The third eye

The pineal gland held fascination since Descartes’ nefarious claim that it is the seat of the soul. There is no evidence of that; he said it might be where the soul resides because he thought the pineal gland was the only solitaire structure in the brain so it must be special. By ‘solitaire’ I mean that all other brain structures come in doublets: 2 amygdalae, 2 hippocampi, 2 thalami, 2 hemispheres etc. He was wrong about that as well, in that there are some other singletons in the brain besides the pineal, like the anterior or posterior comissure, the cerebellar vermis, some deep brainstem and medullary structures etc.

Descartes’ dualism was the only escape route the mystics at the time had from of the demanding of evidence by the budding natural philosophers later known as scientists. So when some scientists noted that some lizards have a third eye on top of their head, the mystics and, later, the conspiracy theorists went nuts. Here, see, if the soul seat is linked with the third eye, the awakening of this eye in people would surely result in heightened awareness, closeness to the Divinity, oneness with Universe and other similar rubbish that can be otherwise easily and reliably achieved by a good dollop of magic mushrooms. Cheaper, too.

Back to the lizards. Yes, you read right: some lizards and frogs have a third eye. This eye is not exactly like the other two, but it has cells sensitive to light, even if they are not perceiving light in the same way the retinal cells form the lateral eyes are. It is located on the top of the skull, so sometimes is called the parietal organ (because it’s in-between the parietal skull bones, see pic).

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Dorsal view of the head of the adult Carolina anole (Anolis carolinensis) clearly showing the parietal eye (small grey/clear oval) at the top of its head. Photo by TheAlphaWolf. Courtesy of Wikipedia. License: CC BY-SA 3.0

It is believed to be a vestigial organ, meaning that primitive vertebrates might have had it as a matter of course but it disappeared in the more evolved animals. Importantly, birds and mammals don’t have it. Not at all, not a bit, not atrophied, not able to be “awakened” no matter what your favorite “lemme see your chakras” guru says. Go on, touch your top of the skull and see if you have some peeking soft tissue there. And no, the soft tissue that babies are born with right there on the top of the skull is not a third eye; it’s a fontanelle that allows for the rapid expansion of the brain during the first year of life.

The parietal organ’s anatomical connection to the pineal gland is not surprising at all for scientists, because the pineal’s role in every single animal that has it is the regulation of some circadian rhythms by the production of melatonin. In humans, the eyes send the information to the pineal that is day or night and the pineal adjusts the melatonin production accordingly, i.e. less melatonin produced during day and more during night. The lizards’ third eye’s main role is to provide information to the pineal about the ambient light for thermoregulatory purposes.

After this long introduction, here is the point: almost twenty years ago Xiong et al. (1998) looked at how this third eye perceives light. In the  human eye, light hitting the rods and cones in the retina (reception) launches a biochemical cascade (transduction) that results in seeing (coding of the stimulus in the brain). Briefly, transduction goes thusly: the photon(s) causes a special protein sensitive to light (e.g. rhodopsin) in the photoreceptor cells in the retina to split into its components (photobleaching), one of these components changes its conformation, then activates a G-protein (transducin), which then activates the enzyme phosphodiesterase (PDE), which then destroys a nucleotide called cyclic guanosine monophosphate (cGMP), which results in the closing of the cell’s ion channels, which leads to less neurotransmitter GABA released, which causes the nearby cells (bipolar cells) to release another neurotransmitter (glutamate), which increases the firing rate of another set of cells (ganglion cells) and from there to the brain we go. Phew, visual transduction IS difficult. And this is the brief version.

It turns out that the third eye retina doesn’t have all the types of cells that the normal eyes have. Specifically, it misses the bipolar, horizontal and amacrine cells, having only ganglion and photoreception cells. So how goes the phototransduction in the third eye’s retina, if at all?

Xiong et al. (1998) isolated photoreceptor cells from the third eyes of the lizard Uta stansburiana. And then they did a bunch of electrophysiological recording on those cells under different illumination and chemical conditions.

