Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP⁺ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP⁺ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells.     

Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion

Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from less active, older adult-born GCs and the major population of dentate GCs generated developmentally. To ascertain whether young and old GCs perform distinct memory functions, we created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts, which was reduced following ablation of young GCs. Furthermore, these mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation requires adult-born young GCs but not old GCs, and older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion.     

Early natural stimulation through environmental enrichment accelerates neuronal development in the mouse dentate gyrus

The dentate gyrus is the primary afferent into the hippocampal formation, with important functions in learning and memory. Granule cells, the principle neuronal type in the dentate gyrus, are mostly formed postnatally, in a process that continues into adulthood. External stimuli, including environmental enrichment, voluntary exercise and learning, have been shown to significantly accelerate the generation and maturation of dentate granule cells in adult rodents. Whether, and to what extent, such environmental stimuli regulate the development and maturation of dentate granule cells during early postnatal development is largely unknown. Furthermore, whether natural stimuli affect the synaptic properties of granule cells had been investigated neither in newborn neurons of the adult nor during early development. To examine the effect of natural sensory stimulation on the dentate gyrus, we reared newborn mice in an enriched environment (EE). Using immunohistochemistry, we showed that dentate granule cells from EE-reared mice exhibited earlier morphological maturation, manifested as faster peaking of doublecortin expression and elevated expression of mature neuronal markers (including NeuN, calbindin and MAP2) at the end of the second postnatal week. Also at the end of the second postnatal week, we found increased density of dendritic spines across the entire dentate gyrus, together with elevated levels of postsynaptic scaffold (post-synaptic density 95) and receptor proteins (GluR2 and GABA(A)Rγ2) of excitatory and inhibitory synapses. Furthermore, dentate granule cells of P14 EE-reared mice had lower input resistances and increased glutamatergic and GABAergic synaptic inputs. Together, our results demonstrate that EE-rearing promotes morphological and electrophysiological maturation of dentate granule cells, underscoring the importance of natural environmental stimulation on development of the dentate gyrus.     

Morphological alterations in newly born dentate gyrus granule cells that emerge after status epilepticus contribute to make them less excitable

Computer simulations of external current stimulations of dentate gyrus granule cells of rats with Status Epilepticus induced by pilocarpine and control rats were used to evaluate whether morphological differences alone between these cells have an impact on their electrophysiological behavior. The cell models were constructed using morphological information from tridimensional reconstructions with Neurolucida software. To evaluate the effect of morphology differences alone, ion channel conductances, densities and distributions over the dendritic trees of dentate gyrus granule cells were the same for all models. External simulated currents were injected in randomly chosen dendrites belonging to one of three different areas of dentate gyrus granule cell molecular layer: inner molecular layer, medial molecular layer and outer molecular layer. Somatic membrane potentials were recorded to determine firing frequencies and inter-spike intervals. The results show that morphologically altered granule cells from pilocarpine-induced epileptic rats are less excitable than control cells, especially when they are stimulated in the inner molecular layer, which is the target area for mossy fibers that sprout after pilocarpine-induced cell degeneration. This suggests that morphological alterations may act as a protective mechanism to allow dentate gyrus granule cells to cope with the increase of stimulation caused by mossy fiber sprouting.     

Combined Role of Seizure-Induced Dendritic Morphology Alterations and Spine Loss in Newborn Granule Cells with Mossy Fiber Sprouting on the Hyperexcitability of a Computer Model of the Dentate Gyrus

Temporal lobe epilepsy strongly affects hippocampal dentate gyrus granule cells morphology. These cells exhibit seizure-induced anatomical alterations including mossy fiber sprouting, changes in the apical and basal dendritic tree and suffer substantial dendritic spine loss. The effect of some of these changes on the hyperexcitability of the dentate gyrus has been widely studied. For example, mossy fiber sprouting increases the excitability of the circuit while dendritic spine loss may have the opposite effect. However, the effect of the interplay of these different morphological alterations on the hyperexcitability of the dentate gyrus is still unknown. Here we adapted an existing computational model of the dentate gyrus by replacing the reduced granule cell models with morphologically detailed models coming from three-dimensional reconstructions of mature cells. The model simulates a network with 10% of the mossy fiber sprouting observed in the pilocarpine (PILO) model of epilepsy. Different fractions of the mature granule cell models were replaced by morphologically reconstructed models of newborn dentate granule cells from animals with PILO-induced Status Epilepticus, which have apical dendritic alterations and spine loss, and control animals, which do not have these alterations. This complex arrangement of cells and processes allowed us to study the combined effect of mossy fiber sprouting, altered apical dendritic tree and dendritic spine loss in newborn granule cells on the excitability of the dentate gyrus model. Our simulations suggest that alterations in the apical dendritic tree and dendritic spine loss in newborn granule cells have opposing effects on the excitability of the dentate gyrus after Status Epilepticus. Apical dendritic alterations potentiate the increase of excitability provoked by mossy fiber sprouting while spine loss curtails this increase.     

Environment matters: synaptic properties of neurons born in the epileptic adult brain develop to reduce excitability

Neural progenitors in the adult dentate gyrus continuously produce new functional granule cells. Here we used whole-cell patch-clamp recordings to explore whether a pathological environment influences synaptic properties of new granule cells labeled with a GFP-retroviral vector. Rats were exposed to a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus, which gave rise to neuronal death, inflammation, and chronic seizures. Granule cells formed after these stimuli exhibited similar intrinsic membrane properties. However, the new neurons born into the pathological environment differed with respect to synaptic drive and short-term plasticity of both excitatory and inhibitory afferents. The new granule cells formed in the epileptic brain exhibited functional connectivity consistent with reduced excitability. We demonstrate a high degree of plasticity in synaptic inputs to adult-born new neurons, which could act to mitigate pathological brain function.     

