New Brain Cells: Torrent, or Trickle?

An important paper just out asks, Could adult hippocampal neurogenesis be relevant for human behavior?

Neuroscientists, and the media, are very excited by hippocampal neurogenesis - the ongoing creation of new neurons in an area called the dentate gyrus of the hippocampus. This is because it was thought, for a long time, that no new neurons were created in the adult brain. It turned out that this was wrong.

There's lots of exciting suggestive evidence that the process is involved in learning and memory, responses to stress, depression, and the action of antidepressants, to name just a few, although this is controversial.

However, there's a big question which has rarely been considered: how much neurogenesis are we talking about? Are there enough new cells that it would be realistic for them to be doing important stuff, or is it just a little trickle?

The most common source of skepticism toward a functional role for adult neurogenesis is the perception that too few new neurons are added in adulthood to have a significant impact. Interestingly, this concern, while valid, is usually raised informally and rarely in the scientific literature. Very few studies have addressed this issue...

The new paper reviews the evidence. Firstly, they point out that in the hippocampus, there's a group of cells called dentate gyrus granule cells which are unusual in that activity in just a few of these cells can have big downstream consequences. And these are the cells that new born neurons turn into.

Each granule cell contacts only 10–15 CA3 pyramidal cells...a single granule cell is able to trigger firing in downstream CA3 targets...Because of this “detonator” action...a single granule neuron can potentially have a large impact despite representing only a tiny fraction of the population.

So new cells may play an important role. But exactly how many are there? They re-analyze data from their own lab in rats, and, making a few assumptions, arrive at the following rough estimate: in 3 month old rats, there are 650k "young" cells less than 8 weeks old; even in 2 year old rats (ancient, for a rat) there are 50k.

This is enough to have a big impact downstream:

Since there are approximately 500,000 CA3 pyramidal cells, and each granule cell contacts 11–15 pyramidal cells, this suggests that even in the oldest animals, each CA3 pyramidal cell could receive a direct contact from a young granule cell

That's all in rats, though. What about humans? It's hard to tell. The problem is that the best way to assess the rate of neurogenesis is to inject a drug called BrdU and then study the brain post-mortem. Unfortunately, this drug can cause cancer so you can't just give it to people for the purposes of science. The only time it's used in humans is (ironically) to help detect cancer.

However, one study did manage to look at BrdU staining in the hippocampus, using people who'd been injected with BrdU for cancer (not brain cancer) and then died. This study found, the authors say, rates of neurogeneis at least as high as in rats, considering the low dose of BrdU, the fact that the patients were old, and stressed (by having cancer).

They admit that this is just one study, and comparing doses between rats and humans is inexact. They nonetheless conclude:

Are these numbers potentially sufficient to exert a functional impact in humans? We feel that the answer to this question is an overwhelming "yes".

Snyder JS, & Cameron HA (2011). Could adult hippocampal neurogenesis be relevant for human behavior? Behavioural brain research PMID: 21736900

Brand New Cortical Neurons

Are new neurons created in the adult brain?

For a long time, everyone thought the answer was "no". Then, about 15 years ago, we learned that neurogenesis does occur in the adult brain, but it was thought to be limited to two very small regions, the dentate gyrus and the sub-ventricular zone. Except in cases of injury, when adult neurogenesis had been reported elsewhere.

Now Guo et al look set to overturn this orthodoxy in a new Journal of Neuroscience paper, as they found ongoing neurogenesis in healthy adult brains in an area called the piriform cortex, part of the cerebral cortex.

The key to the discovery was oligodendrocyte progenitor cells (OPCs). OPCs were previously believed to only be able to turn into cells called oligodendrocytes, which are not neurons, but glia; glia are a kind of support crew for the brain.