They found that the phototransduction in the third eye is different from the lateral eyes in that when they expected to see hyperpolarization of the cell, they observed depolarization instead. Also, when they expected the PDE to break down cGMP they found that PDE is inhibited thereby increasing the amount of cGMP  The fact that G-protein can inhibit PDE was totally unexpected and showed a novel way of cellular signaling. Moreover, they speculate that their results can make sense only if not one, but two G-proteins with opposite actions work in tandem.

A probably dumb technical question though: the human rhodopsin takes about 30 minutes to restore itself from photobleaching. Xiong et al. (1998) let the cells adapt to dark for 10 minutes before recordings. So I wonder if the results would have been slightly different if they allowed the cell more time to adapt? But I’m not an expert in retina science, you’ve seen how difficult it is, right? Maybe the lizard proteins are different or rhodopsin adaption time has little or nothing to do with their experiments? After all, later research has shown that the third eye has its own unique opsins, like the green-sensitive parietopsin discovered by Su et al. (2006).

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REFERENCE:  Xiong WH, Solessio EC, & Yau KW (Sep 1998). An unusual cGMP pathway underlying depolarizing light response of the vertebrate parietal-eye photoreceptor. Nature Neuroscience, 1(5): 359-365. PMID: 10196524, DOI: 10.1038/1570. ARTICLE

Tags: whole-cell electrophysiological recordings, perforated-patch electrophysiological recording, phototransduction, rhodopsin, photoreceptor, retina, G-protein, phosphodiesterase (PDE), cyclic guanosine monophosphate (cGMP), adenylyl cyclase,  3-isobutyl-1-methyl-xanthine (IBMX), parietal eye, third eye, lizard, opsin, G-protein, Uta stansburiana

By Neuronicus, 30 March 2017

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Aging and its 11 hippocampal genes

Aging is being quite extensively studied these days and here is another advance in the field. Pardo et al. (2017) looked at what happens in the hippocampus of 2-months old (young) and 28-months old (old) female rats. Hippocampus is a seahorse shaped structure no more than 7 cm in length and 4 g in weight situated at the level of your temples, deep in the brain, and absolutely necessary for memory.

First the researchers tested the rats in a classical maze test (Barnes maze) designed to assess their spatial memory performance. Not surprisingly, the old performed worse than the young.

Then, they dissected the hippocampi and looked at neurogenesis and they saw that the young rats had more newborn neurons than the old. Also, the old rats had more reactive microglia, a sign of inflammation. Microglia are small cells in the brain that are not neurons but serve very important functions.

After that, the researchers looked at the hippocampal transcriptome, meaning they looked at what proteins are being expressed there (I know, transcription is not translation, but the general assumption of transcriptome studies is that the amount of protein X corresponds to the amount of the RNA X). They found 210 genes that were differentially expressed in the old, 81 were upregulated and 129 were downregulated. Most of these genes are to be found in human too, 170 to be exact.

But after looking at male versus female data, at human and mouse aging data, the authors came up with 11 genes that are de-regulated (7 up- and 4 down-) in the aging hippocampus, regardless of species or gender. These genes are involved in the immune response to inflammation. More detailed, immune system activates microglia, which stays activated and this “prolonged microglial activation leads to the release of pro-inflammatory cytokines that exacerbate neuroinflammation, contributing to neuronal loss and impairment of cognitive function” (p. 17). Moreover, these 11 genes have been associated with neurodegenerative diseases and brain cancers.

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These are the 11 genes: C3 (up), Cd74  (up), Cd4 (up), Gpr183 (up), Clec7a (up), Gpr34 (down), Gapt (down), Itgam (down), Itgb2 (up), Tyrobp (up), Pld4 (down).”Up” and “down” indicate the direction of deregulation: upregulation or downregulation.

I wish the above sentence was as explicitly stated in the paper as I wrote it so I don’t have to comb through their supplemental Excel files to figure it out. Other than that, good paper, good work. Gets us closer to unraveling and maybe undoing some of the burdens of aging, because, as the actress Bette Davis said, “growing old isn’t for the sissies”.

Reference: Pardo J, Abba MC, Lacunza E, Francelle L, Morel GR, Outeiro TF, Goya RG. (13 Jan 2017, Epub ahead of print). Identification of a conserved gene signature associated with an exacerbated inflammatory environment in the hippocampus of aging rats. Hippocampus, doi: 10.1002/hipo.22703. ARTICLE

By Neuronicus, 25 January 2017

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How do you remember?

