My goal in compiling this list was to assess the magnitude of adult neurogenesis in primates. It’s definitely more challenging than assessing the magnitude of neurogenesis in rodents, which we know much more about, and so I had put it off. At this point I haven’t reached a clear conclusion but, in quickly skimming these papers, the number of proliferating cells and/or new neurons averages thousand(s) of cells in the young adult primate hippocampus. The range is much much larger, and many studies cannot be easily compared due to variability in the methods, which is partly understandable since primates are scarce and are often used in multiple studies, thereby limiting the analyses that can be performed.

Download the list

Some studies showing different functions of septal and temporal hippocampus

• Some of the best reviews of the topic are by Bannerman et al from 2004 and 2011.
• A recent and free review article by Fanselow and Dong.
• Classic Moser papers showing spatial memory is more dependent on dorsal hippocampus and anxiety/fear behavior on ventral hippocampus
• A recent paper suggesting that spatial processing in the septal hippocampus meets the behavioral-control functions of the temporal hippocampus to enable rapid spatial learning

History of neurogenesis quantification. So, back in the day, before I even knew what a neuron was, and before it was well-established that there was functional differentiation along the hippocampal axis, people would pick a few sections from the dorsal hippocampus (it’s much more photogenic, gets all the glory), count new neurons, and make it a density measurement. Then the stereology police arrived (seriously, that’s what they’re called) and pointed out that changes in tissue volume or cell packing could change density measurements without there being any differences in numbers of cells. Stereological analyses also prevent any biases that might arise from creating arbitrary boundaries when examining only part of the hippocampus. And so people started doing stereological counts, which require a systematic quantification throughout the entire hippocampus. My guess is that this probably delayed the appreciation that neurogenesis could vary in magnitude and function along the hippocampal axis. Now that we know that stereology is pointless we can get back to business (this is a joke – please don’t arrest me).

Difficulty of quantifying subregions due to curvature of the hippocampus. One of the reasons the hippocampus is such a popular neurobiological model is its anatomy – the dentate gyrus, CA3 and CA1 subfields are all composed of tightly packed cells that are easy to identify. Thinking of the hippocampus along its long axis, one end projects to the septum and the other abuts the temporal lobe, hence “septotemporal” is technically the most accurate way to refer to the different ends of the hippocamus. The hippocampus is curved in such a way that you can actually cut it along any of the 3 spatial planes (X, Y, Z aka coronal, horizontal, sagittal) and hit the hippocampus perpendicular to the septotemporal axis somewhere, giving rise to the classic the trisynaptic circuit. However, because of this same curvature, sectioning the brain in only one of the three planes means that some portion of the hippocampus is not going to be cut perpendicular to the long axis, producing sections in which septotemporal coordinates are hard to define.

The 3D nature of the hippocampus using images from the Allen Brain Explorer:

Figure 1: The dentate gyrus subfield of the hippocampus (i.e. green banana), from its septal pole, extends caudally and laterally and then ventrally. Green axis=dorsoventral, red=rostrocaudal, yellow=mediolateral.

Others on the curvature problem:

So how can we divide the hippocampus? Many people work with coronal sections. Can we delineate boundaries between different hippocampal subregions in coronal sections? Banasr et al. has described a reproducible method for separating dorsal from ventral hippocampus using coronal sections. Here, the dorsal regions would contain a fair bit of mid-septotemporal hippocampus but indeed, only the dorsal sections would contain septal hippocampus and only ventral sections would contain temporal hippocampus:

Jayatissa et al. has horizontally sectioned the rat brain and then used anatomical coordinates to divide dorsal from ventral. This seems to be a good way to isolate pure, septal hippocampus but dorsal measures would again blur together the septal and mid-septal regions.

What if we wanted to separate the septal and temporal ends of the hippocampus? One method, described in Amaral & Witter, 1989 offers a solution:

I have used a similar approach (see here and here). One drawback is that you ruin much of the rest of the brain during the dissection process (insert but-who-cares-about-the-rest-of-the-brain joke here). Here’s a figure from my thesis that illustrates the similar-shaped hippocampal slices obtained with this method:

Another strategy, which I can currently exploring since I’m working with coronally-sectioned brains, isn’t too different from the method of Banasr, above. To get at the septal hippocampus I’m just being a bit more selective and only examining portions of the dorsal hippocampus that extend quite far rostrally. For the caudal sections that contain both dorsal and ventral hippocampus the rhinal fissure seems like a good guide – anything falling on the ventral side I’m counting as ventral.

