http://snyderlab.com/functional-neurogenesis-2009-2013/

In its true form, pattern separation is a neurophysiological computation that is very difficult to measure since we know very little about how information is represented, in terms of action potential firing patterns in assemblies of cells (i.e. how can you measure how information has changed if you don’t have a good handle on what the incoming neural activity meant in the first place?). There has been some progress suggesting the dentate gyrus may pick up on minor changes in the environment and perform such a function. And so behaviourists have been keen to test whether the dentate gyrus and immature neurons are important for this function, using tasks such as discriminative context fear conditioning (is this the place where I received a shock?) or object location tests (did these objects move just a tiny bit since I saw them last?). When the dentate gyrus is compromised, or when neurogenesis is reduced, we sometimes see deficits in these behaviours. If you have a look at the pattern separation blog you’ll see an impressive interdisciplinary discussion of what these findings mean (and don’t mean!). In short, they are consistent with a pattern separation role but they don’t prove that the dentate is actually performing pattern separation at a physiological level.

Here I present some new data on adult neurogenesis, context fear discrimination, and stress hormones. It’s been on my hard drive since 2008. Which is ridiculous since it reflects many long days of putting mice into boxes and the findings are pretty intriguing, if inconclusive.

So finally I wrote it up and have published it on Figshare. Download it there and read along.

The basic idea is that I was training neurogenesis-deficient GFAP-TK mice in a discriminative context fear paradigm. The hypothesis was that, if the dentate gyrus and adult neurogenesis is important for pattern separation, then we would expect that the TK mice would be impaired, and show similar levels of freezing in the so-called “safe” and “shock” contexts. This is now obvious given work by McHugh, Tronel, Sahay, Niibori, Kheirbeck.

To make the discrimination challenging, I started with a discrimination paradigm where the 2 contexts were quite similar and the only difference was the pattern on the walls of the 2 contexts: circles or stripes. During the training session it appeared to be too challenging – the mice showed no discrimination whatsoever. Interesting finding #1: when tested 1 week later, the WT mice did show a discrimination whereas the TK mice did not. To get the most out of the experiment, I re-tested the mice the following day: mice that were tested in the shock context on test 1 were tested in the safe context on test 2 and vice versa. Interesting finding #2: There was a carry over effect such that the WT mice again discriminated, but on test 2 they now froze more in the safe context! On test 2 corticosterone levels were also greater in the mice tested in the safe context.

This experiment (“Circles vs Stripes”) suggests to me that neurogenesis may indeed be involved in some sort of pattern separation function, since the TK mice never successfully discriminated. But it is interesting that WT mice only discriminated during the test. Usually, context fear memories become more generalized with time (see Wiltgen, Biedenkapp, Wang) but here they are becoming more accurate. I don’t have a solid explanation for this but wonder if the simplicity of the context difference plays a role. If mice were able to form a simple stimulus-shock association (circle-shock or stripe-shock association, rather than complex context-shock association) then these memories might not subject to the same generalization/interference processes that typically occur during consolidation. This result is also a reminder that memory may be intact, even when there isn’t behavioural evidence. Regarding the reversal effect, the paradigm is different but reminiscent of findings by Beracochea showing that stress can alter which of 2 context memories dominates at the time of retrieval. It is also worth noting that blood samples were taken 30min after testing for corticosterone measurements, using a submandibular cheek-lancet method. This is a stressful procedure and may have altered the memory retrieved on test #1, and contributed to the carryover effect on test #2.

To see if we could pull out a context discrimination difference during training, I repeated the experiment but changed many more features between the 2 contexts (shape, odours etc). This variation was code named MO DIFF since the contexts were made “more different” and I have kept that name since this isn’t a journal. If anything, the TK mice now did a better job of discriminating (at least during training). Compared to Circles vs Stripes there was weaker discrimination during Mo Diff testing and also fewer reversal/carryover effects between tests #1 and #2. TK mice had huge elevations in corticosterone compared to WT mice at the time of fear memory retrieval.

