It’s fun to zoom out and get the big picture sometimes. This is one such picture I took long ago when I wanted to see if staining for zinc transporter 3 effectively labels the mossy fiber axons of the dentate gyrus. You can see by the perfect overlap with calbindin that it does the job, though the staining wasn’t quite as bright and obvious as calbindin. The abundance of zinc in mossy fiber axons is one of the peculiarities of the DG and it underlies numerous synaptic properties of DG neurons.

I think the goal was to build on previous work by Lipp, Ramirez-Amaya, and Routtenberg showing that spatial learning causes “sprouting” of mossy fibers, though when I found out that this phenomenon does not occur in mice the project was aborted.

But what else can you see in this picture?

• clear differential expression of calbindin: DG (lots) > CA1 > CA3 (none), and a scattering of strongly-positive interneurons (e.g. 5 cells where CA3 and CA1 meet)
  • in CA1 you can see calbindin is expressed only in the lower band of cells (see hi res photo if needed; there is a ref for this, somewhere)
• a thin band of calbindin-positive fibers crossing the corpus callosum (CC)
• A small group of cells that are not contacted by the calbindin-positive mossy fiber axons (i.e. beyond CA3) yet do not express somatic calbindin (as seen in CA1). I’m guessing this may be mysterious and ambiguous field CA2.

Neurons are not born with dendrites and spines – they are acquired during a developmental process that takes many weeks (see here & here). During early development, the pattern of formation of dendrites and spines are sculpted by experience, as might be expected if dendrites and spines are anatomical structures involved in processing and storing sensory information. While a body of work has emerged suggesting adult-born neurons are involved in memory and behavior, no one has yet investigated whether experience is capable of altering the dendritic development of these new neurons. This paper by Tronel et al. is therefore very important because it is the first to look at this phenomenon. They show a dramatic acceleration of dendritic development in response to learning, suggesting a potentially powerful role for new neurons in storing and processing information.

It has been 10+ years since Gould et al. and Kempermann et al. showed that learning and enriched environments can enhance the survival of new neurons. These findings are logical precursors to the current study since, if these new neurons have all the necessary components,  they suggest experience could add to the mnemonic functions of the hippocampus. But subsequent studies indicated that experience could also decrease the survival of new neurons. So perhaps structural changes to new neurons that are more relevant to learning might be worth investigating. For example, in many of my own experiments, I have failed to observe learning-induced changes in the number of new neurons but, if the number of dendrites or spines is increased, then there could still be an enhanced ability to process information. Or there could be the removal of some spines and the formation of others, suggesting a transformation in the type of information processed by new neurons. To get at these possibilities, Tronel et al. used doublecortin (DCX) staining and retroviral-GFP labeling to visualize the dendritic structure of newborn neurons in rats that had either remained in their cage (non-learners) or had learned a spatial memory task, the Morris water maze.

Since the authors had previously shown that water maze learning enhances the survival of 1-week-old cells, they first examined whether water maze learning would also alter the dendritic structure of this same population of neurons. Training rats for 6 days and examining new BrdU+/DCX+ neurons the following day (i.e. when new neurons were 14-days-old) they found that the dendritic length and the number of dendritic branches was doubled compared to rats that sat in their home cage.

More remarkable is the duration that the increased dendritic complexity persisted. To get at this question a GFP retrovirus was used to label new neurons born 1 week before learning, since DCX is eventually downregulated and cannot be used to examine dendritic morphology in neurons more than ~2 weeks old.  They found that even 3 months after learning, maze-trained rats had longer dendrites, more branch points, and more dendritic ends. The differences were not trivial either – maze-trained rats had ~70% increases for all of these measures. The number of spines (and therefore putative synapses) was also elevated, 3-fold, and the proportion of spines that showed a mature, mushroom-shaped morphology was 6-fold greater than naive, untrained rats. Since the dendritic morphology of developmentally-born hippocampal neurons can be altered by learning, physiological changes in hormones, and exercise, it is also worth noting that in this study learning did not affect the dendritic complexity of mature granule neurons (though spines were not analyzed in mature neurons and it is possible that learning caused retraction and formation of spines in mature neurons with no overall effect in spine numbers or morphology), suggesting adult-born neurons are particularly sensitive to learning-related activity.