But Guo et al show convincingly (with the help of genetically modified mice) that OPCs do become neurons in the piriform cortex of adult mice. Once they've been "born", these new neurons mature into functional pyramidal cells - they form synapses with other neurons, and otherwise seem to be perfectly happy, and they survived for hundreds of days (i.e. most of a mouse's lifetime).

The methods they used are complex but the crucial result was that they observed pyramidal cells expression yellow fluorescent protein, in mice genetically modified to express this protein only in OPCs; the picture above this post is one of these "yellow" (I know, it looks green to me) neurons.

This isn't the first paper to report neurogenesis in the adult mouse piriform cortex - a different group did so in 2008, but then two other experiments published later that year failed to confirm the result, so it's remained controversial. Whether this will end the controversy is uncertain but it looks pretty solid to me.

What does it all mean? The piriform cortex is a bit of a weird area, as while it's part of the cerebral cortex, the most "complex" part of the brain, it is evolutionary very old, and quite unlike the neocortex which is by far the largest part of the brain in humans.

The piriform is involved in the sense of smell, which is very important for mice, not so much in humans. We do have a piriform cortex, but it's tiny. Whether adult neurogenesis also occurs in the neocortex is the next big question...

Guo F, Maeda Y, Ma J, Xu J, Horiuchi M, Miers L, Vaccarino F, & Pleasure D (2010). Pyramidal neurons are generated from oligodendroglial progenitor cells in adult piriform cortex. The Journal of neuroscience : the official journal of the Society for Neuroscience, 30 (36), 12036-49 PMID: 20826667

Tickling Rats for Science

"Tickling the rat" has got to be a euphemism for something. But it's also a way of studying the neurobiology of depression.

At least that's what Wöhr et al say in a new paper. They started from the fact that when you tickle rats, some of them seem to enjoy it, and express this by making 50 kHz squeaks of joy. But other rats don't like it, and they make a different sound, much lower at 22 kHz. (These sounds are all too high for most humans to hear, but they can be recorded electronically.)

Whether a given rat is cool with being tickled seems to be a fairly stable individual trait. Some do, some don't. And rats which don't like being tickled tend to be generally anxious and neurotic in lots of other ways. They're the Woody Allens of the rat world, maybe.

Wöhr et al decided to see whether these personality differences were related to the rate of hippocampal neurogenesis in the brain. Stress is known to decrease the rate of neurogenesis, and it may be reduced in clinical depression, while antidepressants increase it (more).

After much tickling (ten rats for five days at ten minutes per day), they did indeed find a pretty strong correlation between tickle-liking and hippocampal neurogenesis. What does this mean? Hard to say. The problem is that rates of neurogenesis were measured after all the tickling was over. (This is because you can only measure it by killing the animals and dissecting their brains. Sorry, rat lovers.) Given that the rats who didn't like being tickled probably must have found the procedure very stressful, and given that we know that stress strongly reduces neurogenesis, maybe it doesn't mean very much. But it must have been a lot more fun than most animal experiments.

Before you rush to submit this paper to NCBI ROFL, I already did. Finally, here's a video of someone tickling a rat: YouTube really does have everything.




Wöhr M, Kehl M, Borta A, Schänzer A, Schwarting RK, & Höglinger GU (2009). New insights into the relationship of neurogenesis and affect: Tickling induces hippocampal cell proliferation in rats emitting appetitive 50-kHz ultrasonic vocalizations. Neuroscience PMID: 19638303

Antidepressants and Neurogenesis in Humans

How do antidepressants work? Some people will tell you that it’s all about neurogenesis. The theory goes that antidepressants increase the rate at which new neurones are created in a region called the dentate gyrus of the hippocampus, and that, somehow, this boom in the number of new hippocampal cells alleviates depression.

To date, however, all of the research linking antidepressants and neurogenesis has involved animals. It was generally assumed that if drugs altered neurogenesis in mice, the same thing happened in humans – but this was an assumption, and clearly a pretty big one. Now a new report from a New York-based team claims that antidepressants do enhance neurogenesis in people - Antidepressants increase neural progenitor cells in the human hippocampus.