But if you’re lazy…

A fast, revolutionary new method for examining the hippocampus along its full septotemporal axis in a single section! It almost sounds too good to be true. In fact, it is. But it provides some interesting pictures for those of you who have stuck with me this far.

Recently, we irradiated a lot of rats to eliminate adult neurogenesis. Before coming to any conclusions about the behavioral data we needed to know whether neurogenesis was completely blocked AND whether it was blocked throughout the entire dentate gyrus. We were too lazy to cut hundreds of sections for each rat so what we did was extract the hippocampus but instead of sectioning perpendicular to its septotemporal axis, we sectioned it parallel to, or along, its septotemporal axis by flattening and freezing it on a microtome stage. With this approach we could cut the entire dentate gyrus in about 30 sections and get sections that had the entire septotemporal length of the dentate gyrus present. We then stained them for NeuN and DCX to visualize neurons and immature neurons, respectively. I think every other section was stained; one example is shown below.

Is it really necessary to divide septotemporally? I guess it depends. Many studies that have focussed more on dorsal vs. ventral have made significant findings. If the anatomical method is well-described and reproducible, what more could you ask for? It’s possible, however, that combining different septotemporal regions into the same analysis could obscure a result. For example, when I examined activation of new neurons after water maze training I found a steadily-increasing amount of activation as I went from septal to temporal (see Figure 7). Had I pooled the 2 septal quartiles together and pooled the 2 temporal quartiles together the observed difference would have been much smaller than when comparing the septalmost quartile with the temporalmost quartile.

Figure 7: The density of ‘activated’ new neurons (i.e. PSA-NCAM+ and Fos+) increased from septal to temporal. Note the mid-septal and mid-temporal regions were similar. Also note that I used D and V nomenclature, for ‘dorsal’ and ‘ventral’, despite repeatedly emphasizing in this post that ’septal’ and ‘temporal’ is more accurate. A reviewer told me to do this (and I listened).

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and now…

Pretty pictures from these sections!

Thanks and credit to Sarah Ferrante for sectioning, staining and imaging the tissue.

]]> http://www.functionalneurogenesis.com/blog/2011/04/dorsoventral-vs-septotemporal-hippocampus/feed/ 4 http://www.functionalneurogenesis.com/blog/2011/01/studies-of-adult-hippocampal-neurogenesis-in-humans/?utm_source=rss&utm_medium=rss&utm_campaign=studies-of-adult-hippocampal-neurogenesis-in-humans http://www.functionalneurogenesis.com/blog/2011/01/studies-of-adult-hippocampal-neurogenesis-in-humans/#comments Fri, 28 Jan 2011 17:28:53 +0000 Jason Snyder http://www.functionalneurogenesis.com/blog/?p=1033 As we accumulate more and more data on adult neurogenesis in rodents I keep asking myself what kind of impact these new cells could have. The dearth of literature on primate and human adult neurogenesis seems to make these questions all the more relevant. As a starting point, I created a Pubmed collection of all the studies of adult hippocampal neurogenesis in humans. They’re also listed below in a Google spreadsheet. Note that human studies often do not directly measure neurogenesis but instead measure 1) cell proliferation (which usually correlates with neurogenesis in rodents, but assumes that proliferation results in surviving neurons in humans), 2) stem cell markers (such as nestin, which correlates with neurogenesis only if they indeed divide and produce new neurons), 3) immature neurons (which, technically speaking, is neurogenesis, but whether these neurons mature and become functional remains to be determined), or 4) other factors that correlate with neurogenesis, such as blood flow or stem cell biomarkers. So, while the conclusions of these studies may be exciting (or depressing), they have to be taken with a grain of salt at this point.

