Age-Dependent Remarkable Regenerative Potential of the Dentate Gyrus Provided by Intrinsic Stem Cells.

Multiple insults to the brain lead to neuronal cell death, thus raising the question to what extent can lost neurons be replenished by adult neurogenesis. Here we focused on the hippocampus and especially the dentate gyrus (DG), a vulnerable brain region and one of the two sites where adult neuronal stem cells (NSCs) reside. While adult hippocampal neurogenesis was extensively studied with regard to its contribution to cognitive enhancement, we focused on their underestimated capability to repair a massively injured, nonfunctional DG. To address this issue, we inflicted substantial DG-specific damage in mice of either sex either by diphtheria toxin-based ablation of >50% of mature DG granule cells (GCs) or by prolonged brain-specific VEGF overexpression culminating in extensive, highly selective loss of DG GCs (thereby also reinforcing the notion of selective DG vulnerability). The neurogenic system promoted effective regeneration by increasing NSCs proliferation/survival rates, restoring a nearly original DG mass, promoting proper rewiring of regenerated neurons to their afferent and efferent partners, and regaining of lost spatial memory. Notably, concomitantly with the natural age-related decline in the levels of neurogenesis, the regenerative capacity of the hippocampus also subsided with age. The study thus revealed an unappreciated regenerative potential of the young DG and suggests hippocampal NSCs as a critical reservoir enabling recovery from catastrophic DG damage.SIGNIFICANCE STATEMENT Adult hippocampal neurogenesis has been extensively studied in the context of its role in cognitive enhancement, but whether, and to what extent can dentate gyrus (DG)-resident neural stem cells drive regeneration of an injured DG has remained unclear. Here we show that DG neurogenesis acts to replace lost neurons and restore lost functions even following massive (>50%) neuronal loss. Age-related decline of neurogenesis is paralleled by a progressive decline of regenerative capacity. Considering also the exceptional vulnerability of the DG to insults, these findings provide a further rationale for maintaining DG neurogenesis in adult life.

Gray-Level Co-Occurrence Matrix Analysis of Granule Neurons of the Hippocampal Dentate Gyrus Following Cortical Injury.

Traumatic brain injury (TBI) is a main cause of death and disabilities in young adults. Although learning and memory impairments are a major clinical manifestation of TBI, the consequences of TBI on the hippocampus are still not well understood. In particular, how lesions to the sensorimotor cortex damage the hippocampus, to which it is not directly connected, is still elusive. Here, we study the effects of sensorimotor cortex ablation (SCA) on the hippocampal dentate gyrus, by applying a highly sensitive gray-level co-occurrence matrix (GLCM) analysis. Using GLCM analysis of granule neurons, we discovered, in our TBI paradigm, subtle changes in granule cell (GC) morphology, including textual uniformity, contrast, and variance, which is not detected by conventional microscopy. We conclude that sensorimotor cortex trauma leads to specific changes in the hippocampus that advance our understanding of the cellular underpinnings of cognitive impairments in TBI. Moreover, we identified GLCM analysis as a highly sensitive method to detect subtle changes in the GC layers that is expected to significantly improve further studies investigating the impact of TBI on hippocampal neuropathology.

Hippocampal Damage During Mechanical Ventilation in Trendelenburg Position: A Secondary Analysis of an Experimental Study on the Prevention of Ventilator-Associated Pneumonia.

We previously corroborated benefits of the Trendelenburg position in the prevention of ventilator-associated pneumonia (VAP). We now investigate its potential effects on the brain versus the semirecumbent position. We studied 17 anesthetized pigs and randomized to be ventilated and positioned as follows: duty cycle (TI/TTOT) of 0.33, without positive end-expiratory pressure (PEEP), placed with the bed oriented 30° in anti-Trendelenburg (control group); positioned as in the control group, with TI/TTOT adjusted to achieve an expiratory flow bias, PEEP of 5 cm H2O (IRV-PEEP); positioned in 5° TP and ventilated as in the control group (TP). Animals were challenged into the oropharynx with Pseudomonas aeruginosa. We assessed hemodynamic parameters and systemic inflammation throughout the study. After 72 h, we evaluated incidence of microbiological/histological VAP and brain injury. Petechial hemorrhages score was greater in the TP group (P = 0.013). Analysis of the dentate gyrus showed higher cell apoptosis and deteriorating neurons in TP animals (P < 0.05 vs. the other groups). No differences in systemic inflammation were found among groups. Cerebral perfusion pressure was higher in TP animals (P < 0.001), mainly driven by higher mean arterial pressure. Microbiological/histological VAP developed in 0%, 67%, and 86% of the animals in the TP, control, and IRV-PEEP groups, respectively (P = 0.003). In conclusion, the TP prevents VAP; yet, we found deleterious neural effects in the dentate gyrus, likely associated with cerebrovascular modification in such position. Further laboratory and clinical studies are mandatory to appraise potential neurological risks associated with long-term TP.