Memory processes like formation, maintenance and consolidation have been the subjects of extensive research and, as a result, we know quite a bit about them. And just when we thought that we are getting a pretty clear picture of the memory tableau and all that is left is a little bit of dusting around the edges and getting rid of the pink elephant in the middle of the room, here comes a new player that muddies the waters again.

DNA methylation. The attaching of a methyl group (CH3) to the DNA’s cytosine by a DNA methyltransferase (Dnmt) was considered until very recently a process reserved for the immature cells in helping them meet their final fate. In other words, DNA methylation plays a role in cell differentiation by suppressing gene expression. It has other roles in X-chromosome inactivation and cancer, but it was not suspected to play a role in memory until this decade.

Oliveira (2016) gives us a nice review of the role(s) of DNA methylation in memory formation and maintenance. First, we encounter the pharmacological studies that found that injecting Dnmt inhibitors in various parts of the brain in various species disrupted memory formation or maintenance. Next, we see the genetic studies, where mice Dnmt knock-downs and knock-outs also show impaired memory formation and maintenance. Finally, knowing which genes’ transcription is essential for memory, the researcher takes us through several papers that examine the DNA de novo methylation and demethylation of these genes in response to learning events and its role in alternative splicing.

Based on these here available data, the author proposes that activity induced DNA methylation serves two roles in memory: to “on the one hand, generate a primed and more permissive epigenome state that could facilitate future transcriptional responses and on the other hand, directly regulate the expression of genes that set the strength of the neuronal network connectivity, this way altering the probability of reactivation of the same network” (p. 590).

Here you go; another morsel of actual science brought to your fingertips by yours truly.

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Reference: Oliveira AM (Oct 2016, Epub 15 Sep 2016). DNA methylation: a permissive mark in memory formation and maintenance. Learning & Memory,  23(10): 587-593. PMID: 27634149, DOI: 10.1101/lm.042739.116. ARTICLE

By Neuronicus, 22 September 2016

Who invented optogenetics?

Wayne State University. Ever heard of it? Probably not. How about Zhuo-Hua Pan? No? No bell ringing? Let’s try a different approach: ever heard of Stanford University? Why, yes, it’s one of the most prestigious and famous universities in the world. And now the last question: do you know who Karl Deisseroth is? If you’re not a neuroscientist, probably not. But if you are, then you would know him as the father of optogenetics.

Optogenetics is the newest tool in the biology kit that allows you to control the way a cell behaves by shining a light on it (that’s the opto part). Prior to that, the cell in question must be made to express a protein that is sensitive to light (i.e. rhodopsin) either by injecting a virus or breeding genetically modified animals that express that protein (that’s the genetics part).

If you’re watching the Nobel Prizes for Medicine, then you would also be familiar with Deisseroth’s name as he may be awarded the Nobel soon for inventing optogenetics. Only that, strictly speaking, he did not. Or, to be fair and precise at the same time, he did, but he was not the first one. Dr. Pan from Wayne State University was. And he got scooped.98.png

The story is at length imparted to us by Anna Vlasits in STAT and republished in Scientific American. In short, Dr. Pan, an obscure name in an obscure university from an ill-famed city (Detroit), does research for years in an unglamorous field of retina and blindness. He figured, quite reasonably, that restoring the proteins which sense light in the human eye (i.e. photoreceptor proteins) could restore vision in the congenitally blind. The problem is that human photoreceptor proteins are very complicated and efforts to introduce them into retinas of blind people have proven unsuccessful. But, in 2003, a paper was published showing how an algae protein that senses light, called channelrhodopsin (ChR), can be expressed into mammalian cells without loss of function.