Download the list

For my part, Functional Neurogenesis is slated to post on the Theme A topic, development. But I will also post on more general topics in the neurobiology of behavior (mainly in animals – e.g. place cells, plasticity, structural or functional correlates of behavior). Posts are to be at least daily, following SFN’s guidelines. Which (if you know my frequency) means I will be taking full advantage of the big brown vats of lukewarm Starbuck’s coffee. No, seriously, I will probably take the streetcar to a cafe in old town to avoid the lines. And still make it to the poster session faster. Oh, also, there will be tweets.

This year’s SFN meeting marks the 1-year anniversary of the decision to startup Functional Neurogenesis. And it feels like things are only getting started – from within the scientific community FN has gotten some great feedback for which I’m thankful. The blogosphere is a whole different story. .

And now, because I haven’t made a list in weeks, and because there will be much coverage by non-official bloggers…

These bloggers will all be at the meeting. No guarantee that they’ll actually be blogging the meeting though. Let me know in the comments or email jasonscottsnyder (gmail) if you should be on here (or not).

“Official”
Functional Neurogenesis / @jsnsndr
Genetic Expressions / @geneticexpns
Blogging on the Brain (@hillaryjoy
Qscience
Onesci
Fresh Eyes
House of Mind / @houseofmind
Pascal’s Pensees / @Pascallisch
Neuromusings / @neurodilettante
David Deriso / @davederiso
Dormivigilia / @Beastlyvaulter
Blogging Behavior / @aechase
Stanford Neuroblog / @stanfordneuro
SFN2010 / @thekhawaja

Unofficial
Fumbling Towards Tenure Track / @doc_becca
Some Lies / @Tideliar
Drugmonkey / @drugmonkeyblog
Neurocritic / @sarcastic_f
Bjorn Brembs / @brembs
Neurokuz / @kuzyx
Juniorprof / @juniorprofblog
Oscillatory Thoughts / @bradleyvoytek
Ferris Jabr / @ferrisjabr
Neuron Culture / David Dobbs
Danio Reri
@BASi_news

Twitter lists of SFN attendees:
@stanfordneuro’s list
@mocost’s list
@noahWG’s list

Below are what I hope to be comprehensive visual collages of all published timecourse experiments, where a certain property of new neurons is examined at multiple (≥ 3) different ages. They are grouped by studies of: 1) cell survival, 2) marker expression, 3) functionality, and 4) miscellaneous studies that do not quite fit into the first 3 categories. I’ve ordered the data roughly chronologically and have included the first author’s name and publication year so you can read deeper, if needed. Indeed, if you know these studies already, a brief look at the graphs will bring back the take home message. However, since the data is stripped of text, if the studies are unfamiliar, you’ll have to go to the original source to figure out what the heck they mean (use Pubmed to at least obtain abstracts for the original studies if I didn’t provide a direct link).

Personally, I like timecourse studies for the same reason I like to have all my music albums or books visible at the same time: at a single glance they provide a lot of information – each individual stage of maturation can be interpreted within a bigger picture. The result of these many hours of work will either be a) that the purpose of adult neurogenesis will become immediately clear, or b) that we’ll all have some fancy collages to pin on our bulletin boards and look intelligent.

The survival timecourse

New neurons are born and then many die. The survival timecourse answers the questions: How many new neurons are born? Where are they born and where do they end up, anatomically? How many of them survive and can their survival be altered? Survival timecourses are typically performed by injecting animals with a mitotic marker that will label new neurons as they’re being born, e.g. ³H-thymidine (old school), BrdU (tried and true – example), or a GFP-expressing retrovirus (new school). At a later date one can then detect these birthdated new neurons and count them, see where they’re located etc.

What do these survival timecourses tell us?