Memory retrieval-induced activation of adult-born neurons generated in response to damage to the dentate gyrus.

The dentate gyrus (DG) is a neurogenic structure that exhibits functional and structural reorganization after injury. Neurogenesis and functional recovery occur after brain damage, and the possible relation between both processes is a matter of study. We explored whether neurogenesis and the activation of new neurons correlated with DG recovery over time. We induced a DG lesion in young adult rats through the intrahippocampal injection of kainic acid and analyzed functional recovery and the activation of new neurons after animals performed a contextual fear memory task (CFM) or a control spatial exploratory task. We analyzed the number of BrdU+ cells that co-localized with doublecortin (DCX) or with NeuN within the damaged DG and evaluated the number of cells in each population that were labelled with the activity marker c-fos after either task. At 10 days post-lesion (dpl), a region of the granular cell layer was devoid of cells, evidencing the damaged area, whereas at 30 dpl this region was significantly smaller. At 10 dpl, the number of BrdU+/DCX+/c-fos positive cells was increased compared to the sham-lesion group, but CFM was impaired. At 30 dpl, a significantly greater number of BrdU+/NeuN+/c-fos positive cells was observed than at 10 dpl, and activation correlated with CFM recovery. Performance in the spatial exploratory task induced marginal c-fos immunoreactivity in the BrdU+/NeuN+ population. We demonstrate that neurons born after the DG was damaged survive and are activated in a time- and task-dependent manner and that activation of new neurons occurs along functional recovery.

Dentate network activity is necessary for spatial working memory by supporting CA3 sharp-wave ripple generation and prospective firing of CA3 neurons.

Complex spatial working memory tasks have been shown to require both hippocampal sharp-wave ripple (SWR) activity and dentate gyrus (DG) neuronal activity. We therefore asked whether DG inputs to CA3 contribute to spatial working memory by promoting SWR generation. Recordings from DG and CA3 while rats performed a dentate-dependent working memory task on an eight-arm radial maze revealed that the activity of dentate neurons and the incidence rate of SWRs both increased during reward consumption. We then found reduced reward-related CA3 SWR generation without direct input from dentate granule neurons. Furthermore, CA3 cells with place fields in not-yet-visited arms preferentially fired during SWRs at reward locations, and these prospective CA3 firing patterns were more pronounced for correct trials and were dentate-dependent. These results indicate that coordination of CA3 neuronal activity patterns by DG is necessary for the generation of neuronal firing patterns that support goal-directed behavior and memory.

Differential origin of the activation of dorsal and ventral dentate gyrus granule cells during paradoxical (REM) sleep in the rat.

We recently demonstrated that granule cells located in the dorsal dentate gyrus (dDG) are activated by neurons located in the lateral supramammillary nucleus (SumL) during paradoxical sleep (PS) hypersomnia. To determine whether these neurons are glutamatergic and/or GABAergic, we combined FOS immunostaining with in situ hybridization of vesicular glutamate transporter 2 (vGLUT2, a marker of glutamatergic neurons) or that of the vesicular GABA transporter (vGAT, a marker of GABAergic neurons) mRNA in rats displaying PS hypersomnia (PSR). We found that 84 and 76 % of the FOS+ SumL neurons in PSR rats expressed vGLUT2 and vGAT mRNA, respectively. Then, we examined vGLUT2 and FOS immunostaining in the dorsal and ventral DG of PSR rats with a neurochemical lesion of the Sum. In PSR-lesioned animals but not in sham animals, nearly all vGLUT2+ fibers and FOS+ neurons disappeared in the dDG, but not in the ventral DG (vDG). To identify the pathway (s) responsible (s) for the activation of the vDG during PS hypersomnia, we combined Fluorogold (FG) injection in the vDG of PSR rats with FOS staining. We found a large number of neurons FOS-FG+, specifically in the medial entorhinal cortex (ENTm). Altogether, our results suggest that SumL neurons with a unique dual glutamatergic and GABAergic phenotype are responsible for the activation of the dDG during PS hypersomnia, while vDG granule neurons are activated by ENTm cortical neurons. These results suggest differential mechanisms and functions for the activation of the dDG and the vDG granule cells during PS.

