The role of N-methyl-D-asparate receptors in neurogenesis

The dentate gyrus continues to incorporate granule neurons during adulthood. Among the factors that we know modulate adult neurogenesis in the dentate gyrus, one of the first studied was the influence of excitatory amino-acids. These neurotransmitters, acting through NMDA receptors, are able to modulate both the proliferation of progenitor cells as well as the rate of neurogenesis in the adult dentate gyrus. However, the mechanisms by which these processes are influenced are not clearly known. Although there is no anatomical evidence of NMDA receptor expression in adult hippocampal progenitor cells or differentiating granule neurons, electrophysiological data and in vitro studies suggest that NMDA receptors may be expressed by certain precursor cells and immature granule neurons. This review summarizes findings on the influence of pharmacological manipulation of NMDA receptors on adult neurogenesis. We also analyze previous studies that have suggested the expression of NMDA receptors in progenitors and immature granule cells and discuss the putative role of these receptors in the regulation of developmental processes such as proliferation, migration, or neurite outgrowth.     

Granule cell dispersion and aberrant neurogenesis in the adult hippocampus of an LIS1 mutant mouse

Human type 1 lissencephaly is a severe brain malformation associated with cognitive dysfunction and intractable epilepsy. Mutant mice with a heterozygous deletion of LIS1 show varying degrees of hippocampal abnormality and enhanced excitability. Whether a reduction of LIS1 function affects adult hippocampal neurogenesis, and if so, whether aberrant neurogenesis contributes to the generation of a disorganized hippocampus remain unknown. Previous reports indicate the presence of multiple pyramidal cell layers and granule cell dispersion in LIS1 mutant mice. Here we observed disruption of the subgranular zone and glial fibrillary acidic protein-immunoreactive radial astrocytes in the dentate gyrus of adult LIS1 mice. Using pulse-chase bromodeoxyuridine (BrdU) labeling combined with neuronal and glial antibody staining we provide evidence for ectopic adult neurogenesis in LIS1 mice. A gradually decreased survival rate for these newborn granule cells was also demonstrated in LIS1 mice 7 days after BrdU injection. This reduced survival rate was associated with impaired neuronal differentiation 28 days after BrdU administration. Thus, LIS1 haploinsufficiency can lead to abnormal cell proliferation, migration and differentiation in the adult dentate gyrus.     

Balance between neurogenesis and gliogenesis in the adult hippocampus: role for reelin

The extracellular matrix protein reelin is essential for the proper radial migration of cortical neurons. In reeler mice lacking reelin, there is a malformation of the radial glial scaffold required for granule cell migration. Immunostaining for glial fibrillary acidic protein (GFAP) reveals abundant radial glial cells with long fibers traversing the granular layer in the wild type, but almost exclusively astrocytes in the reeler mutant. With the concept that radial glial cells are precursors of neurons, we hypothesized that the balance between neurogenesis and gliogenesis is altered in the reeler mutant. To this end, adult reeler mutants and their wild-type littermates were injected with bromodeoxyuridine (BrdU), a marker of newly generated cells. When compared to wild-type animals, we found a reduction in the number of BrdU-labeled cells in the adult reeler dentate gyrus. Moreover, whereas there was a dramatic decrease in the number of newly generated granule cells identified by double labeling for BrdU and NeuN, the number of BrdU-labeled, GFAP-positive astrocytes had increased. Decreased neurogenesis in the adult reeler dentate gyrus was confirmed by immunostaining for doublecortin, a marker of newly generated neurons. These results indicate that adult neurogenesis is altered in the reeler dentate gyrus and that newly generated cells preferentially differentiate into astrocytes.     

