Taken together, our quantitative morphometric analyses show that during the period between 14 days and 2 months, juvenile neurogenesis makes a substantive contribution to the development of the OB and DG by adding approximately 40% and 25%, respectively, of their total adult compliment of granule cells. This addition is made on top of a relatively stable population of embryonically generated neurons that are not replaced in substantive numbers. Our findings further show that total granule cell numbers do not differ significantly at 2, 3, 12, and 24 months of age in both OB and DG, indicating that the total adult compliment of granule neurons remains stable in wild-type mice over their entire adult life span, including aging. Given that the number of embryonically generated neurons is stable and after 2 months the total number of DG and OB neurons does not increase, adult neurogenesis must preferentially replace juvenile or adult generated, rather than embryonically generated, neurons. In addition, we found no detectable sex differences in either the total numbers of granule neurons in mature adults or in the numbers added to the OB or DG by PNN during juvenile development. It deserves emphasis, that in contrast to the OB and DG, which added neurons during the juvenile period in wild-type mice, the medial habenula exhibited a significant decline in neuronal number of 16.5% between 14 days and 3 months in a manner consistent with the canonical developmental model of over-production of neuronal numbers followed by pruning through naturally occurring cell death (Cowan et al., 1984
). Thus, the continual addition of granule neurons in the OB and DG differs markedly from the developmental program that occurs in most other regions of the CNS. Our findings demonstrate that both the OB and DG continually add substantive numbers of neurons throughout periods of important juvenile behavioral development that include weaning, early environmental and social interactions, and sexual maturation.
Our findings are consistent with and extend older studies that used volumetric estimates of cell number to provide evidence for the addition of granule neurons to both OB and DG during the juvenile period (Bayer et al., 1982
). These studies, conducted prior to the advent of modern stereological quantification and transgenic technology, did not provide accurate information for either total neuronal numbers or precise time course of their addition. Surprisingly, in spite of the surge of interest in adult neurogenesis over the past 20 years, little attention has been paid to neurogenesis during the juvenile period or to accurately documenting the degree to which juvenile neurogenesis might contribute in a developmental manner to the total compliment of granule neurons in adult OB or DG. There have been numerous studies documenting the proportional addition of new granule neurons to the OB or DG by using labeling with cell division markers or activation of genetically encoded lineage markers (Cameron and Mckay, 2001
; Petreanu and Alvarez-Buylla, 2002
; Kempermann et al., 2003
; Lagace et al., 2007
; Ninkovic et al., 2007
; Imayoshi et al., 2008
). These studies all differ substantively from ours in that they are reported by the authors to examine only adult (and not juvenile) neurogenesis and they examine only the relative proportions of labeled neurons. None of these previous studies provides information about total numbers of granule neurons or how this total number is influenced by PNN during either juvenile or adult life. Nevertheless, when carefully examining the reported ages of animals used in previous investigations, our findings are compatible with, and markedly extend, previous studies. For example, our findings indicate that “adult” neurogenesis in mice, when strictly defined as that occurring 2 months of age and older, contributes less than 1% of the total population of granule cells in the DG. This is consistent with Lagace et al., 2007
, but is far less than the 5–6% estimate in juvenile rats from Cameron and Mckay, 2001
. Our findings differ from the conclusion of Imayoshi et al., 2008
, who suggest that the structural contribution of PNN differs between the OB and DG, contributing to continually ongoing neuronal replacement in the former, but neuronal addition in the latter. In contrast, our analysis indicates that PNN makes qualitatively similar contributions to both regions, but that the overall structural contribution to the OB is nearly two-fold larger.
Our findings demonstrate that neurogenesis during the juvenile period between 2 weeks and 2 months of age plays an essential structural developmental role by continually and robustly adding to the total adult compliment of granule neurons. This contrasts with neurogenesis in mature adults older than 2 months, in which there is no net addition of neurons and incorporation of newly generated neurons must be balanced by neuronal loss. This difference suggests that juvenile neurogenesis may subserve somewhat different functions from neurogenesis in the fully mature adult brain (e.g., Wei et al., 2011
), and that studies of neurogenesis conducted on juvenile rodents may be examining developmental events. Seemingly conflicting results in the literature may be due to differences in experimental design that involve manipulations of neurogenesis during periods of pronounced juvenile increases in granule neuron number prior to 2 months of age as compared with studies conducted after 3 months of age where there is stability of total granule neuron number. Furthermore, our findings argue that the age of the animal, not just the age of the granule cells, is a critical consideration with regards to the number of proliferating cells that are likely to be affected by a particular manipulation and, ultimately, to inferences about their functional significance. This information is also important for selecting appropriately aged animals for experimental investigation, even for researchers not directly interested in PNN.
We conducted a behavioral analysis of the same transgenic loss of function mouse model used for the morphological experiments (DNMT1-cKO mice), which have a near complete ablation of all PNN. Our analysis indicated that young adult (3–5 months old) DNMT1-cKO mice exhibited a very specific set of sex-dependent cognitive changes. Standard contextual fear conditioning was intact (Figure ), as was fear to an auditory cue (Figure ) and generalization of fear to both similar and distinct contexts (Figures and ). We did, however, observe increased fear generalization to a novel auditory stimulus (Figure ) as well as a sex-dependent phenotype, with an impairment in incidental contextual learning (Figure ), an enhancement in contextual fear discrimination learning (Figures ) and a reduced reliance on olfactory cues (Figures ). We observed sex differences in control mice that were eliminated by loss of PNN, in the case of contextual discrimination learning as well sex-specific effects of the ablation, in the case of incidental contextual learning. The reduced reliance on olfactory cues exhibited both of these patterns, with the former pattern expressed in the novel context and the latter being expressed in the fear conditioned context.
