Another puzzle piece in the autism mystery

Just like in the case of schizophrenia, hundreds of genes have been associated with autistic spectrum disorders (ASDs). Here is another candidate.

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Féron et al. (2016) reasoned that most of the info we have about the genes that are behaving badly in ASDs comes from studies that used adult cells. Because ASDs are present before or very shortly after birth, they figured that looking for genetic abnormalities in cells that are at the very early stage of ontogenesis might prove to be enlightening. Those cells are stem cells. Of the pluripotent kind. FYI, based on what they can become (a.k.a how potent they are), the stem cells are divided into omipotent, pluripotent, multipotent, oligopotent, and unipotent. So the pluripotents are very ‘potent’ indeed, having the potential of producing a perfect person.

Tongue-twisters aside, the authors’ approach is sensible, albeit non-hypothesis driven. Which means they hadn’t had anything specific in mind when they had started looking for differences in gene expression between the olfactory nasal cells obtained from 11 adult ASDs sufferers and 11 age-matched normal controls. Luckily for them, as transcriptome studies have a tendency to be difficult to replicate, they found the anomalies in the expression of genes that have been already associated with ASD. But, they also found a new one, the MOCOS (MOlybdenum COfactor Sulfurase) gene, which was poorly expressed in ASDs (downregulated, in genetic speak). The enzyme is MOCOS (am I the only one who thinks that MOCOS isolated from nasal cells is too similar to mucus? is the acronym actually a backronym?).

The enzyme is not known to play any role in the nervous system. Therefore, the researchers looked to see where the gene is expressed. Its enzyme could be found all over the brain of both mouse and human. Also, in the intestine, kidneys, and liver. So not much help there.

Next, the authors deleted this gene in a worm, Caenorhabditis elegans, and they found out that the worm’s cells have issues in dealing with oxidative stress (e.g. the toxic effects of free radicals). In addition, their neurons had abnormal synaptic transmission due to problems with vesicular packaging.

Then they managed – with great difficulty – to produce human induced pluripotent cells (iPSCs) in a Petri dish in which the gene MOCOS was partially knocked down. ‘Partially’, because the ‘totally’ did not survive. Which tells us that MOCOS is necessary for survival of iPSCs. The mutant cells had less synaptic buttons than the normal cells, meaning they formed less synapses.

The study, besides identifying a new candidate for diagnosis and treatment, offers some potential explanations for some beguiling data that other studies have brought forth, like the fact that all sorts of neurotransmitter systems seem to be impaired in ADSs, all sorts of brain regions, making very hard to grab the tiger by the tail if the tiger is sprouting a new tail when you look at it, just like the Hydra’s heads. But, discovering a molecule that is involved in an ubiquitous process like synapse formation may provide a way to leave the tiger’s tail(s) alone and focus on the teeth. In the authors’ words:

“As a molecule involved in the formation of dense core vesicles and, further down, neurotransmitter secretion, MOCOS seems to act on the container rather than the content, on the vehicle rather than one of the transported components” (p. 1123).

The knowledge uncovered by this paper makes a very good piece of the ASDs puzzle. Maybe not a corner, but a good edge. Alright, even if it’s not an edge, at least it’s a crucial piece full of details, not one of those sky pieces.

Reference: Féron F, Gepner B, Lacassagne E, Stephan D, Mesnage B, Blanchard MP, Boulanger N, Tardif C, Devèze A, Rousseau S, Suzuki K, Izpisua Belmonte JC, Khrestchatisky M, Nivet E, & Erard-Garcia M (Sep 2016, Epub 4 Aug 2016). Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders. Molecular Psychiatry, 21(9):1215-1224. PMID: 26239292, DOI: 10.1038/mp.2015.106. ARTICLE | FREE FULLTEXT PDF

By Neuronicus, 31 August 2016

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Painful Pain Paper

There has been much hype over the new paper published in the latest Nature issue which claims to have discovered an opioid analgesic that doesn’t have most of the side effects of morphine. If the claim holds, the authors may have found the Holy Grail of pain research chased by too many for too long (besides being worth billions of dollars to its discoverers).