They go on to show that these structural changes in adult-born neurons are even more pronounced when rats learn a more challenging version of the water maze task, where the spatial location of the escape platform moves on a daily basis. They also show these effects require NMDA receptors, which are required for many forms of hippocampal-dependent memory. These additional experiments are notable but it is the basic finding – the magnitude and duration of the structural changes – that is most interesting to me. Here are some of the reasons why:

• previous studies have suggested that adult-born neurons reach a plateau in their functional development by ~8+ weeks of age. These data suggest that new neurons still have a long way to go before they become fully mature.
• the 8w developmental plateau in earlier studies could be normal for animals that have not had any significant life experience (what does this mean when the majority of scientific studies of the brain use naive, deprived animals as models?)
• when experience accelerates the dendritic development of new neurons, are those neurons now less plastic and less likely to contribute to future behaviors? In trying to understand why some studies report behavior deficits after neurogenesis ablation whereas others do not, I’m imagining that 6 weeks of neurogenesis ablation could have major effects on behavior if older (>6w) adult-born neurons are less plastic, perhaps because experience (experimenter handling, group housing, previous learning) accelerated maturation in the way Tronel et al. report. In contrast, if animals have been deprived of learning experiences, 6 weeks of neurogenesis ablation might not have any effects on behavior, because older neurons are still relatively immature and able to compensate.
• depending on how you look at it, it is valid to wonder how a relatively small population of new neurons can be important for behavior. If you now consider the fact that 3-month-old cells still have significant amounts of untapped storage capacity, the cumulative numbers of new neurons generated over 3 months no longer seems so small and insignificant

Reference

Tronel S, Fabre A, Charrier V, Oliet SH, Gage FH, & Abrous DN (2010). Spatial learning sculpts the dendritic arbor of adult-born hippocampal neurons. Proceedings of the National Academy of Sciences of the United States of America, 107 (17), 7963-8 PMID: 20375283

Kitamura et al. (2009) Adult Neurogenesis Modulates the Hippocampus-Dependent Period of Associative Fear Memory. Cell. 139:814-827.

It’s great to see this paper finally in print. At SFN 2008 the authors had a poster that generated a lot of excitement, at least in our circles.  And the poster was quite a sight: there was such a profusion of data that the poster poured off the easel, nearly reaching the floor. With 27 (!) supplemental figures in the final article, one has to wonder if this is the final straw that led to this article.

The authors use an ingenious approach to address an idea that has been floating around for a while: that adult neurogenesis regulates memory turnover in the hippocampus. The hippocampus appears to have a temporary role in memory storage — for instance, lesions to the hippocampus often impair recall of recent memories, but have little or no effect on remote memories. One interpretation of this result is that memories are stored in the hippocampus for only a short time and then transferred to other brain structures (likely neocortex) in a process called systems consolidation. This may then free up space in the hippocampus for new memories. Neurogenesis could be a mechanism for this process: for instance, the addition of new neurons to the dentate gyrus of the hippocampus might destabilize old memories and simultaneously provide a fresh substrate for new memories.

LTP lasts longer in irradiated rats (red circles) that lack adult neurogenesis.

Kitamura et al performed a truly heroic number of experiments to provide evidence that adult neurogenesis is involved in memory turnover in the hippocampus. First, they examined whether neurogenesis is important for the persistence of hippocampal long-term potentiation (LTP) in rats. LTP is an activity-dependent increase in the strength of synaptic connections between neurons, and is studied as a cellular model of memory.