The authors took post-mortem brain samples from three groups of people – those with no history of depression, those with depression who were not on antidepressants when they died, and depressed people who were on antidepressants. They counted the number of neural progenitor cells (NPCs) in the hippocampus using a stain which specifically marks these cells (anti-nestin).

Although like all post-mortem studies the sample size was small (n=19 total), depressed people taking antidepressants when they died had much higher NPC numbers, indicating greater neurogenesis, compared to the other two groups. (Control: 360±246; untreated: 1119±752; treated: 17229±3443).

The picture above illustrates this; the brown cells are NPCs, and there are evidently more of them in the antidepressant-taking person on the right compared to the control on the left. The authors presumably picked these images because they look different, so, pinch of salt. But still, as an antidepressant user myself, it's nice to see what might well be going on inside my skull at this moment.

The dentate gyrus of the hippocampus, the area where neurogenesis happens, was also larger in the antidepressant-treated group.

Is this evidence for the neurogenesis theory? Not exactly. It’s fairly good evidence that some antidepressants do boost hippocampal neurogenesis in humans, in accordance with the animal data. But we really don’t know what that means. It could just be a side effect, and nothing to do with how they work. I’ve previously written about some recent animal experiments finding that antidepressants have effects on behaviour even when neurogenesis is completely blocked. And notably, five of the seven antidepressant-treated patients in this study died from suicide. So, to put it bluntly, the drugs didn’t work very well, despite sending neurogenesis through the roof...

Boldrini, M., Underwood, M., Hen, R., Rosoklija, G., Dwork, A., John Mann, J., & Arango, V. (2009). Antidepressants increase neural progenitor cells in the human hippocampus Neuropsychopharmacology DOI: 10.1038/npp.2009.75

Depression, Neurogenesis and Herpes

Previously, I've discussed the neurogenesis theory of depression in two rather skeptical posts. Not that I'm on some kind of anti-neurogenesis theory crusade, but a study just published adds to the evidence that all's not well with that hypothesis.

The paper is Singer et. al.'s Conditional ablation and recovery of forebrain neurogenesis in the mouse. Via some cunning genetic engineering, the authors created mice with a gene for a protein called herpes simplex virus thymidine kinase. As the name suggests, this is a protein normally found in, er, herpes. Ganciclovir is a drug which can be used to treat herpes and related viral infections. And, as you might expect, cells engineered to express the herpes protein die when exposed to ganciclovir.

The authors engineered mice which expressed herpes simplex virus thymidine kinase, but only in neural progenitor cells. These are the cells which eventually become new neurones in the adult brain. They found that injections of gancyclovir devasasted the production of new neurones in the engineered mice. (It had no effect on normal mice, of course, because their brain cells weren't half mouse, half herpes). That's not all that surprising.

However, they also found that gancyclovir treatment had no effect on the ability of 28 days treatment imipramine, an antidepressant, to affect the mice's behaviour. (The measure of antidepressant action was the Tail Suspension Test). That's a result, because a lot of people are interested in the theory that antidepressants work by boosting neurogenesis in the hippocampus. If that were true, blocking neurogenesis should also block the effects of antidepressants.

Some rather exciting experiments found that it does, most famously the much-cited Santarelli et al (2003). But a growing number of other studies, such as this one, have not confirmed this finding. This doesn't mean that Santarelli et al were wrong, but it does suggest that there's more to antidepressants than neurogenesis. The seemingly-contradictory findings of the various studies might be due to important differences in the methods used. For example, the authors of this paper say that Santarelli et al's way of blocking neurogenesis - using x-rays - may have also caused inflammation and blocked the formation of non-neural cells, such as those which go to make up blood-vessels.

Of course, it's easy enough for us to speculate along such lines - rather harder to work out what exactly is going on. With any luck, the next few years will see more progress on this important topic.