The drug, called PZM21, was discovered using structure-based drug design. This means that instead of taking a drug that works, say morphine, and then tweaking its molecular structure in various ways and see if the resultant drugs work, you take the target of the drug, say mu-opioid receptors, and design a drug that fits in that slot. The search and design are done initially with sophisticated software and there are many millions of virtual candidates. So it takes a lot of work and ingenuity to select but a few drugs that will be synthesized and tested in live animals.

Manglik et al. (2016) did just that and they came up with PZM21 which, compared to morphine, is:

1) selective for the mu-opioid receptors (i.e. it doesn’t bind to anything else)
2) produces no respiratory depression (maybe a touch on the opposite side)
3) doesn’t affect locomotion
4) produces less constipation
5) produces long-lasting affective analgesia
6) and has less addictive liability

The Holy Grail, right? Weeell, I have some serious issues with number 5 and, to some extent, number 6 on this list.

Normally, I wouldn’t dissect a paper so thoroughly because, if there is one thing I learned by the end of GradSchool and PostDoc, is that there is no perfect paper out there. Consequently, anyone with scientific training can find issues with absolutely anything published. I once challenged someone to bring me any loved and cherished paper and I would tear it apart; it’s much easier to criticize than to come up with solutions. Probably that’s why everybody hates Reviewer No. 2…

But, for extraordinary claims, you need extraordinary evidence. And the evidence simply does not support the 5 and maybe 6 above.

Let’s start with pain. The authors used 3 tests: hotplate (drop a mouse on a hot plate for 10 sec and see what it does), tail-flick (give an electric shock to the tail and see how fast the mouse flicks its tail) and formalin (inject an inflammatory painful substance in the mouse paw and see what the animal does). They used 3 doses of PZM21 in the hotplate test (10, 20, and 40 mg/Kg), 2 doses in the tail-flick test (10 and 20), and 1 dose in the formalin test (20). Why? If you start with a dose-response in a test and want to convince me it works in the other tests, then do a dose-response for those too, so I have something to compare. These tests have been extensively used in pain research and the standard drug used is morphine. Therefore, the literature is clear on how different doses of morphine work in these tests. I need your dose-responses for your new drug to be able to see how it measures up, since you claim it is “more efficacious than morphine”. If you don’t want to convince me there is a dose-response effect, that’s fine too, I’ll frown a little, but it’s your choice. However, then choose a dose and stick with it! Otherwise I cannot compare the behaviors across tests, rendering one or the other test meaningless. If you’re wondering, they used only one dose of morphine in all the tests, except the hotplate, where they used two.

Another thing also related to doses. The authors found something really odd: PZM21 works (meaning produces analgesia) in the hotplate, but not the tail-flick tests. This is truly amazing because no opiate I know of can make such a clear-cut distinction between those two tests. Buuuuut, and here is a big ‘BUT” they did not test their highest dose (40mg/kg) in the tail-flick test! Why? I’ll tell you how, because I am oh sooo familiar with this argument. It goes like this:

Reviewer: Why didn’t you use the same doses in all your 3 pain tests?

Author: The middle and highest doses have similar effects in the hotplate test, ok? So it doesn’t matter which one of these doses I’ll use in the tail-flick test.

Reviewer: Yeah, right, but, you have no proof that the effects of the two doses are indistinguishable because you don’t report any stats on them! Besides, even so, that argument applies only when a) you have ceiling effects (not the case here, your morphine hit it, at any rate) and b) the drug has the expected effects on both tests and thus you have some logical rationale behind it. Which is not the case here, again: your point is that the drug DOESN’T produce analgesia in the tail-flick test and yet you don’t wanna try its HIGHEST dose… REJECT AND RESUBMIT! Awesome drug discovery, by the way!

So how come the paper passed the reviewers?! Perhaps the fact that two of the reviewers are long term publishing co-authors from the same University had something to do with it, you know, same views predisposes them to the same biases and so on… But can you do that? I mean, have reviewers for Nature from the same department for the same paper?