The seasonal pattern of cell proliferation and neuron number in the dentate gyrus of wild adult eastern grey squirrels

The dentate gyrus is one of two areas in the mammalian brain that produces neurons in adulthood. Neurogenesis (proliferation, survival, and differentiation of new neurons) is regulated by experience, and increased neurogenesis appears to be correlated with improved spatial learning in mammals and birds. We tested the hypothesis that in long-lived mammals that scatter-hoard food, seasonal variations in spatial memory processing (i.e. increased processing during caching season in the autumn) might correlate with changes in neurogenesis and neuron number in the granule cell layer of the dentate gyrus (gcl DG). We investigated the rate of cell proliferation and the total number of neurons in the granule cell layer of wild adult eastern grey squirrels (Sciurus carolinensis) at three different times of the year (October, January and June). We found no seasonal differences in cell proliferation rate or in total neuron number in the granule cell layer. Our findings are in agreement with those of previous studies in laboratory mice and rats, and in free-ranging, food-caching, black-capped chickadees, as well as with current hypotheses regarding the relationship between neurogenesis and learning. Our results, however, are also in agreement with the hypothesis that neurogenesis in the dentate gyrus represents a maintenance system that may be regulated by environmental factors, and that changes in total neuron number previously reported in rodents represent developmental changes rather than adult plasticity. The patterns observed in mature wild rodents, such as free-ranging squirrels, may represent more accurately the extent of hippocampal plasticity in adult mammals.     

NMDA receptor antagonist treatment induces a long-lasting increase in the number of proliferating cells, PSA-NCAM-immunoreactive granule neurons and radial glia in the adult rat dentate gyrus

During adulthood, neural precursors located in the subgranular zone of the dentate gyrus continue to proliferate, leading to the generation of new granule neurons. These recently generated cells transiently express the polysialylated form of the neural cell adhesion molecule, PSA-NCAM, and are supported by radial glia-like cells that are likely to play a role in neuronal migration and differentiation, or even act as their precursors. Previous reports indicate that treatment with NMDA receptor antagonists stimulates adult neurogenesis in the dentate gyrus, and because of the potential therapeutic value of this approach, we were interested in further characterizing the consequences of pharmacologically modulating this process. We treated adult rats with the competitive NMDA receptor antagonist, CGP43487, and examined cell proliferation, PSA-NCAM expression, and changes in the radial glia cell population in the subgranular zone at different time points. In addition, we sought to determine if this treatment led to changes in cell death or gliotic reactions. The number of proliferating cells in the subgranular region of the dentate gyrus was increased significantly 2 days after treatment and it remained elevated 7 days postinjection. PSA-NCAM-immunoreactive granule cells and nestin-expressing radial glia-like cells also increased in number 7 days after the treatment. In contrast, we did not observe any change in granule cell death, and we were unable to detect any microglial or astroglial reaction during the first 7 days after treatment. Thus, NMDA receptor antagonist treatment serves as a valuable tool to increase neurogenesis in the adult hippocampus without undesirable collateral deleterious effects.     

Dentate granule cell neurogenesis after seizures induced by pentylenetrazol in rats

Epileptic seizures originating from the limbic system have been shown to stimulate the proliferation rate of granule cell precursors in the adult brain, but it is not clear if other type(s) of seizures have the similar effects. This study examined the effects of pentylenetrazol (PTZ)-induced generalized clonic seizures on dentate granule cell neurogenesis in adult rats. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we studied the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. The double-label immunofluorescence with confocal microscopy was used to determine the newborn cell phenotypes. Quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neural precursor cells in the dentate gyrus 3, 7, and 14 days after seizures. The number of BrdU-labeled cells in the dentate gyrus returned to baseline levels by 28 days after the initial seizures. Most of newborn cells migrated into the granule cell layer from the subgranular zone, displayed the neuronal phenotype, and developed morphological characteristics of differentiated dentate granule cells. These results indicated that neuron proliferation in the dentate gyrus was enhanced during a time window (3-14 days) after PTZ-induced seizures. Its underlying mechanism is discussed.     

