Happy mice, raised in cages filled with toys and exercise wheels, are better learners and show less anxiety than their less privileged littermates housed in standard bare cage conditions. Happy mice also display more neurogenesis in their hippocampi than standard-caged mice, because exercise and environmental stimulation seems to crank up the birth of new neurons in the subgranular zone of the dentate gyrus (see ARF related news story). These new neurons have been assumed to contribute to environmental enrichment effects. But do they? A paper in today’s Nature Neuroscience online suggests that hippocampal neurogenesis is not required for mice to display better spatial learning and lower anxiety in an enriched environment. The results, from Rene Hen and colleagues at Columbia University in New York, stand in direct contradiction to a study published last year by French researchers, which found that turning off neurogenesis blocked environment-induced memory enhancement in rats (Bruel-Jungerman et al., 2005 ).

While differences between studies, or the existence of different pathways to learning, might account for opposite outcomes, the new evidence means the jury must re-evaluate the role of adult neurogenesis in modifying learning and behavior. The verdict is of interest to Alzheimer researchers, because environmental enrichment has been shown to decrease both amyloid pathology and cognitive defects in mouse models of AD (see ARF related news story and Jankowsky et al., 2005).

To investigate the role of neurogenesis in environment, joint first authors Dar Meshi, Michael Drew, and their colleagues used targeted radiation to kill off neuronal progenitors in the hippocampus before upgrading their mice to deluxe accommodations. The researchers showed that bromodeoxyuridine and doublecortin—neurogenesis markers that otherwise appear in the hippocampus after environmental enrichment—were absent from irradiated animals.

Six weeks later, the irradiated and control mice were put through several behavioral tests, including those for anxiety, habituation to a new environment, and for spatial learning in the Morris water maze. In each case, environmental enrichment improved the animals’ performance, and that improvement was unaffected by previous irradiation. From this the authors concluded that, at least for these tasks, the effects of environment do not require adult neurogenesis.

The findings contrast those of Elodie Bruel-Jungerman and colleagues, who reported that ablating neurogenesis by injecting the anti-mitotic drug methylazoxymethanol acetate (MAM) totally blocked the improvement in long-term memory that followed environmental enrichment in rats. The studies use different species, and different ways of knocking out neurogenesis, leading Meshi et al. to speculate that the systemic administration of MAM versus localized irradiation may be one explanation for the discrepancies. The studies also tested the animals on different tasks, raising the possibility that some behavioral changes require neurogenesis while others do not. In support of this, the Cornell group previously showed that hippocampal irradiation blocks the ability of antidepressant drugs to reduce the anxiety response in mice (Santarelli et al., 2003). For the neurogenesis-independent effects, increased levels of growth factors, dendritic branching, or synaptogenesis are all potential explanations for the environmental enrichment observed in irradiated mice.—Pat McCaffrey.

Reference:
Meshi D, Drew MR, Saxe m, Ansorge MS, David D, Santarelli L, Malapani C, Moore H, Hen R. Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nature Neuroscience. 30 April 2006. Advanced online publication. Abstract

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  1. The interesting part of this story is that the authors can show that learning occurs following irradiation. This seems to indicate that learning can occur independently of hippocampal neurogenesis, but there are a few aspects of the study that make me favor a much more cautious interpretation of the results.

    First, the behavioral data, as presented, raise some concerns. The latency-to-feed measure they use is not a standard measure of anxiety, and I would like to see this data backed up by either an elevated plus maze or open field experiments—these are far more common tests of anxiety in rodents. Second, the water maze data show that the enriched groups start off at noticeably lower levels than the standard groups, although the difference is not significant by their analysis. On day 5 the difference is barely significant, but, in fact, a regression through the learning curves for each group would not reveal a significant difference. All of the groups decrease their path length by about 700 cm in 5 days. It is not clear how the statistics for the last data points were conducted, either, because the degree of freedom suggest an n = 60 animals with only two groups (d.f. = 1, 60).

    Their claim that neurogenesis might do different things in mice and rats is also very unusual (second-last paragraph). There is no evidence I know of to substantiate this speculation, but this statement may well need to be challenged by a more comprehensive study in the future.