Alrighty then… let’s move on to the stats. Or rather not. Because there aren’t any for the hotplate or tail-flick! Now I know all about the “freedom from the tyranny of p” movement (that is: report only the means, standard errors of mean, and confidence intervals and let the reader judge the data) and about the fact that the average scientist today needs to know 100-fold more stats that his predecessors 20 years ago (although some biologists and chemists seem to be excused from this, things either turn color or not, either are there or not etc.) or about the fact that you cannot get away with only one experiment published these days, but you need a lot of them so you have to do a lot of corrections to your stats so you don’t fall into the Type 1 error. I know all about that, but just like the case with the doses, choose one way or another and stick to it. Because there are ANOVAs ran for the formalin test, the respiration, constipation, locomotion, and conditioned place preference tests, but none for the hotplate or tailflick! I am also aware that to be published in Science or Nature you have to strip your work and wordings to the bare minimum because the insane wordcount limits, but you have free rein in the Supplementals. And I combed through those and there are no stats there either. Nor are there any power analyses… So, what’s going on here? Remember, the authors didn’t test the highest dose on the tail-flick test because – presumably – the highest and intermediary doses have indistinguishable effects, but where is the stats to prove it?

And now the thing that really really bothered me: the claim that PZM21 takes away the affective dimension of pain but not the sensory. Pain is a complex experience that, depending on your favourite pain researcher, has at least 2 dimensions: the sensory (also called ‘reflexive’ because it is the immediate response to the noxious stimulation that makes you retract by reflex the limb from whatever produces the tissue damage) and the affective (also called ‘motivational’ because it makes the pain unpleasant and motivates you to get away from whatever caused it and seek alleviation and recovery). The first aspect of pain, the sensory, is relatively easy to measure, since you look at the limb withdrawal (or tail, in the case of animals with prolonged spinal column). By contrast, the affective aspect is very hard to measure. In humans, you can ask them how unpleasant it is (and even those reports are unreliable), but how do you do it with animals? Well, you go back to humans and see what they do. Humans scream “Ouch!” or swear when they get hurt (so you can measure vocalizations in animals) or humans avoid places in which they got hurt because they remember the unpleasant pain (so you do a test called Conditioned Place Avoidance for animals, although if you got a drug that shows positive results in this test, like morphine, you don’t know if you blocked the memory of unpleasantness or the feeling of unpleasantness itself, but that’s a different can of worms). The authors did not use any of these tests, yet they claim that PZM21 takes away the unpleasantness of pain, i.e. is an affective analgesic!

What they did was this: they looked at the behaviors the animal did on the hotplate and divided them in two categories: reflexive (the lifting of the paw) and affective (the licking of the paw and the jumping). Now, there are several issues with this dichotomy, I’m not even going to go there; I’ll just say that there are prominent pain researchers that will scream from the top of their lungs that the so-called affective behaviors from the hotplate test cannot be indexes of pain affect, because the pain affect requires forebrain structures and yet these behaviors persist in the decerebrated rodent, including the jumping. Anyway, leaving the theoretical debate about what those behaviors they measured really mean aside, there still is the problem of the jumpers: namely, the authors excluded from the analysis the mice who tried to jump out of the hotplate test in the evaluation of the potency of PZM21, but then they left them in when comparing the two types of analgesia because it’s a sign of escaping, an emotionally-valenced behavior! Isn’t this the same test?! Seriously? Why are you using two different groups of mice and leaving the impression that is only one? And oh, yeah, they used only the middle dose for the affective evaluation, when they used all three doses for potency…. And I’m not even gonna ask why they used the highest dose in the formalin test…but only for the normal mice, the knockouts in the same test got the middle dose! So we’re back comparing pears with apples again!

Next (and last, I promise, this rant is way too long already), the non-addictive claim. The authors used the Conditioned Place Paradigm, an old and reliable method to test drug likeability. The idea is that you have a box with 2 chambers, X and Y. Give the animal saline in chamber X and let it stay there for some time. Next day, you give the animal the drug and confine it in chamber Y. Do this a few times and on the test day you let the animal explore both chambers. If it stays more in chamber Y then it liked the drug, much like humans behave by seeking a place in which they felt good and avoiding places in which they felt bad. All well and good, only that is standard practice in this test to counter-balance the days and the chambers! I don’t know about the chambers, because they don’t say, but the days were not counterbalanced. I know, it’s a petty little thing for me to bring that up, but remember the saying about extraordinary claims… so I expect flawless methods. I would have also liked to see a way more convincing test for addictive liability like self-administration, but that will be done later, if the drug holds, I hope. Thankfully, unlike the affective analgesia claims, the authors have been more restrained in their verbiage about addiction, much to their credit (and I have a nasty suspicion as to why).