Activation of latent precursors in the hippocampus is dependent on long-term potentiation

The recent discovery of a large latent population of precursor cells in the dentate gyrus of adult mice led us to investigate whether activation of this population is regulated by synaptic activity, thereby explaining the observation that environmental signals can affect neurogenesis. Using a variety of stimulation protocols, we found that only a long-term potentiation (LTP)-inducing protocol activated the latent precursor pool, leading to increased neurogenesis in the dentate gyrus. LTP induced by high-frequency stimulation (HFS) of the perforant pathway in vivo produced a two-fold increase in the number of neurospheres cultured from the stimulated hippocampus, compared with the unstimulated hippocampus. No increase in neurosphere number or neurogenesis was observed when the HFS failed to induce LTP. These results show that LTP can activate latent neural precursor cells in the adult mouse dentate gyrus, thereby providing a direct mechanism for regulating activity-driven neurogenesis. In the future, it may be possible to utilize such learning- or stimulation-induced neurogenesis to overcome disorders characterized by neuronal loss.     

Role of the postnatal radial glial scaffold for the development of the dentate gyrus as revealed by reelin signaling mutant mice

During dentate gyrus development, the early embryonic radial glial scaffold is replaced by a secondary glial scaffold around birth. In contrast to neocortical and early dentate gyrus radial glial cells, these postnatal glial cells are severely altered with regard to position and morphology in reeler mice lacking the secreted protein Reelin. In this study, we focus on the functional impact of these defects. Most radial glial cells throughout the nervous system serve as scaffolds for migrating neurons and precursor cells for both neurogenesis and gliogenesis. Precursor cell function has been demonstrated for secondary radial glial cells but the exact function of these late glial cells in granule cell migration and positioning is not clear. No data exist concerning the interplay between granule neurons and late radial glial cells during dentate gyrus development. Herein, we show that despite the severe morphological defects in the reeler dentate gyrus, the precursor function of secondary radial glial cells is not impaired during development in reeler mice. In addition, selective ablation of Disabled‐1, an intracellular adaptor protein essential for Reelin signaling, in neurons but not in glial cells allowed us to distinguish effects of Reelin signaling on radial glial cells from possible secondary effects based on defective granule cells positioning. GLIA 2013;61:1347–1363     

Origin,maturation, and astroglial transformation of secondary radial glial cells in the developing dentate gyrus

The dentate gyrus is a brain region where neurons are continuously born throughout life. In the adult, the role of its radial glia in neurogenesis has attracted much attention over the past years; however, little is known about the generation and differentiation of glial cells and their relationship to radial glia during the ontogenetic development of this brain structure. Here, we combine immunohistochemical phenotyping using antibodies against glial marker proteins with BrdU birthdating to characterize the development of the secondary radial glial scaffold in the dentate gyrus and its potential to differentiate into astrocytes. We demonstrate that the expression of brain lipid‐binding protein, GLAST, and glial fibrillary acidic protein (GFAP) characterizes immature differentiating cells confined to an astrocytic fate in the early postnatal dentate gyrus. On the basis of our studies, we propose a model where immature astrocytes migrate radially through the granule cell layer to adopt their final positions in the molecular layer of the dentate gyrus. Time‐lapse imaging of acute hippocampal slices from hGFAP‐eGFP transgenic mice provides direct evidence for such a migration mode of differentiating astroglial cells in the developing dentate gyrus. © 2010 Wiley‐Liss, Inc.     

Neurogenesis in the adult mammalian brain

The production of neurons in the mammalian brain is typically restricted to a discrete developmental period ending, for the most part, prior to parturition. However, in certain regions of the brain, including the dentate gyrus, new neurons continue to be produced well into adulthood. In the adult brain, new cells arise from progenitors located within the hilus and subgranular zone of the dentate gyrus, and then migrate into the dentate granule cell layer. Morphological, biochemical, and ultrastructural evidence indicate that many of these new cells become granule neurons, the principal projection neuron of the dentate gyrus. Anatomic studies have demonstrated that adult-generated granule neurons contribute axonal projections to area CA3 of Ammon's horn, while electron microscopy studies have revealed that these cells possess dendritic processes that extend into the dentate molecular layer to form synapses. Collectively, these data indicate that adult-generated granule neurons become functionally incorporated into the pre-existing neural circuitry of the dentate gyrus. The production and survival of adult-generated granule neurons are significantly influenced by experiential and neuroendocrine factors, suggesting that adult neurogenesis represents a substrate by which the environment may affect the structure and function of the adult brain. Although the precise function of adult-generated granule neurons is unknown, the formation of entirely novel neural circuits, and the regulation of this process by neuroendocrine and experiential factors, is likely to represent an important mode of neural plasticity. Moreover, the persistence of neural progenitors within the adult brain provides hope that an understanding of the process of adult neurogenesis may ultimately be of therapeutic relevance.     