For the last experiment I had some mice that had been subjected to chronic stress so I figured why not then test them on Mo Diff? The mice in Mo Diff didn’t remember super well and chronic stress enhances fear conditioning so…we found that these mice indeed discriminated very well during training and testing. No difference between WT and TK mice during training but TK mice discriminated identically on tests #1 and #2. In contrast, WT mice again showed a carryover effect such that there was no discrimination on test #2.

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Final thoughts: This dataset may raise more questions than it answers and for this reason my work with GFAP-TK mice then took a more straightforward route, eliminating memory from the equation and investigating whether new neurons are important for innate responses to psychological stress. In any case:

1. The data support a role for neurogenesis in context discrimination, and potentially pattern separation, but it suggests that new neurons may bias towards both separation or generalization depending on the conditions.
2. New neurons may be important for accurate consolidation of memory
3. Neurogenesis regulates stress hormone levels during memory retrieval
4. Testing order strongly influences whether mice express fear in the appropriate context

Reference: Snyder, Jason; Cameron, Heather A. (2013): Reduced adult neurogenesis alters behavioural and endocrine discriminative fear conditioning. figshare.
http://dx.doi.org/10.6084/m9.figshare.884597

My re-rabidness (re-raBIDniss) was spawned as I was passing on my experimental notes to a student. The notes were in a Google doc so I could either copy and paste them, or just invite them to share the document. And if everyone in the lab used it then we’d all be on the same page (whose rats for which expt, who has expertise in certain protocols, general curiosity about lab projects). And then I wondered, is it a concern for one lab member to see another’s experimental notes? It sounds crazy but given the amount of secrecy involved in producing scientific research and the fact that no ever reads another’s lab book I wondered if it might just “feel weird”. Since in my heart open notebook science is the way to go, and since some actually put this into practice, I figure there are no real issues with simply sharing notes thoughout the lab, and any weirdness will be temporary and far outweighed by the positives.

At the moment I envision something like: A WordPress blog as the lab home page, containing links to Google spreadsheets (colony, orders, etc), documents (individuals’ experimental notes), calendars, files etc. But if this is where we go every day to organize our science, why have a separate site to present ourselves to the world? Why not make our lab notebook and lab website the same thing? And that’s where the blog comes in, where we can share data, pretty pix, random lab occurrences, literature reviews, whatever. There would likely be different views depending on whether you’re logged in (a lab member) or viewing from the outside. So this wouldn’t be completely open notebook science or anything, but I think it would help unify many of our responsibilities as scientists, namely to effectively and intelligently plan experiments, record our findings, communicate and educate (of course, great venue for jokes too).

RSS is Really Simple Syndication. It’s a way to subscribe to your favorite websites so that their new content shows up in your RSS reader. Subscribing is usually as easy as clicking on one of these things:  or copying and pasting a url. Email for the internet, some have said. Instead of having to revisit dozens of websites that update with varying regularity one can simply open their RSS reader, see the sites that have updated content, and read the new content directly in the RSS reader (or at least the title or abstract¹). If you don’t have time to read a full article you can flag items for later viewing². I subscribe to 25 journals and about 50 blogs. I also subscribe to Google Alerts and Pubmed searches, feeds that track citations to my articles, and feeds that tell me when someone comments on a Flickr photo or edits a Google spreadsheet. It’s the only way I know to stay on top of everything, be all knowing, etc³. And I’ve backed up my Google Reader data so that when it closes on July 1 I can spend Canada Day not with my family but instead testing out a bunch of alternative RSS readers. Or at least testing out a bunch of RSS readers on my phone, in close proximity to my family⁴.

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¹some journals only allow you to see the title in the RSS reader, forcing you to click through to their website to see the abstract. Sometimes I refuse to click and say (in my head) “I hate you”.

²for example you can see in the image that I flagged an article on Lunar Mascon Basins, because I’m really into them.