I do sincerely think the drug shows decent promise as a painkiller. Kudos for discovering it! But, seriously, fellows, the behavioral portion of the paper could use some improvements.

Ok, rant over.

EDIT (Aug 25, 2016): I forgot to mention something, and that is the competing financial interests declared for this paper: some of its authors already filed a provisional patent for PZM21 or are already founders or consultants for Epiodyne (a company that that wants to develop novel analgesics). Normally, that wouldn’t worry me unduly, people are allowed to make a buck from their discoveries (although is billions in this case and we can get into that capitalism-old debate whether is moral to make billions on the suffering of other people, but that’s a different story). Anyway, combine the financial interests with the poor behavioral tests and you get a very shoddy thing indeed.

Reference: Manglik A, Lin H, Aryal DK, McCorvy JD, Dengler D, Corder G, Levit A, Kling RC, Bernat V, Hübner H, Huang XP, Sassano MF, Giguère PM, Löber S, Da Duan, Scherrer G, Kobilka BK, Gmeiner P, Roth BL, & Shoichet BK (Epub 17 Aug 2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature, 1-6. PMID: 27533032, DOI: 10.1038/nature19112. ARTICLE 

By Neuronicus, 21 August 2016

The FIRSTS: Theory of Mind in non-humans (1978)

Although any farmer or pet owner throughout the ages would probably agree that animals can understand the intentions of their owners, not until 1978 has this knowledge been scientifically proven.

Premack & Woodruff (1978) performed a very simple experiment in which they showed videos to a female adult chimpanzee named Sarah involving humans facing various problems, from simple (can’t reach a banana) to complex (can’t get out of the cage). Then, the chimps were shown pictures of the human with the tool that solved the problem (a stick to reach the banana, a key for the cage) along with pictures where the human was performing actions that were not conducive to solving his predicament. The experimenter left the room while the chimp made her choice. When she did, she rang a bell to summon the experimenter back in the room, who then examined the chimp’s choice and told the chimp whether her choice was right or wrong. Regardless of the choice, the chimp was awarded her favorite food. The chimp’s choices were almost always correct when the actor was its favourite trainer, but not so much when the actor was a person she disliked.

Because “no single experiment can be all things to all objections, but the proper combination of results from [more] experiments could decide the issue nicely” (p. 518), the researchers did some more experiments which were variations of the first one designed to figure out what the chimp was thinking. The authors go on next to discuss their findings at length in the light of two dominant theories of the time, mentalism and behaviorism, ruling in favor of the former.

Of course, the paper has some methodological flaws that would not pass the rigors of today’s reviewers. That’s why it has been replicated multiple times in more refined ways. Nor is the distinction between behaviorism and cognitivism a valid one anymore, things being found out to be, as usual, more complex and intertwined than that. Thirty years later, the consensus was that chimps do indeed have a theory of mind in that they understand intentions of others, but they lack understanding of false beliefs (Call & Tomasello, 2008).

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References:

1. Premack D & Woodruff G (Dec. 1978). Does the chimpanzee have a theory of mind? The Behavioral and Brain Sciences, 1 (4): 515-526. DOI: 10.1017/S0140525X00076512. ARTICLE

2. Call J & Tomasello M (May 2008). Does the chimpanzee have a theory of mind? 30 years later. Trends in Cognitive Sciences, 12(5): 187-192. PMID: 18424224 DOI: 10.1016/j.tics.2008.02.010. ARTICLE  | FULLTEXT PDF

By Neuronicus, 20 August 2016

THE FIRSTS: The word ‘scientist’ (1834)

Scientist, by any other name…

History of science is, unfortunately, not among the mandatory classes required for earning a diploma that allows oneself to be called a scientist. Worrisomely, nor is Logic as a formal class. All the more the pity because in the Middle Ages, when the word science entered the English language, to have scientific knowledge meant you have arrived at it by following the Aristotelian way of logical reasoning (a.k.a deductions and inductions). To be fair, the word existed already in Romance languages with the same meaning: new knowledge obtained by applying the rules of Aristotelian syllogisms. By the way, Aristotle is also the guy to whom we owe the basis of the scientific method, but that’s a story for another day.