They showed that blocking adult neurogenesis via irradiation extends the duration of hippocampal LTP: LTP lasts less than 2 weeks in control rats but lasts more than 3 weeks in irradiated rats (left). Thus, at a cellular/synaptic level, it would seem that neurogenesis causes memories to be cleared from the hippocampus.

Inactivating the hippocampus with TTX impairs memory in irradiated mice (red circles).

Then, in a series of fear conditioning experiments, the authors demonstrated that adult neurogenesis is in fact involved in the clearance of hippocampal memories in mice. In these experiments neurogenesis was blocked with irradiation or a genetic method prior to conditioning. On the surface, both control and neurogenesis-arrested mice acquired fear conditioning normally, and both groups remembered for at least a month. However, a critical difference emerged when the hippocampus was inactivated during remote recall testing: one month after training, hippocampus inactivation failed to impair recall in control mice, replicating a classic effect and suggesting the memory had been transferred to other brain regions. In contrast, in neurogenesis-arrested mice, hippocampal inactivation did impair recall of the remote memory, indicating that the memory still resided in the hippocampus. Importantly, hippocampal inactivation did impair recall in controls 1 day after conditioning, when memories had not yet been consolidated into the neocortex. Collectively, these findings suggest that adult neurogenesis contributes to the consolidation of memory to extra-hippocampal structures.

The authors then go ahead and answer some of the exciting new questions raised by these findings: For example, what happens if, instead of having reduced neurogenesis, you have increased levels of neurogenesis? Well, consistent with their hypothesis, mice that were given access to running wheels had increased neurogenesis (as expected) but as soon as 7 days after conditioning their memory was no longer hippocampal-dependent, unlike controls. Thus, increasing neurogenesis accelerated memory consolidation.

Another interesting finding was that the hippocampal-dependence of memory was prolonged when mice were irradiated only 11 days before conditioning. Since 11-day-old neurons do not even have functional (excitatory) synapses this suggests that it may not be functional young neurons present at the time of learning, but instead the synaptic integration of new neurons after learning that facilitates clearance of memory from the hippocampus.

Why else is this paper cool? Well, to date, adult neurogenesis has been implicated in many different types of behavior but it has been hard to identify common threads – this paper makes a new prediction that neurogenesis is specifically involved in consolidation. It also reconciles a discrepancy in the field – whether or not young neurons are involved in long-term memory. Some studies have found that reducing neurogenesis impairs long-term memory (Snyder 2005, Jessberger, 2009), but others have not observed this effect (Saxe, 2006). Kitamura’s data shows that even if long-term memory appears normal at a behavioral level, reducing adult neurogenesis will have altered the neural substrate of the memory. And so could these memories really be equivalent? This will be an exciting avenue for future studies to explore: how is hippocampus-dependent long-term memory (in neurogenesis-arrested animals) different from hippocampus-independent long-term memory?

It will also be interesting to further explore how arresting neurogenesis affects memory acquisition. If clearing memories from the hippocampus makes room for new memories, then shouldn’t the acquisition of new memories be hindered when clearance stops? Kitamura’s failure to observe significant impairments in memory acquisition may mean that neurogenesis is involved less in memory clearance than in reorganizing memory networks to include extra-hippocampal structures. That is, instead of simply expunging hippocampal memories, new neurons may facilitate the generation of additional memory representations in extra-hippocampal structures.

Another interesting issue is how the apparent give-and-take relationship between hippocampus and neocortex (presumably) arises. The data seem to suggest that the presence of a hippocampal memory representation prevents a cortical memory representation from arising. It’s as if the memory is a discrete entity that can be passed from hippocampus to cortex: as long as the memory remains in the hippocampus, the cortex is deprived. It would be great to know how –and why– this happens.

Kitamura T, Saitoh Y, Takashima N, Murayama A, Niibori Y, Ageta H, Sekiguchi M, Sugiyama H, & Inokuchi K (2009). Adult neurogenesis modulates the hippocampus-dependent period of associative fear memory. Cell, 139 (4), 814-27 PMID: 19914173