    Finally, our data (van Praag et al., 2002), suggest that new neurons take at least 5 weeks to become functional units in the CNS. This raises a curious point: Could new neurons actually be incorporated into and take part in a behavioral task that is only learned over 5 days? Probably not, though it might be claimed that an “existing pool” of new neurons might offer the means with which to learn more quickly. Obviously, this is not the case following the irradiation, which seems to have knocked out neurogenesis. In contrast, the original studies by Greenough and colleagues showed that environmental enrichment is also strongly associated with enhanced synaptogenesis. Our own studies (Eadie et al., 2005; Redila et al., 2006) show that both neurogenesis and synaptogenesis are increased by exercise, a major component of their enriched environment. Thus, it may be that this enhanced synaptogenesis is the main player in environmental enrichment’s benefits for learning and not neurogenesis. Unfortunately, the authors did not investigate this and leave us with a tantalizing conundrum that warrants a more involved investigation.

    References:

    . Increased density of multiple-head dendritic spines on medium-sized spiny neurons of the striatum in rats reared in a complex environment. Neurobiol Learn Mem. 1996 Sep;66(2):93-6. PubMed.

    . Voluntary exercise alters the cytoarchitecture of the adult dentate gyrus by increasing cellular proliferation, dendritic complexity, and spine density. J Comp Neurol. 2005 May 23;486(1):39-47. PubMed.

    . Hippocampal cell proliferation is reduced following prenatal ethanol exposure but can be rescued with voluntary exercise. Hippocampus. 2006;16(3):305-11. PubMed.

  2. The new study by Meshi et al. convincingly demonstrates that hippocampal neurogenesis is not required for behavior improvements following environmental enrichment. The experimental design is exceptionally clean, and the work is beautifully done. In fact, the only significant hitch is that the results fly in the face of what we perhaps had expected these newborn neurons to do in the brain, and that this outcome is in direct opposition to the findings of a previously published study of enrichment-induced neurogenesis.

    The previous study from Bruel-Jungerman et al., 2005 describes an essentially similar experiment to address the role of enrichment-associated neurogenesis in the hippocampus. Using a chemical antimitotic agent rather than X-irradiation to suppress hippocampal neurogenesis, Bruel-Jungerman et al. found that enrichment-associated improvements in long-term recognition behavior were eliminated in the treated mice. In contrast to Meshi et al., Bruel-Jungerman et al. concluded that enhanced neurogenesis resulting from enrichment is crucial for improved behavioral performance.

    This set of papers is not the first instance of two seemingly similar experiments arriving at diametrically opposed results. Having been in the middle of one such conflict, I would suggest that rather than trying to identify who is right and who is wrong, we instead take the opportunity to recognize what the studies may reveal about the underlying biology. Given that both studies were carefully controlled and well designed, we might be able to refine our understanding of the significant variables and consequent outcomes through a careful comparison of the two methodologies.

    There are several major differences between the experiments of Meshi and Bruel-Jungerman that may be significant in assessing these studies. The most obvious differences are in the species and gender of the animals under study: Meshi tested female mice, while Bruel-Jungerman used male rats. These differences may not be trivial: we currently understand very little about how species or gender interact with environment to affect outcome.

    Second, Meshi used focal X-irradiation to ablate neural progenitors; Bruel-Jungerman used systemic methazoxy methanol acetate (MAM). Each method of suppressing cell division comes with its own benefits and limitations. X-irradiation allows nearly complete removal of the dividing progenitors and can be focally applied so that systemic effects are substantially reduced. However, irradiation is also associated with a prolonged inflammatory response in nearby tissue, and Meshi et al. found that the brain contained elevated CD68-positive microglia/macrophage staining for more than 1 month after irradiation treatment. Although the authors were careful not to begin their study until the overt signs of inflammation had resolved, the presence of prolonged microglial activation may change the local brain environment in ways we do not yet recognize.

    In contrast, Bruel-Jungerman et al. used systemic MAM administration to knock down hippocampal progenitor proliferation during exposure to enrichment. The main advantages to MAM are that it doesn't require expensive equipment such as an x-ray machine, and that it is not known to induce an inflammatory response (although this has not been carefully examined). There are at least two main drawbacks to MAM. First, it is delivered systemically. While it does not grossly alter motor activity or general health, it likely has subacute effects on many systems that require active cell proliferation (gut, blood, etc.). Second, MAM interrupts proliferation by methylating DNA during cell division; one could imagine that it may also act to silence gene expression in postmitotic cells as chromatin opens for transcription. Gene transcription is required for the formation of new synapses, and this process may be interrupted along with neurogenesis in the enriched mice.