• many newborn neurons die between 1w and 4w of age but after that they all survive
  • neurons born during infancy are an exception as they DO die off many months after their birth (Dayer 2003), lending support to the sexy-but-underexplored idea that neuronal turnover might underlie memory turnover in the hippocampus
• the number of new cells labeled with a birthdating marker (e.g. BrdU) grows between 2 hours and several days after the birthdating marker is administered
  • this is caused by continued division of the stem cell or precursor cell that took up the marker in the first place (see expression timecourse, below). After a few cell divisions the marker gets diluted to undetectable levels.
• the general timecourse of cell death is similar in young and aged animals (McDonald 2005) and in mice and rats, although more cells die in mice (Snyder 2009)
• the addition and culling of newborn neurons in the monkey hippocampus (Gould 2001) follows a delayed timecourse compared to rodents
• CREB signalling is critical for neurons to survive between 5-7 days old (Jagasia 2009), NMDA receptors are critical for survival from 14-21 days (Tashiro 2006)
  • thus, CREB signalling would appear to regulate survival before new neurons have formed excitatory connections and are functional (see below) and NMDA receptors regulate survival during the early phase of excitatory synapse formation, when new neurons are just beginning to be able to contribute to behavior. Knowing how to regulate neuronal survival has obvious implications for disorders where reduced neurogenesis might be a causative factor.
• learning increases the survival of new neurons (Leuner 2004)
• learning does not increase the survival of new neurons (Snyder 2005)

The expression timecourse

All cell types within the body express different genes/proteins that serve the cell’s function. Since a muscle cell has a completely different function than a skin cell, it will naturally express different proteins. In like manner, a 1 week-old neuron is functionally distinct from a 4 week-old neuron and the two will also express different proteins (to some extent). Many people have taken advantage of this, using these different proteins as markers that identify a new cell as a neuron vs. a glial cell or, more specifically, an immature neuron vs. a mature neuron. By simultaneously visualizing (via immunohistochemistry) both the birthdating marker (e.g. BrdU) and these phenotypic markers, one can know both the exact age of the neuron and its general degree of maturity. For a 10 sec guide to cell labeling with BrdU and phenotypic markers, see here.

What do these expression timecourses tell us?

• some markers (proteins) are increasingly expressed as new neurons mature over 4 weeks (NSE, NeuN, calbindin)
• other markers are mainly expressed when new neurons are < 4 weeks-old (DCX, PSA-NCAM, calretinin)
• most studies have used the same markers (e.g. DCX, NeuN) to simply demonstrate that new cells are neurons, but some have examined expression of markers that are associated with a more specific function, such as glucocorticoid receptors (Cameron 1993, Garcia 2004) or vascular markers (Palmer 2000)
• BrdU (or other birthdating markers) labeled cells express cell division markers (e.g. Ki67) several days after BrdU is administered. This does not mean newborn neurons are dividing – what it represents is the continued division of the stem cell, or precursor cell, that was originally labelled. (therefore you can never know the exact age of a new cell, but pretty close)

The functional timecourse

The previous timecourses are all well and good but sheer numbers of cells say nothing about whether the new neurons actually work. And markers like DCX or NeuN may give a general hint at the maturity of a neuron but not much more. Functional timecourses address these gaps. A direct measure of function would be whether a new neuron has electrophysiological properties that enable it to process information (e.g. input and output synapses, action potentials). Less direct signs of function can be inferred from the morphology of a new neuron and whether a new neuron is capable of expressing activity-dependent immediate early genes.

What do these functional timecourses tell us?

• electrophysiology (Esposito 2005; Ge 2006), anatomy/morphology (Hastings 1999; Zhao 2006; Toni 2007; Toni 2008), and activity-dependent gene expression (Jessberger 2003; Snyder 2009) all point to new neurons forming their first synapses at 2-4 weeks of age
• around 4 weeks of age, new neurons go through a phase where they have enhanced synaptic plasticity (Ge et al 2007) and enhanced activation during behavior (Snyder 2009)
  • thus, at a young age, new neurons are more modifiable by experience than mature neurons. This may enable them to make a greater impact on behavior, either at this age or by shaping their further integration into the circuitry so they can alter brain function when fully mature and functional
• young neurons have distinct neurotransmitter profiles: initially they receive GABAergic inputs and later receive excitatory glutamatergic inputs (Esposito 2005). Notably, GABA depolarizes immature neurons (Ge 2006), unlike its typically-inhibitory effects on mature neurons. Also, immature neurons have a unique form of the NMDA receptor (NR2B), which endows them with their enhanced plasticity (Ge 2007).
• blocking CREB signalling (Jagasia 2009) or the depolarizing effects of GABA (Ge 2006) inhibits the functional maturation of new neurons
• by 8 weeks of age, new neurons are pretty much fully developed, though Zhao 2006, Toni 2007 and Toni 2008 show that 8-10 week-old neurons are still slightly underdeveloped, presynaptically and postsynaptically
  • does this mean that 8-10 week-old neurons, despite no longer having enhanced synaptic plasticity or enhanced activation during behavior, might still function differently than fully mature neurons?

