inducible factor stabilization leads to lasting improvement of hippocampal memory in healthy mice

Selegiline

Rating: ★★. This is partially a longevity/health supplement, but acute effects are to be expected, and I didn’t notice too much besides increased ADHD-like symptoms, probably from an acute tyrosine hydroxylase inhibition that’s seen. It’s controversial whether it’s worth taking especially now that we know MAO-B isn’t selective for dopamine like originally thought, and instead is part of GABA biosynthesis (Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition). It’s an interesting one though.

• Irreversible inhibitor of MAO-B, in the sense that MAO-B must be resynthesized. MAO-A is slightly inhibited in high doses (20+mg/day). The standard clinical dose is something like 10mg/day oral.
• Catecholamine release enancer
• Upregulates GDNF→VMAT2
• Anti-Cholinergic - so it can cause mydriasis (pupil dilation - is this the same as lsd?)
• Too much MAO-B inhibition can wreck sleep in the short term.
• Decently decreases amphetamine induced oxidative stress. Amphetamine still causes neurotoxicty through other mechanisms.
• Reversible transvestic fetishism in a man with Parkinson’s disease treated with selegiline ==WTF==
• Pharmacological aspects of the neuroprotective effects of irreversible MAO‑B inhibitors, selegiline and rasagiline, in Parkinson’s disease
  • Increases NGF. Not sure if that’s just in strokes
• [The effect of 6-months l-deprenyl administration on pineal MAO-A and MAO-B activity and on the content of melatonin and related indoles in aged female Fisher 344N rats]
  • 0.25mg/kg (HED ~2.6mg) for six months. MAO-A significantly inhibited but only after 5 hours of darkness. After 14 hours, before lights off, 5-HT, NAS, 5-HIAA and 5-HTOL levels did not differ.
• Chronic selegiline administration transiently decreases tyrosine hydroxylase activity and mRNA in the rat nigrostriatal pathway.
  • This is observed in the corpus Striatum - not substantia nigra.
  • It decreased at 3/7 days but recovered by 14. And then at 21 days, TH mRNA was raised by 3x.
• Evidence that Formulations of the Selective MAO-B Inhibitor, Selegiline, which Bypass First-Pass Metabolism, also Inhibit MAO-A in the Human Brain
  • ‘Zelepar’ disintegrates and is absorbed through the buccal mucosa.
  • 2.5mg/day wasn’t nothing. But in some people, the activity was actually raised.
  • DAT levels were measured in 10mg group by means of cocaine binding. It’s hypothesized selegiline might inhibit it. There were only 3 subjects but yeah not a significant change.
• Inhibition of Bupropion Metabolism by Selegiline: Mechanism-Based Inactivation of Human CYP2B6 and Characterization of Glutathione and Peptide Adducts
  • CYP2B6 substrates (that which it metabolizes) also include Ketamine and Sertraline (inhibitor) and some others. Selegiline inhibits, and other inhibitors are memantine, raloxifene, curcumin, fluoxetine, fluvoxamine
• Effects of selegiline on antioxidant systems in the nigrostriatum in rat
  • 2mg/kg = 22 hed.
• Effect of low‐dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine‐induced dopamine release in vivo

• Effect of long-term treatment with selective monoamine oxidase A and B inhibitors on dopamine release from rat striatum in vivo
  • found no change of dopamine concentration in striatum acutely treated with selegiline and a small increase of dopamine on chronic treatment.

Semax

Rating: ★. Felt nothing.

• Its mechanism of action is unclear. May act through Melanocortin Receptors which has an inhibitory action on D2 I believe.

• Increases TrkB insofar as the Adrenocortocotropic Hormone system is working well. I think it basically mimics ACTH-10.

  • Downregulation warrants cycling.

• Antidepressant and axiolytic; attenuates behavioral effects of chronic stress.

• Semax and Selank Inhibit the Enkephalin-Degrading Enzymes of Human Serum

• Semax, an analog of ACTH(4-10) with cognitive effects, regulates BDNF and trkB expression in the rat hippocampus

  • 50μg/kg (HED 581): 1.4-fold increase of BDNF protein levels accompanying with 1.6-fold increase of TrkB tyrosine phosporylation levels, and a 3-fold and a 2-fold increase of exon III (gene thing) BDNF and trkB mRNA levels, respectively, in the rat hippocampus
  • Upregulates TrkB, unlike noopept. I mean think about it, that’s probably why it has reverse tolerance. The question of course is how long that lasts especially if you ragequit after you notice shedding and your shit is still upregulated.

• Effects of Semax on Dopaminergic and Serotoninergic Systems of the Brain

  • For the fans: Yeah decent increase in Hypothalamic Dopamine concentrations. Looks like a MAO-B inhibitor would do it well considering the dopac+HVA increase but this is probably the case with literally anything that increases dopamine to be honest. Basically no increase in 5-HT but a spike in 5-IAA I believe is a nice sign?

• Another study showed 50% decrease in frontal cortex BDNF expression.

• Semax, An ACTH(4-10) Analogue with Nootropic Properties, Activates Dopaminergic and Serotoninergic Brain Systems in Rodents (Eremin et al., 2005)

Reverse tolerance!

Other Forms

• N-Acetyl-Semax: Possibly anxiogenic.

• N-Acetyl-Semax Amidate: ~33% longer half life. Anxiolytic.

• Adamax: 2x the half life of NAS, and better absorption.

SkQ1

Rating: ★★. I don’t notice anything.

• Part of SkQ (which includes -R1, 2, 2M, 3,4,5, -berb, -palm, C12TPP, and MitoQ) which are mitochondrial Antioxidants. They’re lipophilic cations so they penetrate through membranes. They inhibit ROS directly due to plastoquinone oxidation, or reducing ΔΨ.

• I suppose they’re pretty popular for Longevity. Mice in a study lived 335 days vs. 290.

• Behavioral Effects Induced by Mitochondria-Targeted Antioxidant SkQ1 in Wistar and Senescence-Accelerated OXYS Rats

  • SkQ1-treated rats of both strains displayed significantly higher locomotor and exploratory activity in the open field (OF) and less anxiety in the elevated plus-maze (EPM)
  • SkQ1-treated Wistar rats exhibited slower learning in the Morris water maze (MWM) task comparison to the control group… may be associated with differences in redox homeostasis.

• Mitochondria-targeted antioxidant SkQ1 improves impaired dermal wound healing in old mice

  • SkQ1 treatment resulted in accelerated resolution of the inflammatory phase, formation of granulation tissue, vascularization and epithelization of the wounds. Wounds contained increased amount of myofibroblasts which produce extracellular matrix proteins and growth factors mediating granulation tissue formation.
  • Earlier we have found that SkQ1 stimulated production of active TGFβ by fibroblasts
  • The TGF-β produced by SkQ1-treated fibroblasts was found to stimulate motility (and tubulogenesis) of endothelial cells in vitro, an effect which may underlie pro-angiogenic action of SkQ1 in the wounds. Movement of the epitheliocytes into the in vitro “wound” was directly stimulated by SkQ1
    • Scavenging of reactive oxygen species in mitochondria induces myofibroblast differentiation
      • Scavenging of ROS activated the Rho-ROCK-LIMK pathway, and stimulated MMP9 secretion, which activated TGF-β, which mediated differentiation of subcutaneous Fibroblasts to myofibroblasts. NAC inhibited MMP activity.
  • Prevention of excessive reaction of endothelium to the pro-inflammatory cytokine(s) might account for anti-inflammatory effect of SkQ1.

• Important to support Electron Transport Chain complex IV to prevent peroxide buildup.

Store in the fridge. Can be injected or administered intranasal (~33 μg/spray)

SR9009

Rating: ★★★. My first cycle of 10mg daily, it reduced my sleep drive/need by a whole REM cycle. But I don’t get this anymore. The benefits seem endless and I can’t think of any negatives besides the fact it is untested in humans and we don’t know if something bad could crop up.

• Rev-Erb-α ligand. This is epic because it basically potentiates proper cicadian rhythm.
• Pharmacological targeting of the mammalian clock regulates sleep architecture and emotional behaviour this is one of the main studies, I believe.
  • I need to take a close look at the comparison between the 2. They might have slightly new effects?
• SR9009 has REV-ERB–independent effects on cellproliferation and metabolism
  • SR9009 can decrease cell viability, rewire cellular metabolism, and alter gene transcription in hepatocytes and embryonicstem cells lacking both REV-ERBαand -β.
• Regulation of Circadian Behavior and Metabolism by Synthetic REV-ERB Agonists
  • In addition to the decrease in fat mass we also observed a12% decrease in plasma triglycerides (TGs) and a 47% decrease in plasma total cholesterol (Chol) (Fig. 5c). Plasma non-esterified fatty acids (NEFA) were also reduced (23%) along with plasma glucose (19%) in the SR9009 treated animals (Fig. 5c). There was also a trend toward a decrease in plasma insulin levels (35%). Consistent with the decrease in adipocity we also noted an 80% decrease in plasma leptin and a decrease (72%) in the proinflammatory cytokine IL-6
  • Treatment resulted increased glucose oxidation in addition to fatty acid oxidation
• SR9009 inhibits lethal prostate cancer subtype 1 by regulating the LXRα/FOXM1 pathway independently of REV-ERBs
  • Somehow induces LXRα → blocks FOXM1, which is an inducer of PCS1 (a driver of prostate cancer) amongst other things.
    • (This could be awesome since LXR in the brain is neurogenic)
• No effect on insulin sensitivity. Lowers cholesterol.
• Strong anxiolytic without any depressive/GABAergic effect.
  • Could be due to Rev-Erb inhibiting NPAS2
• REV-ERBβ is required to maintain normal wakefulness and the wake-inducing effect of dual REV-ERB agonist SR9009
  • REV-ERBβ-deficient mice administered with dual REV-ERB agonist SR9009, failed to show drug-induced wake increase
• Rev-erbα agonist SR9009 protects against cerebral ischemic injury through mechanisms involving Nrf2 pathway (Mar 2023)
• Identification of a small molecule SR9009 that activates NRF2 to counteract cellular senescence (2021)
  • Activates NRF2 pathway.
    • RNA sequencing data confirmed that the elevated SASP factors were reduced by SR9009, including IL-1α, IL-1β, IL-8, IL-6, and CXCL-1.
  • Attenuated p38.
  • Weakened NF-κB and PI3K-AKT pathways.
• REV-ERB Agonist SR9009 Regulates the Proliferation and Neurite Outgrowth/Suppression of Cultured Rat Adult Hippocampal Neural Stem/Progenitor Cells in a Concentration-Dependent Manner
  • Inhibited the growth of these AHPs.
  • 9009 activated Rev-Erbβ downstream genes like Ccna2 and Sez6.
    • Ccna2 = Cyclin A2. Promotes proliferation.
    • Sez6 promotes neurite outgrowth
• REV-ERB agonism improves liver pathology in a mouse model of NASH
  • Recruits Ncor1 (Nuclear receptor co-repressor 1).
• Pharmacological and Genetic Modulation of REV-ERB Activity and Expression Affects Orexigenic Gene Expression (2016)
  • Not only orexin, but it also strongly represses transcription of the orexin receptors.
  • Orexin signaling via OX1R in the striatum and nucleus accumbens is associated with reward feeding and addictive behavior toward nicotine and other drugs. SR9009 thus prevents this.
  • I wonder it this provides some insight into inconsistency from Modafinil, given how the wakefulness-promoting effect is enhanced in orexin-KO and everything.
• mTOR inhibitor.
• SR9009 induces a REV-ERB dependent anti-small-cell lung cancer effect through inhibition of autophagy
  • Via what exactly? Repressing Atg5
• Identification of a small molecule SR9009 that activates NRF2 to counteract cellular senescence
  • SASP suppressor (via activating NRF2) , inhibiting senescence.
• Rev-erb-α regulates atrophy-related genes to control skeletal muscle mass
  • KO mice have increased expression of muscle atrophy-related genes (atrogenes), while over-expression = increased muscle size.
• Suppression of atherosclerosis by synthetic REV-ERB agonist
  • reduced REV-ERBα expression in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis
  • Reduced the polarization of bone marrow-derived mouse macrophages to proinflammatory M1 macrophage while increasing the polarization of BMDMs to anti-inflammatory M2 macrophages
• REV-ERBα Regulates TH17 Cell Development and Autoimmunity Reduces incidence of autoimmunity.

• According to tyw’s citiation that is 404, it peaks around 10 PM - noon? ZT0 (zeitgeber time) = midnight to the system being studied, which is the opposite between mice and humans I suppose.
  • (This post is a fantastic writeup on SR9009 though)

Metabolism

• No reported negative side effects afaik brotha.
• α IC50 = 670 nM and αβ IC50 = 800
• In vitro metabolism of the REV-ERB agonist SR-9009 and subsequent detection of metabolites in associated routine equine plasma and urine doping control samples
  • To the best of the authors’ knowledge, this is the first report of an adverse analytical finding in an equine sample for SR-9009 or its metabolites in equine doping control.
    • Adverse meaning what exactly? Besides having evidence of administration. Since some studies it was undetected in urine.
• CYP stuff. A further insight into the metabolic profile of the nuclear receptor Rev-erb agonist, SR9009

SR9011

• There are ohter rev-erb-α ligands. For one thing, this has better bioavailability. Sonething like 5x? Idk if this matters when intranasal, though aerin has recommended intranasal for all of them.
• (α IC50 = 970 nM and αβ IC50 = 790)
• Far less studied. Idk, I’ll check it out some other time maybe? It’s hard to say what the differences are without going deep asf. I guess I’ll see what’s different between α and β.

SR10067 (1380548-06-2)

Also has strong Rev-Erb-β affinity. And something like 6-8x more potent in general. IIRC, this is orally available.

Dose

• 6-20mg intranasal. 4 hour half-life. Very poor oral bioavailability.

Nasal spray in caprylic acid (it’s lipophilic) is what you won’t see a lot of people discussing on the internet.

Rev-Erb

Rev-Erbα (NR1D) and Rev-Erbβ (NR1D2) (aka αβ). The latter was originally thought to be redundant, but it does have some more novel research into distinct actions.

Nuclear receptor transcription factors for the circadian clock.

• Rev-Erbα is also expressed in muscle, adipose tissue, and liver. Regulates Fatty Acid Synthase.
• TLR4 antagonist.
• Detects Heme, its endogenous ligand. Whatever that’s worth.
• Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy
  • Activates the Stk11–AMPK–SIRT1–PGC-1α signaling pathway.
    • Indeed, STK11 (aka LKB1 (liver kinase B)) activates AMPK. mRNA levels are Increased by ERα activation.
  • Inhibits mitophagy. (Thereby increasing mitochondrial content and oxidative capacity of myocytes)
  • A Rev-erb-α/Ppargc1-α cross-talk pathway regulates heme synthesis in hepatic cells:
  • increase in glutamate-malate-stimulated and ADP-driven respiration in absence and presence of succinate of permeabilized fibers
  • Represses genes involved in autophagosome formation and lysosomal degradation.
• Nuclear receptor Rev-erbα: up, down, and all around (2014)
• Nuclear receptor Rev-erbα: a heme receptor that coordinates circadian rhythm and metabolism (2010)
  • Rev-Erb α dimers on RORE recruit NCoR (nuclear corepressor 1) in a Heme-dependent manner. NCoR recruits HDAC3
  • Heme binding to Rev-Erbα induces feedback inhibition of its own synthesis (reducing ALAS1, via NPAS2/PGC-1α)
  • In Brown Adipose (but not the liver), it promotes UCP1.
  • Rev-erbα and Rev-erbβ coordinately protect the circadian clock and normal metabolic function
    • Peaks in the liver (and brown adipose tissue. Wonder about the brain?) at mouse ZT 10 (and α is a bit higher) but so what, is this equivalent to 2 hours (before? After?) waking for humans? Considering that’s probably when they get up to start the day night.
  • Behavioral Changes and Dopaminergic Dysregulation in Mice Lacking the Nuclear Receptor Rev-erbα
    • Deficient mice had upregulation of Tyrosine Hydroxylase, leading to increased hippocampal dopamine turnover. This lead to hyperlocomotion.
      • This one is kind of surprising, maybe. Why does it repress TH?
    • Decreased habituation in novel object paradigms and impaired short- and long-term memory.

(S)unifiram

Stimulates CAMK II and PKCα.

• Antagonizes barbituate-induced inhibition of glucose transport (as do racetams)
• [Pharmacological characterization of DM232 (unifiram) and DM235 (sunifiram), new potent cognition enhancers.]
  • Increase Acetylcholine release in rat cerebral cortex.
  • Unifiram increases fEPSP amplitude in rat hippocampal slices.

TAK-653

Rating: ★★★★. The impressionistic subjective report would be something like it makes everything slightly simpler, obvious, lubricated. Neboglamine can be described as quite similar except on the more behavioral side of things rather than cognitive. You can pretty much think of it like if observing the connections between things as nodes overlapping venn diagrams. TAK makes the overlapping bigger/more obvious. And realistically this corresponds nicely to what it’s doing physiologically. But for the first few days, it’s kind of a mess and can induce sensory overload and a dissociative feeling, because the context of those nodes is also part of the overlap. I.e., it’s hard to consciously parse these things into a more generalizable form if you know what I mean. (This might be mediated by A dentate gyrus-CA3 inhibitory circuit promotes evolution of hippocampal-cortical ensembles during memory consolidation) Whereas without TAK things feel more context/‘scale’-invariant, but as one might assume, that’s ultimately at the cost of nuance no? But after a few days though you just magically get used to it.