So, in 2004, Pan got a colleague from Salus University (if Wayne State University is a medium-sized research university, then Salus is a really tiny, tiny little place) to engineer a ChR into a virus which Pan then injected in rodent retinal neurons, in vivo. After 3-4 weeks he obtained the expression of the protein and the expression was stable for at least 1 year, showing that the virus works nicely. Then his group did a bunch of electrophysiological recordings (whole cell patch-clamp and voltage clamp) to see if shining light on those neurons makes them fire. It did. Then, they wanted to see if ChR is for sure responsible for this firing and not some other proteins so they increased the intensity of the blue light that the ChR is known to sense and observed that the cell responded with increased firing. Now that they saw the ChR works in normal rodents, next they expressed the ChR by virally infecting mice who were congenitally blind and repeated their experiments. The electrophysiological experiments showed that it worked. But you see with your brain, not with your retina, so the researchers looked to see if these cells that express ChR project from retina to the brain and they found their axons in lateral geniculate and superior colliculus, two major brain areas important for vision. Then, they recorded from these areas and the brain responded when blue light, but not yellow or other colors, was shone on the retina. The brain of congenitally blind mice without ChR does not respond regardless of the type of light shone on their retinas. But does that mean the mouse was able to see? That remains to be seen (har har) in future experiments. But the Pan group did demonstrate that they can control neurons by light.

All in all, a groundbreaking paper. So the Pan group was not off the mark when they submitted it to Nature on November 25, 2004. As Anna Vlasits reports in the Exclusive, Nature told Pan to submit to a more specialized journal, like Nature Neuroscience, which then rejected it. Pan submitted then to the Journal of Neuroscience, which also rejected it. He submitted it then to Neuron on November 29, 2005, which finally accepted it. Got published on April 6, 2006. Deisseroth’s paper was submitted to Nature Neuroscience on May 12, 2005, accepted on July, and published on August 14, 2005… His group infected rat hippocampal neurons cultured in a Petri dish with a virus carrying the ChR and then they did some electrophysiological recordings on those neurons while shining lights of different wavelengths on them, showing that these cells can be controlled by light.

There’s more on the saga with patent filings and a conference where Pan showed the ChR data in May 2005 and so on, you can read all about it in Scientific American. The magazine is just hinting to what I will say outright, loud and clear: Pan didn’t get published because of his and his institution’s lack of fame. Deisseroth did because of the opposite. That’s all. This is not about squabbles about whose work is more elegant, who presented his work as a scientific discovery or a technical report or whose title is more catchy, whose language is more boisterous or native English-speaker or luck or anything like that. It is about bias and, why not?, let’s call a spade a spade, discrimination. Nature and Journal of Neuroscience are not caught doing this for the first time. Not by a long shot. The problem is that they are still doing it, that is: discriminating against scientific work presented to them based on the name of the authors and their institutions. Personally, so I don’t get comments along the lines of the fox and the grapes, I have worked at both high profile and low profile institutions. And I have seen the difference not in the work, but in the reception.

That’s my piece for today.

Source:  STAT, Scientific American.

References:

1) Bi A, Cui J, Ma YP, Olshevskaya E, Pu M, Dizhoor AM, & Pan ZH (6 April 2006). Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration. Neuron, 50(1): 23-33. PMID: 16600853. PMCID: PMC1459045. DOI: 10.1016/j.neuron.2006.02.026. ARTICLE | FREE FULLTEXT PDF

2) Boyden ES, Zhang F, Bamberg E, Nagel G, & Deisseroth K. (Sep 2005, Epub 2005 Aug 14). Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neuroscience, 8(9):1263-1268. PMID: 16116447. DOI: 10.1038/nn1525. doi:10.1038/nn1525. ARTICLE 

By Neuronicus, 11 September 2016

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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

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

Fructose bad effects reversed by DHA, an omega-3 fatty acid

Despite alarm signals raised by various groups and organizations regarding the dangers of the presence of sugars – particularly fructose derived from corn syrup – in almost every food in the markets, only in the past decade there has been some serious evidence against high consumption of fructose.

A bitter-sweet (sic!) paper comes from Meng et al. (2016) who, in addition to showing some bad things that fructose does to brain and body, it also shows some rescue from its deleterious effects by DHA (docosahexaenoic acid), an omega-3 fatty acid.