Singer, B., Jutkiewicz, E., Fuller, C., Lichtenwalner, R., Zhang, H., Velander, A., Li, X., Gnegy, M., Burant, C., & Parent, J. (2009). Conditional ablation and recovery of forebrain neurogenesis in the mouse The Journal of Comparative Neurology, 514 (6), 567-582 DOI: 10.1002/cne.22052

Prozac and Old Mice

A while back, I wrote about an important paper which cast doubt on the "neurogenesis hypothesis" of antidepressant drug action, which I summarized as

...the proposal that antidepressants work by promoting the survival and proliferation of new neurones in certain areas of the brain - the "neurogenesis hypothesis". Neurogenesis, the birth of new cells from stem cells, occurs in a couple of very specific regions of the adult brain, including the elaborately named subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. Many experiments on animals have shown that chronic stress, and injections of the "stress hormone" corticosterone, can suppress neurogenesis, while a wide range of antidepressants block this effect of stress and promote neurogenesis. (Other evidence shows that antidepressants probably do this by inducing the expression of neurotrophic signalling proteins, like BDNF.)

It's a popular theory at the moment, not least because it's the only real alternative to the older, much-maligned and certainly incomplete monoamine hypothesis of antidepressants. But the neurogenesis hypothesis has problems of its own. A new paper claims to add to what seems like a growing list of counter-examples: Ageing abolishes the effects of fluoxetine on neurogenesis.

The researchers, Couillard-Despres et. al. from the University of Regensburg in Germany, found that fluoxetine (Prozac) enhances hippocampal neurogenesis in mice - as expected - but found in addition that this only holds true in young mice. In middle-aged and older mice, there was no such effect. That's a new finding, and a very important one.

More specifically, the (male) mice were given injections of Prozac for two weeks each. Compared to mice given placebo injections, the mice on Prozac showed

increased survival and the frequency of neuronal marker expression in newly generated cells of the hippocampus in the young adult group (that is 100 days of age) only. No significant effects on neurogenesis could be detected in fluoxetine-treated adult and elderly mice (200 and over 400 days of age).

For mice, 100 days old corresponds to a human age of about 20 years; 200 days is 35 and 400 days is 65 years. The graph here shows the number of BrdU-labelled cells in the dentate gyrus, a measure of neural progenitor cell survival. As you can see, although Prozac robustly increased BrdU+ cell counts in the 100 day old mice, this effect was much less prominent (although perhaps still present a bit?) in the older mice.

It's already well known that hippocampal neurogenesis is age dependent. Young animals (and people) have lots of new neurones being generated, but the rate progressively and inevitably declines with age. This has always been a problem for the simple hypothesis that reduced neurogenesis causes depression, because if that were the case, we'd all be paralyzed by despair by the age of 50. Despite this, it remained plausible that antidepressants worked by increasing neurogenesis, but this new evidence suggests otherwise.

Or does it? What if it turns out that fluoxetine has no antidepressant-like effects in old rodents? In that case, the neurogenesis hypothesis would be supported, not weakened, by this evidence. The author's of the paper don't even consider this possibility, which is a little odd. They do note that antidepressants are effective in older people with depression, but given that this is a paper about mice that's not the same thing. Someone needs to find out whether Prozac has anti-depressant-like effects in the same kind of old mice as those used in this study. If so, the neurogenesis hypothesis will be looking pretty fragile.

This should also serve as a reminder that lab mice are animals, not research robots. They get old, like the rest of us, and research done only on young mice, or male mice, or a certain breed of mice, may not be applicable to others. I have two cats: if you stroke the grey one on the belly, she'll purr contentedly. But if you foolishly assume that the tabby one is the same, you'll get bitten pretty quickly...