³On the other hand, I simply cannot actually read everything I come across. So another approach is to use something like Google Scholar’s “My Updates” feature as it selectively notifies you of papers that are similar to your own. Given the number of grey haired academics with Google Scholar profiles, the effectiveness of Scholar’s citation data, and the fact that it’s Google I’ve often wondered why they don’t extend its functionality just a bit, so you can follow other scientists and their papers etc.

⁴So that when my son sees a float at the parade and says “Look daddy!” I can glance up and feign excitement for a moment and go back to my phone when he’s distracted again⁵.

⁵I’m so kidding⁶.

⁶Okay, 80% kidding.

The study was a bit of a fun learning experience for me for several reasons. As many of you know I recently changed labs and will be starting my own lab soon. So things are on the go and I haven’t had the time to dive deep into a study that is going to take several years to complete. But some research projects can be done quickly and still are able to produce very useful results. As I prepare for my own lab I was probably thinking, “What kind of projects could a Master’s student accomplish??”. And indeed we had a strong postbaccalaureate fellow in the lab for about a year who fit this description pretty well (she’s the middle author). Also, we had lots of tissue remaining from a recent study where we compared neurogenesis in mice and rats that could be used to answer other questions, thereby saving time, money and importantly, animals. So we decided to ask whether the maturation and survival of adult-born neurons differ between the aforementioned functionally-distinct hippocampal subregions.

The basic idea of the study.

BrdU was injected to birthdate+label new neurons and at various intervals kainate was given to “activate” all the neurons that had integrated into the circuitry, and formed synapses. This integration (a major step in the new neuron maturation process) can then be measured by immunohistochemically staining for gene products such as Arc that are expressed after synaptic activity. Our tissue was cut in the coronal plane, however, which is not ideal for isolating the septal and temporal ends of the hippocampus (though others have found that there are meaningful differences along this rostrocaudal axis). Since the rostral sections are purely septal (see blue portions, above) this subregion was not a problem. We then decided to basically “cut” the caudal sections in half and by analyzing only the ventral portion we were able to specifically target neurons located in the far temporal dentate gyrus (shown in red). We were also able to investigate whether new neurons are more likely to survive in one subregion than another, by counting the number of cells present before (at 7d old) and after (at 28d old) the period of cell death.

Since this article is completely open access you can see the actual data for yourself here. The short story is that new neurons matured faster in the septal dentate gyrus (all cells could express Arc by 3w of age whereas in the temporal dentate gyrus this didn’t occur until somewhere between 4-10w of age). The other new finding was that there were many more new neurons initially added to the infrapyramidal blade of the dentate gyrus but these neurons were much less likely to survive than neurons born in the suprapyramidal blade.

What is the significance of these findings? To me, they provide a framework for future studies. If you want to investigate new neurons specifically during their immature stage (when they might have unique functions) you certainly have to consider the anatomical location. But what if the main function for new neurons is not realized until they are fully mature? Well, if you plan to ablate or silence new neurons and examine behavioural effects, then you might want to wait longer if you’re planning on investigating emotion/stress-related behaviours that might rely more heavily on the temporal hippocampus. They also suggest that new neurons in the temporal dentate gyrus might have an extended unique role since they remain immature for longer. The peculiar survival difference between the infrapyramidal and suprapyramidal blades doesn’t do much to clarify the functions of these two regions, but it adds to the ever-growing list of differences that suggests they are truly distinct.

Often findings across labs do not match up as well as one would like so I am rather happy that Piatti et al. have found similar maturation differences in another species (mice) and using different methods (electrophysiological recordings from virally-labelled new neurons). So this is probably for real!

Lastly, a reviewer pointed out something very helpful, which is that it can be very difficult to discern the two blades of the dentate gyrus in caudal coronal sections (like this or those slightly more caudal where the dentate gyrus is essentially a blob). I spent a fair bit of time looking perplexed as I played with 3D paper models of the dentate gyrus but felt pretty cool doing so because similar strategies have been used by some of the foremost neuroanatomists of our time – see here. A 3D computer model that incorporated the blades of the dentate gyrus would have been very convenient (talking to you, Allen). In any case, we decided to remove our caudal blade analyses from the paper and instead only focussed on the septal infrapyramidal vs. suprapyramidal differences.