The prominent sex interactions observed here are consistent with the sexually dimorphic nature of the DG, which exhibits sex differences in synaptic connectivity and plasticity as well as differences in hormone receptors and sensitivity to neurosteroids (Parducz and Garcia-Segura, 1993
; Maren et al., 1994
; Galea, 2008
). It is also consistent with recent findings that OB PNN is important for sex-specific functions such as maternal behavior and male aggression (Sakamoto et al., 2011
). Cahill (2006
) pointed to the necessity of exploring sex differences in neural function “to fully understand a host of brain disorders with sex differences in their incidence and/or nature.” Adolescence is a “core risk” period for the development of mental disorders, including anxiety disorders and depression, that emerge in a sexually dimorphic manner with an average age of onset that corresponds quite well to the transition from juvenile to adult neurogenesis (Hayward and Sanborn, 2002
; Beesdo et al., 2009
). We found that the major structural contribution of PNN occurs during sexual maturation, suggesting that juvenile neurogenesis is likely a key mediator of sex differences in brain function both in healthy individuals and in psychopathological conditions.
Increased fear generalization to a novel auditory stimulus is consistent with a role for impaired PNN in psychopathology, as this is a hallmark of anxiety-related disorders (Sahay et al., 2007
; Lissek et al., 2010
). This finding may also provide evidence for a pattern separation role for PNN, as has recently been proposed (Sahay et al., 2011b
). The enhancement of contextual discrimination learning, however, indicates exactly the opposite, i.e., it is suggestive of improved pattern separation ability. Enhanced contextual discrimination learning in DNMT1-cKO mice is opposite to recent findings that disruption of adult neurogenesis impairs context discrimination (Scobie et al., 2009
; Tronel et al., 2010
) and increasing adult neurogenesis improves it (Sahay et al., 2011a
). Our near complete ablation of PNN, together with the greater difficulty of the discrimination and the absence of odor as a discriminative stimulus (see below) may have contributed to the opposite findings in the present study. Furthermore, our findings suggest that potential involvement of PNN in pattern separation may differ depending on whether the pattern to be separated is a discrete stimulus or a more complex multimodal stimulus. This would be consistent with the different roles that the hippocampus plays in cue and contextual conditioning (e.g., Kim and Fanselow, 1992
Ablation of adult neurogenesis has been previously shown to enhance working memory in a radial arm maze task when repetitive information was presented in a single day (Saxe et al., 2007
). This was interpreted as evidence for a temporal integration function, whereby events experienced close in time are more strongly bound together due to the enhanced plasticity of a cohort of immature granule cells (Aimone et al., 2006
; Deng et al., 2010
). This process interferes with distinguishing between events experienced close in time and release from this interference after ablation of adult neurogenesis allows for enhanced discrimination. The present findings of enhanced contextual discrimination learning after ablation of PNN may, therefore, provide further evidence for a temporal integration function, as both contexts were highly similar and were presented on each day of discrimination training.
The specific impairment of incidental contextual learning indicates that PNN may be particularly important for contextual learning in the absence of the amygdala-dependent neuromodulatory effects foot-shock (Akirav and Richter-Levin, 1999
; McGaugh, 2004
; Huff et al., 2006
). This could be mediated by the enhanced capacity for synaptic plasticity of the immature postnatally generated granule cells (Snyder et al., 2001
; Saxe et al., 2006
; Ge et al., 2007
), and/or their potential preferential recruitment into learning and memory circuits (Ramirez-Amaya et al., 2006
; Kee et al., 2007
), although see (Stone et al., 2010
). Additionally, this finding may provide further evidence for a temporal integration function of PNN. In this case, temporal integration may be necessary to bind together a brief, weakly encoded exploratory experience into a stable contextual representation.
The reduced reliance on olfactory cues we observed after ablation of PNN is consistent with previous findings of impaired olfactory-based fear conditioning after adult neurogenesis ablation when odor was used as an explicit cue (Valley et al., 2009
). In the present experiment odor was only one of several sensory modalities that defined the training and novel contexts. The normal level of contextual fear and equivalent generalization prior to the odor switch indicates that PNN ablation may have increased reliance on auditory, visual, and tactile cues at the expense of olfactory cues. These findings also provide cautionary evidence against purely hippocampus-based interpretations of transgenic methodologies that affect both OB and DG PNN, particularly in contextual discrimination tasks that use different odors to define the contexts (Scobie et al., 2009
; Tronel et al., 2010
; Sahay et al., 2011a
). In the present study, we attempted to avoid this confound as much as possible by keeping olfactory cues constant for the hippocampus-based tasks.
Recent computational work has argued that as postnatally generated granule cells mature they come to support high information encoding of the stimulus dimensions that were encountered during their immature stage (Aimone et al., 2011
). Viewed in the context of the critical structural role of juvenile neurogenesis, such a process takes on new meaning: it provides a mechanism for early postnatal experience to optimize information encoding in the adult brain. This basic concept was proposed in the earliest reports of PNN (Altman and Das, 1965
) and indicates that future research focused on juvenile neurogenesis may add important new insights into the functional significance of PNN as a whole.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.