Traumatic Brain Injury Causes Aberrant Migration of Adult-Born Neurons in the Hippocampus.

Traumatic brain injury (TBI) promotes neural stem/progenitor cell (NSC) proliferation in an attempt to initiate innate repair mechanisms. However, all immature neurons in the CNS are required to migrate from their birthplace to their final destination to develop into functional neurons. Here we assessed the destination of adult-born neurons following TBI. We found that a large percentage of immature neurons migrated past their normal stopping site at the inner granular cell layer (GCL), and became misplaced in the outer GCL of the hippocampal dentate gyrus. The aberrant migration of adult-born neurons in the hippocampus occurred 48 hours after TBI, and lasted for 8 weeks, resulting in a great number of newly generated neurons misplaced in the outer GCL in the hippocampus. Those misplaced neurons were able to become mature and differentiate into granular neurons, but located ectopically in the outer GCL with reduced dendritic complexity after TBI. The adult-born neurons at the misplaced position may make wrong connections with inappropriate nearby targets in the pre-existing neural network. These results suggest that although stimulation of endogenous NSCs following TBI might offer new avenues for cell-based therapy, additional intervention is required to further enhance successful neurogenesis for repairing the damaged brain.

The role of the ventral dentate gyrus in olfactory pattern separation.

Dorsoventral lesion studies of the hippocampus have indicated that the dorsal axis of the hippocampus is important for spatial processing and the ventral axis of the hippocampus is important for olfactory learning and memory and anxiety. There is some evidence to suggest that the ventral CA3 and ventral CA1 conduct parallel processes for pattern completion and temporal processing, respectively. Studies have indicated that the dorsal dentate gyrus (DG) is importantly involved in processes reflecting underlying pattern separation activity for spatial information. However, the ventral DG is less understood. The current study investigated the less-understood role of the ventral DG in olfactory pattern separation. A series of odor stimuli that varied on only one level, number of carbon chains (methyl groups), was used in a matching-to-sample paradigm in order to investigate ventral DG involvement in working memory for similar and less similar odors. Rats with ventral DG lesions were impaired at delays of 60 sec, but not at delays of 15 sec. A memory-based pattern separation effect was observed performance was poorest with only one carbon chain separation between trial odors and was highest for trials with four separations. The present study indicates that the ventral DG plays an important role in olfactory learning and memory processes for highly similar odors. The results also indicate a role for the ventral DG in pattern separation for odor information, which may have further implications for parallel processing across the dorsoventral axis for the DG in spatial (dorsal) and olfactory (ventral) pattern separation.

Mechanisms of neurogenesis in the normal and injured adult brain.

Even in the adult brain, new neurons are continuously generated from endogenous neural stem cells that reside in two restricted regions: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus of the hippocampus. These new neurons are integrated into the mature neuronal circuitry and become involved in various functions, thereby contributing to structural and functional plasticity in the adult brain. In this review, we summarize our recent findings on the regulatory mechanisms of SVZ neurogenesis under physiological and pathological conditions in various animal models. Some of these findings were presented in the Kitazato Prize Lecture at Keio University School of Medicine in 2011.

TGF-ß superfamily gene expression and induction of the Runx1 transcription factor in adult neurogenic regions after brain injury.

Traumatic brain injury (TBI) increases neurogenesis in the forebrain subventricular zone (SVZ) and the hippocampal dentate gyrus (DG). Transforming growth factor-ß (TGF-ß) superfamily cytokines are important regulators of adult neurogenesis, but their involvement in the regulation of this process after brain injury is unclear. We subjected adult mice to controlled cortical impact (CCI) injury, and isolated RNA from the SVZ and DG at different post-injury time points. qPCR array analysis showed that cortical injury caused significant alterations in the mRNA expression of components and targets of the TGF-ß, BMP, and activin signaling pathways in the SVZ and DG after injury, suggesting that these pathways could regulate post-injury neurogenesis. In both neurogenic regions, the injury also induced expression of Runt-related transcription factor-1 (Runx1), which can interact with intracellular TGF-ß Smad signaling pathways. CCI injury strongly induced Runx1 expression in activated and proliferating microglial cells throughout the neurogenic regions. Runx1 protein was also expressed in a subset of Nestin- and GFAP-expressing putative neural stem or progenitor cells in the DG and SVZ after injury. In the DG only, these Runx1+ progenitors proliferated. Our data suggest potential roles for Runx1 in the processes of microglial cell activation and proliferation and in neural stem cell proliferation after TBI.