• A more selective TAK-137. Finally a bona-fide ampakine, as it has basically no agonist activity.
• Central nervous system effects of TAK-653, an investigational alpha-amino-3-hydroxy-5-methyl-4-isoxazole receptor (AMPAR) positive allosteric modulator in healthy volunteers (Sep 2022)
  • Improves Stroop test (proxy of Executive Function apparently): TAK-653 0.5 mg but not 6 mg decreased the number of correct responses in incongruent trials
  • demonstrated a psychostimulant-like pharmacodynamic profile on the NeuroCart consistent with previously reported increase of cortical excitability following Transcranial Magnetic Stimulation (TMS) of the human motor cortex.
• [Pharmacokinetic and pharmacodynamic properties of the investigational AMPA receptor positive allosteric modulator TAK-653 after single and multiple rising doses in healthy volunteers]
  • Tried 0.3-18mg.
  • Maximum plasma concentrations were attained within 1.25 h to 5 h after dosing, the terminal half-life varied from 33.1 h to 47.8 h and cerebrospinal fluid concentrations were suggestive of rapid brain penetration
• Strictly regulated agonist-dependent activation of AMPA-R is the key characteristic of TAK-653 for robust synaptic responses and cognitive improvement (Suzuki et al. 15 July 2021)
  • 3 and 10mg/kg in mice. The former had like a 20% greater effect on BDNF release after AMPA treatment.
  • Did not show prominent subunit selectivity for homomeric AMPA-R
  • In monkeys, the beneficial effect (~20%) on “delayed match-to-sample” task was maintained for 24 hours after administration. Its half life is 9.4 $\pm$ 3.8 hours at 0.03 mg/kg p.o in monkeys.
  • Enhanced sustained attention in poor-performing mice. Increased novel objection recognition test results by like 10%. 0.03-0.3 mg/kg were all pretty similar.
  • Ameliorated abnormal social interaction in models of schizophrenia (poly-I:C. No idea, but it made them complete antisocial and then back to near baseline)
  • 10 μM in prefrontal cortex had the best effect on AMPAR-mediated EPSPs in the PFC: tripling EPSP duration and 6x the number of spikes.
• TAK-653, an AMPA receptor potentiator with minimal agonistic activity, produces an antidepressant-like effect with a favorable safety profile in rats (Dec 2021)
  • 0.1 or 1 mg/kg p.o. The time to reach peak plasma concentration after the oral administration of TAK-653 at 1 mg/kg was between 1 and 2 h
  • “Increased” phosphorylated and active forms of mTOR, P70S6, and AKT and ERK.
  • Stimulated mTOR and BDNF production.
  • Despite expectations, no relevant clinical effects have been demonstrated for the CX series.
  • In vitro for 10 minutes wih 1μM tripled ERK phosphorylation. Nothing tooo crazy otherwise
  • Virtually no agonist activity. Steric repulsion by GluR2 Ser750 of the closed AMPAR caused TAK-653 only to bind in the presence of a ligand.
    • Otherwise, there would be some kind of bell-shaped dose-response curve whereby excess glutamate release could get you in big trouble.
    • GluR2 Ser750 = GluR1 Ser743.
• [Novel AMPA Receptor Potentiators TAK-137 and TAK-653 as Potential Rapid-Acting Antidepressants (2021)]

THC

• Partial agonist of CB1 (25.1nM Ki), CB2 (35.2nM Ki), as well as other Endocannabinoid receptors like:
  • ‘Orphan receptors’:
    • GPR18 (0.96nM Ki)
      • (N-Arachidonyl glycine receptor (indeed that’s a ligand, a metabolite of Anandamide) (but guess what it may not even an agonist))
      • Resolvin (metabolite of DHA D2 is an agonist. Have I not heard about PUFAs and intraocular pressure?
        • GPR18 ‘resolves’ inflammatory-responses.
    • GPR55 (8.1nM Ki) (G13 G-protein coupled.)
      • Forms heteromers with CB2
    • Maybe GPR119 (endogenous ligand is Oleoylethanolamine but also binds is anandamide etc.)
      • Expressed predominantly in the pancreas and GI; activation reduces food intake and weight gain.
    • TRPV1/2, TRPA1
• When cannabis is decarboxylated, tetrahydrocannabinolic acid is converted into THC.
• When THC is taken orally, it is metabolized into 11-hydroxy-THC (then into 11-nor-9carboxy-THC), which are glucuronidated and excreted into the urine.
• Blocking both LTP and LTD makes sense why it’s used for escapism, no?
• PPAR-γ agonist.
• Δ9-Tetrahydrocannabinol Disrupts Estrogen-Signaling through Up-Regulation of Estrogen Receptor β
  • Suppresses Estradiol-induced MCF-7 cell proliferation. Inhibits E2-liganded ERα activation. Upregulates ERβ.
• Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling
  • Mediated by the βγ of CB1.
  • Ablation of COX-2 also eliminates Δ9-THC-impaired hippocampal long-term synaptic plasticity, spatial, and fear memories.
    • I’ve heard of problems regarding STAT3 though.
• Chronic cannabis promotes pro-hallucinogenic signaling of 5-HT2A receptors through Akt/mTOR pathway
  • Supersensitive coupling of 5-HT2A toward inhibitory Gαi/o/etc. G proteins was observed, without alteration in canonical Gq signaling.
• Dissociation of the Pharmacological Effects of THC by mTOR Blockade
  • Acute anxiogenic and amnesic-like effects were prevented by subchronic mTOR inhibition. But hypothermia, anxiolysis, etc.
  • a clear tolerance to THC-induced anxiolysis, hypothermia, hypolocomotion, and antinociception was observed after chronic treatment, but not to its anxiogenic- and amnesic-like effects.
• The Role of Cannabinoids in Neuroanatomic Alterations in Cannabis Users (2016)
• Cannabinoid modulation of hippocampal long-term memory is mediated by mTOR signaling
  • THC → GABAergic inhibition → NMDA signalling (!) → mTOR aberrant protein synthesis
  • THC long-term memory deficits were mediated by CB1Rs expressed on GABAergic interneurons through a glutamatergic mechanism, as both the amnesic-like effects and p70S6K phosphorylation were reduced in GABA-CB1R knockout mice and by NMDA blockade.
• Hyper-priming in cannabis users: a naturalistic study of the effects of cannabis on semantic memory function
  • Finding connections between unrelated concepts is due to ‘hyper-priming’ causing ‘fast and loose’ patterns of spreading activity.
• Bayesian causal network modeling suggests adolescent cannabis use accelerates prefrontal cortical thinning

Opioid

• Cannabis-Induced Hypodopaminergic Anhedonia and Cognitive Decline in Humans: Embracing Putative Induction of Dopamine Homeostasis
  • it is possible to induce ‘dopamine homeostasis,’ that is, restore dopamine function with dopamine upregulation with the proposed compound and normalize behavior in chronic cannabis users with cannabis-induced hypodopaminergic anhedonia (depression) and cognitive decline
  • I believe they’re referring to ‘Pro-Dopamine Regulator’ (KB220(Z)), which is an Enkephalinase inhibitor. As well as screening for risk alleles, and balancing dysfunctional dopamine with pro-dopamine reward genes - and they actually cite DAT1, D2, D4, and COMT.
• Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common mu1 opioid receptor mechanism (Tanda 1997)
  • Increased extracellular dopamine concentrations selectively in the shell of the nucleus accumbens; these effects were mimicked by the synthetic cannabinoid agonist WIN55212-2.
  • SR141716A, an antagonist of central cannabinoid receptors, prevented the effects of THC but not those of heroin. Systemic naloxone, or naloxonazine (selective μ-1 antagonist) infusion into the ventral tegmentum, prevented the action of cannabinoids and heroin on dopaine transmission.
• [Cannabinoid receptor and WIN 55 212‐2‐stimulated [35S]‐GTPγS binding in the brain of mu‐, delta‐ and kappa‐opioid receptor knockout mice (Latimer 1987)]: The efficacy of CB1 receptor activation by the cannabinoid agonist WIN 55 212-2 was dramatically reduced in the caudate-putamen of MOR knockout animals
• Cannabinoid withdrawal syndrome is reduced in double mu and delta opioid receptor knockout mice
  • Acute effects and physical dependence were not modified in single deletion of μ-opioid receptor, δ-opioid receptor, or κ-opioid receptor.
  • Antinociception and hypolocomotion induced by acute THC administration remained unaffected, whereas the hypothermic effect was slightly attenuated in these double knockout mice.
  • During chronic THC treatment, knockout mice developed slower tolerance to the hypothermic effect, but the development of tolerance to antinociceptive and hypolocomotor effects was unchanged.
  • The rewarding properties of THC, measured in the conditioned place preference paradigm, were reduced in knockout mice.
  • Interestingly, the somatic manifestations of THC withdrawal were also significantly attenuated in mutant mice, suggesting that a cooperative action of MOR and DOR is required for the entire expression of THC dependence.

Neurotransmitters

I’d say it’s contentious.

• [Marijuana and Cholinergic Dynamics (Cheney 1981)]
  • Modertate doses reduce acetylcholine release from the cat cortex, and reduces the rate of biosynthesis in the hypothalamic and striatal slices.
• Increases total brain Acetylcholine levels R R R; inhibits AChE
  • Inhibitory Effects of Cannabinoids on Acetylcholinesterase and Butyrylcholinesterase Enzyme Activities
• Pre-treatment with D1 antagonist (barely) reduced THC-induced ACh release in PFCx and hippocampus. THC has been shown to increase DA release in Nucleus Accumbens via μ-Opioid Receptor-mediated mechanism in the VTA R
• Increases Pregnenolone synthesis via stimulation of CB1 (Gi/o), with it acting as an inhibitor reducing THC’s effects. R
  • Related: Prenatal THC exposure produces a hyperdopaminergic phenotype rescued by pregnenolone
• Delta(9)-tetrahydrocannabinol decreases extracellular GABA and increases extracellular glutamate and dopamine levels in the rat prefrontal cortex: an in vivo microdialysis study wtf?
  • Delta(9)-THC (1 mg/kg, i.v.) significantly increased extracellular dopamine and glutamate levels and decreased GABA levels. These effects were prevented by the cannabinoid antagonist SR141716A

Prenatal

• Prenatal THC exposure raises kynurenic acid levels in the prefrontal cortex of adult rats
• [Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects (Jiang et al 2005)] wtf?
• Prenatal marijuana exposure and neonatal outcomes in Jamaica: an ethnographic study
  • The neonates of heavy-marijuana-using mothers had better scores on autonomic stability, quality of alertness, irritability, and self-regulation and were judged to be more rewarding for caregivers.

Tropisetron

Rating: ★★★★. Laser focus, and it delivers every time. Not to mention it seems to be healthy and neuroprotective.

• https://old.reddit.com/r/NooTopics/comments/uw3y4v/tropisetron_is_one_of_the_best_nootropics_v2/

• https://old.reddit.com/r/NooTopics/comments/uu7se7/tropisetron_analysis_best_nicotinic_nootropic/

• Inhibits IL-2 synthesis and transcription in stimulated T cells. Potent inhibitor of PMA+ionomycin-induced NF-κB activation, but TNF-α mediation is not affected.

• Very anxiolytic. Also a lowering of rumination is confirmed in studies for the treatment of OCD.

  • Fluvoxamine combination therapy with tropisetron for obsessive-compulsive disorder patients: A placebo-controlled, randomized clinical trial

• Tropisetron protects against brain aging via attenuating oxidative stress, apoptosis and inflammation: The role of SIRT1 signaling (+SIRT1)

• Tropisetron sensitizes α7 containing nicotinic receptors to low levels of acetylcholine in vitro and improves memory-related task performance in young and aged animals

  • ~2.4 μM EC50 and 50 Imax for α7 nAChR partial agonism and 1.5 μM EC50 and 34 Imax @ α7β2 nAChR. And 20μM for α4 nAChR.
  • Apparently has α3β4 nAChR blocking properties.
  • 3 nΜ Ki for 5-HT3.

• [The 5-HT3 antagonist tropisetron (ICS 205-930) is a potent and selective alpha7 nicotinic receptor partial agonist]

  • 
    • 55 Ki on α4β2 nAChR isn’t nothing, I suppose.
  • so 36% efficacy for α7, where ACh has 100%. Nicotine has 53% actually, but good luck getting it to ever bind.
  • 1.3 μM EC50 for α7.

• Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor

  • The carbonyl oxygen in the ester linkage between the indole and tropane moieties contributes to stabilize a network of four water molecules located in the apical portion of the interface.

• 5-HT–induced alteration in the autophagy proteins LC3-II and p62 was significantly blocked by the 5-HT3 receptor antagonist tropisetron,…

• Tropisetron modulation of the glycine receptor: femtomolar potentiation and a molecular determinant of inhibition

  • Inhibitory effects occur at micromolar concentrations, whereas the potentiating effects are shown here to occur at femtomolar concentrations at the homomeric alpha1 (glycine) receptor. Potentiation implying it only occurred in the presence of glycine.

• Adenylyl Cyclase (AC) Mediates the Antidepressant-Like Effects of Tropisetron on a Mouse Model of Maternal Separation Stress

  • Administration of tropisetron at doses of 3 and 5 mg/kg (2x of 3) significantly increased the grooming activity time following the splash test. Forskolin showed similar results with 10 and 25 respectively. This is a nondepressive behavior.

• Effect of tropisetron on circulating catecholamines and other putative biochemical markers in serum of patients with fibromyalgia

  • U-shaped curve for dopaminergic properties, with >5mg being the turning point.. This is what causes constipation, I think.
  • Also interesting how there’s a Calcitonin increase after prolnged use?

• Tropisetron A Review of its Pharmacodynamic and Pharmacokinetic Properties, and Therapeutic Potential as an Antiemetic

  • Minor 5-HT4 antagonism. This is what causes constipation, I think.
    • This however showed partial agonism: 5HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle
  • Reduced the incidence of, or completely abolished, vomiting and retching induced by cytotoxic drugs such as cisplatin. Cytotoxic drugs cause gastrointestinal cellular damage which may result in the release of serotonin, activating vagal and possibly splanchnic afferent neurons which elicit the vomiting response. Tropisetron probably prevents emesis induced by cytotoxic agents by antagonising the effects of serotonin both at a peripheral (vagal) site and in the central nervous system at the terminus for vagal afferent fibres.
  • Increased plasma and urine 5-HIAA in emetic patients.
  • 52% for 20mg dose and 66% for 100mg dose Fischer et al 1992)

• Serotonin receptor type 3 antagonists improve obesity-associated fatty liver disease in mice.

  • (.06 mg/kg HED) Tropisetron treatment significantly reduced liver fat content (-29%), liver inflammation (-56%), and liver cell necrosis (-59%) in ob/ob mice.
  • Four-week-old ob/ob mice and lean controls were treated for 6 weeks orally with tropisetron or palonosetron at 0.2 mg/kg per day.
  • Reduced liver fat content (-29%), liver inflammation (-56%), and liver cell necrosis (-59%). Decreased plasma Alanine Aminotransferase, and portal vein Endotoxin levels. Caused a reduction of elevated serotonin levels and an increase of SERT in the duodenum.

• The 5-HT3 receptor antagonist ondansetron potentiates the effects of the acetylcholinesterase inhibitor donepezil on neuronal network oscillations in the rat dorsal hippocampus

  • Prolonged the theta and gamma currents

• The acute effect of tropisetron on ECG parameters in cancer patients

  • Did not result in QT interval, etc. @ 5mg IV.

• https://www.medsafe.govt.nz/profs/Datasheet/t/tropisetronaftinj.pdf

  • At very high repeated doses, visual hallucinations and, in patients with pre-existing hypertension, an increase in blood pressure, have been observed.
  • At the 5 mg dose, constipation and, less frequently, dizziness, fatigue, somnolence, and gastrointestinal disorders, such as abdominal pain, diarrhoea and anorexia were observed as well.

Neuroprotection

• Tropisetron and its targets in Alzheimer’s disease (Hashimoto 2015)
  • Promoted greater improvements in memory than current AD therapeutic drugs, such as memantine and Donepezil.
• WTF! The multi-functional drug tropisetron binds APP and normalizes cognition in a murine Alzheimer’s model
• Tropisetron as a neuroprotective agent against glutamate-induced excitotoxicity and mechanisms of action
  • Neuroprotection against glutamate-induced excitotoxicity is mediated by α7 nAChR activation.
  • caused internalization of NR1A
    • activation of nAChRs in fetal rat cortical neurons by treatment with Nicotine and Donepezil caused internalization of glutamate receptors, resulting in attenuation of the glutamate induced Ca2+ influx, reduction in Caspase 3 activation, and protection of cells from glutamate-induced excitotoxicity.
  • Decreases p38 levels.
    • Several studies have suggested that Ca influx through activated nAChRs affects phosphorylation level of the p38 MAPK and Akt intracellular signaling pathways resulting in neuroprotection
• The effect of tropisetron on oxidative stress, SIRT1, FOXO3a, and claudin-1 in the renal tissue of STZ-induced diabetic rats
  • Reversed decrease in SIRT1 and increase in NF-κB, p-FOXO3 and claudin-1.
• The multi-functional drug tropisetron binds APP and normalizes cognition in a murine Alzheimer’s model
  • Incresed sAPPα/Aβ ratio; it binds directly to APP.
    • Soluble amyloid precursor protein, which is actually a trophic factor. Inhibits caspase activation, cell eath, and neurite retraction.
  • Aβ monomers eventually aggregate into soluble oligomers and insoluble plaques.
• Reduces IL-1, IL-6, TNF-α, NF-κB and I’m sure other things in rat models of colitis. R
• Tropisetron upregulates cannabinoid CB1 receptors in cerebellar granule cells: Possible involvement of calcineurin
  • It significantly inhibits calcineurin, which regulates the CB1 expression. Calcineurin is highly expressed in Granule Cells, and they do not express 5-HT3. Indeed, other calcineurin inhibitors like FK506/tacrolimus also increase CB1 expression.
• Constipating at ~10mg+.

Vinpocetine

• 

• PDE1 inhibitor, improving peripheral Vasodilation.

• Compare to Ginkgo or Reserpine.

• Inhibits The NF-κB cascade via inhibiting IKK.

  • 
  • 

• Potent antioxidant. Used as a treatment for tinnitus.

• Reduces neuronal calcium influx without blocking presynaptic Ca2+ channels.

• [Effects of several cerebroprotective drugs on NMDA channel function: evaluation using Xenopus oocytes and [3H]MK-801 binding]

  • These results suggest that the inhibition of NMDA channels by vinpocetine shows a similarity to the action of Zn2+ which closes the gate of the NMDA channel

• [Vinpocetine preferentially antagonizes quisqualate/AMPA receptor responses: evidence from release and ligand binding studies.]

  • Reduced the efflux of dopamine and acetylcholine evoked by glutamate, quisqualate and NMDA, but not that evoked by kainate**

• Vinpocetine reduces cisplatin-induced acute kidney injury through inhibition of NF–κB pathway and activation of Nrf2/ARE pathway in rats

  • Seems to increase HO-1 and NF-κB inthis injury model unless I’m reading this wrong.

• Unique alterations to the rheological properties of Red Blood Cells. Reduced MDA and increased GSH levels in models of neurological damage

• Characterization of vinpocetine effects on DA and DOPAC release in striatal isolated nerve endings

  • Does not modify baseline DA release or exocytotic release evoked by K+; it inhibits DA release evoked by veratridine reversal of DAT.
  • Increases DOPAC levels suggesting an augmentation of dopamine metabolism, likely indepdendent of VSSC blockage, via increasing availability of the cytoplasm extravesicular DA; Impairs vesicular storage of Dopamine.
    • Does not enhance MAO, rather it acts like reserpine (VMAT2 inhibitor (it’s over)) which decreases the monoaminergic tone.
  • Inhibits voltage-gated sodium channel permeability, selectively inhibiting the transporter-mediated release of all neurotransmitters.

• [Vinpocetine and Ischemic Stroke]

  • Cerebral vasodilation enhances supply of oxygen & glucose, and ATP production.

• [Psychopharmacological Effects of Vinpocetine in Normal Healthy Volunteers]

  • Reported a lasting increase of cerebral 5-HIAA levels after treatment and transitorily enhanced 5-HT levels 2 h following (i.p.) treatment. Catecholamine levels were similarly increased 4-6 h following administration of vinpocetine.
  • Twelve females age 25-40 had dose-dependent decrease in reaction time in this memory test thing. Placebo 600 and plumetted from 560 to 420 after 20=>40mg.

Essentially, you can thank the monastic culture of (understandably) shying away from sharing attainments for the relative obscurity of the entire project and how tangibly progress along the path is demarcated, whereby full enlightenment (when we’re going off the four path model right out of the suttas) is just the final step on it.

There are discrete, well-catalogued steps along the way: ~12-16 of them leading up to the first stage of enlightenment out of 4, known as stream entry. See: The Progress of Insight by Mahasi Sayadaw or 30. The Progress of Insight - MCTB, and Four stages of awakening - Wikipedia. Roger Thisdell has one of the best (especially considering it’s brief) explanations I’ve ever seen of the 4 stages of enlightenment: Stage Theory of Enlightenment (it has “theory” in the title, but I dare say I’m dogmatic enough to say that it’s hardly “theoretical”.)

When you are ready, I highly recommend reading Daniel Ingram’s Mastering the Core Teachings of the Buddha. This is the closest thing to a ‘bible’ for awakening insofar as it’s a great starting place that could get the average person from start to finish, especially in that it also recommends other books, teachers, etc. I also highly recommend Frank Yang’s videos on Youtube to get a more visceral idea of how deep this really goes; how profoundly your perception changes. He’s documented his entire journey from the first sit to 4th path.

Again, I’m not very good at convincing people of things. I’m just writing here to share resources and research. As for the scientific section, my goal in the future is for us to understand the neurophysiological underpinnings of meditative progress and democratize it; in other words, develop neuroprosthetic, pharmacological, or otherwise technological interventions that facilitate progress without the tedious work of meditation. Not (just) for a wealthy elite, but also eventually for people who lack have the timenergy for meditation in the first place. I anticipate that this will, ideally, have profound effects on the global political organization of the human species and will exponentially promote cooperation that’s totally forward-thinking and increasingly pareto-efficient, and ultimately bring an end to all war and conflict. That’s mostly not a joke.

Or at the very least, use the neuroscience in order to understand the path and the process of meditation itself a little better. And as far as I’m aware, we’re still quite a ways off from determining any neural substrate for progress. For now we’ll just sit our asses down and meditate.

Cf. Zen and the art of speedrunning enlightenment

Meditation

I’m no meditation teacher, but if there’s any advice I could give to my younger self a few years ago, the main thing would be to stick to one technique—the technique you’re best at—which in my case is noting, and to stick with it without deviation, and maintain a minimum of ~90 minutes of sitting practice daily. The more the better. 2 hours is apparently a good sweet spot. Meditate at the same time of day, too. Intuitively speaking, this would promote hebbian learning to the highest possible extent (besides adding more time, such as in a retreat(-like) context) and it’ll gradually train your mind to unconsciously just know that now is a good time you can totally let go. Not being able to just let go has been my #1 problem, and letting go is practically the entire path in a nutshell.

But yeah, I think you should stick to the technique you’re best at, because what else are you going to have the motivation to do? You need to be haveng fun, actually. You need to be energetic, inquisitive, vigilant, etc. If you hate the technique you’re doing right from the get-go I think you’re probably screwed. There’s the sentiment that you should work on what you’re weak at, but frankly I don’t know how skillful that is. There’s also the notion that the duality expansion and contraction eventually collapses; some people may have a proclivity for contractive practices, some for expansion.

