Since it is reasonably well established that hippocampal progenitors are activated by injury resulting in increased numbers of new neurons within the dentate gyrus, the primary purpose of this study was to establish the relevance of this phenomenon. The possibilities regarding the significance of injury-induced neurogenesis include the fact that the generation of new neurons might be beneficial and contribute to recovery of learning and memory and possibly other functions impaired by brain injury. However, neurogenesis may contribute to TBI-related morbidity such as epilepsy, which is relatively common following moderate and severe TBI. Finally, this reservoir of progenitors may be nothing more than a developmental remnant that is incapable of providing functionally relevant neurons into the sophisticated hippocampal circuitry. Although we demonstrate here that injury-induced neurogenesis occurs in a long-lasting manner, we also note that the progenitor pool itself may become depleted (). Since there is an apparently permanent depletion of GFP-expressing early progenitors following injury, a number of questions remain open. There may either be damage to the ipsilateral dentate gyrus that impairs its ability to sustain baseline neurogenesis, or the injury itself may accelerate depletion of the overall progenitor pool. Either possibility suggests that recurrent injuries may not result in such a robust neurogenic response and that this ability to self-repair may be limited.
A number of strategies have emerged to test whether hippocampal neurogenesis has physiologic relevance. Methods to inhibit neurogenesis include systemic or local administration of anti-mitotic agents such as Ara-C that are known to non-specifically target all dividing cells including neural progenitors (Doetsch et al., 1999
; Lau et al., 2009
). Another is to perform cranial irradiation directed to neurogenic zones such as the subventricular zone and dentate gyrus in order to more selectively impair neurogenesis by better controlling what cells are exposed (Hellstrom et al., 2008
; Naylor et al., 2008
; Clelland et al., 2009
; Noonan et al., 2010
). Problems with these approaches include both their lack of specificity and potentially toxic side effects that lead to unwanted immune activation that may affect other mediators of recovery or damage not related to neurogenesis. Thus far, the data supporting a role for adult neurogenesis in improving cognitive function in the uninjured state are compelling, but rely almost exclusively on these various imperfect techniques.
Recently, genetically engineered mice have become available that can regulate neurogenesis in a more precise and temporally controlled manner. One strategy is to inducibly express diptheria toxin or its receptor in progenitor cells, which can then be ablated in a temporally controlled manner (Luquet et al., 2005
; Durieux et al., 2009
). One confounder with this approach is that all progenitors, not just dividing ones, are ablated and therefore the pool becomes depleted and cannot be reactivated. An alternative approach is a transgenic mouse that expresses the herpes simplex virus thymidine kinase (HSV-TK) under the control of the glial fibrillary astrocytic protein (GFAP) promoter. This allows for the inducible ablation of dividing cells that express GFAP, which includes early type 1 hippocampal progenitor cells as well as dividing reactive astrocytes (Sofroniew et al., 1999
; Morshead et al., 2003
; Saxe et al., 2006
). Recently, more specific modified versions of HSV-TK have been shown in nestin-expressing progenitor cells to inducibly ablate early progenitors during normal states as well as after injury (Yu et al., 2008
; Clelland et al., 2009
It remains both controversial and unclear whether adult hippocampal neurogenesis contributes to memory formation during normal adult brain maturation. Some of the conflicting results regarding the impact of neurogenesis on memory have been attributed to the heterogeneous manner in which ablation occurs (Deng et al., 2010
). However, even with the most recently developed genetic techniques, there remains evidence for hippocampal-based Morris water maze and contextual fear conditioning deficits in some models of ablated neurogenesis, but not in others (Dupret et al., 2008
; Imayoshi et al., 2008
; Zhang et al., 2008
; Deng et al., 2009
). In our present study, we note no differences in fear conditioning in mock-injured animals with or without neurogenesis intact () and only very modest differences in Morris water maze-based spatial memory (–). Although contextual fear conditioning is generally thought to be a hippocampal-dependent behavior, it is possible that other brain regions required for this task, such as the amygdala (Goosens and Maren, 2001
; Onishi and Xavier, 2010