HIV-1, chemokines and neurogenesis

HIV-1 infection of the brain results in a large number of behavioural defecits accompanied by diverse neuropathological signs. However,it is not clear how the virus produces these effects or exactly how the neuropathology and behavioural defecits are related. In this article we discuss the possibility that HIV-1 infection may negatively impact the process of neurogenesis in the adult brain and that this may contribute to HIV-1 related effects on the nervous system. We have previously demonstrated that the development of the dentate gyrus during embryogenesis requires signaling by the chemokine SDF-1 via its receptor CXCR4. We demonstrated that neural progenitor cells that give rise to dentate granule neurons express CXCR4 and other chemokine receptors and migrate into the nascent dentate gyrus along SDF-1 gradients. Animals deficient in CXCR4 receptors exhibit a malformed dentate gyrus in which the migration of neural progenitors is stalled. In the adult, neurogenesis continues in the dentate gyrus. Adult neural progenitor cells existing in the subgranlar zone, that produce granule neurons, express CXCR4 and other chemokine receptors, and granule neurons express SDF-1 suggesting that SDF-1/CXCR4 signaling is also important in adult neurogenesis. Because the cellular receptors for HIV-1 include chemokine receptors such as CXCR4 and CCR5 it is possible that the virus may interfere with SDF-1/CXCR4 signaling in the brain including disruption of the formation of new granule neurons in the adult brain.     

Depletion of norepinephrine decreases the proliferation,but does not influence the survival and differentiation,of granule cell progenitors in the adult rat hippocampus

The dentate gyrus region retains the ability to generate neurons throughout adulthood. A few studies have examined the neurotransmitter regulation of adult hippocampal neurogenesis and have shown that this process is regulated by serotonin and glutamate. Given the strong noradrenergic innervation of the adult hippocampus and the ability of norepinephrine to influence proliferation during development, we examined the influence of norepinephrine on adult hippocampal neurogenesis. Our study indicates that depletion of norepinephrine by the selective noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromo benzylamine hydrochloride (DSP-4), results in a 63% reduction in the proliferation of dentate gyrus progenitor cells identified through 5-bromo-2'-deoxyuridine (BrdU) labelling. In contrast, the survival of BrdU-positive cells labelled prior to treatment with DSP-4 is not influenced by norepinephrine depletion. The differentiation of BrdU labelled progenitors into neurons or glia was also not sensitive to noradrenergic depletion. These results indicate that the proliferation, but not the survival or differentiation, of adult hippocampal granule cell progenitors is affected by norepinephrine depletion.     

N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke

Neurogenesis in the adult rat dentate gyrus was studied following focal ischemic insults produced by middle cerebral artery occlusion (MCAO). Animals were subjected to either 30 min of MCAO, which causes damage confined to the striatum, or 2 h of MCAO, which leads to both striatal and cortical infarction. When compared to sham-operated rats, MCAO-rats showed a marked increase of the number of cells double-labelled for 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU; injected during 4-6 days postischemia) and neuronal-specific antigen (NeuN; a marker of postmitotic neurons) in the ipsilateral dentate granule cell layer and subgranular zone at 5 weeks following the 2 h insult. Only a modest and variable increase of BrdU-labelled cells was found after 30 min of MCAO. The enhanced neurogenesis was not dependent on cell death in the hippocampus, and its magnitude was not correlated to the degree of cortical damage. Systemic administration of the N-methyl-D-aspartate (NMDA) receptor blocker dizocilpine maleate (MK-801) completely suppressed the elevated neurogenesis following 2 h of MCAO. Our findings indicate that stroke leads to increased neurogenesis in the adult rat dentate gyrus through glutamatergic mechanisms acting on NMDA receptors. This modulatory effect may be mediated through changes in the levels of several growth factors, which occur after stroke, and could influence various regulatory steps of neurogenesis.     