This is obviously not a comprehensive list, but some of the more robust techniques that really produce results would be include, in rough order from shamatha-focused to vipassana (take the exact order with a a grain of salt)

• Mantras: TM (David Lynch, lol), Vajrayana, etc.
• Visualizations
• Anapanasati (breath): The Mind Illuminated, Rob Burbea, Leigh Brasington, Sutra-inspired style “awareness”, Thai Forest, and many more.
• Kasina: with a candle (fire kasina), a bowl, a disk of construction paper, a body of water, etc.
  • Daniel Ingram claims that fire kasina and mahasi noting seem to be similar in terms of how quickly one goes through the progress of insight. But don’t get it twisted, this develops some serious concentration, and easily—like easier than anapanasati (I have heard it expressed in multiple contexts (example) that it just seems better than anapana and we might be leaps and bounds ahead in the meditation world had kasina been the default ‘concentration’ or ‘meditation’ practice). And, importantly, it’s fun, and it can get seriously trippy (for better or worse—don’t underestimate that; see some of the podcasts below). It’s also great at increasing lucidity of dreams (dream yoga!)—clearly it’s is a cool practice to do at night.
  • See: https://firekasina.org/, and Daniel Ingram’s interviews on the topic:
    • Deconstructing Yourself - Meditation, Magick, and the Fire Kasina, with Daniel Ingram
    • Deconstructing Yourself - The Liberating Practice of the Fire Kasina – with Daniel Ingram
    • Guru Viking - Ep166: Fire Kasina Mystic - Daniel Ingram
  • Kasinas - Discussion - The Dharma Overground
  • Fire Kasina Hints for Shinzen Meditators
  • How to blast through dullness into clarity and How to Practice Kasina Meditation: A Guide for Beginners from duffstoic.
  • The Yogi Toolbox: Candle Flame Kasina Practice
• Mettā: TWIM, Rob Burbea
  • Roger Thisdell - How to Meditate: Metta (loving-kindness)
  • Mettā is actually the opposite of craving , not ‘hatred’ (whose opposite is equanimity). From this, it should be a little more clear how it’s a perfectly valid object for attaining jhana.
  • Practicing Love - Tasshin
• MIDL (Mindfulness in Daily Life) Insight Meditation
• Vipassanā Body Scanning: S.N. Goenka (U Ba Khin lineage)
• Fast noting: Daniel Ingram, Shinzen Young
  • Roger Thisdell - How to Meditate: Noting
• Detailed noting: Kenneth Folk

Rather outside of that continuum are:

• Shikantaza (“just sitting”)/"Do nothing". Possibly not the best for complete beginners to meditation, but it is a complete path.

  • Roger Thisdell - How to Meditate: Do Nothing

• ‘Nondual’ type practices like Dzogchen which are generally more ‘advanced’, becoming more intuitive after stream entry.

• Dream Yoga; the practice of meditation inside lucid dreams (and obviously relevant in the general practice is inducing lucid dreams in the first place).

• There all all kinds of different styles of the above techniques. Anapanasati is probably taught by tens of thousands of different people, though I’m bad at numbers. The Mind Illuminated is recommended, but worrying and over-efforting about the stages is something I had trobule with, though YMMV. Rob Burbea is great and so are his dharma talk recordings on the jhanas, etc. A lot of people have written about the Jhanas:

  • Discovering Bliss States - Tasshin

Also yeah, I find it kind of hilarious how the concept of jhana infiltrated the hyper-materialist memplex of LessWrong, EA, e/acc, etc. In its wake there are (increasingly secular) efforts like Jhourney that aim to popularize its existence and relative accessibility. Here’s hoping the dharma-free™ methodology of this memeplex doesn’t prove to be a stopgap game of telephone that draws people away from the path to awakening, but I don’t really see that as a possibility, because jhanas are profound, and that sure as hell primes somebody for the rest of the teachings that Buddhism provides. There are more things in heaven and earth, Horatio, than are dreamt of in your philosophy. My simple request to this strawman of mine is that one should keep in mind (you may see this coming) that the Buddha was not satisfied even after achieving all 8 jhanas, for jhana alone will not liberate you from suffering. But again: not a thing wrong with it really. Wild descriptions of peak experiences are probably what initially got me interested in meditation.

Neurofeedback, Neuromodulation, etc.

It’s clear that there’s a hierarchy of sorts with meditation → neurofeebdack → neuromodulation via tACS/tDCS, rTMS, ultrasound, etc. in order of difficulty and effort, and I wonder if this isn’t very important for facilitating certain neurogenic changes. What I mean is:

• Meditation involves focusing one’s attention on a certain object via (what one would assume are primarily prefrontal) efferents (which is really in concert with myriad other regions) which also controls the activity of other regions. Meditation also involves the activation of self-monitoring regions making sure that you’re actually following the technique properly and conducively—that is to say, following the seven factors of awakening. Now you could say that the monitoring and the technique are one and the same (experientially or otherwise), but at the end of the day it also still seems to be a discrete process distributed in different networks across spacetime. I’d have to think about that though. But this is essentially the distinction made in The Mind Illuminated between attention and (peripheral) awareness.

• Neurofeedback (and very involved guided meditations, perhaps) still involves following a technique, but the monitoring component is offloaded to the feedback mechanism whose very purpose is to be more concrete than the meditator ‘interpreting’ the noisy wetware of their brain. I think as a result, the scope of the technique and what you’re monitoring for has been limited quite significantly; there’s way less mistaking which qualia are positive signals. Either the brain regions responsible for monitoring are now focused on the feedback (as the brain with the protocol such as a certain level of prefrontal alpha wave activity, a noise goes from loud to quiet, or a light goes from dim to bright), or perhaps it’s a different regions entirely.

• Neuromodulation then attenuates the conscious volition component, and not only that but essentially makes monitoring extraeneous. (Assuming here the montages are placing electrodes on regions of the prefrontal cortex)

But is it really the same as tried and true meditation as one goes down the hierarchy? This is what I ask here. The idea either is to train one’s natural ability to enter a given state, andor align one’s default state/qEEG with the protocol (much better for our purposes here, of course). If you take the helmet off and you’re the exact same as before, it’d be pretty gimmicky. Momentary stress relief or what have you is obviously not really the goal with (insight) meditation, or really any neurofeedback protocol worth its salt. So if you’re training the default activity, you’re trying to elicit a certain general cascade of plasticity: it’s hard to say it would be the exact same as meditation—is that important, or no?

Furthermore, another one of the biggest questions is if the brain changes seen in meditators are causative, such that instantly copy+pasting the analogous connection from awakened/experienced brain A → novice brain B (even if such a thing could be done rapidly or semi-reliably with a certain technological intervention) would place person B’s default state into a level of insight just like person A (or mostly there, or leaves them in the same state but places them on the fast lane with minimal ensuing effort, etc., since our brains are all different and certain steps may be close to impossible at least without extremely sophisticated brain-remodeling technology), or are these changes merely an arbitrary end result—an epiphenomenon—perhaps even from something that goes beyond the current scientific paradigm or even our own comprehension? Because on the one hand there’s the undeniable fact brain activity gives rise/plays a role to somebody’s current activity, wakefulness, etc. yet the experiential reality tells us that it’s actually consciousness that’s giving rise to all of reality including the brain.

I’m thinking out loud a lot here. But I think a lot of important questions come out of this.

• https://tagsync.com/ is one of the coolest things ever. He devised a neurofeedback program for facilitating the cross-frequency coupling between theta, alpha, and gamma. This ménage à trois might represent a key component of the brain’s higher functions: “TAG Sync is a phenomenon seen in the brain waves (EEG) during learning, insight, problem solving, & the near death experience”.

Dharma Links/Resources

You will (probably) never in your entire life attain profound insight just from reading something. Meditation is all that matters (broadly speaking). That being said, you must learn how to practice effectively and in accordance with your skills, which is easier said than done for most people.

• Kenneth Folk: his E-book Contemplative Fitness, which is absolutely fantastic (this book is a great all-in-one guide for introducing somebody to pragmatic dharma as a whole—because also you could read it in a day if you really wanted) as well as his Youtube videos, which are also informative.

  • What would I say if I had just five minutes to give comprehensive instructions for awakening?

• Daniel Ingram and his book Mastering the Core Teachings of the Buddha, free to read online. Masterpiece. Tl;dr of the entire book is to observe The Three Characteristics faster and faster and at a more and more minute scale. See here:

  1. Not trying to practice, lost in our stuff, spacing out, mindfulness weak.
  2. Mindfulness weak, lost in our stuff, but at least attempting some technique or just basic attention to what is happening at times, even if we can’t actually do it for long. People spend whole retreats at this level, unfortunately.
  3. Better able to practice, albeit with frequent interruptions, and to follow basic instructions such as noting, body scanning, or whatever you are trying to do.
  4. Able to do a specific vipassana technique or set of techniques that allows you to stay with aspects of your sensate reality as it presents with few interruptions.
  5. Able to apply those techniques or practices uninterruptedly.
  6. Able to perceive directly the three characteristics of objects in the center of attention consistently and directly regardless of whether we are using a more specific technique. In short, if you can do this, for that time, however long that lasts, whether you use a more formal technique is irrelevant.
  7. Able to perceive directly and continuously the sensations that make up the coarse background components also in that same light of strong, direct vipassana awareness, meaning direct comprehension of the three characteristics of not only the foreground objects, but also those in the background or periphery, such as rapture, equanimity, fear, doubt, frustration, analysis, expectation, and other sensations, as well as other objects as they arise, such as thoughts and the component sensations of feelings, as well as the primary object or objects (assuming we at this point are still using primary objects, which is not necessary).
  8. … and then add core processes, such as the sensations that seem to make up attention itself, intention itself, memory itself, questioning, effort, surrender, subtle fear, space, consciousness, and everything that seems to be subject or observer or self all the way through the skull, neck, chest, abdomen, and all of space such that nothing is excluded from this comprehensive, cutting, piercing, instantly comprehending clarity that is synchronized with all phenomena (or is just about to be).
  9. Able to do #8 naturally, effortlessly, and clearly due to diligent efforts to write that wiring on the mind as our new baseline default mode of perception.
  10. … Conformity Knowledge: we comprehend simultaneously two of the three characteristics of our entire sense field, including space, consciousness, and everything else in that volume as an integrated whole, and so attain Conformity, and then Change of Lineage, Path, and Fruition (to be detailed shortly). That’s what we are shooting for if we are at least going for stream entry, and it even works well for the sort of complete mindfulness that effects higher paths.

• Mahasi Sayadaw: especially Practical Insight Meditation and (his translation/interpretation of) The Progress of Insight as mentioned above.

• Shinzen Young’s videos on Youtube are probably what I would refer a more agnostic, secular skeptic to, but there’s also a lot of videos to wade through. His book The Science of Enlightenment is pretty good; a lot of his material more or less assumes you know nothing about whta meditation even is, and in some of his PDFs a significant amount of length is spent discussing trivialities for the sake of symmetry/completion and a lot of techniques/explanations that are frankly way too pedestrian for somebody trying to radically change their reality. And yet there’s also a lot of advanced stuff out there. He does not shy away from claiming attainments/enlightenment (which I respect greatly; transparency) (i.e 4th path unless I’m totally mistaken) and knows a thing or two about comparative religion, philology/etymology, etc.

  • This blog post annotates a section of his instructional PDF “What is mindfulness?” that denotes a 10-step progression in meditation ability.

• Ron Crouch’s outline of the progress of insight

  • Frankly I’m not absolutely positive this is accurate—but if so—I think this framework is one of the best and most concise explanations of the path there is. This wavelet represents a single sensation, and at each stage, your attention is able to ‘sync up’ further down its lifespan (the macrocosmic following suit).

• Just read Wikipedia! Topics pertaining to Buddhism are very comprehensively written, with a historical and etymological background and everything. It’s not like it comes with advice though, and it’s not always practical researching terms outside the framework of a certain system; Wikipedia is undestandably more philosophical.

  • http://tibetanbuddhistencyclopedia.com is also good, but not as comprehensive.

• /r/streamentry

  • Naturally, there are some prominent posters with really informative threads. /u/duffstoic or /u/adivader for instance.

• https://dharmaseed.org/

• https://thehamiltonproject.blogspot.com/

• The Dharma Overground, Daniel Ingram’s forum.

  • DhO Dharma Wiki
  • Daniel’s Posts Compilation
  • Shargrol’s Posts Compilation
  • A Reformed Slacker’s Guide to Stream Entry

• https://mandala-of-love.com/

Scientific Links/Resources

• Qualia Research Institute

• Qualia Computing | Revealing the computational properties of consciousness

• https://qualiaresearch.miraheze.org/wiki/Effective_Wellbeing_Engineering

• https://opentheory.net/

• Karl Friston & Robin Carhart-Harris. Free energy principle, entropic brain theory, etc.

• Guru Viking | Trialogue Series - Shinzen, Chelsey, and Jay some of the most important questions are brought up in this series. Though I’ll say most of them aren’t just gone and answered—a few instances “oh my God” though. And then you also get to hear Shinzen Young talk about how profound category theory supposedly is, lol. Apparently that will be important going forward.

  • https://semalab.arizona.edu/
  • https://www.jaysanguinetti.com/
  • 

• Orch OR and quantum consciousness

• Deconstructing Mindfulness - YouTube

• https://blankhorizons.com/

• https://meditationstuff.wordpress.com/ and https://meditationbook.page/

  • Review: Meditation from Cold Start to Complete Mastery

• https://smoothbrains.net/

• https://www.bltc.com/

Studies

• Neurobiology of Spirituality (3)

  • The volitional aspect of non-guided meditation appears to be guided by the Prefrontal Cortex, somewhat moreso on the right, and the Cingulate gyrus.
  • (T)he hallmark of altered states of consciousness is the subtle modification of behavioural and cognitive functions that are typically ascribed to the prefrontal cortex… Hyperfunction of the medial PFC, on the other hand, will lead to rigid conformity with rules and customs, excessive concern over oneself and one’s existence and excessive interpretation of the mind of others, all of which results in heightened religiosity (hyperreligiosity). R
  • Functional neuroanatomy of altered states of consciousness: The transient hypofrontality hypothesis
  • The PFC, when activated via the glutamatergic projections, can activate the Thalamus, especially the reticular nucleus, as part of a more global attentional network. The thalamus mediates the flow to the cortex of sensory information both visual and information needed to determine the body’s spatial orientation via the lateral geniculate body (LGB) and the lateral posterior nucleus (LPN), respectively.
    • The visual information is relayed via LGB to the striate (visual) cortex and the spatial information is relayed via LPN to the PSPL (posterior superior parietal lobule). When excited, the reticular nucleus via inhibitory GABAergic projections to the LGB and LPN cuts the input to the striate cortex and the PSPL (especially right). This functional deafferentation means a decrease in the arrival of distracting stimuli to the striate cortex and PSPL, enhancing the sense of focus during meditation.
      • The PSPL is involved in the analysis and integration of higher order visual, auditory and somaesthetic information and is also part of the complex attentional network including the PFC and thalamus. The PSPL helps construct a complex three-dimensional image of the body in space, helps distinguish objects and helps identify objects that can be grasped and manipulated. The PSPL deafferentation is supported by three neuroimaging studies, all of which showed decreased activity in the region during intense meditation
  • Evidence of mutual activation of PSNS and SNS, possibly due to the more characteristic PSNS activation causing a ‘breakthrough’/‘discharge’ to the SNS:
  • Serotonin, via the mechanism of inhibition of the LGB (lateral genticulate body), greatly reduces the passage of visual information and this results in visual hallucinations.

• The neural basis of the complex mental task of meditation: neurotransmitter and neurochemical considerations

  • 

• Neural correlates of religious experience - comparison between religious and non-religious subjects during a psalm recitation thing. Religious subjects did not have activated limbic areas (including Amygdala and Orbitofrontal Cortex), while non-religious subjects did show acivation in left amygdala.

• Shifting brain asymmetry: the link between meditation and structural lateralization

• Beyond the veil of duality—topographic reorganization model of meditation (11 Oct 2022) - recent, modern metaanalysis. Seems to have most of the important stuff and in context.

  • As meditation becomes effortful, that is when the Default Mode Network becomes rewired appropriately and coactivates with the central Executive Network, rather than relying on it.

• Neuromodulation of the mind-wandering brain state: the interaction between neuromodulatory tone, sharp wave-ripples and spontaneous thought

  • We advance the hypothesis that the hippocampal sharp wave-ripple (SWR) is a compelling candidate for a brain state that can trigger mind-wandering episodes.
    • It propagates activity throughout the Default Mode Network: recollective, associative, imagination and simulation processes

• Functional Connectivity of Prefrontal Cortex in Various Meditation Techniques – A Mini-Review

The Gray Matter of Grey Matter

There are myriad studies on the effect of various meditation paradigms and the gross anatomical changes they induce in the brain. Some hypertrophy here, some atrophy there. Some activation here, some deactivation there. Honestly I don’t know how relevant the changes in and of themselves are to progress, and who’s to say there’s a universal phenotype, even when you use the same technique?

• Increased ability to decouple regions in the brain associated with cognitive apprasial of pain: Anterior Cingulate Cortex and Dorsolateral Prefrontal Cortex. And, Ventromedial Prefrontal Cortex for analgesic effects.

• Experienced meditators had stronger coupling between Posterior Cingulate Cortex, Dorsal Anterior Cingulate Cortex, and Dorsolateral Prefrontal Cortex both when meditating and when not.

• Frontopolar Prefrontal Cortex: Grey/white matter increase ¹

• Sensory cortex: Grey/white matter increase ¹

• Orbitofrontal Cortex: Grey/white matter increase ¹

• Putamen: Experienced meditations experience slower age-related decline in Grey Matter

• Insular Cortex: Correlated with thickness, notably right anterior, correlating with accuracy/detection of stimuli. ¹, and activation ²

• Anterior Cingulate Cortex

  • Vipassana, et al.: Heightened activity, notably rostral.
    • This suggests that meditators have a stronger processing of conflict/distraction and are more engaged in emotional regulation. However, as the meditators become more efficient at focused attention, regulation becomes unnecessary and consequentially decreases activation of ACC in the long term"
  • Zen: decreased activity (Ritskes et al., 2003)

• Dorsal Anterior Cingulate Cortex: Increased cortical thickness/Grey Matter

• Posterior Cingulate Cortex

  • Samatha/metta/choiceless awareness: Deactivation.

• Cingulate Cortex: Increased activity during zen

• Prefrontal Cortex: increased activation increases free synaptic glutamate in the brain. ³

• Frontal Cortex: Increased activity ²

• Prefrontal Cortex:

  • Vipassana: Increased activity, specifically the dorsal medial prefrontal area, and correlated with thickness.

• Dorsomedial Prefrontal Cortex

  • Increased activation and cortical thickness/Grey Matter.

• Dorsolateral Prefrontal Cortex: Increased activation of right DLPFC

  • Increases in the right dorsolateral prefrontal cortex and decreases the rostral prefrontal cortex activation after-8 weeks of focused attention based mindfulness meditation

• Medial Prefrontal Cortex

  • Samatha/metta/choiceless awareness: Deactivation.

• Occipital Lobe: Metta: enhanced Gamma. Increased activation, notably lateral ²

• Parietal Lobe: Increase in grey matter and stimulation ². Vipassana: Significant enhancement of Gamma in experienced meditators.

• Locus Coeruleus: decreased activation (and its concomitant production of Noradrenaline ³

• Reticular Nucleus: activation is the chief mechanism responsible for (the) increase in GABA . ³

• Amygdala: activity reduced, especially in the right hemiphere/right basolateral.

  • Reduced right amygdala activity for mindful attenton, but (basically) Metta increased it more than control R
  • Impact of short- and long-term mindfulness meditation training on amygdala reactivity to emotional stimuli
    • Decreased right amygdala response to pos-neum but heightened connectivity between amygdala and Ventromedial Prefrontal Cortex: I reckon the vmPFC activity could be inhibitory projections.
      • Similarly, there is a negative correlation between Bipolar and Ventral Prefrontal Cortex-amygdala activity.

• Brain Stem: Increased grey matter in the Brain Stem. This improves communication between the cortex and all other areas within the brain. R

• Caudate: strengthened.

• Medulla: Increased grey matter

• Precuneus: Increased grey matter

• Hippocampus: Increase in left grey matter.