), were able to compensate and successfully support the contextual fear learning and memory. For example, even if mice were unable to develop an integrated representation of the context, it is possible that they were able to use a combination of discrete cues in the environment to remember specific aspects of the learning context. In addition, the dentate gyrus has been recently linked to pattern separation within the hippocampus (Deng et al., 2010
), and neither contextual fear conditioning nor our Morris water maze task specifically tested pattern separation. Our Morris water maze task might not have been sensitive enough to detect the subtle differences that ablation of neurogenesis impart on hippocampal function, and it is possible that we might detect a stronger effect of neurogenesis ablation with more challenging behavior parameters or a spatial learning task that expressly tests pattern separation. Although the most recent studies do suggest functional roles for adult neurogenesis within some aspects of memory formation, they may become most readily apparent when the experimental animals undergo induced induction of neurogenesis with an acquired injury as demonstrated here (Deng et al., 2010
Although injury-induced neurogenesis clearly occurs in response to a wide variety of injuries, the mechanisms underlying it need more investigation. The injured brain releases numerous extracellular proteins and ions that may play roles in regulating neurogenesis. Two of the best studied of these, potassium chloride and glutamate, have been implicated in enhancement of proliferation in immature cells while at the same time directing toxicity in more mature cell types (Shi et al., 2007
; Mattson, 2008
). In addition, the injured brain activates both astrocytes and microglia, which are both known to secrete a variety of growth factors as well as immune modulators that may affect progenitor proliferation and survival (Myer et al., 2006
; Bessis et al., 2007
). Finally, the progenitor cells themselves make physical contact with the vasculature so circulating factors such as cytokines and growth factors that increase after injury may also direct some of these effects (Mignone et al., 2004
). Thus, the mechanisms underlying TBI-induced neurogenesis are likely not straightforward nor easily worked out, and therefore remain compelling targets to study, particularly since our data suggest that blockade of such neurogenesis can exacerbate the cognitive deficits results from traumatic brain injury.
Our data show that pharmacogenetic ablation of neurogenesis after brain injury results in impaired learning and memory in the Morris water maze, but ablation of neurogenesis did not modulate the effect of brain injury on motor coordination and learning
(as assessed by the rotarod) or fear learning and memory (as assessed by cued and contextual fear conditioning). It is not surprising that the influence of neurogenesis ablation on the behavioral effects of brain injury was limited to spatial learning and memory, given that the most robust neurogenesis within rodents is known to occur within the hippocampus. While further study is needed to determine why neither the ablation of neurogenesis nor brain injury alone affected contextual fear conditioning, which is generally thought to be a hippocampal-dependent behavior, it is possible that there was compensation of other brain regions (as discussed above). There is also evidence that different sub-regions of the hippocampus are required for learning in the contextual fear conditioning and Morris water maze tasks (Richmond et al., 1999
), and this could account for the different results in these two tasks. In addition, the modeling demonstrated here affects subventricular zone neurogenesis as well. Although it is less likely that these progenitors contribute to the hippocampal-specific behaviors tested here, it remains formally possible that our observations are confounded by ablation of subventricular zone progenitors.
In multiple measures of learning and memory in the Morris water maze, brain-injured mice lacking neurogenesis (Injured + Gan) exhibited impaired performance compared to all control mice, including mice with either brain injury alone or ablation of neurogenesis alone. As discussed in the Results secion, brain-injured mice lacking neurogenesis (Injured + Gan) also showed increased thigmotaxic behavior during learning in the Morris water maze, suggesting that these mice were unable to progress from the initial use of a thigmotaxic search strategy to the use of an efficient spatial strategy to find the hidden platform (Lipp and Wolfer, 1998
; Powell et al., 2004
). Although we interpret this as additional evidence for a learning deficit, we acknowledge that we cannot conclusively rule out the possibility that the increased thigmotaxic behavior could be an indication of other possible cognitive deficits, such as increased anxiety-like behavior. Even if that is the case, however, our data still demonstrate that ablation of adult neurogenesis exacerbates the general cognitive deficits resulting from brain injuries and suggest that enhancing hippocampal neurogenesis after injury might be a viable therapeutic strategy.