S Couillard-Despres, C Wuertinger, M Kandasamy, M Caioni, K Stadler, R Aigner, U Bogdahn, L Aigner (2009). Ageing abolishes the effects of fluoxetine on neurogenesis Molecular Psychiatry DOI: 10.1038/mp.2008.147

Prozac Made My Cells Spiky

A great many neuroscientists are interested in clinical depression and antidepressants. We're still a long way from understanding depression on a biological level - and if anyone tries to tell you otherwise, they're probably trying to sell you something. I've previously discussed the controversies surrounding the neurotransmitter serotonin - according to popular belief, the brain's "happy chemical". My conclusion was that although clinical depression is not caused by "low serotonin" alone, serotonin does play an important role in mood at least in some people.

A paper published recently in Molecular Psychiatry makes a number of important contributions to the literature on depression and antidepressants; I haven't seen it discussed elsewhere, so here is make take on it. The paper is by a Portuguese research group, Bessa et. al., and it's titled The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling. The findings are right there in the title, but a little history is required in order to appreciate their significance.

For a long time, the only biological theory which attempted to explain clinical depression and how antidepressants counteract it was the monoamine hypothesis. During the early 1960s, it was noticed that early antidepressant drugs, such as imipramine, all inhibited either the breakdown or the removal (reuptake) of chemicals in the brain called monoamines, including serotonin. This led many to conclude that antidepressants improve mood by raising monoamine levels, and that depression is probably caused by some kind of monoamine deficiency. For various reasons (not all of them good ones), it was later decided that serotonin was the crucial monoamine involved in mood, although for several years another, noradrenaline, was favored by most people.

This "monoamine hypothesis" was always a little shaky, and over the past decade or so, an alternative approach has become increasingly fashionable. If you were so inclined, you might even call it a new paradigm. This is the proposal that antidepressants work by promoting the survival and proliferation of new neurones in certain areas of the brain - the "neurogenesis hypothesis". Neurogenesis, the birth of new cells from stem cells, occurs in a couple of very specific regions of the adult brain, including the elaborately named subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus. Many experiments on animals have shown that chronic stress, and injections of the "stress hormone" corticosterone, can suppress neurogenesis, while a wide range of antidepressants block this effect of stress and promote neurogenesis. (Other evidence shows that antidepressants probably do this by inducing the expression of neurotrophic signaling proteins, like BDNF.)

The literature on stress, neurogenesis, and antidepressants, is impressive and growing rapidly. For good reviews, see Duman (2004) and Duman & Monteggia (2006). However, the crucial question - do antidepressants work by boosting hippocampal neurogenesis? - remains a controversial one. The hippocampus is not an area generally thought of as being involved in mood or emotion, and damage to the human hippocampus causes amnesia, not depression. Given that the purpose (if any) of adult neurogenesis remains a mystery, it's entirely possible that neurogenesis has nothing to do with depression and mood.

To establish whether neurogenesis is involved in antidepressant action, you need to to manipulate it - for example, by blocking neurogenesis and seeing if this makes antidepressants ineffective. This is practically quite tricky, but Luca Santarelli et. al. (2003) managed to do it by irradiating the hippocampi of mice with x-rays. They found that this made two antidepressants (fluoxetine, aka Prozac, and imipramine) ineffective in protecting the animals against the detrimental effects of chronic stress. This was a landmark result, and raised a lot of interest in the neurogenesis theory.

This new paper, however, says differently. The authors gave lab rats a six-week Chronic Mild Stress treatment, a Guantanamo Bay-style program of intermittent food deprivation, sleep disruption, and confinement. Chronic stress has various effects on rats, including increased anxiety and decreased time spent grooming leading to fur deterioration. These behaviours and others can be quantified, and are treated as a rat analogue of human clinical depression - whether this is valid is obviously debatable, but I'm willing to accept it at least until a better animal model comes along.