Although words like scientific or science were altogether frequently used with regards of the scholarly endeavors of the ladies and gentlemen of the early 19th Century (yes, there were ladies too that dabbled into the sciences, even if sometimes it was only to write about the spectacular discoveries and controversies of their time), the term scientist has been officially coined in 1834 by William Whewell. A man truly blessed in the art of words, being credited with coining a lot of other famous words like anode and physicist, he proposed the word in a review of a science popularization book written by one Mrs. Somerville. The circumstance of how this came to be is masterly imparted to us by Sydney Ross in a superb historical account of the word scientist, published in 1962.

For the rounded scientist or for the merely curious, I truly recommend the lecture of the referenced papers. They’re delightful!

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Reference 1. [Whewell W] (1834). Art. III. [Review of] On the Connexion of the Physical Sciences. By Mrs. Somerville. The Quarterly Review, 51: 58-61. FULLTEXT PDF at GoogleBooks

Reference 2. Ross S (1962). Scientist: The story of a word, Annals of Science, 18:2, 65-85, DOI: 10.1080/00033796200202722. FREE FULLTEXT PDF

P.S. I checked and Wikipedia is correct with the following statement:

“To be exact, the person coined the term scientist was referred to in Whewell 1834 only as “some ingenious gentleman.” Ross added a comment that this “some ingenious gentleman” was Whewell himself, without giving the reason for the identification. Ross 1962, p.72.”

Even if, by some very slim chance, the “ingenious gentleman” was not Whewell himself, Whewell did propose the term scientist in a more formal manner six years later in 1840 bringing more than just linguistic justifications, like the diversity of those engaged in scientific endeavors and how they don’t call themselves natural philosophers anymore.

By Neuronicus, 9 August 2016

Can you tickle yourself?

As I said before, with so many science outlets out there, it’s hard to find something new and interesting to cover that hasn’t been covered already. Admittedly, sometimes some new paper comes out that is so funny or interesting that I too fall in line with the rest of them and cover it. But, most of the time, I try to bring you something that you won’t find it reported by other science journalists. So, I’m sacrificing the novelty for originality by choosing something from my absolutely huge article folder (about 20 000 papers).

And here is the gem for today, titled enticingly “Why can’t you tickle yourself?”. Blakemore, Wolpert & Frith (2000) review several papers on the subject, including some of their own, and arrive to the conclusion that the reason you can’t tickle yourself is because you expect it. Let me explain: when you do a movement that results in a sensation, you have a pretty accurate expectation of how that’s going to feel. This expectation then dampens the sensation, a process probably evolved to let you focus on more relevant things in the environment that on what you’re doing o yourself (don’t let your mind go all dirty now, ok?).

Mechanistically speaking, it goes like this: when you move your arm to tickle your foot, a copy of the motor command you gave to the arm (the authors call this “efference copy”) goes to a ‘predictor’ region of the brain (the authors believe this is the cerebellum) that generates an expectation (See Fig. 1). Once the movement has been completed, the actual sensation is compared to the expected one. If there is a discrepancy, you get tickled, if not, not so much. But, you might say, even when someone else is going to tickle me I have a pretty good idea what to expect, so where’s the discrepancy? Why do I still get tickled when I expect it? Because you can’t fool your brain that easily. The brain then says; “Alright, alright, we expect tickling. But do tell me this, where is that motor command? Hm? I didn’t get any!” So here is your discrepancy: when someone tickles you, there is the sensation, but no motor command, signals 1 and 2 from the diagram are missing.

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Fig. 1. My take on the tickling mechanism after Blakemore, Wolpert & Frith (2000). Credits. Picture: Sobotta 1909, Diagram: Neuronicus 2016. Data: Blakemore, Wolpert & Frith (2002). Overall: Public Domain

Likewise, when someone tickles you with your own hand, there is an attenuation of sensation, but is not completely disappeared, because there is some registration in the brain regarding the movement of your own arm, even if it was not a motor command initiated by you. So you get tickled just a little bit. The brain is no fool: is aware of who had done what and with whose hands (your dirty mind thought that, I didn’t say it!) .