Impaired fear memory and decreased hippocampal neurogenesis following olfactory bulbectomy in rats

It has been proposed that a decrease in adult hippocampal neurogenesis provides a biological and cellular basis for major depression. The olfactory bulbectomy (OB) in rats is widely accepted as an animal model of depression. In the present study, we investigated the effect of OB on memory formation in the memory tasks related to the hippocampal function and adult hippocampal neurogenesis. OB induced a behavioural syndrome, which was characterized by an increased activity in the open-field test and impairment in passive avoidance behaviour and contextual fear conditioning. The behavioural changes, following OB, were accompanied by a decrease in the number of proliferating cells in the dentate gyrus. Furthermore, the differentiation of the newly born cells, into mature calbindin-positive neurons, was also retarded. Stereological analysis revealed a decrease in the total granule neuron numbers within the granule cell layer of the dentate gyrus, without a significant decrease in volume of the dentate gyrus. Although a relationship between altered neurogenesis and behavioural syndrome, induced by OB, is not established yet, our results suggest that decreased neurogenesis might at least partly contribute for behavioural deficits following OB.     

Seizures preferentially stimulate proliferation of radial glia-like astrocytes in the adult dentate gyrus: functional and immunocytochemical analysis

Kainate-induced seizures increase hippocampal neurogenesis. Glial fibrillary acidic protein-positive astrocytes with radial processes in the dentate gyrus share many of the characteristics of radial glia and appear to act as precursor cells for adult dentate neurogenesis. Using the chemoconvulsant kainate and transgenic mice with human glial-fibrillary acidic protein (hGFAP) promoter-controlled enhanced green fluorescent protein (EGFP) expression, we examined the proliferation, morphology and electrophysiological properties of astrocytes in the neurogenic subgranular zone of the dentate gyrus in control animals and upon the induction of seizure-induced cell proliferation, three days post-kainate. EGFP-positive cells with and without radial processes could easily be distinguished. Kainate treatment caused a significant increase in the total number of proliferating EGFP-positive cells, particularly a tenfold elevation in the number of proliferating radial glia-like astrocytes, and also caused a preferential shift in the dividing cell population towards cells expressing EGFP. Immunohistochemical analysis revealed a surprisingly low proportion of cells coexpressing the astroglial marker S100beta and EGFP. Kainate increased the number of EGFP-positive, S100beta-positive and S100beta-positive-EGFP-positive astrocytes in the subgranular zone. We also report a subset of faintly EGFP-positive cells expressing markers of early neuronal differentiation. Patch-clamp analysis revealed the presence of three functionally different populations of EGFP-positive cells in both kainate and control tissue. We conclude that there is an early increase in proliferating radial glia-like astrocytes in the dentate after kainate-induced seizures, consistent with a recruitment of precursors for seizure-induced neurogenesis.     

Unique expression patterns of cell fate molecules delineate sequential stages of dentate gyrus development.