Insulin growth factor-I promotes functional recovery after a focal lesion in the dentate gyrus.

The adult brain is plastic and able to reorganize structurally and functionally after damage. Growth factors are key molecules underlying the recovery process and among trophic molecules, Insulin-Like Growth Factor-I (IGF-I) is of particular interest given that it modulates neuronal and glial responses in the hippocampus including neurogenesis, which has been proposed as a mechanism of neurorepair. In this study we analyzed the effect of intracerebroventricular chronic infusion of IGF-I on functional recovery and morphological restoration after the induction of an excitotoxic lesion in the dentate gyrus (DG) of young-adult rats. Our results show that the lesion impairs contextual fear memory which is a DG dependent task, but not cued fear memory or performance in the open field motor task, which are independent of the DG integrity. Chronic administration of IGF-I, but not vehicle, promotes functional recovery to control levels in injured subjects. Analysis in NeuN immunoprocessed tissue revealed that the lesion volume was not different between groups and that the DG was not evidently restructured in the IGF-I treated group. Glial fibrillary acidic protein (GFAP) analysis revealed an increased astrocytic response in the injured region in both groups and Doublecortin (DCX) analysis showed a similar increase in number of newly born neurons in both groups. However, a remarkable increase in young neurons dendritic arborization was observed in the IGF-I treated group. These results provide evidence for IGF-I as a molecule mediating functional and cellular plasticity during a reorganization process after damage to a neurogenic niche.

Excitatory amino acid transporter 2 and excitatory amino acid transporter 1 negatively regulate calcium-dependent proliferation of hippocampal neural progenitor cells and are persistently upregulated after injury.

Using a transgenic mouse (Mus musculus) in which nestin-expressing progenitors are labeled with enhanced green fluorescent protein, we previously characterized the expression of excitatory amino acid transporter 2 (GltI) and excitatory amino acid transporter 1 (Glast) on early neural progenitors in vivo. To address their functional role in this cell population, we manipulated their expression in P7 neurospheres isolated from the dentate gyrus. We observed that knockdown of GltI or Glast was associated with decreased bromodeoxyuridine incorporation and neurosphere formation. Moreover, we determined that both glutamate transporters regulated progenitor proliferation in a calcium-dependent and metabotropic glutamate receptor-dependent manner. To address the relevance of this in vivo, we utilized models of acquired brain injury, which are known to induce hippocampal neurogenesis. We observed that GltI and Glast were specifically upregulated in progenitors following brain injury, and that this increased expression was maintained for many weeks. Additionally, we found that recurrently injured animals with increased expression of glutamate transporters within the progenitor population were resistant to subsequent injury-induced proliferation. These findings demonstrate that GltI and Glast negatively regulate calcium-dependent proliferation in vitro and that their upregulation after injury is associated with decreased proliferation after brain trauma.

Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus.

Hippocampal injury-associated learning and memory deficits are frequent hallmarks of brain trauma and are the most enduring and devastating consequences following traumatic brain injury (TBI). Several reports, including our recent paper, showed that TBI brought on by a moderate level of controlled cortical impact (CCI) induces immature newborn neuron death in the hippocampal dentate gyrus. In contrast, the majority of mature neurons are spared. Less research has been focused on these spared neurons, which may also be injured or compromised by TBI. Here we examined the dendrite morphologies, dendritic spines, and synaptic structures using a genetic approach in combination with immunohistochemistry and Golgi staining. We found that although most of the mature granular neurons were spared following TBI at a moderate level of impact, they exhibited dramatic dendritic beading and fragmentation, decreased number of dendritic branches, and a lower density of dendritic spines, particularly the mushroom-shaped mature spines. Further studies showed that the density of synapses in the molecular layer of the hippocampal dentate gyrus was significantly reduced. The electrophysiological activity of neurons was impaired as well. These results indicate that TBI not only induces cell death in immature granular neurons, it also causes significant dendritic and synaptic degeneration in pathohistology. TBI also impairs the function of the spared mature granular neurons in the hippocampal dentate gyrus. These observations point to a potential anatomic substrate to explain, in part, the development of posttraumatic memory deficits. They also indicate that dendritic damage in the hippocampal dentate gyrus may serve as a therapeutic target following TBI.

NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.

We present a model for the study of injury-induced neurogenesis in the dentate gyrus (DG) in murine organotypic hippocampal slice cultures (OHCs). A brief exposure of 8-day-old hippocampal slice cultures to the glutamate receptor agonist N-methyl-d-aspartate (NMDA; 20-50µM for 30 min) caused a selective excitotoxic injury in the CA1 subfield of the hippocampus that matured over a period of 24h. The insult resulted in a prominent up-regulation of proliferating nuclei within the OHC dentate gyrus (DG), and a corresponding increase in Ki67/doublecortin double-positive cells in the SGZ of the dentate gyrus. 5-bromo-2-deoxyuridine (BrdU)-labelling of the OHCs for three days subsequent to the NMDA exposure revealed significantly increased BrdU incorporation within the DG (SGZ and GCL) of the hippocampus. Doublecortin immunofluorescence indicated a concurrent up-regulation of neuronal precursor cells specifically in the SGZ and GCL. Significantly increased BrdU incorporation could be detected up to 6-9 days after termination of the NMDA exposure. The model presented here enables easy manipulation and follow-up of injury-induced neuroblast proliferation in the DG that is amenable to the study of transgenic mice.

Dentate gyrus is necessary for disambiguating similar object-place representations.

Objects are often remembered with their locations, which is an important aspect of event memory. Despite the well-known involvement of the hippocampus in event memory, detailed intrahippocampal mechanisms are poorly understood. In particular, no experimental evidence has been provided in support of the role of the dentate gyrus (DG) in disambiguating such events, even though computational models suggest otherwise. In the current study, rats encountered multiple objects in different locations and were required to discriminate the object-place paired associates for reward. Specifically, two different objects appeared in one of two locations (arms in a radial maze) that were relatively close to each other. Different objects were rewarded depending on the arm in which the objects appeared. The rats with colchicine-based, dorsal DG (dDG) lesions showed severe and sustained impairment in disambiguating the objects compared with controls (Experiment 1). The dDG-lesioned rats were normal, however, in discriminating four different objects presented (Experiment 2) in the same locations as in Experiment 1. Finally, when the two different objects used in Experiment 1 were presented at two remote locations (Experiment 3) involving less overlap between arm-associated contextual cues, the dDG-lesioned animals showed initial deficits in discriminating the objects, but gradually relearned the task, in contrast to the sustained deficits observed in Experiment 1. These results collectively suggest that the DG is necessary when the similarity is maximal between object-place paired associates due to overlapping object and/or spatial information, whereas its role becomes minimal as the overlap in either object or spatial information decreases.

Object exploration and reactions to spatial and nonspatial changes in dentate gyrus lesioned rats.

In order to investigate the possible involvement the DG in spatial and object recognition memory, we have opted for a non-associative task where no explicit reward was present. Colchicine was used for bilateral DG lesions for its well-known specificity for DG lesion. Colchicine-induced lesions produce severe damage in the granule cells of DG, while minimally affecting pyramidal cells in CA1 and CA3. The main results are as follows: The overall habituation to the familiar environment in DG lesioned rats was decreased than in sham operated rats. There was no significant impairment in detecting spatial novelty. Lesions of the DG did not affect the detection of a novel object placed in a familiar location. Considering both the impaired habituation and the generally intact detection of spatial changes, we suggest that exploratory activity in relation to the entire environment and to the particular objects is thought to be subserved by diverse nervous substrate, and testing in the given conditions allows for their differential estimation.

Expression of protein phosphatase 2B (calcineurin) subunit A isoforms in rat hippocampus after traumatic brain injury.

Calcineurin (CaN) is a calcium/calmodulin-dependent phosphatase directly activated by calcium as a result of neuronal activation that is important for neuronal function. CaN subunit isoforms are implicated in long-term potentiation (LTP), long-term depression (LTD), and structural plasticity. CaN inhibitors are also beneficial to cognitive outcomes in animal models of traumatic brain injury (TBI). There are known changes in the CaN A (CnA) subunit following fluid percussion injury (FPI). The CnA subunit has two isoforms: CnAalpha and CnAbeta. The effect of moderate controlled cortical impact (CCI) on distribution of CnA isoforms was examined at 2 h and 2 weeks post-injury. CnA distribution was assayed by immunohistochemistry and graded for non-parametric analysis. Acutely CnA isoforms showed reduced immunoreactivity in stratum radiatum processes of the ipsilateral CA1 and CA1-2. There was also a significant alteration in the immunoreactivity of both CnA isoforms in the ipsilateral dentate gyrus, predominantly within the hidden blade. Alterations in CnA isoform regional distribution within the CA1, CA1-2, and dentate gyrus may have significant implications for persistent hippocampal dysfunction following TBI, including dysfunction in hippocampal plasticity. Understanding alterations in CnA isoform distribution may help improve the targeting of current therapeutic interventions and/or the development of new treatments for TBI.