The authors had 3 groups of rodents: one group got fructose in their water for 6 weeks, another group got fructose and DHA, and another group got their normal chow. The amount of fructose was calculated to be ecologically valid, meaning that they fed the animals the equivalent of 1 litre soda bottle per day (130 g of sugar for a 60 Kg human).

The rats that got fructose had worse learning and memory performance at a maze test compared to the other two groups.

The rats that got fructose had altered gene expression in two brain areas: hypothalamus (involved in metabolism) and hippocampus (involved in learning and memory) compared to the other two groups.

The rats that got fructose had bad metabolic changes that are precursors for Type 2 diabetes, obesity and other metabolic disorders (high blood glucose, triglycerides, insulin, and insulin resistance index) compared to the other two groups.

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The genetic analyses that the researchers did (sequencing the RNA and analyzing the DNA methylation) revealed a whole slew of the genes that had been affected by the fructose treatment. So, they did some computer work that involved Bayesian modeling  and gene library searching and they selected two genes (Bgn and Fmod) out of almost a thousand possible candidates who seemed to be the drivers of these changes. Then, they engineered mice that lacked these genes. The resultant mice had the same metabolic changes as the rats that got fructose, but… their learning and memory was even better than that of the normals? I must have missed something here. EDIT: Well… yes and no. Please read the comment below from the Principal Investigator of the study.

It is an ok paper, done by the collaboration of 7 laboratories from 3 countries. But there are a few things that bother me, as a neuroscientist, about it. First is the behavior of the genetic knock-outs. Do they really learn faster? The behavioral results are not addressed in the discussion. Granted, a genetic knockout deletes that gene everywhere in the brain and in the body, whereas the genetic alterations induced by fructose are almost certainly location-specific.

Which brings me to the second bother: nowhere in the paper (including the supplemental materials, yeas, I went through those) are any brain pictures or diagrams or anything that can tell us which nuclei of the hypothalamus the samples came from. Hypothalamus is a relatively small structure with drastically different functional nuclei very close to one another. For example, the medial preoptic nucleus that deals with sexual hormones is just above the suprachiasmatic nucleus that deals with circadian rhythms and near the preoptic is the anterior nucleus that deals mainly with thermoregulation. The nuclei that deal with hunger and satiety (the lateral and the ventromedial nucleus, respectively) are located in different parts of the hypothalamus. In short, it would matter very much where they got their samples from because the transcriptome and methylome would differ substantially from nucleus to nucleus. Hippocampus is not so complicated as that, but it also has areas with specialized functions. So maybe they messed up the identification of the two genes Bgn and Fmod as drivers of the changes; after all, they found almost 1 000 genes altered by fructose. And that mess-up might have been derived by their blind hypothalamic and hippocampal sampling. EDIT: They didn’t mess up,  per se. Turns out there were technical difficulties of extracting enough nucleic acids from specific parts of hypothalamus for analyses. I told you them nuclei are small…

Anyway, the good news comes from the first experiment, where DHA reverses the bad effects of fructose. Yeay! As a side note, the fructose from corn syrup is metabolized differently than the fructose from fruits. So you are far better off consuming the equivalent amount on fructose from a litre of soda in fruits. And DHA comes either manufactured from algae or extracted from cold-water oceanic fish oils (but not farmed fish, apparently).

If anybody that read the paper has some info that can help clarify my “bothers”, please do so in the Comment section below. The other media outlets covering this paper do not mention anything about the knockouts. Thanks! EDIT: The last author of the paper, Dr. Yang, was very kind and clarified a bit of my “bothers” in the Comments section. Thanks again!

Reference: Meng Q, Ying Z, Noble E, Zhao Y, Agrawal R, Mikhail A, Zhuang Y, Tyagi E, Zhang Q, Lee J-H, Morselli M, Orozco L, Guo W, Kilts TM, Zhu J, Zhang B, Pellegrini M, Xiao X, Young MF, Gomez-Pinilla F, Yang X (2016). Systems Nutrigenomics Reveals Brain Gene Networks Linking Metabolic and Brain Disorders. EBioMedicine, doi: 10.1016/j.ebiom.2016.04.008. Article | FREE fulltext PDF | Supplementals | Science Daily cover | NeuroscienceNews cover

By Neuronicus, 24 April 2016

Autism cure by gene therapy

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Nothing short of an autism cure is promised by this hot new research paper.