• Corpus Callosum: Grey/white matter increase ¹

• Cerebellum: Activation ²

• Thalamic nuclei: activation ²

• Basal Ganglia: activation ²

• Super longitudinal fasciculus: Grey/white matter increase ¹

Cognitive/Psychological

• Experience was found to correlate negatively with reaction times on an Eriksen flanker test.
• Participants with at least 6 years of experience performed better on the Stroop test, and reduced P3 ERP amplitude following the test. Increased amplitude in the N2 ERP. LPP ERP is larger in amplitude.
• Kundalini yoga in 81 participants aged >=55 improved recall/visual memory.
• Mindfulness Training Improves Working Memory Capacity and GRE Performance While Reducing Mind Wandering
  • Cognitive regulation: having control over giving attention to a particular stimuli, or by changing the response to it. Achieved through reapprasial (interpreting the stimulus in a more positive manner) and extinction (reversing the response).
  • Behavioral regulation: inhibiting the expression of certain behaviors in response to a stimulus.
  • Lateral Prefrontal Cortex is important for selective attention, and ventral prefrontal cortex is involved in hibiting response.
  • Attentional blink is reduced. P3b ERP amplitude decreased in those who completed a mindfulness retreat.
    • Btw, attention residue is when cognitions about a certain task persist even when one has transitioned to another. Clearly then, performance is inhibited. Fuck multitasking.
• Transcendental meditation and improved performance on intelligence-related measures: A longitudinal study
  • Twice-daily TM increased CFIT scores by 8 and 9 points over 4 years for university students, and 9 IQ points in high schoolers over only 3.5 months. There are plenty of other studies showing an increase in IQ.
    • CFIT has a standard deviation of 24 points. It correlates alright with other IQ tests.
• Fluid intelligence and brain functional organization in aging yoga and meditation practitioners
  • Pretty interesting how it takes certain interesting parameters. Yoga > meditation pretty consistently.
    • They measured cognitive decline:
    • Fluid intelligence (Raven’s), ‘Brain functional network resilience for targeted damage of 15 nodes’, Global efficiency, and characteristic path length:

Neural Oscillations

• Meditation States and Traits: EEG, ERP, and Neuroimaging Studies (Cahn & Polich 2006)

  • Has a mega list of previous studies from 1957-2005.
  • Theta and alpha activation related to proficiency of practice.
  • Only a few studies, namely Tibetans with metta/dzogchen, or Zen, increased Gamma. Experience probably fills in the gaps for whatever technique they’re using - that is, masters doing normally relaxing may show an increase in Hz if they intend for it to be somehow stimulating.

• Kundalini/kriya yoga/chakra stuff is implicated with gamma and beta waves.

• Linked with lower frequency Alpha waves and with Theta waves.

• Increase in alpha range frequencies, alpha band power, and overall slowing (frequency reduction) in EEG activity, during meditation. For most techniques that is.

• Beta waves are implicated in thinking, suppressing alpha/gamma.

• Decreased (even plummetted) frontal, and slight increase in central Delta waves is associated with reduced processing of self-referential thoughts.

• Nondirective meditation: Significantly increased theta power was found while meditating when averaged across all brain regions, but specifically in frontal and temporal-central regions. Significant increase in alpha power, specifically in the posterior region, which I suppose is complementary to the the theta in the other 2 regions. R

• Before and after 3-month training. Transcendental meditation. Theta power decreased in left hemisphere, but not right. Increased theta coherence was found overall and in central, temporal, and occipital areas. (There’s more stuff but idk the terminology) R

• Lutz et al. (2004)

  • Subjects: 8 long-term (15-40 years; 10k-50k hours) Tibetan GOATs, and 10 student volunteers who underwent 1 week training @ 1h/day. 30 seconds rest, and 20+60 seconds meditation for 4 times. They did metta!
  • Long-term buddhist practitioners induced sustained high-amplitude gamma-band oscillations and phase-synchrony, in particular over lateral frontoparietal electrodes. The students didn’t have shit on the yogis.
  • Ratio of gamma-band activity (25-42 Hz) to slow (4-13 Hz) is inititally higher in the resting baseline for practitioners than controls over medial frontoparietal electrodes.
  • Very unique in its findings, but considering the subjects, and the meditation technique at that, one can see why. All the Tibetan studies show increases in Gamma, either because dzogchen is epic or whatever, or because the researchers have been finding masters.

Note-Taking Broadly Considered; How I Research Things

Obviously, the practice of spaced repetition is to technoprosthetize your long-term memory. In the same way, I view the practice of taking notes as a way of extending your working memory into the digital.

I use the program Obsidian for writing notes.

I never took notes in school; as a whole I only started the practice in ~2020 once I began somewhat seriously studying nutrition, computer science, western philosophy, and a few other topics, and realized that the sheer scope of these various constellations had since expanded past the celestial sphere of my immediate stream of consciousness, warranting some kind of concretization that allowed me to pick up this or that train of thought again when the appropriate occasion returned, something indeed many a man allows to remain a spontaneous and entirely mental process.

I tried Notion, OneNote, and finally used Emacs Org Mode for a while (which I in retrospect consider to have pretty much been a complete waste of time), coinciding with an interest in free software and Linux (time well spent). But I never got serious, with my vault consisting of <20 pages compared to the 1639 in the biology directory alone. But once I used Obsidian, I didn’t look back. It’s hard to say if it lacks anything the aforementioned programs have; Obsidian has a lot of features, and you can safely eschew any of them if they don’t fit your way of learning things. Tags? Never used them, yet I’m sure some people can’t live without them. The graph view? Maybe once in a blue moon to see how far gone I am. Et cetera. In fact, I highly recommend the Hider and Style Settings plugins to remove all UI elements associated with features you don’t use or would otherwise distract you.

‘Zettelkasten’ is the obligatory buzzword when we’re talking about Obsidian and similar programs in the sphere. But technically, I have a wiki; I think the hard sciences like biology are probably better suited as such until you’re at a researcher-tier level of abstraction (but even then it’s usually just ultimately a matter of syntax). What I mean is that the individual notes that constitute a zettelkasten proper are, generally speaking, atomic and often take on a ‘declarative’ form. Andy Matuschak’s ‘working notes’ are a great example of this, with notes titled things like “Evergreen notes permit smooth incremental progress in writing” I originally tried making cards this way for a bit, but I found it to be pretty useless and way too fragmented, and so at this point there are precisely 0 cards with a ‘declarative’ form remain; all my notes refer to nouns, much like how the pages on Wikipedia are. Mere concepts even at the most abstract.

Safe to say the concept of [[linking]] probably represents the pièce de résistance of Obsidian’s features. But I’m using it less and less. (For whatever reason I think being minimalist as possible is the most fruitful…) But the reason is because the backlinks section exists. You don’t need to have the [[]] syntax. At best it’s just a 2-tier prioritization system of what meets my eye first, or pragmatic reasons like one-time aliasing something, e.g. not wanting to abridge synonyms for syntactical reasons, etc. You can alias a page and easily hardlink it as such, or expand its repertoire.

I have the advantage of writing my vault in English though, which uses little to no conjugation/declension. Even plurals (i.e. affixin a word with s) will preclude a string’s inclusion in the backlinks pane. So indeed, I often [[hard link]] a page for the sole purpose of affixing an s to it, or aliasing a page with its plural equivalent. So I hope one day there will be a plugin that extends the syntax of unlinked mentions, because you’re probably syntactically screwed if you’re writing in an agglutinative language like Japanese, even if you use hard links as much as you can. Frankly leaving things unlinked it’s only slightly just a convenience, but it’s good knowing that the backlinks pane probably does have every mention of the note you’re looking at without having to go and search for it, where you maybe once thought something was insignificant so as not to warrant a link but are now glad you see it. It’s not like I’m going to hard link (which makes a kind of ‘empty note’ whose link(s) have a different color and shows up amongst your notes marked as uncreated when searching your vault) every single novel concept I write about. This happens often when I read about some obscure protein, and the topic finally comes up later in an entirely different context, and then I look at the previous context in the unlinked mentions, and upon that moment I can just feel my brain expanding.

Every now and then I’ll have, say, Glycogen as a page, with a Brain subheading, but eventually I have so much information I’ll eventually cut and paste that content into a new Brain Glycogen note. That separation move there risks excessive fragmentation (which sacrifices the readability of any macro- or medium-level topic) and that the branched concept should hold its own ground, so to say. Imagine if I made a note for every little thing, like “Brain glycogen prefers aerobic glycolysis regardless of oxygen status.” This is a true fact and is totally interesting and everything, but it’s hard to say how beneficial the fragmentation would be, rather than just worrying about improving my understanding of the primitives, like how glycolysis works, in hopes that some of these deeper concepts would eventually make more sense passively, rather than worrying about minutae (unless I really do just find it interesting!), because that seems less likely to facilitate transfer learning.

Or a better example: when I first started, I had Hippocampus and would perhaps alias it as CA1, CA2, CA3 such that any mention of those strings of text in my vault would appear in the backlinks. But eventually CA1, CA2, and CA3 became perfectly legitimate individual objects of research once my understanding of the hippocampus grew (inception), and thus they became their own new pages, and links/mentions to Hippocampus are obviously now only about the structure more broadly speaking. I even occasionally alias something as a technically disparate topic(s), like aliasing Ferroptosis as Ferritinophagy because for all intents and purposes my research on the subjects so far has seamlessly overlapped (owing to temporal coincidence). Aliasing HMG-Coa Reductase as Statin, Statins and so on. Get the idea?

As you can see, studies obviously link a lot of disparate topics. Now when I Go to [[Lipid Raft]] or [[TGF-α 1]] to just go and get an overview of what I know about it (yes, wouldn’t it be bad if I didn’t do that often enough?) or just write something down and look in the corner of my eye at least, I have this new information to look at, just like I can read about a study about amyloid β that mentioned GLT-1.

All in all, I’ve found Obsidian absolutely instrumental in saving studies and annotations on them. My Scattered Notes posts (e.g. Scattered Notes: Deferoxamine) are (very slightly abridged versions of) some particularly massive individual notes I have.

Anki, or: How I Learned To Stop Worrying and Love the Flashcard

But of course, periodically returning to notes is only a weak form of spaced repetition, especially considering it doesn’t make use of the testing effect. These notes predate my use of Anki by at least a year or so, and it’s made subtle changes not to the form of my Obsidian vault (so far at least) but it has reduced the frequency of it, in a way.

Strangely enough I find myself wanting to delete certain things if I successfully ‘ankified’ them—why would I need them taking up space in my notes? Maybe one day I’ll make a Water page where I list everything I know about water, but for now I’ll simply trust my implicit understanding of it, you know? Just the same, it’s perfectly obvious to me that it’s the enzyme aromatase that catalyzes the conversion of testosterone to estradiol, and thus, there’s no explicit mention of this fact anywhere. Obviously if the topic needs further elaboration I’ll return to writing about it, but biology is not so concrete that it warrants explicitly stating everything an argument may be technically predicated upon, you know what I mean?

And we can pretend I know that because I made an Anki card for it, because that’s the process I’m trying to convey here; the evolution beyond mere notetaking. If a concept is hard to grasp, creating a properly formulated flashcard requires not only enough dopamine to feel like writing it, but a surprisingly nuanced and often higher-order level of understanding than what merely understanding a given fact requires. For example (I see this faux pas in medical Anki decks a lot) I could make a card “Q: How is estradiol synthesized? A: From testosterone via aromatase”, but things are never so simple in biology: estradiol can also be synthesized from estrone via 17β-HSD. So to formulate it the same way, it immediately has to be something like “Q: What are the *2* ways estradiol is synthesized” and that’s definitely bordering on not being atomic enough to remember, since now that’s actually 6 things in the answer to remember. Tedious work ensues trying to make a set of cards that accurately reflects everything you want to know (and sometimes, you don’t even know what that even is). For instance, how do you remember the fact that ]α7 nicotinic acetylcholine receptors colocalize with NMDA receptors in the dlPFC](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3718126/)? You’re going to need to have that be implied in between the lines with a bunch of other cards that require their own cognitive effort to create.

To make matters wose, sometimes these things are far from obvious facts. Sometimes even the first link between two things is entirely theoretical, perhaps based off an experiment in a single study. You could say fine, let’s do a literature review to make sure, but hold on—what are you talking about? A literature review to make a couple of flashcards? The thing is though, I do that often! But make no mistake, I’m moreso going for the low-hanging fruit in associated topics, and the information that’s interesting yet evades my complete understanding is simply written down for future. Rinse and repeat until your vault has thousands of files. This is the power of the wiki format of Obsidian, and I call this process something like ‘the funnel’ since it allows you to seamlessly jump between topics (cf. the idea of Learntropy such that it’s all ultimately subordinate to a PKM aggregation system. And then eventually, scrapbook fragments of knowledge may coalesce into ankifiable relationships that can be written into lucid flashcards without needing massive cognitive overhead anymore. Even if this isn’t entirely an issue of information but one’s mindset; more implicit things about a subject. Once again: amgituity is a bitch. So clearly, it can be really easy to just not even bother writing a card yet about this or that specific subject until you understand it really well. And continuing on that train of thought: knowledge does not exist in a vacuum, especially in biomedical science, but instead in the context of studies; articles; opinions of one person, but not necessarily universal fact. The fact of the matter is Anki shines with universal facts that are simple Q/A associations.

And but so¹ it became a real paradigm shift when I got over a mental blockage of making cards about studies. For whatever reason, I was originally highly reluctant, no doubt because the idea of cognitive overhead itself was literally the matter at hand. So maybe it was just too many meta-levels of thinking for my brain to handle at the time. I wasn’t sure if it was a good idea to do cards that pertained to specific studies, but that seems pretty silly not to in retrospect, even if I’m not a researcher or whatever that has to keep track of citations. I wondered if it wouldn’t just result in pattern matching? I think it does to a certain extent, but I don’t see that as an entirely bad thing, since that comes with the bonus of keeping track of references, not infrequently a boon to fluidity of thought, which is the name of the game with spaced repetition (the idea that in our digital age you can technically look something up if you forget it, but that A) the time spent doing that would exceed the time spent reviewing a flashcard over your lifetime, and B) it makes a train of thought exponentially better when there is no time gap between ideas caused by poor memory of a certain fact and having to look it up or having to ‘buffer’ uncertainty). But in line with the 20 rules, what else would it be besides something so simple as giving a screenshot of the title? So that’s what I did, and it represents one of a few major improvements I can think of in my card-making heuristics in my journey to become an Anki virtuoso.

I might make up to say, 10 cards like this for an interesting study. Or, maybe just a single card asking for the key finding.

Here are some regular cards. You probably could’ve just used your imagination to figure out what these look like though.

So at this point, I’m still learning little personal heuristics here and there. But YMMV. A word on pre-made decks: I think they’re okay. It’s generally well-recognized in the Anki community that you remember best when you make your own cards (though in medical school anki it’s a whole different ballgame where you do not have time for that). I’ve downloaded a lot of pre-made decks for pretty much any subject I can think of and, much like how I explaind above as for why wrote this article and why I read so many other people’s articles, I basically only download them to get an idea of what other people have done. I rarely just copy+paste cards from their deck to mine; it’s moreso to get an idea of a ‘curriculum’ for a given topic; to see how someone else went through a topic is if nothing else… reassuring? (Kind of like watching experts at work facilitating learning of tacit knowledge) But at this point, I have a few dozen crammed to the bottom of my collection never to be reviewed, and I occasionally search my entire collection for keywords I want to learn, and often there’s a match, and I’ll often look at what else is near that card in its relative position in its deck to know what else I ought to know, you know. The underlying ideas behind the cards should all look familiar (“Do not learn if you do not understand”) and may all be worded/formulated in ways you would never write cards, but there’ll be a recognition that maybe you overlooked something or its connections. And so, the best pre-made decks for you to check out are those that start from the ground up so that you can retrace topics you previously covered yourself from a slightly different perspective. For instance, Brian Spiering’s public decks on CS/math topics satisfy everything I’ve talked about.

So I look forward to GPT/AI flashcard creation that automatically does the heavy lifting for you of phrasing things right, and things like that. I’ve experimented with it with ChatGPT (for instance automatically creating cards from a (large) body of text (et al.)) but I haven’t figured out how to really make it any better or faster than doing things yourself. The next step is having an AI that actively keeps up with your current knowledge base (cf. some of Paul Bricman’s prototypes) and is able to seamlessly curate a daily digest of cards with novel information in whichever direction you decide. (For example in the Japanese/language learning community, they have the idea of i+1 sentence cards, where there is only 1 word you don’t know, and these can be detected (in ‘sentence mining’ via reading/watching media) or even automatically created, so long as every word you know is somehow logged, e.g. MorphMan).

Anki Tips

• Use the FSRS algorithm, which is still kind of under the radar, but I think it represents a pretty big step forward. Here is a breakdown of how the creator devised the algorithm—it’s pretty cool.

  • A great feature this has is being able to set different target retentions for each of your decks if you want. For example my default is 0.86, but math deck is at 0.9 since that benefits a lot from not never forgetting the basics, while my IT deck is at 0.82, since it has a lot of trivial knowledge like what this or that program does.

• Nobody is too good for mnemonics. If you have leeches that you really want to remember but can’t think of any way to make it stick any further, this could be an ace up the sleeve.

  • For example, I kept mixing up Tay Sach’s disease being a lack of hexosaminidase activity, and Farber disease being a lack of acid ceramidase activity. I asked ChatGPT to make some mnemonics to help me differentiate these 2 (technically 4, since they’re reversed) cards, and got the association of Sach’s = six = hex(osaminidase) and Farber = brrrr = AC = acid ceramidase. Trust me, this stuff works. Just like that, I never forget them now.

• Basic cards > all. Cloze and image occlusion have their uses, but whenever possible, I find standard Q/A format to easily be the most robust format with minimal pattern matching. I don’t care what it is: even anatomy benefits from asking you to visualize—at least as a kind of equivalent to reversed/double-sided cards.

  • This is one reason why I’m a bit skeptical of RemNote. Interesting in theory, and probably more featureful than most apps, but I think it can easily lead to unnecessary pattern matching, as well as not really being atomic enough. I’ll have to elaborate on that, though. And like I mentioned in Note-Taking Broadly Considered; How I Research Things, wouldn’t it be pointless to have stuff that’s supposed to be in your LTM still in your notes? Notes are a workspace, not exactly a database per se in that way.

• Focused sessions. Having it open on another monitor or just doing it in those little empty moments in the day like on the toilet, etc. are okay, but I find that if I sit down with the intention of doing Anki, I get done exponentially faster than sporadic reviewing. I just get in the zone.

• Get all the Add-ons you think you need. The majority of mine are kind of just cosmetic, but that can make a big difference.

  • 
  • Opening the same window multiple time is great when you’re really at the point where Anki serves as a workspace for you to jump between topics. Saving certain queries/browsing certain decks in multiple browser windows, adding several cards at once. I can’t tell you how many times I wanted to ankify a piece of knowledge only to swiftly realize it’s predicated on several other engrams in order to make understanable a properly-formulated card (or to make properly-formulated a set of understandable engrams…) and sometimes this goes down to the nth level across several different sources (especially when the modafinil is really hitting… thank you tunnel vision.)
    • When I’m studying hard I expect to eventually see something like this emerge on my monitor, though I usually leave the card adding windows floating+pinned to top.
  • I use PassFail. I learned about this from the Japanese/language learning community’s way of of preventing ’ease hell’ as well as overthinking. This lets you get done with reviews a lot quicker, and it lets you use the volume button on your phone, and other devices: During a period of desperately figuring out ways to deal with RSI (ultimately cured with wrist extensor training, but that’s another conversation), I bought a 4-key external keyboard to use and bind Pass/Fail/Undo/Sync to, so that I could use it in any position I please, even standing with my hands down—without having to strain my wrist and whatnot keeping my hand static over a space bar.
  • Review Heatmap: I don’t see the point at all. You’ll learn the brutal repurcussions of skipping a day; I don’t think you need some tesselated eyesore under your decks to ‘motivate’ you.

Resources/Further Reading

I’ve read all of these, in hopes of gleaning even a modicum of insight in process, since best practices with this stuff will pay off handsomely in the long run. (Watching experts at work)

• Andy Matuschak is the best place to start:

  • Why: Why books donʼt work
  • How: How to write good prompts: using spaced repetition to create understanding
  • His ‘working notes’ are fantastic, with the occasional really interesting original idea in there, like OS-level spaced repetition systems (along the lines of how everything is an interfaceable file in Plan 9. This kind of thinking is what drives real paradigm shifts, and he talks about that, too.)

• The next mandatory reading: Augmenting Long-term Memory by Micheal Nielsen, who is an Anki fanatic, and has other writings about it:

  • Using spaced repetition systems to see through a piece of mathematics
  • https://michaelnotebook.com/tag/memory.html
  • https://michaelnielsen.org/

• Spaced Repetition for Efficient Learning · Gwern.net delivers like usual. He actually has a lot of links. However! He’s missing a few—and if nothing else, a list like that is what I intended for this post to be.