This mechanism of comparing sensation with movement of self and others appears to be impaired in schizophrenia. So when these patients say that “I hear some voices and I can’t shut them up” or ” My hand moved of its own accord, I had no control over it”, it may be that they are not aware of initiating those movements, the self-monitoring mechanism is all wacky. Supporting this hypothesis, the authors conducted an fMRI experiment (Reference 2) where they showed that that the somatosensory and the anterior cingulate cortices show reduced activation when attempting to self-tickle as opposed to being tickled by the experimenter (please, stop that line of thinking…). Correspondingly, the behavioral portion of the experiment showed that the schizophrenics can tickle themselves. Go figure!

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Reference 1: Blakemore SJ, Wolpert D, & Frith C (3 Aug 2000). Why can’t you tickle yourself? Neuroreport, 11(11):R11-6. PMID: 10943682. ARTICLE FULLTEXT

Reference 2: Blakemore SJ, Smith J, Steel R, Johnstone CE, & Frith CD (Sep 2000, Epub 17 October 2000). The perception of self-produced sensory stimuli in patients with auditory hallucinations and passivity experiences: evidence for a breakdown in self-monitoring. Psychological Medicine, 30(5):1131-1139. PMID: 12027049. ARTICLE

By Neuronicus, 7 August 2016

Transcranial direct current stimulation & cognitive enhancement

There’s so much research out there… So much that some time ago I learned that in science, as probably in other fields too, one has only to choose a side of an argument and then, provided that s/he has some good academic search engines skills and institutional access to journals, get the articles that support that side. Granted, that works for relatively small questions restricted to narrow domains, like “is that brain structure involved in x” or something like that; I doubt you would be able to find any paper that invalidates theories like gravity or central dogma of molecular biology (DNA to RNA to protein).

If you’re a scientist trying to answer a question, you’ll probably comb through some dozens papers and form an opinion of your own after weeding out the papers with small sample sizes, the ones with shoddy methodology or simply the bad ones (yes, they do exists, even scientists are people and hence prone to mistakes). And if you’re not a scientist or the question you’re trying to find an answer for is not from your field, then you’ll probably go for reviews or meta-analyses.

Meta-analyses are studies that look at several papers (dozens or hundreds), pool their data together and then apply some complicated statistics to see the overall results. One such meta-analysis concerns the benefits, if any, of transcranial direct current stimulation (tDCS) on working memory (WM) in healthy people.

tDCS is a method of applying electrical current through some electrodes to your neurons to change how they work and thus changing some brain functions. It is similar with repetitive transcranial magnetic stimulation (rTMs), only in the latter case the change in neuronal activity is due to the application of a magnetic field.

Some people look at these methods not only as possible treatment for a variety of disorders, but also as cognitive enhancement tools. And not only by researchers, but also by various companies who sell the relatively inexpensive equipment to gamers and others. But does tDCS work in the first place?

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Mancuso et al. (2016) say that there have been 3 recent meta-analyses done on this issue and they found that “the effects [of tDCS on working memory in healthy volunteers] are reliable though small (Hill et al., 2016), partial (Brunoni & Vanderhasselt, 2014), or nonexistent (Horvath et al., 2015)” (p. 2). But they say these studies are somewhat flawed and that’s why they conducted their own meta-analysis, which concludes that “the true enhancement potential of tDCS for WM remains somewhat uncertain” (p.19). Maybe it works a little bit if used during the training phase of a working memory task, like n-back, and even then that’s a maybe…

Boring, you may say. I’ll grant you that. So… all that work and it revealed virtually nothing new! I’ll grant you that too. But what this meta-analysis brings new, besides adding some interesting statistics, like controlling for publication bias, is a nice discussion as to why they didn’t find nothing much, exploring possible causes, like the small sample and effects sizes, which seem to plague many behavioral studies. Another explanation which, to tell you the truth, the authors do not seem to be too enamored with is that, maybe, just maybe, simply, tDCS doesn’t have any effect on working memory, period.

Besides, papers with seemingly boring findings do not catch the media eye, so I had to give it a little attention, didn’t I 😉 ?

Reference: Mancuso LE, Ilieva IP, Hamilton RH, & Farah MJ. (Epub 7 Apr 2016, Aug 2016) Does Transcranial Direct Current Stimulation Improve Healthy Working Memory?: A Meta-analytic Review. Journal of Cognitive Neuroscience, 28(8):1063-89. PMID: 27054400, DOI: 10.1162/jocn_a_00956. ARTICLE

 By Neuronicus, 2 August 2016