Reference: Snyder JS, Ferrante SF, Cameron HA (2012) Late Maturation of Adult-Born Neurons in the Temporal Dentate Gyrus. PLoS ONE 7(11): e48757. PMID: 23144957

¹in rodents

²in humans

There’s one week remaining before the  Society for Neuroscience annual meeting begins. That means you have about 6 days before you really really have to start tallying a list of presentations. Of course, WITH HUBBIAN*, you actually could put it off that long and still gather a great list of presentations. This is because Hubbian allows you to see related content, popular presentations, and presentations that are generating discussion. The key ingredient, which was missing until now, was the ability to save a list of presentations you’ve discovered on Hubbian.

DID I MENTION THE UNTIL NOW PART??

That’s right. Go nuts. Search and save.

I WANT YOU IN MY LAB!

The lab’s goal is to identify the role of adult neurogenesis in memory and stress-related behaviours. We inhibit neurogenesis with transgenic animals in order to understand how they contribute to these behaviours, viral tools for labelling and modifying neurons, immunohistochemistry to quantify and characterize the neurogenesis process, and in vitro electrophysiology to understand the circuit mechanisms by which these new neurons regulate behaviour. The neurobiology of behaviour extends far beyond adult neurogenesis, however, and so we are also generally interested in how neurons throughout the dentate gyrus, hippocampus, and related structures interact to guide behaviour.

I’m excited about the science but I’m also excited about doing it in the open. Discoveries exist well before they’re printed in a journal but in most cases people don’t appreciate this, since discoveries are rarely shared as they happen. I’d like to do things a little differently and get our science out in the open. Early. To assist others and stimulate discussion. I’d like to see undergrads in my lab have have their data available online in a citable format. You don’t need a peer-reviewed publication or a graduate degree to contribute something valuable to the scientific record (and perhaps your CV).

If this sounds like the bomb:

Potential postdocs can email me (jasonsnyder@psych.ubc.ca) directly to inquire about joining the lab. External funding deadlines are approaching and would go a looong way at this point.

Potential graduate students can contact me and apply through the Psychology or Neuroscience programs. Deadlines for a September 2013 start date are December 15 and January 30, respectively. There is also a June/July deadline for starting Neuroscience graduate studies in January 2014.

Potential undergraduates that are interested can email me directly. Previous lab experience is not a prerequisite to join the lab!

The lab will officially open in January 2013 and, after setting up, will be ready for real business around summertime. Oh, and I will be at the Society for Neuroscience meeting if interested folks would like to chat in person.

THIS IS EXCITING. READERS THAT ARE PREGNANT / HAVE HEART PROBLEMS STOP NOW CLICK HERE.

Over 30,000 people attend the annual Society for Neuroscience meeting and for this reason alone people either love it or hate it. On one hand, you can learn about any type of neuroscience research imaginable. On the other hand, it can be extremely difficult to find the meaningful stuff. The online itinerary planner. It works. Kind of. Did you search the right keywords? What about that new technique you’re interested in* – it’s mentioned in 100s of abstracts – which ones are worth checking out? What are other neuroscientists looking at? And in case you’ve missed something interesting you now find yourself at the meeting, aimlessly scanning titles, just a brainstem….being pulled towards the larger crowds…failing to penetrate the bobbling masses…

BUT THE KILLER APP THAT WILL SAVE YOUR LIFE HAS ARRIVED → Hubbian ←

SEARCH THE ABSTRACTS FOR WHATEVER TERMS YOU LIKE. ABSTRACT CONTENT, AUTHOR NAMES, EVEN PRESENTATION & POSTER BOARD NUMBERS.

SCROLL THROUGH THE SEARCH RESULTS, SELECT AN ABSTRACT AND, IF YOU LIKE IT, VOTE FOR IT! BETTER YET, LEAVE A COMMENT!