• https://supermemo.guru/wiki/SuperMemo_Guru ‘gold mine’ might be an understatement. If you love to learn, then this wiki is a must-read. I’m positive you’ll have fun perusing this site.

  • The 20 rules of knowledge formulation is also mandatory reading for upping your Anki skill. Matuschak talks about a lot of this though.

• Paul Bricman these are only prototypes (years dated, at that—and notably pre-ChatGPT), but I think this guy is a genius. Seems to be at least a decade ahead of the curve on using spaced repetition as a new pedagogic/software paradigm and taking what we know to its logical extreme. He’s working on alignment now, though.

• https://wiki.issarice.com/wiki/Category:Spaced_repetition absolutely fantastic, especially for math. Big ass zettelkasten site on a mediawiki instance.

  • A fan of mathematics, he has a deck on Tao’s Analysis (he actually made it in Orbit). He has a lot to say on making proof-based or otherwise mathematical cards: https://wiki.issarice.com/wiki/Spaced_proof_review as well as the idea of finding out the best practices for ‘Big cards’ (which on one hand go against standard best practices for flashcards, but on the other hand, the potential utility is apparent).
  • https://wiki.issarice.com/wiki/Add_all_permutations_of_a_card_to_prevent_pattern-matching
  • He also created r/FlashcardCrafting
  • (And other cool stuff: https://machinelearning.subwiki.org/wiki/User:IssaRice)

• https://old.reddit.com/r/AnkiComputerScience/comments/jyd0wq/how_do_you_build_cards_to_learn_proofs/gd2qu4w/ this guy knows what he’s talking about if you stalk his posts. Found this just today.

• Chasing 10X: How Anki Saved My Software Career and its sequel Strategies, Tips, and Tricks for Anki

  • This is one of my favorites. But as of today it looks like most of the images are in the afterlife. Nonetheless, I really appreciated how in-depth he went in explanining his exact thought processes. A notion you may familiarize yourself with is that flashcard creation is a virtuosic process that rewards having a nuanced metacognition. Again, this is why writing your own cards is the best.

• Effective Spaced Repetition | borretti.me

  • Also one of my favorites. Does an exemplary job at breaking down and explaining the ‘syntactical’/‘mathematical’ essence of how a desired piece of knowledge is properly turned into all the necessary flashcards.
  • 

• Make Memory a Choice, Not Chance. | by Kyle Clements | Medium

• Artem Kirsanov, a computational neuroscience researcher, has a few videos on his worflow for knowledge acquisition, which primarily consists of Obsidian, Zotero, and Anki.

  • From How to REMEMBER what you read 🧠: a pretty basic overview of Anki, but interestingly he shows some examples of how he makes research cards:
    • 
    • 
    • 
    • Notice how he simply refers to the paper as <Name, year> since indeed as a researcher that’s something he needs to remember, unlike my format shown above. These are also probably decks sorted by a certain class or a certain video where there are only few key papers; if there’s no additional context like that, having hundreds of citations denoted like this in a single deck seems like a nightmare.

• Anki as Learning Superpower: Computer Science Edition some advice for breaking down mathematics proofs atomically as possible.

• https://nintil.com/massive-input-spaced-repetition

• https://www.jackkinsella.ie/articles/janki-method for programming-oriented tips.

• https://controlaltbackspace.org/categories/memory/

• There’s a wealth of posts on LessWrong relating to using SRS to study (textbooks):

  • DirectedEvolution/AllAmericanBreakfast’s posts:
    • (Trying To) Study Textbooks Effectively: A Year of Experimentation
    • Goldfish Reading
      • The Multi-Tower Study Strategy
    • Progressive Highlighting: Picking What To Make Into Flashcards
  • Turntrout’s posts:
    • Becoming Stronger
      • Lessons I’ve learned from self-teaching

• Is Expanded Retrieval Practice a Superior Form of Spaced Retrieval? A Critical Review of the Extant Literature. - ‘Spaced retrieval’ here referring to fixed intervals. (The answer is yes.)

• Retrieval practice facilitates learning by strengthening processing in both the anterior and posterior hippocampus

• https://github.com/tianshanghong/awesome-anki

• https://old.reddit.com/r/Anki/comments/w4pui6/i_think_the_spaced_repetition_community_should_up/ih3isq5/ Too long intervals > too short?

• https://twitter.com/andy_matuschak/status/1621276086779912192 reviewing 1 day after lapsing, rather than some percentage, is the way (this is default on Anki)

• https://warosu.org/sci/thread/S10439507

  • This one guy opens ebook textbooks in GIMP (which opens each page as a layer) and deletes pages that have been successfully ‘extracted’, leaving behind stuff you don’t really get or isn’t explained well. Go back a 2nd time after finishing and you’ll absorb it all. Very interesting, though kind of weird reading in GIMP, lol. That would be cool to implement in a PDF reader, with a function to delete the current page, if nothing else.

In any case, there’s considerable variation in how well different people respond to it, which is reflected in its rather ‘dirty’ pharmacology. It’s one of the most effective nootropics I’ve ever tried, yet some people are complete non-responders. So am I some days, when the heavens decide the time isn’t ripe, I guess.

Anyways, here we are. I won’t just proceed to tabulate every effect ever known and reiterate everything that’s on Wikipedia, since they said it better than I can. I’ll focus on the more obscure MOAs.

Pharmacology

• NDRI:

  • (This is in Rhesus monkeys. For the human equivalent dose, divide by ~3.1. So for a 75kg human, 5 on the graph corresponds ~120 mg.)

• Indeed, its wake-promoting effects are at least partly mediated via dopamine, considering:

  • Its wake-promoting effects were aboloished in DAT knckout mice: Dopaminergic Role in Stimulant-Induced Wakefulness
  • Its wake-promoting effects were enhanced in orexin knockout mice: Modafinil more effectively induces wakefulness in orexin-null mice than in wild-type littermates. This one confuses me a lot, and definitely doesn’t take orexin out of the equation, once you think about things like rs121912617, the infamous ‘short sleeper gene’ that increases orexin via weakening DEC2 repression of MyoD.
    • Orexin knockout also abolishes modafinil’s promotion of histamine¹ release (another major effect): Action of modafinil through histaminergic and orexinergic neurons though it’s also been shown (R) that modafinil inhibits GABAergic input in a 5-HT3 agonism-dependent manner from the ventrolateral preoptic nucleus → tuberomamillary nucleus also facilitates histamine release.

• α1B adrenergic agonist.

• Upregulates mGluR2/mGluR3, and downregulating mGluR1/5.

• Modafinil exerts anti-inflammatory and anti-fibrotic effects by upregulating adenosine A2A and A2B receptors granted this is in a model of liver fibrosis, which induces downregulation of A2A/A2B.

• Clinical pharmacokinetic profile of modafinil

  • Metabolism is largely via amide hydrolysis, with lesser contributions from cytochrome P450 (CYP)-mediated oxidative pathways.:
    • Induction of CYP1A2, CYB2B6 and CYP3A4.
    • Suppression/reversible inhibition of CYP2C9 and CYP2C19.

• Pharmacogenetics of Modafinil After Sleep Loss: Catechol-O-Methyltransferase Genotype Modulates Waking Functions But Not Recovery Sleep low COMT (thus high baseline dopaminergic signaling) can expect not to feel much from modafinil.

  • “Two‐time 100 mg modafinil potently improved vigor and well‐being, and maintained baseline performance with respect to executive functioning and vigilant attention throughout sleep deprivation in Val/Val genotype subjects but was hardly effective in subjects with the Met/Met genotype”
  • Effects of modafinil on the sleep EEG depend on Val158Met genotype of COMT

• The wakefulness promoting drug Modafinil causes adenosine receptor-mediated upregulation of receptor activator of nuclear factor κB ligand in osteoblasts: Negative impact of the drug on peak bone accrual in rats

  • Modafinil → A2A and A2B → RANKL expression, and a higher RANKL:OPG (osteoprotegerin) ratio results in increased osteoclast number per bone surface. Noradrenergic drugs are not favorable for bone health, ladies and gentlemen.

• Impact of Astroglial Connexins on Modafinil Pharmacological Properties

  • Flecainide (VGSC blocker; CYP2D6 inhibitor?) enhanced the wake-promoting and pro-cognitive effects of modafinil. Decreased number and duration of transitions to NREM, i.e. attenuated narcolepsy from orexin knockout.

• Modafinil shifts human locus coeruleus to low-tonic, high-phasic activity during functional MRI

Cognition

There are numerous reviews on its efficacy. Frankly I’m not sure how appreciated any annotations or spamming links would be. But anyways:

• Modafinil: A Review of Neurochemical Actions and Effects on Cognition

• Modafinil for cognitive neuroenhancement in healthy non-sleep-deprived subjects: A systematic review (2015)

• Stimulating meditation: a pre-registered randomised controlled experiment combining a single dose of the cognitive enhancer, modafinil, with brief mindfulness training

  • Modafinil acutely increased state mindfulness and improved sustained attention.. No effects on mind-wandering. Participants receiving modafinil engaged in more ‘strategy’ (mindfulness) practice across strategy conditions during follow-up. Modafinil acutely mimicked the effects of brief mindfulness training on state mindfulness but did not enhance the effects of this training.

• Cognitive enhancing effects of modafinil in healthy volunteers (2003)

  • Enhanced performance on tests of digit span, visual pattern recognition memory, spatial planning and stop-signal reaction time.
  • … no significant effects of drug on spatial memory span, spatial working memory, rapid visual information processing or attentional set-shifting. No effects on paired associates learning were identified.

• Effects of modafinil on non-verbal cognition, task enjoyment and creative thinking in healthy volunteers (2013)

  • Improvements under modafinil were seen on spatial working memory, planning and decision making at the most difficult levels, as well as visual pattern recognition memory following delay. Subjective ratings of enjoyment of task performance were significantly greater under modafinil compared with placebo, but mood ratings overall were not affected.

• Caffeine and Modafinil Ameliorate the Neuroinflammation and Anxious Behavior in Rats during Sleep Deprivation by Inhibiting the Microglia Activation

• Acute Effects of Modafinil on Brain Resting State Networks in Young Healthy Subjects

• Moral decision making under modafinil: a randomized placebo-controlled double-blind crossover fMRI study

• The Effects of Modafinil on Convergent and Divergent Thinking of Creativity: A Randomized Controlled Trial

Mechanism

• Modafinil (and bupropion, as well) binds to DAT in what is a safer/more sustainable way than cocaine, methylphenidate, monoamine releasing agents like amphetamine, etc. and is non-addictive: The Atypical Stimulant and Nootropic Modafinil Interacts with the Dopamine Transporter in a Different Manner than Classical Cocaine-Like Inhibitors There are 3 conformations that neurotransmitter sodium symporters (SLC6*) (which includes DAT, NET, SERT, usw.) may assume. Modafinil facilitates the ‘open-to-out’ conformation that accepts neurotransmitters from the extracellular space like usual, while amphetamines etc. facilitate the ‘open-to-in’ conformation of releasing substrate into the cytoplasm of the presynaptic neuron.

• Mass Spectrometry Imaging Shows Modafinil, A Student Study Drug, Changes the Lipid Composition of the Fly Brain (wtf is this image?)

  • They show that modafinil decreases the abundance of phosphatidylcholine and sphingomyelin, in favor of phosphatidylethanolamine and phosphatidylinositol. In other words, decreases cylindrical-shaped phospholipids in favor of the conical ones, which would elicit membrane curvature. Combined electrochemistry and mass spectrometry imaging to interrogate the mechanism of action of modafinil, a cognition-enhancing drug, at the cellular and sub-cellular level: This has numerous interesting effects, such as slowing exocytotic events, thereby incerasing the number of catecholamines in each. This potentiates the action of VMAT-2, which is significantly neuroprotective.
    • cf. bupropion, which may work in this way identical to modafinil: Bupropion increases striatal vesicular monoamine transport where interestingly they also noted: “bupropion-induced increase in vesicular DA uptake was prevented by pretreatment with eticlopride, a DA D2 receptor antagonist, but not by SCH23390, a DA D1 receptor antagonist.”
      • Indeed, as is mentioned on Wikipedia, armodafinil (but not esmodafinil) is a D2 partial agonist (R)—let alone functioning as a DRI along with facilitating the action of VMAT-2.
    • The work of Philipsen wrt. vesicular membrane composition is worth looking into: Mass Spectrometric Imaging of Plasma Membrane Lipid Alteration Correlated with Amperometrically Measured Activity-Dependent Plasticity in Exocytosis: “We found that increasing high-curvature lipid species and decreasing low-curvature lipids in the cell membrane favor the formation of a longer-lasting exocytotic fusion pore, resulting in higher release fraction for individual exocytotic events”.

• One would assume this membrane-stabilizing effect is downstream of α1A, 5-HT2A (themselves part of a feedback loop with the increased vesicular monoamine content), H1, etc. → Gαq → PLA2 (which could also potentially liberate choline for methylation)

Analogues

Armodafinil and Modafinil Have Substantially Different Pharmacokinetic Profiles Despite Having the Same Terminal Half-Lives

‘Armodafinil’, if you were unaware, is just the R-enantiomer. It has a longer half-life of ~12-15 hours and is generally more potent than ordinary racemic modafinil.

The action of S-Modafinil is pretty obscure. I was originally interested in it for recreational use (i.e. does not cause insomnia due to a long half life, considering its half life is ~3-5 hours) but there’s always the possibility that it’s actually pharmacologically inactive, and that the lower AUC is simply due to taking a lower effective dose of armodafinil. I haven’t seen anything to suggest the S enantiomer is in any way unique, especially considering there are no studies on it (all information being hidden in the context of studies on modafinil broadly), since why would you want a narcolepsy drug with a half life of only a couple hours, anyways?

Adrafinil seems to be retroactively obsoleted, beyond the fact it’s more legal due to being a prodrug. There are concerns (which frankly could be overblown) about liver toxicity (if for no other reason than the fact that the effective dose is 2x that of modafinil, which I think means your urine will smell of asparagus twice as much) so it’s advised to avoid. Fladrafinil is a similar deal.

Enter the halogenated analogues. Like adrafinil, these are nice because they’re not scheduled substances, and so can be purchased on research chemical sites like Alkonchem.

Elucidation of Structural Elements for Selectivity across Monoamine Transporters: Novel 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues shows that para-halogen substitution increases the affinity for DAT over NET and SERT (such that H < F < Br = Cl). From this, F-, 4F-, Cl-, 4-Cl, 3-Br—and maybe some others—enter the scene. But Fl-, 4Cl, and 3Br-Modafinil are probably the most popular:

Flmodafinil (aka CRL-40,940) Its dose is ~½ that of armodafinil. Again, one would assume it is a bit more dopaminergic. This is the most well-studied halogenated analogue, I believe.

3Br-Modafinil lacks NET affinity. The anecdotes I’ve read considered it to be underwhelming in terms of stimulation and wake-promotion, and is surpassed by 4Cl/Armoda. Dose is ~¾ of armodafinil.

4Cl-Modafinil has slight NET affinity, but still significantly lower than armodafinil. It still lasts 8-12 hours. Anecdotes include it not being as wakefulness-promoting as regular armodafinil, instead being more for ‘well-being’. Dose: ~¾ of armodafinil? Try 50-100 mg.

Heterocyclic Derivatives

• There are 3 main ones I’m aware of:
  • (S)-MK-26: currently undergoing clinical trials.
    • A Novel and Selective Dopamine Transporter Inhibitor, (S)-MK-26, Promotes Hippocampal Synaptic Plasticity and Restores Effort-Related Motivational Dysfunctions
  • CE-158: A mere 1-2 hour half life.
  • CE-123, the most researched. Fucking Red Bull has a patent for it. Coming to a gas station near you?

Pragmatics

/r/modafinil and /r/afinil for most questions you may have. Not a lot of discussion of neuroscience or anything there, though. The former is mostly people who are being prescribed modafinil for legitimate reasons like narcolepsy, shift work disorder, etc. and the latter seems to be more about recreational usage.

Modafinil · Gwern.net is a classic. He also has a tag that’ll give you fair bit of studies.

As far as sourcing goes, /r/modareviewsnotbought used to be the go-to place, but it’s since been banned, and as far as I can tell, the entire modafinil sourcing scene on Reddit is heavily astroturfed. Your best bet is asking people who you know have it where they got it. And that being said, I’m 90% sure it’s legal for me here to vouch for ModafinilXL. I promise this post is not secretly one big advertisement for them. I’m spilling the beans about them because I can tell you right now that I’ve seen them being denounced on Reddit by highly questionable accounts in favor of this or that highly questionable vendor. It’s vicious out there.

What does it feel like? Cold and emotionless, usually. You sustain a bare (“clean” almost says too much) level of alertness and awareness. Whatever you want to do is doable and reduced to a list of actionable steps to grind through. Like the world is a field of data, wherever you aim your lance of vigilance subordinate to analysis. It’s usually slightly euphoric, especially when provding ‘event-induced’ reward at getting stuff done. At best, you feel like you could probably win the nobel prize if you felt like this for solid few years. But potentially dysphoric when there’s nothing to do and focus on.

Getting sidetracked, yet remaining 100% focused while doing so. Yet on the same side of the coin, tasks with multiple complex steps can be mentally coordinated more easily thanks to this, letting you jump ship to and from slightly different topics/contexts in service of a overarching goal.

Insomnia, and the potential virtues of intranasal administration

As I mentioned earlier, insomnia poses a serious problem with armodafinil’s ~12-15 hour half life. The wakefulness-promoting effect doesn’t seem to have a tolerance, unlike some of the other effects. This is perfectly beneficial for narcoleptics, medical students pulling all-nighters (working against their long-term health, of course) etc. but plenty of us out here only want a functional stimulant that noticeably lasts perhaps at most 8-10 hours? It’s a ridiculous experience having an insanely productive day thanks to taking a high dose of modafinil, then bedtime hits, and you damn near feel like you’re ready to start the day, not end it.

As is well known in the ‘drug world’, snorting something effectively compresses its duration of effects (and also bypasses first-pass metabolism, which could maybe be beneficial? Would have to research this though.)

Snorting powders is caustic, so clearly not sustainable for the long term. Modafinil is lipid soluble, hence: Google patents - Intranasal delivery of modafinil for potential formulation of a lipid microemulsion nasal spray. This uses extra virgin olive oil along with phosphatidlyserine, + sonicated sodium succinate for pH regulation. But perhaps simple caprylic acid is a candidate. 4Cl-modafinil is also soluble in propylene glycol.

• So apparently there are two opposing models: The ‘high cholesterol model’ assumes the presence of cholesterol→lipid rafts allows for colocalization of APP and BACE1.

  • cf. the entire ‘lipid raft signaling hypothesis’: Lipid raft microdomains and neurotransmitter signalling, i.e the idea that (caveolar) lipid raft microdomains form kinetically favorable interactions for protein complexes, or rather, perhaps they inhibits them by separating. Or both, of course.

• The low cholesterol model assumes APP is located in non-raft membrane regions, with lipid rafts keeping it separated from BACE1. Furthermore, they note that activation of plasminogen to plasmin is a raft-associated event, and a reduction would somehow increase the poroduction of amyloid for some reason.

• So, seems to me that this is essentialy predicated on where APP likes to associate, which could just be due to its chemical structure or whatever, which is yet another thing that makes me wonder about its differences with AC-rER or other peptides.

• LRP1 is essential for ApoE endocytosis+catabolism. LRP1 expression seems to be under negative transcriptional control of APP or its intracellular domain: Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1

• Most of the brain cholesterol is translocated to the circulation across the BBB in the form of the oxidized derivative 24S-hydroxycholesterol. This is a measure of Cholesterol turnover in the CNS, since cholesterol 24-hydroxylase is responsible for ~40% of cholesterol turnover/catabolism in the brain. Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model. 27OH-Chol originates from the periphery and crosses the BBB - so, vascular damage will see decreased CNS 24S-hydroxycholesterol and increased 27OH-Chol, which is seen in late AD (in early AD, 24SOH-Chol increases in CSF and periphery, reflecting increased brain cholesterol turnover.)