Other timecourses

There are some timecourse-ish studies that, instead of examining new neurons of different ages, have examined the final fate of same-aged neurons that had been manipulated at different stages of their development. From the first 3 figures we can see that specific stages during a new neuron’s development are associated with enhanced plasticity, unique neurotransmitter profiles and increased likelihood of cell death. Therefore, it is very possible that the ultimate fate of an adult-born neuron depends on when experiences occur, relative to these different stages.

What do these timecourses tell us?

Several show us that experience can modify the number of new neurons, but that the magnitude and direction of the change depends on how old the neurons are when the animal undergoes the experience. For example:

• environmental enrichment enhances survival of new neurons mainly when the neurons are 1-2 weeks old (Tashiro 2007)
• spatial learning in the water maze enhances survival when the neurons are 5-10 days old (Epp 2007) or 1-2 weeks old (Kee 2007)
• the stress hormone corticosterone decreases new neuron survival when administered for 18-day, but not 9-day, stints during new neuron development (Wong 2006)
• Dobrossy 2003 and Olariu 2005 show the interesting but difficult-to-interpret findings that neuron addition can be increased or decreased depending on the extent of learning and the age of the neuron relative to the learning experience
• The Kee 2007 data suggests that fully mature, 10-week-old neurons are activated during memory retrieval only if they were old enough, at the time of  learning, to be involved in forming the original memory

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Hopefully it’s now clear that a 3 day-old neuron differs from a 3-week-old neuron from a 3-month-old neuron. We have seen examples where the developmental stage of a new neuron influences it’s functional maturation and survival, clues that could someday be used to manipulate adult neurogenesis for therapeutic purposes.

Lastly, comparing many of these timecourses side by side, I’m reminded why I like them so much: I trust them. By examining the same thing at multiple time points, each study inherently has a lot of controls. If you take a single time point out of some of these studies, say the % of new neurons that have excitatory glutamatergic inputs at 14 days-old in Esposito 2005 and Ge 2006, you might wonder, who’s right here? One finds 5%, the other finds 70%. I am curious as to why they differ but I still trust both of these studies and refer to them often because, within their respective timecourses, both 14-day data points make sense and, across studies, the 2 timecourses themselves do generally agree, even if they are slightly shifted.

I’ve always enjoyed making lists. As a kid I can remember writing lists of rhyming words, lists of all the Ocean Pacific clothes I owned, lists of all the people I knew. Many years later, I hope I’ve now made a list that is actually useful.

Adult neurogenesis is now undisputed. Pretty much on a weekly basis there is a new paper that examines both levels of adult hippocampal neurogenesis and behavior, attempting to draw a functional connection. The good news is that the argument for a behavioral function for adult neurogenesis continues to get stronger. The bad news is that there’s a massive pileup of data, and it’s becoming hard to filter through the relevant studies – first you have to find them amongst the 1000+ studies of adult neurogenesis. Then you have to read them. What behaviors are examined? Is there an effect of reducing or enhancing neurogenesis? What method is used to manipulate neurogenesis? What do other studies find that performed a similar analysis?

In this spreadsheet I’ve tried to provide summary answers to these questions. The data can be sorted by the type of behavior examined (e.g. depressive behaviors, memory etc), how neurogenesis was manipulated (e.g. via irradiation, transgenic tools or exogenous factors like anti-mitotic drugs), and behavioral effect.

It should be noted that I essentially took authors’ claims at face value and nothing here should be blindly accepted as evidence for or against a behavioral function for neurogenesis – read the papers! Task, neuronal age, and other methods should all be considered. Also, at this time, I have only entered data for a fraction of the studies, namely those that have claimed to use a technique specific for reducing neurogenesis. In reality, no such technique exists and I’d like to enter the same data for all studies that correlate neurogenesis with behavior, even those that have manipulated neurogenesis using methods that have widespread effects in the nervous system (e.g. exercise, enriched environment).