Experimental mild traumatic brain injury induces functional alteration of the developing hippocampus.

It is estimated that approximately 1.5 million Americans suffer a traumatic brain injury (TBI) every year, of which approximately 80% are considered mild injuries. Because symptoms caused by mild TBI last less than half an hour by definition and apparently resolve without treatment, the study of mild TBI is often neglected resulting in a significant knowledge gap for this wide-spread problem. In this work, we studied functional (electrophysiological) alterations of the neonatal/juvenile hippocampus after experimental mild TBI. Our previous work reported significant cell death after in vitro injury >10% biaxial deformation. Here we report that biaxial deformation as low as 5% affected neuronal function during the first week after in vitro mild injury of hippocampal slice cultures. These results suggest that even very mild mechanical events may lead to a quantifiable neuronal network dysfunction. Furthermore, our results highlight that safe limits of mechanical deformation or tolerance criteria may be specific to a particular outcome measure and that neuronal function is a more sensitive measure of injury than cell death. In addition, the age of the tissue at injury was found to be an important factor affecting posttraumatic deficits in electrophysiological function, indicating a relationship between developmental status and vulnerability to mild injury. Our findings suggest that mild pediatric TBI could result in functional deficits that are more serious than currently appreciated.

Excitability changes within transverse lamellae of dentate granule cells and their longitudinal spread following orthodromic or antidromic activation.

The functional organization of the perforant path input to the dentate gyrus of the exposed hippocampus was studied in adult rabbits anesthetized with urethane and chloralose. Electrical stimulation of perforant path fibers caused excitation of granule cells along narrow, nearly transverse strips (lamellae) of tissue. Stimulation of granule cell axons (mossy fibers) in CA3 caused antidromic activation of granule cells along similar strips. Paired-pulse stimulation revealed marked changes in granule cell excitability both within a lamella (on-line) and for several mm off-line along the septo-temporal axis of the dentate gyrus. After the first pulse, granule cells were inhibited for up to about 100 ms and then facilitated for up to hundreds of ms. Feedback activity along mossy fiber collaterals exciting local inhibitory and excitatory neurons appeared to dominate in producing on- and off-line inhibition and facilitation. Neurons mediating these effects could be inhibitory basket cells and other inhibitory interneurons targeting granule cells on- and off-line. In addition, excitatory mossy cells with far reaching, longitudinally running axons could affect off-line granule cells by exciting them directly or inhibit them indirectly by exciting local inhibitory interneurons. A scheme for dentate gyrus function is proposed whereby information to the dentate gyrus becomes split into interacting transverse strips of neuronal assemblies along which temporal processing occurs. A matrix of neuronal assemblies thus arises within which fragments of events and experiences is stored through the plasticity of synapses within and between the assemblies. Similar fragments may then be recognized at later times allowing memories of the whole to be created by pattern completion at subsequent computational stages in the hippocampus.

Roles of continuous neurogenesis in the structural and functional integrity of the adult forebrain.

Neurogenesis occurs continuously in the forebrain of adult mammals, but the functional importance of adult neurogenesis is still unclear. Here, using a genetic labeling method in adult mice, we found that continuous neurogenesis results in the replacement of the majority of granule neurons in the olfactory bulb and a substantial addition of granule neurons to the hippocampal dentate gyrus. Genetic ablation of newly formed neurons in adult mice led to a gradual decrease in the number of granule cells in the olfactory bulb, inhibition of increases in the granule cell number in the dentate gyrus and impairment of behaviors in contextual and spatial memory, which are known to depend on hippocampus. These results suggest that continuous neurogenesis is required for the maintenance and reorganization of the whole interneuron system in the olfactory bulb, the modulation and refinement of the existing neuronal circuits in the dentate gyrus and the normal behaviors involved in hippocampal-dependent memory.