So you’ve scanned the abstracts a bit, maybe noticed the ‘related abstracts’ tool and stumbled across some presentations you’d never have found (awesome)…but now what?

HOW ABOUT GIVING YOUR BRAIN A REST. LET THE APP TELL YOU WHAT’S INTERESTING**

Do a search and sort the results by the number of views, votes or comments. Go back and sort by ‘best match’ to find content that is closest to your particular query. Sorting by time will tell you which presentations are coming up next. And you can always click the links at the very top of the page to see the most popular presentations of the entire meeting.

SHARE IT WITH OTHER NEUROSCIENTISTS THAT ARE NOT PREGNANT & DO NOT HAVE HEART PROBLEMS!

*Golgi staining

**or at least which abstracts are getting the most attention

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@Hubbian on Twitter

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Extra details for aficionados

• the development crew, which I was lucky enough to join
• database contains abstract content only
• got feedback? let us know!

• there is a blog!

• will be using Twitter hashtag #sfn12

• when you go to http://hubbian.com you’ll be automatically redirected to the mobile or desktop version. You can also visit the desktop version directly at http://hubbian.com/desktop.html – the mobile version is at http://hubbian.com/mobile.html

• Internet Explorer is not supported

Well, it could…and we did end our paper with the following:

Because the production of new granule neurons is itself strongly regulated by stress and glucocorticoids, this system forms a loop through which stress, by inhibiting adult neurogenesis, could lead to enhanced responsiveness to future stress. This type of programming could be adaptive, predisposing animals to behave in ways best suited to the severity of their particular environments. However, maladaptive progression of such a feed-forward loop could potentially lead to increased stress responsiveness and depressive behaviours that persist even in the absence of stressful events.

We had to end it somehow – I was just happy that after 3 years of work we were DONE²! But our final speculation makes it clear that, while this chapter may be done, the story is not. And this fact was rightly pointed out in a recent commentary by Lucassen et al. in Molecular Psychiatry³, where they continue the discussion and bring up some good points. Here is a loose elaboration on some of the outstanding issues they bring up.

Is a feed-forward cascade plausible? In other words, is it possible that stress reduces neurogenesis, which leads to a hyperactive HPA response, which further reduces neurogenesis, thereby additionally increasing the stress response etc etc, eventually damaging the brain and leading to depression? As Lucassen et al point out, stress typically reduces neurogenesis by only ~30% which is much smaller than the 100% reduction seen in our transgenic mice. Could a 30% reduction be enough to initiate this vicious cycle? What if it was chronic? People have looked in the past and not observed HPA alterations after smaller (but also equally large) reductions in neurogenesis, so the answer might appear to be negative. But there are many important differences between studies, including when stress hormones were measured (e.g. baseline or after stress) as well as factors such as life history, genetic makeup, and stressor controllability, as is suggested in the commentary. Of course, in reality, chronic stress has multiple effects throughout the hippocampus (and brain) and so the development of depression is certainly due to additive effects (this is both their sentiment and mine). And so perhaps in the real world a more modest reduction in neurogenesis does have the potential to tip the scales towards depression, if it is coupled with other (which?) pathologies.

How could so few adult-born neurons regulate the HPA axis? Depending on my mood, my thoughts on this question fluctuate quite a bit. Half of the time I think “This is ridiculous” and the other half of the time I look at the evidence and think “Heck yeah. Maybe⁴.” (see our recent review for more detailed arguments on the heck yeah side of things). In our study we reduced neurogenesis for up to 12 weeks, preventing about 50,000 cells from being added or 10% of the total population. More important than sheer numbers, however, is the ever-increasing evidence that adult-born neurons are different from mature neurons – more plastic, more excitable, uniquely neuromodulated. Some of the most intriguing evidence comes from one of the authors themselves who has shown that even 4 month old neurons (but potentially older?) undergo extensive structural modification following learning whereas perinatal-born cells do not. We have estimated that ~40% of the total granule cell population is added in adulthood in the rat and so my point is that in the real world we have to consider that there are probably cumulative effects of adult neurogenesis over years, and even decades in humans. And so the population of adult-born cells, and its functionality, may not be so small in the end.