Among many thousands of proteins that a neuron needs to make in order to function properly there is one called SHANK3 made from the gene shank3. (Note the customary writing: by consensus, a gene’s name is written using small caps and italicized, whereas the protein’s name that results from that gene expression is written with caps).

This protein is important for the correct assembly of synapses and previous work has shown that if you delete its gene in mice they show autistic-like behavior. Similarly, some people with autism, but by far not all, have a deletion on Chromosome 22, where the protein’s gene is located.

The straightforward approach would be to restore the protein production into the adult autistic mouse and see what happens. Well, one problem with that is keeping the concentration of the protein at the optimum level, because if the mouse makes too much of it, then the mouse develops ADHD and bipolar.

So the researchers developed a really neat genetic model in which they managed to turn on and off the shank3 gene at will by giving the mouse a drug called tamoxifen (don’t take this drug for autism! Beside the fact that is not going to work because you’re not a genetically engineered mouse with a Cre-dependent genetic switch on your shank3, it is also very toxic and used only in some from of cancers when is believed that the benefits outweigh the horrible side effects).

In young adult mice, the turning on of the gene resulted in normalization of synapses in the striatum, a brain region heavily involved in autistic behaviors. The synapses were comparable to normal synapses in some aspects (from the looks, i.e. postsynaptic density scaffolding, to the works, i.e. electrophysiological properties) and even more so in others (more dendritic spines than normal, meaning more synapses, presumably). This molecular repair has been mirrored by some behavioral rescue: although these mice still had more anxiety and more coordination problems than the control mice, their social aversion and repetitive behaviors disappeared. And the really really cool part of all this is that this reversal of autistic behaviors was done in ADULT mice.

Now, when the researchers turned the gene on in 20 days old mice (which is, roughly, the equivalent of the entering the toddling stage in humans), all four behaviors were rescued: social aversion, repetitive, coordination, and anxiety. Which tells us two things: first, the younger you intervene, the more improvements you get and, second and equally important, in adult, while some circuits seem to be irreversibly developed in a certain way, some other neural pathways are still plastic enough as to be amenable to change.

Awesome, awesome, awesome. Even if only a very small portion of people with autism have this genetic problem (about 1%), even if autism spectrum disorders encompass such a variety of behavioral abnormalities, this research may spark hope for a whole range of targeted gene therapies.

Reference: Mei Y, Monteiro P, Zhou Y, Kim JA, Gao X, Fu Z, Feng G. (Epub 17 Feb 2016). Adult restoration of Shank3 expression rescues selective autistic-like phenotypes. Nature. doi: 10.1038/nature16971. Article | MIT press release

By Neuronicus, 19 February 2016

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The Firsts: Anandamide (1992)

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Cannabis, the plant whose psychoactive tetrahydrocannabinol (THC) binds to the same receptors in the brain as anandamide.

A rare tragedy took place in France a few days ago when a Phase I clinical trial for a new drug destined to improve mood and alleviate pain has resulted in one person dead and five other hospitalized. Phase I means that the drug successfully passed all animal tests and was being tried for the first time in humans to test its safety (efficacy and potency are tested in phase II and III, respectively).

The trial has been suspended and an investigation is on the way. So far, it appears that both the manufacturer (Bial) and the testing company (Biotrial) have followed all the guidelines and regulations. The running hypothesis is that the drug (BIA 10-2474) is acting on an unexpected target. What does that mean?

BIA 10-2474 is a FAAH inhibitor (fatty acid amide hydrolase). This enzyme breaks down anandamide, which is an endocannabinoid. In other words, is a neurotransmitter in the brain that binds to the same receptors as THC, the main active component of marijuana. So, if you give someone BIA 10-2474, the result would be an increase in the availability of anandamide, presumably with anxiolytic and analgesic effects (yes, similar to smoking weed).

There are other FAAH inhibitors out there that had been previously tried in humans and they were never marketed not because they were unsafe, but because they were ineffective in producing the desired results.