High Cholesterol Model

• Amyloidogenic processing of the Alzheimer β-amyloid precursor protein depends on lipid rafts
  • BACE1 seems to require intact rafts for activity, and BACE1 outside of rafts appears to be inactive.
  • They demonstrated a decrease of cellular cholesterol inhibited Aβ generation, consistent with something like Cholesterol-dependent formation of GM1 ganglioside-bound amyloid beta-protein, an endogenous seed for Alzheimer amyloid : Increases in not only GM1 but also cholesterol contents in the lipid bilayers facilitated the binding of Abeta to the membranes by altering the binding capacity but not the binding affinity.
    • GM1/Aβ is never found in the normal brain, only in those exhibiting early pathological changes of AD
    • Generation of GM1/A b is facilitated by the combination of cholesterol and sphingomyelin
    • Enrichment of cholesterol of the host membranes facilitated the generation of GM1/Aβ via formation of a GM1 “cluster” that acts as a binding site of Aβ.
  • γ-Secretase is also lipid raft associated: Gamma-secretase activity is present in rafts but is not cholesterol-dependent
    • An explanation for Wahlre 2002’s report that it is cholesterol-dependent is not clear. Perhaps: they treated the cells with a γ-secretase inhibitor prior to harvest. Using the parent APP-transfected CHO cells, two of the present authors (H.M. and Y.I.) initially made an observation suggesting that the cellular cholesterol levels can modulate γ-cleavage of APP. When the cells were treated with 25-hydroxycholesterol, which reduces the levels of cholesterol and elevates those of Sphingomyelin, the levels of intracellular Aβ‚ significantly decreased, while those of β-CTF increased.
      • This ultimate effect is very similar to that of a potent γ-secretase inhibitor. However, we found no suppression of Aβ production by membranes isolated from 25-hydroxycholesterol-treated cells. Thus, these data raise the possibility that the treatment of in vitro cultured cells with a certain inhibitor prior to cellular fractionation may perturb additional cellular especially lipid) parameters to compensate for aberrant metabolism, and as a result thus-prepared membranes may not necessarily represent the membrane under normal metabolic conditions. In other words, the γ-secretase activity would be modulated or influenced by many factors that may be involved mostly in membrane integrity.
• However, most of these data were obtained in cells heavily perturbed by chemical treatment with methyl-β-cyclodextrin or lovastatin. The former extracts cholesterol from the plasma membrane, thus heavily affecting lipid rafts, and is a general endocytosis inhibitor, whereas statins have been shown to alter APP processing by modifying cell cholesterol distribution, rather than total levels, with its increase in the exofacial membrane leaflet.
  • Cholesterol distribution, not total levels, correlate with altered amyloid precursor protein processing in statin-treated mice (Burns et al. 2006)
    • All three statins caused CNS cholesterol to translocate from the cytofacial leaflet to the exofacial leaflet! (AKA intra vs. extracellular-facing)
      • There might be differences in expression between the two: [Cholesterol—A Janus-Faced Molecule in the Central Nervous System] says exofacial has PC, SM, PE, and cytofacial has PE, PS, and PI.
    • Only lovastatin significantly reduced total cholesterol in isolated plasma membranes.
    • Nontransgenic mice. Reduced Aβ40/42 and decreased β-CTF.
  • Referencing: Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10 (Kojiro et al.)
    • “See commentary: A fluid connection: Cholesterol and Aβ”
      • ADAM10 associates with membranes that are cholesterol-poor, like those with Phospholipids.
      • These guys were pretty influential as well: Simvastatin strongly reduces levels of Alzheimer’s disease β-amyloid peptides Aβ42 and Aβ40 in vitro and in vivo (Fassbender et al. 2001). This has caveats being some kind of megaddose though
        • Did not reduce cholesterol, but did reduce lathosterol (the precursor to cholesterol) and Aβ by about 50%. The reduction of Aβ occurring in absence of any change in cholesterol could be explained by a minor cholesterol compartment in neurons that changes more rapidly, but whose size is too small to be reflected in measures of total brain cholesterol. Alternatively, it is also possible that the critical species regulating Aβ production is another lipid in the cholesterol biosynthetic pathway.
      • Kojiro observed no significant increase in α-secretase until the reduction in cholesterol production is greater than 50%.
        • Although α-secretase activity is a separate activity than Aβ production, this result raises the possibility that Aβ will decrease only when a threshold of cholesterol reduction is achieved. If this possibility is true, then why did simvastatin reduce Aβ production despite little, if any, change in total cholesterol levels? The answer might be that a precursor of cholesterol regulates Aβ production in vivo.
          • The former, right?
    • Sites of APPsα production occur in membrane regions with low cholesterol content and high fluidity
    • Constitutive and regulated α-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease
      • After cholesterol depletion, a 3-6x increase in APPsα was observed.
  • Nonetheless this study still recognizes that APP happens on the cell surface, and that cholesterol-rich caveolae microdomains may play a role in a-secretase mediated proteolysis of PAPP in vivo: Caveolae, plasma membrane microdomains for alpha-secretase-mediated processing of the amyloid precursor protein
    • Alpha secretase processing was significantly promoted by recombinant overexpression of caveolin.
    • ApoE promotes cholesterol and phosphatidylcholine! efflux from astrocytes/neurons.
      • Related: Evidence of altered phosphatidylcholine metabolism in Alzheimer’s disease
    • Lovastatin increases ADAM10 expression by unknown mechanisms!
• [Cholesterol depletion inhibits the generation of β-amyloid in hippocampal neurons (Simons 1998)]
• Cholesterol decreases secretion of the secreted form of amyloid precursor protein by interfering with glycosylation in the protein secretory pathway
  • Ganglioside GM1 efficiently decreases the membrane microviscosity
• Re-assessing the relationship between cholesterol, statins and Alzheimer’s disease (Wolozin et al. 2006)
  • Statins inhibit isoprenylation as it is an extension of the cholesterol biosynthetic pathway (from Farnesylpyrophosphoric acid), and it is required for APP processing (via ras, rac, rho.).
  • Clinical studies suggest that high doses of statins can reduce Ab load, but the same clinical studies suggest that statins delay the progression of AD rather than preventing the incidence of AD. Our own studies using neuropathology raise the possibility that statins prevent the progression of AD by inhibiting inflammation.
• Elevated dietary cholesterol uptake increased amyloid plaque formation in rabbits and transgenic mice:
  • Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic mouse model This is also high fat diet so that’s lovely
  • APP intracellular domain is increased and soluble Abeta is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer disease
• Similarly: Alzheimer’s disease: the cholesterol connection
  • A hallmark of Alzheimer’s is an abnormal accumulation of Aβ. Nay, its accumulation is the initiating factor.
  • Elevated cholesterol levels increase Aβ in cellular and most animals models of AD, and drugs that inhibit cholesterol synthesis lower Aβ in these models.
  • The identification of a variant of the Apolipoprotein E gene as a major genetic risk factor for AD is also consistent with a role for cholesterol in the pathogenesis of AD.
• The Mevalonate Pathway in Alzheimer’s Disease — Cholesterol and Non-Sterol Isoprenoids BIG thing on Cholesterol synthesis!
  • down-regulation of the mevalonate pathway may play an important role in the increased rates of cognitive decline in AD. Studies at the subcellular level suggest that SREBP-2 may be posttranslationally regulated in AD. We demonstrated that oAβ42 inhibit SREBP-2 maturation in cultured neurons. We also discovered that the levels of (M)SREBP-2 are reduced in the frontal cortex of the AD CRND8 mouse

Low Cholesterol Model

• The beneficial effects of low brain cholesterol resulting from this model are strongly questioned by several studies carried out on experimental systems close to the physiological and pathological situations. Actually, many data suggest that cholesterol loss in neuronal membranes enhances amyloid peptide generation.

  • The conflicting role of brain cholesterol in Alzheimer’s disease: lessons from the brain plasminogen system (2005)
    • It has not been shown that higher neuronal cholesterol increases Abeta production… it has not been demonstrated that neurons in AD have more cholesterol than control neurons. On the contrary, the brains of AD patients show a specific down-regulation of Seladin-1, a protein involved in cholesterol synthesis, and low membrane cholesterol was observed in hippocampal membranes of ApoE4 (apolipoprotein E4) AD cases.
      • What would you know, there is controversy on seladin-1’s role.
    • In female mice, the most brain-permeant statin induces neurodegeneration and high amyloid production
      • It appears more likely that the advantageous role of statins arises from improved brain oxygenation
  • Plasma membrane cholesterol controls the cytotoxicity of Alzheimer’s disease AβP (1–40) and (1–42) peptides
    • PC12 cells become resistant to the cytotoxic action of AβP when incubated in a medium that enriches cholesterol levels of the surface membrane. On the other hand, making cholesterol-deficient membranes by either cholesterol extraction with cyclodextrin or by inhibiting de novo synthesis of cholesterol makes PC12 cells more vulnerable to the action of Aβ
    • Increasing cholesterol content of PS liposomes also suppresses Aβ-dependent liposome aggregation

• Neuronal membrane cholesterol loss enhances amyloid peptide generation (2004)

  • Much higher levels of BACE 1–APP colocalization is found in hippocampal membranes from AD patients or in rodent hippocampal neurons with a moderate reduction of membrane cholesterol!
    • The ultimate question is where cholesterol plays a role in this causality. It’s no mystery that increased ceramides play a role. But what about ganglioside?
    • γ-Secretase executes an intramembranous cleavage, which is apparently difficult and uncommon. They compare it to SREBP and Notch which are also intramembraneous, and indeed the linkage is kind of directly especially with the latter.
      • See:

• Structural membrane alterations in Alzheimer brains found to be associated with regional disease development; increased density of gangliosides GM1 and GM2 and loss of cholesterol in detergent-resistant membrane domains

• The role of DHCR24 in the pathogenesis of AD: re-cognition of the relationship between cholesterol and AD pathogenesis (Zhang 2022)

  • According to previous data and our, reduced cholesterol level in plasma membrane and intracellular compartments by the deficiency of DHCR24 activity obviously was involved in β-amyloid generation, tau hyperphosphorylation, apoptosis.
  • Universally regulated by sterols/steroids, ACTH, thyroid, neurotrophins, and xenobiotics.
  • Apparently it is no paradox that impaired cholesterol catabolism and trafficking leads to a loss in brain cholesterol!

Etc. Notes

• “I believe that the reduced secretion of sappa from the nucleus due to a failure in autophagesome formation may be more reflective of the phenotype observed in sappa overexpression models or in autism”
  • Here we have Insulin→mTORC2 and overall ceramide accumulation which can be thanks to abberant Acid Sphingomyelinase from an Aβ feedback loop, ROS, or a feedback loop in general from ceramide displacing cholesterol.
• Alzheimer’s Disease as a Membrane Disorder: Spatial Cross-Talk Among Beta-Amyloid Peptides, Nicotinic Acetylcholine Receptors and Lipid Rafts
  • a) β-Secreatase is present in both raft and non-raft domains, but needs to be in raft domains to be functional.
  • b) β-secretase in raft domains corresponds to an incative pool that needs to relocate to non-raft domains to be functional.

• Notice the fully intramembrane α-secretase. Real? Can’t be. Look at the cleavage site.

sAPP (Secreted/Soluble APP)

• promotes mitochondrial protein synthesis and the traffixking of glua1 ampa, nr2a and nr2b nmda receptors to the cell surface by improving mitochondrial transport to synaptic vesicles to dwliver glutamate receptors to the cell surface.
• APP endocytosis and loss of alpha secretase processing/trafficking to synaptic vesicles and the accumulation of app intracellular domain presynaptically with increased processing at gamma and beta secretase leading to the formation of plaques, decline in mitochondrial homeostasis with endolysosomal accumulation of gangliosides, cholesterol, and iron s well as defects in the extracellular matrix lead to synaptic collapse, cell death, endothelial senescence as well as senescence throughout the body.This leads to the induction of alzheimers disease as well as aging itself
• Diseased neurons have less cholesterol and synthesize less neurosteroids because mitochondrial function breaks down and app undergoes endocytosis to the nucleus
• how we learn from activity dependant synaptic nmda receptor modulation is all dependant on sappa
• Ganglioside formation and cholesterol accumulation in the cell along with iron prevent app from being transported to synapses
• Cholesterol and ammonia accumulation within thw lipid membrane of neurons also reduced the axonal transport of sappa and may mediate an increase in soluble app alpha intracellular domain accumulation in the lipid membrane which promotes synaptic dysfunction
• In neurons this synaptic transport of sappa intracellularly acts presynaptically to increase gaba a receptor expression on the cell surface, reduces the expression of sodium transporters reducing exitability, and delivers nmda(?!) and ampa receptors to the synapse.

• sAPP-α is cleaved from APP via α-secretase (non-amyloidogenic processing).
• Iron dysregulates APP processing accompanying with sAPPα cellular retention and β-secretase inhibition in rat cortical neurons
  • sAPPα remained in the cellular lysates instead of being secreted into the extracellular milieu. SAPPβ and Aβ levels were decreased.
• Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum
  • Hyperammonemia increases mAPP (mature) content in astrocytes.
  • BACE1 and γ-secretase are expressed in the endoplasmic reticulum.
• Lipoprotein receptors and cholesterol in APP trafficking and proteolytic processing, implications for Alzheimer’s disease
  • Cholesterol and ammonia accumulation within the lipid membrane of neurons also reduced the axonal transport of sappa and may mediate an increase in soluble app alpha intracellular domain accumulation in the lipid membrane which promotes synaptic dysfunction. Removal of cellular cholesterol in senescent skin cell lines reverses the progression of cellular senescence and restores sappa transport to the cell membrane: Overexpression of Amyloid Precursor Protein Promotes the Onset of Seborrhoeic Keratosis and is Related to Skin Ageing
• Amyloid Precursor Protein (APP) Mediated Regulation of Ganglioside Homeostasis Linking Alzheimer’s Disease Pathology with Ganglioside Metabolism
  • Ganglioside clusters in neuronal membranes take part in the formation of amyloid fibrils; GM1 increases Aβ production.
• Therapeutic Potential of Secreted Amyloid Precursor Protein APPsα (Mockett 2017)
• Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission
  • Directly bound the sushi 1 domain specific to GABA-B α1. Binding suppressed synaptic transmission and enhanced short-term facilitation via inhibition of synaptic vesicle release in mouse hippocampal synapses.
• Lentivirus-Mediated Expression of Human Secreted Amyloid Precursor Protein-Alpha Promotes Long-Term Induction of Neuroprotective Genes and Pathways in a Mouse Model of Alzheimer’s Disease (Ryan 2021)
  • Upregulated key neuroprotective genes.
• Viral gene transfer of APPsalpha rescues synaptic failure in an Alzheimer’s disease mouse model (Fol 2016)
• From synaptic spines to nuclear signaling: nuclear and synaptic actions of the amyloid precursor protein
  • AICD regulates transcription: promotes p53, GSK-3β, BACE1, and - this one is good - neprilysin, a membrane metallo-endopeptidase. It degrades Amyloid β, as well as glucagon, enkephalin, substance P, neurotensin, oxytocin, etc.

Plasticity

• Synaptic NMDA Receptor Activation Stimulates α-Secretase Amyloid Precursor Protein Processing and Inhibits Amyloid-β Production
  • A pool of APP is localized to the postsynaptic compartment in cortical neurons and observed partial overlap of APP with both NR1 and PSD-95.
  • Increase in nonamyloidogenic APP processing, as expected, was inhibited by calcium chelation, NMDAR antagonist, or alpha secretase inhibitor, but not AMPAR antagonist or L-type calcium channel blocker.
  • (NMDAR ->) SAP-97 mediates α-secretase ADAM10 trafficking and promotes its activity
• Glutamate Receptor Trafficking and Protein Synthesis Mediate the Facilitation of LTP by Secreted Amyloid Precursor Protein-Alpha (Mockett 2019)
  • Exposure converts short-lasting E(early)-LTP into protein-synthesis-dependent late LTP in hippocampal slices from male rats. These results are consistent with other studies suggesting that constitutive protein synthesis before LTP induction may be an important mechanism supporting L-LTP.
  • Associated with CAMK II-mediated Ser831 enhancement. Ser845 was unaltered! Perhaps basal levels simply are sufficient. Yet, In a CAMK II and Protein Kinase G dependent manner, it promotes exocytosis of GluR2-lacking AMPARs, and NMDARs.
    • CAMK II, ERK, and PKG-dependent stimulation of synaptic protein synthesis in synaptoneurosomes Secreted amyloid precursor protein-α upregulates synaptic protein synthesis by a protein kinase G-dependent mechanism (Claasen et al., 2009)
      • So, PKG probably stimulates accessory proteins for AMPA’s surface accumulation, rather than Ser845.
      • The secreted form of the Alzheimer’s beta-amyloid precursor protein stimulates a membrane-associated guanylate cyclase
        • Inhibition of the formation of nitric oxide or carbon monoxide did not affect the ability of sAPP to lower rapidly intraneuronal calcium levels or elevate cGMP, suggesting that sAPP does not activate a soluble (cytosolic) guanylate cyclase.
  • Cell surface receptor accumulation and LTP facilitation were present even after sAPPα washout and inhibition of receptor trafficking or protein synthesis, revealing a metaplastic capability of endogenous sAPPα administration.
    • The idea is that the synapse’s previous history of activity determines its current plasticity.
    • (Unlike BDNF) sAPPα does not by itself cause synaptic potentiation even though it is a potent stimulator of protein synthesis. Rather, it engages metaplasticity mechanisms, specifically a coordinated de novo protein synthesis and glutamate receptor trafficking that puts synapses in a state of readiness for future LTP and, presumably, learning
      • Metaplasticity: tuning synapses and networks for plasticity
  • No increase in the cell-wide levels of receptor subunits was detected following 30 min exposure to sAPPα, despite an increase of GluA1 and NR1/NR2..
  • had no significant effect on LTD, indicating specificity for LTP modulation that bears a similarity to the pull–push effects of neuromodulation on LTP/LTD in visual cortex: PULL-PUSH NEUROMODULATION OF LTP AND LTD ENABLES BIDIRECTIONAL EXPERIENCE-INDUCED SYNAPTIC SCALING IN VISUAL CORTEX
    • Previous studies have shown that Gs-coupled receptors directly promote LTP induction and Gq11-coupled receptors promote LTD.
    • LTP and LTD An Embarrassment of Riches (2004) (>3k citations)
      • Apparently E-LTP lasts ~60 minutes.
      • They comment on the now dying idea that LTP involves presynaptic changes like via retrograde messengers like NO or AA. They think adhesion molecules are probably the right candidate if these presynaptic changes are worth looking at.
      • LTP is induced by things like PKA/CAMKa IV/MAPK which activates CREB and Egr-1, etc.
        • Secreted amyloid precursor protein β and secreted amyloid precursor protein α induce axon outgrowth in vitro through Egr1 signaling pathway
  • sAPPα may promote LTP by acting as a positive allosteric modulator at α7 nAChR! Distinct in vivo roles of secreted APP ectodomain variants APPsα and APPsβ in regulation of spine density, synaptic plasticity, and cognition (Ritcher 2018) (“The C terminal 16 amino acids that differentiate sAPPα from sAPPβ”)
• Secreted Amyloid Precursor Protein-Alpha Promotes Arc Protein Synthesis in Hippocampal Neurons (Livingstone 2019)
  • In vivo LTP in the dentate gyrus was depressed by ~50% by intra- hippocampal infusion of antibodies directed at sAPPa.
• Increase of synaptic density and memory retention by a peptide representing the trophic domain of the amyloid beta/A4 protein precursor
  • Obviously the question is if this includes Ac-RER’s 3 amino acid sequences and what the differences are.
  • Resulted in an 18% increase in the number of presynaptic terminals in the frontoparietal cortex. At the behavioral level, 17-mer-infused animals with nonimpaired learning capability showed an increased memory retention that seemed to interfere with reversal learning performance.
• Secreted form of beta-amyloid precursor protein shifts the frequency dependency for induction of LTD, and enhances LTP in hippocampal slices (Ishida 1997)
  • LTD is harder to induce and LTP is easier to induce.
  • Pretreatment of slices with 8-bromo-cyclic GMP mimicked the effect of sAPP alpha on LTD suggesting a role for cGMP in modulation of LTD. - this goes back to a dependence on PKG.
  • Intrahippocampal infusion of sAPPα antibodies reduced LTP in the DG of adult rats by ~50%.
• [Acute function of secreted amyloid precursor protein fragment APPsα in synaptic plasticity]
• Memory-enhancing effects of secreted forms of the β-amyloid precursor protein in normal and amnestic mice
  • The effects of APPss on LTD induction are in agreement with their cGMP-mediated ability to potently stimulate potassium currents, and hence decrease intracellular calcium levels: Activation of K+ channels and suppression of neuronal activity by secreted beta-amyloid-precursor protein - mimicked by cGMP analogue
    • Signaling events regulating the neurodevelopmental triad. Glutamate and secreted forms of beta-amyloid precursor protein as examples (1997)
      • counteract effects of glutamate on growth cone behaviors, and increase synaptic complexity. Acute actions of sAPPs appear to be transduced by cGMP which promotes activation of K+ channels and reduces [Ca2+]i.
• Metabotropic glutamate receptor subtype mGluR1alpha stimulates the secretion of the amyloid beta-protein precursor ectodomain!
  • mGluR1. It is coupled to APP processing via protein kinases and PLA2 (so probably Gp1’s Gq->Ca2+)
• Endogenous secreted amyloid precursor protein-alpha regulates hippocampal NMDA receptor function, long-term potentiation and spatial memory
  • Pharmacological inhibition of alpha-secretase and other a-disintegrin-and-metalloproteases by TAPI-1 reduced both LTP and tetanus-evoked NMDA receptor-mediated currents in dentate granule cells… ==indicating that sAPPα is a key contributor to plasticity!==
  • High concentrations of exogenous sAPPα strongly reduced LTP induction, indicating that signaling pathways inhibitory to LTP can also be activated
  • The neuroprotective effects (PI3K/AKT, p42/p44, NK-κB) may be mediated by the N-terminal, while the Guanylate Cyclase/cGMP effects may be mediated by the C-terminal (which RER is not part of) - The effect of a secreted form of β‐amyloid‐precursor protein on intracellular Ca2+ increase in rat cultured hippocampal neurones (Koizumi 1998) Ca2+i elevation mimicked by amino terminal peptides of APPS, but not by carboxy terminal peptides. This sounds like the opposite of what they just said…