    A third difference of note between the two studies is the measure each study used to assess what role the enrichment-associated neurogenesis had in learning and memory. Meshi et al. tested their mice in two behavioral tasks: the standard Morris water maze and a novelty-suppressed feeding test. Bruel-Jungerman et al. assessed animals using a novel-object recognition task. The three tasks vary greatly in nature, and likely tap different neural networks outside of the hippocampus. Comparing the results between studies, therefore, becomes almost impossible; instead, each must be assessed on its own terms. However, it should be noted that Bruel-Jungerman et al. observed the greatest differences between MAM-treated and saline-injected enriched animals at the longest retention intervals (48 hours). The Morris water maze has no such long-term memory requirement: mice are trained and tested over 9 consecutive days during which the task is rehearsed 3 times per day. It is possible that the addition of a final probe trial 24-48 hours after the last training session would reveal neurogenesis-dependent memory that was not needed to perform the task during the training period.

    Enrichment is a variable protocol, and each study uses slightly different parameters to produce their enriched housing. In particular, Meshi et al. included exercise wheels, while Bruel-Jungerman et al. did not. Access to exercise may increase the extent of angiogenesis resulting from enrichment, and running alone was shown to be sufficient to reproduce the effects of full-scale enrichment on neuronal survival in the hippocampus (van Praag et al., 1999). The duration of enriched housing before behavioral testing also differed between the Meshi and Bruel-Jungerman studies. The longer enrichment period provided by Meshi et al. may have allowed for other functional and/or morphological changes associated with enrichment (synaptogenesis, increased LTP, greater dendritic branching, etc. (for review, see van Praag et al., 2000), to compensate for the loss of newborn neurons.

    In summary, the studies by Meshi et al. and Bruel-Jungerman et al. show that even the most seemingly straightforward experiments are often more complicated than we realize. Biology is usually not simple, and sometimes it’s the messy conflicting data that will in time provide the most useful insight.

    References:

    . New neurons in the dentate gyrus are involved in the expression of enhanced long-term memory following environmental enrichment. Eur J Neurosci. 2005 Jan;21(2):513-21. PubMed.

    . Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci. 1999 Mar;2(3):266-70. PubMed.

    . Neural consequences of environmental enrichment. Nat Rev Neurosci. 2000 Dec;1(3):191-8. PubMed.

  3. Activity level and respiration do co-vary when rodents play in an enriched environment. And Kheirandish et al. (2005) showed that hypoxia adversely affected working memory specially in male rats, and the dendritic branching and dopamine transport in the frontal cortex—not the hippocampus—of those male rats.

    The implications of the above finding for Alzheimer's and depression can be extrapolated from this study. Thomas et al. (2006) found a decrease in serotonin transporter (a dopamine precursor) binding in the prefrontal cortex in Alzheimer disease subjects compared to both control and, ironically, depressed elderly, postmortem. They found no difference, however, in serotonin transporter binding between the depressed and the control subjects. That also held true when comparing Alzheimer disease subjects with and without depression. Serotonin transporter binding reduction does not increase in Alzheimer patients who also have major depression.

    References:

    . Intermittent hypoxia during development induces long-term alterations in spatial working memory, monoamines, and dendritic branching in rat frontal cortex. Pediatr Res. 2005 Sep;58(3) PubMed.

    . A study of the serotonin transporter in the prefrontal cortex in late-life depression and Alzheimer's disease with and without depression. Neuropathol Appl Neurobiol. 2006 Jun;32(3):296-303. PubMed.

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References

News Citations

  1. Exercise Helps Mouse Elders Learn, Generate New Neurons
  2. Sorrento: More Fun, Less Amyloid for Transgenic Mice

Paper Citations

  1. . New neurons in the dentate gyrus are involved in the expression of enhanced long-term memory following environmental enrichment. Eur J Neurosci. 2005 Jan;21(2):513-21. PubMed.
  2. . Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer's disease. J Neurosci. 2005 May 25;25(21):5217-24. PubMed.
  3. . Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science. 2003 Aug 8;301(5634):805-9. PubMed.
  4. . Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nat Neurosci. 2006 Jun;9(6):729-31. PubMed.

Further Reading

Papers

  1. . Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. Eur J Neurosci. 2003 May;17(10):2042-6. PubMed.
  2. . Environmental enrichment mitigates cognitive deficits in a mouse model of Alzheimer's disease. J Neurosci. 2005 May 25;25(21):5217-24. PubMed.
  3. . Effects of voluntary exercise on synaptic plasticity and gene expression in the dentate gyrus of adult male Sprague-Dawley rats in vivo. Neuroscience. 2004;124(1):71-9. PubMed.
  4. . Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nat Neurosci. 2006 Jun;9(6):729-31. PubMed.

Primary Papers

  1. . Hippocampal neurogenesis is not required for behavioral effects of environmental enrichment. Nat Neurosci. 2006 Jun;9(6):729-31. PubMed.