Do new neurons sense detect stress through glucocorticoids? One common assumption is that glucocorticoid receptors are necessary for inhibition of the HPA axis…but must that always be the case? By 1-2 weeks of age the majority of adult-born neurons do express MRs and GRs and therefore certainly could directly detect glucocorticoid levels and initiate a shutting down of the HPA axis. But perhaps new neurons process stressful information that is relayed through glutamatergic pathways. In this scenario new neurons might not be required for sensing glucocorticoids and initiating negative feedback. Instead, after being stressed, they could be required for biasing the animal away from the negative experience (akin to perceiving a change in context, similar Opendak & Gould‘s proposal for reconciling stress and memory hypotheses of new neuron function) which might reduce CNS drive on the HPA axis. This opens the door to the possibility that the HPA and behavioural roles of adult neurogenesis are somewhat dissociable, in which case a role for new neurons in the development of depression is not (only) through the feedforward cascade hypothesis but through direct effects on behaviour. This might also help explain the inconsistent links between stress hormones, hippocampal volume, and depression that plague the stress-depression literature.

So, we showed that adult-born neurons are required (in mice) for normal stress responses and emotional behaviour. Our final speculation was, well, just that – a leap towards the next big question. Maybe realistic, maybe not. Maybe a component of the real picture. In any case, the route has been mapped.

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References
Lucassen PJ, Fitzsimons CP, Korosi A, Joels M, Belzung C, & Abrous DN (2012). Stressing new neurons into depression? Molecular psychiatry PMID: 22547116

Snyder JS, Soumier A, Brewer M, Pickel J, & Cameron HA (2011). Adult hippocampal neurogenesis buffers stress responses and depressive behaviour. Nature, 476 (7361), 458-61 PMID: 21814201

Footnotes

¹You thought this footnote was going to be some comment on human depression vs. depressive-like behaviour in animal models but instead I just wanted to mention that, now that I’ve moved back to Canada, I feel compelled to use ‘behaviour’ instead of ‘behavior’ and it feels silly because the same people read this stuff no matter which country I’m in when I write it.

²Is there an emoticon for dusting the dirt off your hands, blowing smoke away from the tip of a revolver, or enjoying a refreshing beverage after chopping a bunch of wood or giving birth? Insert it here.

³One of the authors having actually commented on this blog (!), and who is 1st author on what looks to be a very interesting paper that is related to this whole discussion.

⁴I may be a pessimist. Realist? A guy with a healthy amount of skepticism?

⁵The photo? It’s a depression.

Now, on with the pictures! As always, I recommend high-res viewing (click on the image to view it, bigger, on Flickr).

Using a retrovirus, which infects dividing cells, I made the amazing discovery of four adult-born cells which all had the exact same shape and were located right next to each other!

More dentate granule cells, infected with Herpes Simplex Virus and expressing GFP.

In the process of learning the surgeries required to stereotactically inject the virus, you inevitably target the wrong regions. Which is fun because then you get a glimpse of something new. Here are GFP+ CA1 pyramidal neurons. You can see their axons projecting down and to the left, towards the subiculum.

More CA1 pyramidal neurons from the same animal but an adjacent section. I love the fanning of the dendrites. Click on the image and you can see spines in a higher resolution version.

Every day I see a retrovirally-labelled, non-neuronal (?) cell that looks more beautiful than the last one I imaged. Makes me want to cry.

Here are dentate granule cell axons, the mossy fibers, flowing through CA3 pyramidal neuron cell bodies, like a river gushing over rocks. In the woods. The balls on a string appearance is caused by the infamously large presynaptic boutons, which are distributed along the axon (like balls on a string, thin rope, or even dental floss).

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