Neurogenesis

• beta-Amyloid precursor protein binds to the neurite-promoting IKVAV site of laminin
  • sAPPα interupts APPL1-APPL2 dimerization, and this is how dendritic outgrowth occurs.
    • Wtf are those?: Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription
      • Effectors of Rab5. Complexes with Reptin, β-catenin, HDAC1, and HDAC2.. Overexpression of either APPL protein relieves Reptin-dependent transcriptional repression and correlates with the reduced amounts of HDACs and β-catenin associated with Reptin as well as with the lower levels of Reptin and HDAC1 on the promoters of β-catenin target genes.
• Secreted amyloid precursor proteins promote proliferation and glial differentiation of adult hippocampal neural progenitor cells (Baratchi 2012)
• Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone (Caille 2004)
  • The SVZ is major sAPP binding site, where binding occurs on EGFR-containing Progenitor Cells (not on the receptors themselves) which are the Type C cells for SVZ neurogenesis. They self-renew in the presence of EGF and differentiate into neurons and glia upon EGF removal. sAPP brinding sites are also pressent on Type A, but not B or ependymal cells.
  • EGF stimulates sAPP secretion by NS cells and sAPP is required for full EGF mitogenic activity.
  • Alzheimer’s shows very early odor discrimination defects! As do mice with reduced SVZ neurogenesis.
• The amyloid precursor protein controls adult hippocampal neurogenesis through GABAergic interneurons (Wang 2014)
  • Selective deletion of GABAergic, but not glutamatergic (or progenitor cells) APP disrupts adult hippocampal neurogenesis.
  • In case I needed reminding, this is important for maintaining excitatory-inhibitory balance of Granule Cells in the DG via these interneurons.
    • Tonic GABAergic currents control network excitability in dentate gyrus: Variations on an inhibitory theme: phasic and tonic activation of GABA-A receptors (Farrant 2005)
    • Important for Pattern Separation as well: Performance on a pattern separation task by Alzheimer’s patients shows possible links between disrupted dentate gyrus activity and apolipoprotein E ∈4 status and cerebrospinal fluid amyloid-β42 levels
  • GABA neurotransmission have been shown to influence key steps of hippocampal adult neurogenesis, ranging from progenitor proliferation to newborn neuron maturation and integration:
    • Adult neurogenesis: from precursors to network and physiology (Abrous 2005)
    • GABA sets the tempo for activity-dependent adult neurogenesis (Ge 2007)
    • GABAergic control of neurite outgrowth and remodeling during development and adult neurogenesis: general rules and differences in diverse systems (Sernagor 2010)
  • GABAergic APP, possibly through the regulation of adult hippocampal neurogenesis, controls a mouse’s cognitive ability to discriminate between similar contexts… this study addressed the question of in vivo actions of APP: increased proliferation of neural progenitor cells in APP−/− mice, and this is accompanied by reduced tonic GABA current recorded from granule cell.
    • This view is in agreement with the recent finding that the activity of Parvalbumin+ interneurons regulates stem cell quiescence via GABA-A receptor-mediated tonic GABA signaling: Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision (Song 2012)
    • In light of the reduced long-term survival of newborn neurons, it is possible that increased progenitor proliferation represents a compensatory response to diminished survival. (in APP-/- mice)
  • Earlier work has shown that abolishing GABA-induced depolarization can result in defects in dendritic growth and synapse formation during adult neurogenesis:
    • Excitatory actions of gaba during development: the nature of the nurture (Ben-Ari 2002)
    • Is there more to gaba than synaptic inhibition? (Owens 2002)
  • Amyloid Precursor Protein Regulates Cav1.2 L-type Calcium Channel Levels and Function to Influence GABAergic Short-Term Plasticity (Yang, Wang et al. 2009)
  • In contrast, APP has not been shown as an essential regulator in controlling excitatory synaptic strength as a few earlier studies reported normal efficacy of excitatory hippocampal synapses both in dentate gyrus and area CA1 of APP−/− mice (Seabrook 1999, Ring 2007, Jedlicka 2012)
• Secreted forms of beta-amyloid precursor protein modulate dendrite outgrowth and calcium responses to glutamate in cultured embryonic hippocampal neurons (1994)
  • Intracellular messengers in the generation and degeneration of hippocampal neuroarchitecture (1988)
    • Micro levels of glutamate caused regression of dendrites but not axons. Millimolar levels caused cell death.
    • Calcium ionophore and PKC activator caused regression of dendrites and axons.
    • Forskolin enhanced outgrwoth rates of dendrites and axons.
    • Co2+ (cobalt? wtf?) and trifluoperazine each significantly reduced glutamate-induced dendritic regression and neurotoxicity suggesting that calcium influx and/or PKC activation mediated glutamate’s actions.
• [Amyloid precursor protein regulates differentiation of human neural stem cells (Kwak 2006)]
  • sAPP binds to the surface of neural stem cells (And neuroblasts) (in the SVZ) - and regulates their proliferation and differentiation
• Activity requires soluble amyloid precursor protein alpha to promote neurite outgrowth in neural stem cell-derived neurons via activation of the MAPK pathway
  • “Activity”…?
  • Required active NMDAR + MAPK/ERK recruitment to induce neurite outgrowth.
    • Acute depolarization led to a sharp increase in phosphorylated ERK (with fast kinetics) and such an increase was abolished by blockade of NMDAR.
      • And continuous generation of sAPPα was necessary for this! Though acute sAPPα administration did not lead to ERK activation.
      • This shows that basal levels of p-ERK are mostly due to endogenous activation of NMDAR and that, as in more mature neurons, also in neurons differentiating from neural stem cells ERK phosphorlation is a downstream event of NMDAR activation.
    • Acute application of sAPPα did not directly affect ERK phosphorylation either in the presence or in the absence of depolarization. However, in the absence of depolarization, sAPPa enhanced the effect of APV (an NMDAR antagonist) in preventing ERK phosphorylation
  • APP gene family encompasses APP and two other related proteins, amyloid precursor-like protein 1 (APLP1) and APLP2.
    • “soluble amyloid precursor-like protein 2 (sAPLP2),” but not sAPLP1 is functionally redundant to sAPP in this neurite outgrowth stuff.
  • The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling
    • Cleavage of APP to release sAPPα is enhanced by membrane depolarization andor activation of cell surfface receptors like M1 AChR/M3 AChR (Via Gq) and EGFR. BDNF→TrkA & p75 respectively increase processing and trascriptional regulation of APP.
      • And indeed α-secretase activity is increased by Protein Kinase C activation - and possibly so does depolarization if it means Ca2+ influx.
      • Brothers in arms: proBDNF/BDNF and sAPPα/Aβ-signaling and their common interplay with ADAM10, TrkB, p75NTR, sortilin, and sorLA in the progression of Alzheimer’s disease
        • looks like BACE is restricted to APP, but interesting how ADAM10 cleaves TrkB!!
• Secreted Amyloid Precursor Protein Alpha, a Neuroprotective Protein in the Brain Has Widespread Effects on the Transcriptome and Proteome of Human Inducible Pluripotent Stem Cell-Derived Glutamatergic Neurons Related to Memory Mechanisms (2022) - interesting figures on protein expression changes.
  • The amyloid precursor protein is a conserved Wnt receptor (Liu 2021)
    • Binds WNT3a and WNT5a. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels
    • Loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons.
  • Regulates IGF-2: [Secreted amyloid precursor protein-α mediates neuroprotection and gene expression (Morris 2011)] - this paper is nowhere to be seen.
  • 
  • 
    • Arp C1A (ARP 2/3 complex subunit 1A) up:
    • Some of these things are a bit strange. PSD-95 down?
• Not neurogenesis, but we even see thyrocyte proliferation: Growth regulation of rat thyrocytes (FRTL-5 cells) by the secreted ectodomain of beta-amyloid precursor-like proteins

Autism

• Pax says this is something of a misunderstanding regarding the endocytosis of APP: tied to defects in mitochondrial function from cholesterol and ammonia handling issues with failure of autophagy mediated secretion of sappa to synaptic vesicles. The sappa is high but it stays in the mitochindria and never leaves the nucleus
• Autism genes and the leukocyte transcriptome in autistic toddlers relate to pathogen interactomes, infection and the immune system. A role for excess neurotrophic sAPPα and reduced antimicrobial Aβ
  • There are 206 ‘Autworks’ genes (i.e. implicated in Autism) that are localized in the immune system. Many autism genes converge on APP processing, tilting this axis: in contrast to Alzheimer’s disease, levels of the antimicrobial peptide beta-amyloid are decreased and the levels of the neurotrophic/myelinotrophic soluble APP alpha are increased in autism, together with an increased activity of α-secretase.
  • sAPPα induces an increase in glutamatergic and a decrease in GABA-ergic synapses creating and excitatory/inhibitory imbalance that has also been observed in autism.
  • 
    • Interesting substrates here for ADAM10…Neuroligin1/3, EAAT1
    • SLC1A1:
    • SLC6A8: Sodium- and chloride-dependent creatine transporter 1
• Gene expression profiling in the stress control brain region hypothalamic paraventricular nucleus reveals a novel gene network including Amyloid beta Precursor Protein
  • 
    • ADAM10 is differentially down/upregulated in C57BL/6J and DBA/2J mice respectively. DBA/2J also expresses 1.44x APP and -2x GABA-A α subunit.
  • 
    • PDE1C downregulation during stress is interesting…
• Autism as early neurodevelopmental disorder: evidence for an sAPPα-mediated anabolic pathway
  • sAPPa simulates both neuroprotection and microglial activation - leading to neuronal overgrowth + infallatmion simultaneously.
  • 
  • Note FMRP downregulation leading to depression and a rise in APP->autism pipeline: Autistic behavior in children with fragile X syndrome: prevalence, stability, and the impact of FMRP 25% of boys with FXR had ’tism.
    • Not to say sAPPα has an effect on FMRP. It’s all downstream.
    • FMRP mediates mGluR5-dependent translation of amyloid precursor protein and indeed as the figure shows, sAPPα and sAPPβ/Aβ is elevated in autism.
      • mGluR5 displaces FMRP from negatively regulating APP transcription! In the absence of FMRP (so FXS), APP synthesis is constitutively increased and nonresponsive to mGluR-mediated signaling.
    • Genetic downregulation of mGluR5 signaling has reversed behavioral deficits in fmr-1 knockout mice
• Increased Secreted Amyloid Precursor Protein-α (sAPPα) in Severe Autism: Proposal of a Specific, Anabolic Pathway and Putative Biomarker (2011)

FMRP

• 
  • Astrocyte FMRP plays an important role in shaping the neuron morphology and synaptic protein profiles. It inhibits excess protein synthesis, leading to increased branching and thus the abundance of immature spines (so maybe not necessarily ‘over-mature’ ones)
  • Indeed it is synthesized in neurons, meaning both Gp1 mGluR are implicated. Is the transcription it not just calcium influx regulated? What’s special about mGluR?
    • Roles of Calcium-Stimulated Adenylyl Cyclase and Calmodulin-Dependent Protein Kinase IV in the Regulation of FMRP by Group I Metabotropic Glutamate Receptors (2008) this is in the ACC.
      • Ca2+->CaM->AC->PKA->CAMK IV->CREB->Fmr1.
      • Ca2+->CaM->CREB
    • Metabotropic glutamate receptors activate FMRP in the anterior cingulate cortex through calcium-dependent signaling pathways (2007)
    • FMRP Phosphorylation Reveals an Immediate-Early Signaling Pathway Triggered by Group I mGluR and Mediated by PP2A
      • Rapid mGluR1 (<1 min) -> PP2A -> rapid dephosphorylation.
      • Extended mGluR activation (1-5 mins) -> mTOR inhibiting PP2A, facilitating FMRP rephosphorylation.
• Fragile Xsyndrome is the second most common form of mental retardation, second only to down syndrome overall.
• Gq-dependent: The mGluR theory of fragile X mental retardation (Gp1 = postsynaptic group 1, aka mGluR1/mGluR5.)
  • FMRP is synthesized in response to mGluR5 activation. When mGluR5 upregulated (it has beentheorized that could be the case in Autism, at least in some areas. Might be more to do with sAPPα) or FMR1 is knocked out (FXS) then FMRP-mediated translation repression is overshadowed/prevented, and results in protein synthesis-dependent LTD:
    • Increased synapse loss and/or turnover, both requiring mRNA translation, and if exaggerated could account for the delay in synaptic maturation and elongated spines, i.e.:
  • I am a bit confused. mGluR is just like anything else; transient activation is LTP, but prolonged agonism is LTD? They’re being weird about it.
  • Just makes me wonder what these proteins are that are upregulated?? This has been hard to investigate apparently.
  • What’s so special about mGluR1/5? What about other modes of Ca2+ influx?

(Ac-)rER

• NH2-(D-arg)-(L-glu)-(D-arg)-COOH. Ac/NAc = N-acetylated. These are AAs 328-330 of APP. Not the exact same thing as RER.
  • N-terminal acylation of RER protects it against rapid degradation.
  • Deacetylated, the first Arg is L, I think. But notice it is palindromic.
  • It is such a small sequence, it associates with mitochondria.
• Soluble amyloid precursor protein: a novel proliferation factor of adult progenitor cells of ectodermal and mesodermal origin
• The Tripeptide RER Mimics Secreted Amyloid Precursor Protein-Alpha in Upregulating LTP (Morrissey 2019)
  • 
  • Protects ’newly formed engrams’ from interference.
  • Also binds to HSC71 (heat shock cognate 71) and Syntaxin binding protein STXBP1. HSC71 hyperphosphorylation is seen in AD.
  • sAPPα is too large to crossthe cell membrane.
  • Defective neurite extension is caused by a mutation in amyloid beta/A4 (A beta) protein precursor found in familial Alzheimer’s disease
    • Deletion of RERMS (328-332) sequencing from sAPPα removes its ability to protect against Aβ insult although this may have disrupted the structure of the coiled coil where the RER resides in the E2 domain.
• The peptide sequence Arg-Glu-Arg, present in the amyloid precursor protein, protects against memory loss caused by A beta and acts as a cognitive enhancer (2004) nothing special; some old references on plasticity in discussion section
  • RER binding is displaced from longer peptides derived from APP’s external domain, but not Aβ. (So like sAPP and Aβ?) likely competing for binding to a putative receptor.
• The memory enhancing effect of the APP-derived tripeptide Ac-rER is mediated through CRMP2
  • It is binding partners with CRMP2, Syntaxin binding protein 1, and Hsp70.
  • Collapsin Response Mediator Protein-2 (CRMP2) is a Plausible Etiological Factor and Potential Therapeutic Target in Alzheimer’s Disease: Comparison and Contrast with Microtubule-Associated Protein Tau
• Secreted Amyloid Precursor Protein-Alpha Enhances LTP Through the Synthesis and Trafficking of Ca2+-Permeable AMPA Receptors (Livingstone 2021)

Dosing

Orally active. 12 hour half life.

• Picogram doses - wtf.

https://warosu.org/sci/?task=search2&ghost=false&search_text=&search_subject=&search_username=&search_tripcode=%21%21cu2HYeafqTG&search_email=&search_filename=&search_datefrom=&search_dateto=&search_media_hash=&search_op=all&search_del=dontcare&search_int=dontcare&search_ord=new&search_capcode=all&search_res=post

• Heavy metal chelator, especially 3+ ions (Ferric Iron, Aluminum, lead, mercury, chromium, zinc, copper, etc.) limited affinity for ferrous iron.

  • Deferoxamine has a very high affinity and specificity for the ferric iron and chelates it in a 1:1 molar ratio; that is, 100 mg of deferoxamine will bind to and eliminate 8.5 mg of elemental iron
  • Binds free iron in the bloodstream as well as intralysosomal ferritin/siderin. Does not readily bind iron from transferrin, hemoglobin, myoglobin, or cytochrome (not typo).
  • Fe-DFO = ferrioxamine B, which is water soluble and is excreted in the kidneys and feces. Half-life of ~10 hours.
  • Al-DFO = aluminoxamine, which increases blood aluminum concentrations. According to pubchem, 100mg dfo can bind ~4.1mg aluminum.

• AFAIk, it targets or at least effects every organelle: cyotosol, endolysosome, etc. However it isn’t membrane-permeable? I really want to know what this means for its pharmcokinetics?

• Ferritin accumulates as a consequence of endolysosomal deacidification promoting expression of Ferritin Heavy Chain. Failure of ferritin turnover within our braincells leads to the decline of the autophagesome and defective autophagy that’s present in alzheimers, down syndrome, Autism sprectrum disorder, Parkinsons, Huntingtons disease, nathman pick disease, etc.

  • This makes sense, since H+-ATPase mediates the acidification, and if respiration is deficient then there’s no ATP, immediately creating a deadly cycle.
  • Iron leaks out of mitochondria dislodging from the mitochondrial machinery and into the LIP whereby it generates ROS.
  • DFO should rescue ferritin turnover, and shuttle it towards mitochondria if needed. In certain disorders i.e. where autophagy is compromised, the iron in ferritin simply doesn’t leave.
  • Utilization alongside EPO andor ferritin pore unfolding peptide:Apart from Lipid rafts, extracellular atp, and loss of neural stem cells and senescence this would spell the end for neurodegeneration
    • We can target inflammatory purine receptors with p2x7 inhibition, reduce degeneration of our lipid rafts by promoting insulin sensitivity, reducing sphingolipid incorporation with inhibitors, and promoting their breakdown with glucocerebosidase activators. We promote better liver health which reduces ammonia, and bilrubin accumulation that aggravates endozapine release and further damages the lipid rafts of neuronal cells. Then we can increase our neuronal stem cell pools by increasing insulin sensitivity and activation with insulin/IGF2 and reducing oxidative stress with powerful mitochondrial targeted antioxidants.

• Lysosomal deacidifcation also increases iron intake into hepcidin and ferritin.

• As we age, the activity of Heme Oxygenase increases as a means of attempting to mitigate oxidative stress and that breaks down hemoglobin into bilrubin, carbon monoxide, and free iron.

  • So the process which iron continues to accumulate within the brain becomes more aggressive and this is partially responsible for the dysfunction of Erythropoietin activity and increased levels of epo within the brain in Alzheimer’s or Parkinson’s disease. Epo can’t exert its functions on mitochondria promoting mitochondrial biogenesis and it also leads to the breakdown of neuronal insulin sensitivity and ferritin turnover.

• In CSF, 5-HT level is significantly decreased and the levels of iron and transferrin are dramatically increased in fatigue group

• iron dysbiosis even leads to the failure of phageocytosis of microglial cells (THEIR degradation, pretty sure) and that means that microglial cells can nolonger properly break down neuronal progenitor cells that dont incorporate into neuronal networks or consume dead cells and clearance of associated cellular wastes. Iron dysbiosis even supports the survival and reactivation of latent bacteria within the brain that increase inflammation through endotoxemia and it all feeds forward

• The mitochondria in this instance is called the Endosome; what’s outside of mitochondria where all other organelles of our cells are is called the lysosome

• Potent induction of angiogenesis can make it a topical for Hair Loss?