Anyway, some of the rats were injected with antidepressants during the final two weeks of the stress procedure. As expected, these rats coped better with the stress at the end of six weeks. This graph shows the effects of stress and antidepressants on the rat's behaviour in the Forced Swim (Porsolt) Test. Higher bars indicate more "depressed" behaviour. The second pair of bars, representing the stressed rats who got placebo injections, is a lot higher than the first pair of bars representing rats who were not subjected to any stress. In other words, stress made rats "depressed" - no surprise. The other four pairs of bars are pretty much the same height as the first pair; these are rats who got antidepressants, showing that they were resistant to the effects of stress.

The crucial finding is that the white and the black bars are all pretty much the same height. The black bars represent animals who were given injections of methylazoxymethanol (MAM), a cytostatic toxin which blocks cell division (rather like cancer chemotherapy). As you can see, MAM had no effect at all on behaviour in the swim test. It had no effect on most other tests, although it did seem to make the rats more anxious in one experiment.

However, MAM powerfully inhibited neurogenesis. This second graph shows the number of hippocampal cells expressing KI-67, a protein which is a marker of neuroproliferation. As expected, stress reduced neurogenesis and antidepressants increased it. MAM (black bars again) reduced neurogenesis, and in particular, it completely blocked the ability of antidepressants to increase it.

But as we saw earlier, MAM did not stop antidepressants from protecting rats against stress. So, the authors concluded, neurogenesis is not necessary for antidepressants to work. This contradicts the landmark finding of Santarelli et. al. - why the discrepency? There are so many differences between the two experiments that there could be any number of explanations - the current study used rats, while Santarelli used mice, for one thing, and that could well be important. Whatever the reason, this result suggests at the least that neurogenesis is not the only mechanism by which antidepressants counteract the effects of stress in animals.

The most interesting aspect of this paper, to my mind, was an essentially unrelated new finding. Stress was found to reduce the volume of several areas of the rat's brain, including the hippocampus and also the medial prefrontal cortex (mPFC). Unlike the hippocampus, this is an area known to be involved in motivation and emotion. Importantly, the authors found that following stress, the mPFC did not shrink because neurones were dying or because fewer neurones were being born, but rather because the existing neurones were changing shape - stress caused atrophy of the dendritic spines which branch out from neurones. Dendrites are essential for communication between neurones.

As you can see in the drawings above, stress (the middle column) caused shrinking and stunting of the dendrites in pyrimidal neurones from three areas relative to the unstressed rats (left), while those rats recieving antidepressants as well as stress showed no such effect (right). The cytostatic MAM had no effect whatsoever on dendrites. Further work found that antidepressants increase expression of NCAM1, a protein which is involved in dendritic growth.

So what does this mean? Well, for one thing, it doesn't prove that antidepressants work by increasing dendritic branching. Cheekily, the authors come close to implying this in their choice of title for the paper, but the published evidence shows no direct evidence for this. To find out, you would have to show that blocking the effects of antidepressants on dendrites also blocks their beneficial effects. I suspect this is what the authors are now working hard to try to do, but they haven't done so yet.

It also doesn't mean that taking Prozac will change the shape of your brain cells. It might well do, but this was a study in rats given huge doses of antidepressants (by human standards), so we really don't know whether the findings apply to humans. On the other hand, if Prozac changes the shape of your cells, this study suggests that stressful situations do too - and Prozac, if anything, will put your cells back to "normal".

Finally, I don't want to suggest that the neurogenesis theory of depression is now "dead". In neuroscience, theories never live or die on the basis of single experiments (unlike in physics). But it does suggest that the much-blogged-about neurogenesis hypothesis is not the whole story. Depression isn't just a case of too little serotonin, and it isn't just a case of too little neurogenesis or too little BDNF either.


J M Bessa, D Ferreira, I Melo, F Marques, J J Cerqueira, J A Palha, O F X Almeida, N Sousa (2008). The mood-improving actions of antidepressants do not depend on neurogenesis but are associated with neuronal remodeling Molecular Psychiatry DOI: 10.1038/mp.2008.119