The dentate gyrus of the hippocampus is uniquely organized with a displaced proliferative zone that continues to generate dentate granule cells throughout life. We have analyzed the expression of Notch receptors, Notch ligands, and basic helix-loop-helix (bHLH) genes during dentate gyrus development to determine whether the need to maintain a pool of undifferentiated precursors is reflected in the patterns of expression of these genes. Many of these genes are expressed diffusely throughout the cortical neuroepithelium at embryonic days 16 and 17 in the rat, just preceding the migration of newly born granule cells and dentate precursor cells into the dentate anlage. However, at this time, Mash1, Math3, and Id3 expression are all concentrated in the area that specifically gives rise to granule cells and dentate precursor cells. Two days later, at the time of migration of the first granule cells and dentate precursor cells, cells expressing Mash1 are seen in the migratory route from the subventricular zone to the developing dentate gyrus. Newly born granule cells expressing NeuroD are also present in this migratory pathway. In the first postnatal week, precursor cells expressing Mash1 reside in the dentate hilus, and by the third postnatal week they have largely taken up their final position in the subgranular zone along the hilar side of the dentate granule cell layer. After terminal differentiation, granule cells born in the hilus or the subgranular zone begin to express NeuroD followed by NeuroD2. This study establishes that the expression patterns of bHLH mRNAs evolve during the formation of the dentate gyrus, and the precursor cells resident in the mature dentate gyrus share features with precursor cells found in development. Thus, many of the same mechanisms that are known to regulate cell fate and precursor pool size in other brain regions are likely to be operative in the dentate gyrus at all stages of development.     

Increased granule cell neurogenesis in the adult dentate gyrus following mossy fiber stimulation sufficient to induce long-term potentiation

Neurons are continually added at a low rate to the granule cell layer of the dentate gyrus during adulthood in rats. The functional significance of this unusual feature is not completely understood, although recent studies suggest continued granule cell neurogenesis is essential for normal learning and memory. We report here that, in the adult rat, stimulation of the granule cell mossy fibers sufficient to induce long-term potentiation (LTP) increases the number of newly formed granule cells in the dentate gyrus, indicating that granule cell neurogenesis is regulated by efferent activity and, possibly, the induction of LTP.     

Glucocorticoid regulation of glial responses during hippocampal neurodegeneration and regeneration

Glucocorticoids can prevent or accelerate neurodegeneration in the adult rat hippocampus. To investigate these actions of glucocorticoids, we previously cloned genes from the hippocampus. Adrenalectomy specifically increased glial fibrillary acidic protein and transforming growth factor (TGF)-β1 mRNAs in the dentate gyrus and these effects were dependent on induced apoptosis. Corticosterone treatment prevented apoptosis, and decreased glial activation and the influx of activated microglia. Since these effects are opposite to injury and neurodegeneration, we propose that they represent adaptive actions of glucocorticoids, preventing cellular defense mechanisms from overshooting. We used adrenalectomy as a model to investigate how adult granule neurons die in vivo and the effects of neurotrophic factors in protecting against apoptosis. Neurotrophin-4/5 and TGF-β1 protected granule neurons against adrenalectomy-induced apoptosis. Since neurogenesis is also greatly increased in the dentate gyrus following adrenalectomy, we compared the time course of birth and death with glial responses. TGF-β1 mRNA increased before the detection of dying cells in the dentate gyrus, which was coincident with increased proliferation in the neurogenic zone. Glucocorticoids also increased Ndrg2 mRNA in glia in the neurogenic zone; Ndrg2 is a member of a novel gene family involved in neural differentiation and synapse formation. Therefore, studying the effects of glucocorticoid manipulation on the dentate gyrus is increasing our understanding of how mature neurons die by apoptosis and the role of glia in induced apoptosis and neurogenesis. Discovering how endocrine and inflammatory responses regulate neuron birth and survival is important for developing successful neuron replacement strategies to treat neurodegenerative diseases.     

Milestones of neuronal development in the adult hippocampus

Adult hippocampal neurogenesis originates from precursor cells in the adult dentate gyrus and results in new granule cell neurons. We propose a model of the development that takes place between these two fixed points and identify several developmental milestones. From a presumably bipotent radial-glia-like stem cell (type-1 cell) with astrocytic properties, development progresses over at least two stages of amplifying lineage-determined progenitor cells (type-2 and type-3 cells) to early postmitotic and to mature neurons. The selection process, during which new neurons are recruited into function, and other regulatory influences differentially affect the different stages of development.