So we don’t know yet why BIA 10-2474 killed people, but the bet is that in addition to FAAH, it also binds to some other protein. Why they didn’t discover this in animal trials, is a mystery; perhaps the unknown protein is unique to humans? By the looks of the drug’s structure, I think is computer generated, meaning is composed of a bunch of functional groups that someone put together in the hopes that it would fit neatly on the target binding site; but so many functional groups thrown in together might bind unexpectedly to other places than the intended. More on the story in Nature.

Anyway, that was the very long intro to today’s featured paper: the discovery of anandamide. Which happened very recently, in 1992, by the Mechoulam group at Hebrew University of Jerusalem, Israel. Anandamide is the first endocannabinoid to be isolated. Mechoulam’s postodcs, William Devane and Lumir Hanus, used mass spectroscopy and NMR (the counterpart of MRI) to identify and isolate the molecule in a pig brain. And then they named it, fittingly, the “amide of bliss”…

Of note, members of the same Mechoulam group identified two more of the six known endocannabinoids. The three pages paper is highly technical, but I am assured (by a chemist) that is an easy-peasy read for any organic chemist.

Reference: Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, & Mechoulam R (18 Dec 1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science, 258(5090):1946-9. PMID: 1470919, DOI: 10.1126/science.1470919.  Article | Research Gate Full Text

By Neuronicus, 18 January 2016

CCL11 found in aged but not young blood inhibits adult neurogenesis

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Portion of Fig. 1 from Villeda et al. (2011, doi: 10.1038/nature10357) describing the parabiosis procedure. Basically, under complete anesthesia, the peritoneal membranes and the skins of the two mice were sutured together. The young mice were 3–4 months (yellow) and old mice were 18–20 months old (grey).

My last post was about parabiosis and its sparse revival as a technique in physiology experiments. Parabiosis is the surgical procedure that joins two living animals allowing them to share their circulatory systems. Here is an interesting paper that used the method to tackle blood’s contribution to neurogenesis.

Adult neurogenesis, that is the birth of new neurons in the adult brain, declines with age. This neurogenesis has been observed in some, but not all brain regions, called neurogenic niches.

Because these niches occur in blood-rich areas of the brain, Villeda et al. (2011) wondered if, in addition with the traditional factors required for neurogenesis like enrichment or running, blood factors may also have something to do with neurogenesis. The authors made a young and an old mouse to share their blood via parabiosis (see pic.)

Five weeks after the parabiosis procedure, the young mouse had decreased neurogenesis and the old mouse had increased neurogenesis compared to age-matched controls. To make sure their results are due to something in the blood, they injected plasma from a old mouse into a young mouse and that also resulted in reduced neurogenesis. Moreover, the reduced neurogenesis was correlated with impaired learning as shown by electrophysiological recordings from the hippocampus and from behavioral fear conditioning.

So what in the blood does it? The authors looked at 66 proteins found in the blood (I don’t know the blood make-up, so I can’t tell if 66 is a lot or not ) and noticed that 6 of these had increased levels in the blood of ageing mice whether linked by parabiosis or not. Out of these six, the authors focus on CCL11 (unclear to me why that one, my bet is that they tried the others too but didn’t have enough data). CCLL11 is a small signaling protein involved in allergies. So the authors injected it into young mice and Lo and Behold! there was decreased neurogenesis in their hippocampus. Maybe the vampires were onto something, whadda ya know? Just kidding… don’t go around sucking young people’s blood!

This paper covers a lot of work and, correspondingly, has no less than 23 authors and almost 20 Mb of supplemental documents! The story it tells is very interesting and as complete as it gets, covering many aspects of the problems investigated and many techniques to address those problems. Good read.

Reference: Villeda SA, Luo J, Mosher KI, Zou B, Britschgi M, Bieri G, Stan TM, Fainberg N, Ding Z, Eggel A, Lucin KM, Czirr E, Park JS, Couillard-Després S, Aigner L, Li G, Peskind ER, Kaye JA, Quinn JF, Galasko DR, Xie XS, Rando TA, Wyss-Coray T. (31 Aug 2011). The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature. 477(7362):90-94. doi: 10.1038/nature10357. Article | FREE PDF

By Neuronicus, 6 January 2016