• Covid is a hepcidin memetic. Severity has been shown to be related to how well it is metabolized, i.e. in younger people.

• What I want to find out is its cellular localization and its dynamics on mitochondria and cellular respiration. Main concerns really if you look at good/ugly.

• Iron chelation down-regulates dopamine transporter expression by decreasing mRNA stability and increasing endocytosis in N2a cells

  • Cellular iron depletion elevated protein levels of the early endosomal marker Rab5.

The Good

• Significantly inhibits BACE1

• Deferoxamine: a reversible S-phase inhibitor of human lymphocyte proliferation

  • Potent inhibitor of DNA synthesis by T Cells and B Cells Prevents cells from completing the S phase of the cell proliferation cycle.

• Deferoxamine pharmacokinetics

  • 50 mg/kg/d, and initial elimination half-life of 0.28/h and steady-state concentration of 7μM/L were observed.

• Increased iron levels in the lung reduced function and worsened pulmonary fibrosis, and was prevented with DFO. Lung fibrosis enters the iron age

• Red Cells, Iron, and Erythropoiesis: Specific iron chelators determine the route of ferritin degradation (Nov 2009)

  • Deferoxamine ‘induces autophagy’, which leads to Ferritin entry into Lysosomal lumen.
    • DFO-treated cells resulted in cytosolic accumulation of LC3B, while DFX/DFP, or ferrioxaxmine, did not.
    • Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, resulted in degradation of ferritin by the proteasome.
    • This process of feritin turnover is important for rechanneling iron into the proteome which autophagy-promiting compounds favor.
  • The membrane-permeable chelators desferasirox (DFX) and deferiprone diverted ferritin degradation towards the proteasomal pathway.
  • Did not induce pH changes in the lysosome.
  • Inhibition of endocytosis prevents DFO-induced ferritin degadation, supporting the importance of the lysosomal localization of Deferoxamine.
    • Furthermore, it might be specific to siderophores or sometihng, because another impermeable chelator, bathophenanthroline did not induce autophagy.
  • Rapamyci] can induce macroautophagy but does not induce Ferritinophagy.
  • Some kinda PI3K inhibitors inhibit lysosomal ferritin degradation.
  • our results show that ferritin can be degraded either by the lysosome or the proteasome
  • Lysosomal deferoxamine can induce Ferritinophagy even when entry of ferritin into the lysosome is prevented.
  • A combination of DFO and a permeable chelator provides a shuttle mechanism by which DFO takes iron from the permeable chelator for it to again bind more iron.

• A recent study has shown that DFO and DFX increased autophagy via inhibiting mTOR. Their differences may be cell-type dependent.

• The hypoxia memesis is by means of… for one, removing Fe2+ from Prolyl Hydroxylase, which marks HIF-1α and HIF-2α for ubiquination.

  • Stimulation of HIF-1alpha, HIF-2alpha, and VEGF by prolyl 4-hydroxylase inhibition in human lung endothelial and epithelial cells
    • Prolyl Hydroxylase inhibition enhances VEGF release and glucose consumption

Insulin

• iron accumulates in astrocytes, reduces insulin sensitivity and that prevents insulin increasing the transcription and prevents the release of Erythropoietin within the brain.
  • This would support why neuronal levels of epo decline and systemic levels of epo rise with age or could even support why neuronal epo might be higher in dementia as a consequence of failure of epo to exert its actions on mitochondrial biogenesis due to the rise of hepcidin, and ferritin within the cytosol of neurons, and microglial cells
  • Tl;dr, DFO fixes this by lowering GSK, thereby Tau. Increases HIF and thereby insulin signaling.
• Iron Deposition Leads to Hyperphosphorylation of Tau and Disruption of Insulin Signaling
  • Via decreased phosphorylation levels of IR-β (unable to detect changes in levels) IRS-1, and PI3K.
  • Treatment of insulin within a short time led to a rapid and transient hyperphosphorylation of tau
  • Cognition is impaired and causes abnormal tau phosphorylation in mice fed high-iron chow.
• Iron Depletion by Deferoxamine Up-Regulates Glucose Uptake and Insulin Signaling in Hepatoma Cells and in Rat Liver

Neuronal

• Reversion of age-related recognition memory impairment by iron chelation in rats

  • Neonatal iron supplementation induces selective accumulation in certain brain regions, notably the basal ganglia, associated with memory impairments in adulthood. It also induces lipid peroxidation and protein carbonylation in substantia nigra.

• Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells

  • Neuroinflammation from Endotoxins etc. activates Microglia release of TNF-α, IL-1β, etc. This leads to:
    • Hepcidin expression is increased in astrocytes/microglia
    • Ferroportin is decreased in all 3 cell types.
    • DMT1 expression is increasd in all 3 cell types
  • So, iron accumulates in neurons and microglia (but not astrocytes) with hepcidin binding to iron otherwise present in mitochondria.
    • Aging is associated with increased brain iron through cortex-derived hepcidin expression
      • Here, we measured the levels of iron in different tissues of aged mice, and demonstrated that while cytosolic non-heme iron is increased in the liver and muscle tissue, only the aged brain cortex exhibits an increase in both the cytosolic and mitochondrial non-heme iron

• Protection from Oxidative Stress–Induced Apoptosis in Cortical Neuronal Cultures by Iron Chelators Is Associated with Enhanced DNA Binding of Hypoxia-Inducible Factor-1 and ATF-1/CREB and Increased Expression of Glycolytic Enzymes, p21waf1/cip1, and Erythropoietin

  • Iron chelators pick up redox-active iron… would that mean only Fe2+?
  • 100μM pentupled Lactate Dehydrogenase acivity.
  • Not too much erythropoiesis. 500μM doubled.

• Hypoxia-inducible factor-1α upregulates tyrosine hydroxylase and dopamine transporter by nuclear receptor ERRγ in SH-SY5Y cells

  • Deferoxamine-induced TH upregulation (and DAT upregulation apparently) was attenuated by ERRγ inverse agonist.

• Neuroprotective effect of deferoxamine on erastin-induced ferroptosis in primary cortical neurons

  • Inhibits the ferroptotic pathway by upregulating the Cystine-Glutamate Antiporter system light chain (xCT) and Glutathione Peroxidase 4.

• Deferoxamine-mediated up-regulation of HIF-1α prevents dopaminergic neuronal death via the activation of MAPK family proteins in MPTP-treated mice

  • Upregulated Tyrosine Hydroxylase. Downregulated DMT1+IRE and TfR1
  • Inhibited MPTP-induced phosphorylation of JNK; enhanced phosphorylation of ERK and p38.

• Desferrioxamine enhances hypoxic ventilatory response and induces tyrosine hydroxylase gene expression in the rat brainstem in vivo

• Iron chelation down-regulates dopamine transporter expression by decreasing mRNA stability and increasing endocytosis in N2a cells

  • Chelation via DFO decreased the stability of DAT mRNA, increasing its phosphorylation and ubiquination. Increased its localization into the endosome.
  • Rab5 increased 3-fold and Rab11 decreased 5-fold.
  • Protein Kinase A -62%, Protein Kinase B -81%, Protein Kinase Cγ -32%. Protein Kinase Cδ -60%, and Protein Kinase Cζ -40%, but Protein Kinase D increased 250%.
  • Down-regulation of dopamine transporter by iron chelation in vitro is mediated by altered trafficking, not synthesis

• Morphine-Induced Modulation of Endolysosomal Iron Mediates Upregulation of Ferritin Heavy Chain in Cortical Neurons

  • Both in vitro and in vivo, FHC upregulation was accompanied by loss of mature dendritic spines, which was also dependent on μ-Opioid Receptor and Gαi-protein signaling.
    • Deferoxamine blocked morphine-mediated FHC upregulation. Iron chelation with Deferoxamine (100 µM, 24 h) did not significantly change basal FHC or FLC expression
  • The same morphine treatment significantly increased cytoplasmic iron levels as measured by phen green SK, which was blocked by chelating endolysosomal iron with DFO
  • Morphine’s ability to reduce dendritic spine density was blocked by chelation of endolysosomal Iron with Deferoxamine, but not affected by extracellular iron chelation with DTPA, demonstrating the importance of endolysosomal iron for this pathway. Morphine-mediated reduction of mature dendritic spines requires endolysosomal iron. This is massive. At the end of the day, DFO blocked morphine-induced neurological dysfunction.
    • morphine and FAC significantly reduced thin and mushroom spines, and this effect was similarly blocked by DFO, but not DTPA;
  • Morphine and FAC both significantly reduced overall dendritic spine density by the same amount. Morphine’s ability to reduce dendritic spine density was blocked by chelation of endolysosomal iron with DFO.

• Lysosomal iron modulates NMDA receptor-mediated excitation via small GTPase, Dexras1

  • NMDAR activation can induce Iron movement into neurons via DMT1 (Divalent metal transporter), and under pathological conditions like excitotoxicity, contributes to metal-catalyzed ROS generation.
    • S-nitrosylation of Dextras1 by Nitric Oxide induces DMT1.
  • Genetic and pharmacological ablation of this neuronal iron pathway (decreasing cytosolic iron) in the mice increased glutamatergic transmission and synaptic plasticity, due to synaptic modification of NMDA receptor activity via modulation of the PKC/Src/NR2A pathway.
    • Lysosomal iron serves as a main source for intracellular iron signaling modulating glutamatergic excitability
  • In wild type CA1 slices (no toxicity no nothing) 80% increase in spontaneous EPSC after application of pyridoxl isonicotinoyl hydrazine, a membrane-permeable iron chelator.

• Deferoxamine regulates neuroinflammation and iron homeostasis in a mouse model of postoperative cognitive dysfunction

• Translocation of iron from lysosomes into mitochondria is a key event during oxidative stress‐induced hepatocellular injury pretty interesting stuff actually. Just read it for localization info.

  • ROS formation initiates mitochondrial permeability transition (MPT) that opens nonspecific permeability transition pores in the Inner Mitochondrial Membrane These pores conduct all solutes up to 1500 Da. Sounds like suicide!
  • Es gibt ein non-chelatable iron pool in ferritin, proteins, Fe-S clusters, etc. that chelators like deferoxamine cannot reach - only free iron, and iron bound less strongly to anions.
  • DFO is poorly permeable, not impermeable.
    • Evidence that desferrioxamine cannot enter cells by passive diffusion
      • Deferoxamine enters cells by endocytosis, and then localizes to lysosomes/endosomes.
    • What transporter takes it up??
  • Mitochondria accumulate Fe2+ electrogenically via the mitochondrial Ca2+ uniporter, while Fe3+ is not transported.

• Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

  • Prevents peroxynitrite-mediated oxidations and attenuates Nitric Oxide and oxygen radical-dependent oxidative damage.

Amyloid

• Deferoxamine enhances alternative activation of microglia and inhibits amyloid beta deposits in APP/PS1 mice

  • Induced M2 activation of Microglia and inhibited M1 activation, in the hippocampus of APP mice.
    • This means TGF-β→AKT
  • 0.56-fold Caspase 3.

• Deferoxamine reduces amyloid-beta peptides genesis and alleviates neural apoptosis after traumatic brain injury

• Aβ is capable of accumulating Iron (III) within amyloid aggregates, with this process resulting in Aβ-mediated reduction of iron(III) to a redox-active iron(II). The presence of Aluminum increases the reductive capcity.

The Bad

• Chronic Energy Depletion due to Iron Deficiency Impairs Dendritic Mitochondrial Motility during Hippocampal Neuron Development

  • This paper is about mitochondrial trafficking. That’s important for arborization and outgrowth as they go from old to new areas I suppose
  • At 11 days in vitro, DFO reduced average mitochondrial speed (kinesin/dynein and adaptor proteins) by increasing the pause frequency of individual dendritic mitochondria. Time spent in retrograde motion was reduced.
  • We have previously shown that chronic iron deficiency (ID) impairs mitochondrial respiration and dendritic complexity without severely impairing overall neuronal health: Iron Deficiency Impairs Developing Hippocampal Neuron Gene Expression, Energy Metabolism, and Dendrite Complexity

• Deferoxamine deconditioning increases neuronal vulnerability to hemoglobin This is after blood vessel rupture, which causes an elevation in tissue iron. 3-4x neuronal loss after Hemoglobin exposure, since it releases iron upon Heme Oxygenase.

• Deferoxamine produces nitric oxide under ferricyanide oxidation, blood incubation, and UV-irradiation (2020)

  • Similar compounds with nitroxyl groups/hydroxylamines are known to produce nitric oxide under oxidative conditions. DFO incubation with sheep adult/fetal blood resulted in formation of HNO into Nitric Oxide, forming iron nitrosyl hemoglobin. This is still just under oxidative conditions, with ferricyanide. NO is only generated when DFO is injected directly with FeCN.
    • The reaction does not happen in pure deionized water. Spontaneously nitrite formation is observed in DFO + $\ce{CO3^{2-}}$ even without FeCN.
  • Reducing agent $\ce{I3^-}$ didn’t lead to any NO generation.
  • DFO at >1 mM concentrations can produce a nitroxide radical via oxidation by a hydroxyl radical produced by photolysis of H2O2.
  • NO generation was eliminated upon DFO pre-treatment with anaerobically prepared FeSO4
  • Production of NO from DFO+FeCN was not affected by pre-treatment with 100 μM H2O2, contrary to expectations.
  • Plasma concentrations of DFO are reported to reach between 80 and 130 μM following 3 min of intravenous injection

• Desferrioxamine decreases NAD redox potential of intact red blood cells: evidence for desferrioxamine as an inducer of oxidant stress in red blood cells

  • Its stability constant of 10^31 for ferric iron contrasts significantly with those for other ions such as zinc (10^11), calcium (10^2) and magnesium (10^4).
  • Since certain compounds liberate iron from ferritin, iron excess can occur without ‘iron overload’.
    • Ferritin, lipid peroxidation and redox-cycling xenobiotics
    • Oxygen-based free radical generation by ferrous ions and deferoxamine
      • DFO accelerates autoxoidation of Fe2+ into Fe3+, associated with uptake of oxygen; fenton reactions, and with a fall in pH.
      • DFO may directly scavenge $\ce{OH^.}$ and $\ce{O2^{.-}}$.
    • DFO conjugated with starch (purpose is simply to increase molecular weight, I think) is unable to penetrate into RBC (as if DFO wasn’t impermeable enough), and decreases NAD redox potential. Thus this effect probably is borne of the extracellular realm, yet the NAD is inside the erythrocyte.
  • 500μL of whole blood with 0-6mM. That’s crazy compared to the ~10μM/L in clinical use in vivo. Not bad for a 15% drop in redox potential.
  • Able to induce oxidation of ferrous iron? They say it’s likely to be involved but I fail to recognize how it wouldn’t just be spontaneous

• Recent insights into interactions of deferoxamine with cellular and plasma iron pools: Implications for clinical use

  • Its permeability is favored by its positive charge.
  • Its uptake into hepatocytes is 800x faster into red blood cells at 37°C.
  • Tommy: Causes a rebound in non-Tf-bound iron if delivered at high doses.
  • Removes ~1/3 of non-Tf-bound iron rapidly, and increased dosing does not increase this removal, instead leading to greater rebound on cessation.
  • Technique used is stabilizing DFO with aluminum ions so as to prevent non-Tf-bound iron shuttling to it after collecting plasma.

The Ugly

• Deferoxamine Enhanced Mitochondrial Iron Accumulation and Promoted Cell Migration in Triple-Negative MDA-MB-231 Breast Cancer Cells Via a ROS-Dependent Mechanism
  • Under iron-deficient conditions, increased mitochondrial iron levels in these cancer cells increases ROS generation. DFO is an anticancer drug - I think this ROS is a good thing or some shit.
• Cell penetrating peptide (CPP)-conjugated desferrioxamine for enhanced neuroprotection: synthesis and in vitro evaluation
  • The applications of DFO are limited because of its inability to access intracellular labile iron. Honestly, WTF? Where does it go then?
    • This is a lie. I mean, maybe it doesn’t touch it, but it certainly decreases LIP: R
  • Its limited permeability is due to high hydrophobicity and high molecular weight.
  • Bidentate (3:1 stabilizing iron) ligands are likely to be toxic.

Intranasal studies

• Mechanisms of Intranasal Deferoxamine in Neurodegenerative and Neurovascular Disease

• Intranasal Administration of Deferoxamine to Iron Overloaded Patients

• [Intranasal deferoxamine improves performance in radial arm water maze, stabilizes HIF-1α, and phosphorylates GSK3β in P301L tau transgenic mice (Fine 2012)]

• Intranasally-administered deferoxamine mitigates toxicity of 6-OHDA in a rat model of Parkinson׳s disease (Fine 2014)

• Intranasal deferoxamine affects memory loss, oxidation, and the insulin pathway in the streptozotocin rat model of Alzheimer’s disease (Fine 2017)

  • I mean this is still an Alzheimer’s model that reverses the consequences of NFTs. Nonethleess it increased morris water maze performance before streptozotocin treatment.

• Intranasal deferoxamine engages multiple pathways to decrease memory loss in the APP/PS1 model of amyloid accumulation (Fine 2015)

  • Both pGSK-3β and β-Catenin were significantly increased by approximately 50% in the DFO-treated mice

• Intranasal deferoxamine can improve memory in healthy C57 mice, suggesting a partially non-disease-specific pathway of functional neurologic improvement (Fine 2019)

  • Not only are they healthy, but these are 7 week old mice. That’s like 5 year old kids. The iron accumulation has hardly begun! Though, perhaps, maybe neither has natural chelation from perhaps high prenatal levels (if that even happens).
  • DFO may slow the process of retinal degeneration (Obolensky et al., 2011) and improve aspects of human cerebrovascular function, namely vasoreactivity and autoregulation, from baseline (Sorond et al., 2015).
  • 100mg/kg = ~600 mg HED. It was administered daily for over a month! 18g would be nuts mate…
  • Significantly decreases GSK-3β activity via phosphorylation at Ser9. In this study: 68% increase in HIF-1α, 200% increase in pGSK3β, ~40% increase in β-Catenin. Contrary to expectations, ~30% decrease in GLUT1.
  • Treatment results in phosphorylation of AKT. Along with VEGF I can think of, yet another mechanism to decrease GSK-3β. The inhibition of it is neuroprotective in brain injury.
  • Decreased Amyloid β and Tau in AD, and preservation of striatal DA neurons in PD.
  • Chronic intranasal deferoxamine ameliorates motor defects and pathology in the α-synuclein rAAV Parkinson’s model
    • Iron can compromise the solubility of α-synuclein. DFO did not protect against Parkinson’s model dopaminergic cell death, but it decreased the number of pathological α-synuclein formations at the terminal level.
  • Lack of weight loss or ‘physical abnormalities’.

• Intranasal Deferoxamine Provides Increased Brain Exposure and Significant Protection in Rat Ischemic Stroke
  • 
• Serial Studies of Auditory Neurotoxicity in Patients Receiving Deferoxamine Therapy
  • An analysis of Jhe clinical data showed that most members Of the affected group were younger, had lower serum ferritin value, and were receiving higher doses of the drug per kilogram of body weight. These people are 3-27 years, mean 12.
  • *Most of these reports had dealt with a variety of ocular changes ranging from blurring of vision [5], loss of acuity (4,6-81, loss of central vision [4], night blindness [9], pigmentary retinopathy [4,9,10], and optic neuropathy [4,9,11]. *
  • Most cases of acute visual loss were reversible after stopping therapy with deferoxamine, but anatomic abnormalities such as optic neuropathy and pigmentary changes remained unchanged.
    • In our four symptomatic patients [4], two presented with a marked decrease in central vision, eccentric fixation, and severely impaired visual acuity. The symptoms completely reversed within four weeks after withdrawal of deferoxamine, but optic atrophy and thinning of the nerve fiber layer persisted. A third patient had markedly decreased visual acuity, a left afferent pupillary defect, and asymmetric optic atrophy: after withdrawal of the drug, acuity improved but the atrophy was unchanged. The fourth patient also had decreased visual acuity, abnormal color vision, and translucent swelling of the optic disk; after stopping the drug, acuity and color vision partly improved. Abnormal pigmentary changes were observed in five other patients [4], but there were no abnormalities of visual acuity or color vision in these cases.

Other chelator considerations

Deferiprone

• A Combined Drug Treatment That Reduces Mitochondrial Iron and Reactive Oxygen Levels Recovers Insulin Secretion in NAF-1-Deficient Pancreatic Cells
  • Memebrane-permeable baby. And coadministrated with NAC.

Dosage

Lasts reconstituted for 2 days.