The synaptic scaffolding protein CNKSR2 interacts with CYTH2 to mediate hippocampal granule cell development

CNKSR2 is a synaptic scaffolding molecule that is encoded by the CNKSR2 gene located on the X chromosome. Heterozygous mutations to CNKSR2 in humans are associated with intellectual disability and epileptic seizures, yet the cellular and molecular roles for CNKSR2 in nervous system development and disease remain poorly characterized. Here, we identify a molecular complex comprising CNKSR2 and the guanine nucleotide exchange factor (GEF) for ARF small GTPases, CYTH2, that is necessary for the proper development of granule neurons in the mouse hippocampus. Notably, we show that CYTH2 binding prevents proteasomal degradation of CNKSR2. Furthermore, to explore the functional significance of coexpression of CNKSR2 and CYTH2 in the soma of granule cells within the hippocampal dentate gyrus, we transduced mouse granule cell precursors in vivo with small hairpin RNAs (shRNAs) to silence CNKSR2 or CYTH2 expression. We found that such manipulations resulted in the abnormal localization of transduced cells at the boundary between the granule cell layer and the hilus. In both cases, CNKSR2-knockdown and CYTH2-knockdown cells exhibited characteristics of immature granule cells, consistent with their putative roles in neuron differentiation. Taken together, our results demonstrate that CNKSR2 and its molecular interaction partner CYTH2 are necessary for the proper development of dentate granule cells within the hippocampus through a mechanism that involves the stabilization of a complex comprising these proteins.     

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.     

小鼠海马内HDAC2的表达及其与NMDA受体、PSD-95的共定位

目的探讨组蛋白去乙酰化酶2（HDAC2）在成年C57BL/6小鼠海马内的分布及其与突触后致密区（PSD）蛋白成员的共定位,为揭示HDAC2与PSD蛋白复合物之间的内在联系及在海马相关的学习记忆过程中可能起到的调控作用提供形态学依据。方法应用免疫组化方法观察HDAC2在C57BL/6小鼠海马各区的表达分布。应用免疫荧光双标技术研究HDAC2与PSD蛋白成员N-甲基-D-天冬氨酸（NMDA）受体亚单位1（NR1）、PSD-95之间是否存在共定位。结果 HDAC2在小鼠海马CA1～CA3区锥体细胞和齿状回颗粒细胞均具有明显表达,而在各区的始层、辐射层、腔隙-分子层以及齿状回多形细胞层表达均较少。免疫荧光双标染色图片的重叠表明,HDAC2与NR1、PSD-95在小鼠海马CA1～CA3区锥体细胞层和齿状回颗粒细胞层内均可见显著共表达现象,其他区域偶见散在分布的双染神经元。结论 HDAC2在小鼠海马锥体细胞层和颗粒细胞层表达丰富,并与PSD蛋白成员间存在共定位现象。本实验结果为探讨HDAC2对谷氨酸能突触后神经元依赖的突触可塑性的调节机制提供了形态学依据。     

The neuronal primary cilium: Driver of neurogenesis and memory formation in the hippocampal dentate gyrus?

Considerable attention has recently been focused on the postnatal persistence of neurogenesis in the dentate gyrus of the hippocampus and the roles of signals from the primary cilium in the different functions of an increasing number of tissues and their malfunctions. Here we summarize the evidence that ties sonic hedgehog-triggered proliferogenic signaling from the primary cilia on granule cell progenitors in the adult dentate subgranular zone to maintain a pool of new “blank slate” dentate granule cells. These can be recruited to bundle and encode novel inputs flowing from various regions of the brain into the dentate gyrus via the entorhinal cortex without altering and erasing the synaptic patterns from previous inputs inscribed on older granule cells.     

Properties of doublecortin-(DCX)-expressing cells in the piriform cortex compared to the neurogenic dentate gyrus of adult mice

The piriform cortex receives input from the olfactory bulb and (via the entorhinal cortex) sends efferents to the hippocampus, thereby connecting the two canonical neurogenic regions of the adult rodent brain. Doublecortin (DCX) is a cytoskeleton-associated protein that is expressed transiently in the course of adult neurogenesis. Interestingly, the adult piriform cortex, which is usually considered non-neurogenic (even though some reports exist that state otherwise), also contains an abundant population of DCX-positive cells. We asked how similar these cells would be to DCX-positive cells in the course of adult hippocampal neurogenesis. Using BAC-generated transgenic mice that express GFP under the DCX promoter, we studied DCX-expression and electrophysiological properties of DCX-positive cells in the mouse piriform cortex in comparison with the dentate gyrus. While one class of cells in the piriform cortex indeed showed features similar to newly generated immature granule neurons, the majority of DCX cells in the piriform cortex was mature and revealed large Na+ currents and multiple action potentials. Furthermore, when proliferative activity was assessed, we found that all DCX-expressing cells in the piriform cortex were strictly postmitotic, suggesting that no DCX-positive "neuroblasts" exist here as they do in the dentate gyrus. We conclude that DCX in the piriform cortex marks a unique population of postmitotic neurons with a subpopulation that retains immature characteristics associated with synaptic plasticity. DCX is thus, per se, no marker of neurogenesis but might be associated more broadly with plasticity.     

Properties of doublecortin expressing neurons in the adult mouse dentate gyrus

The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses.     

Cell death,gliosis, and synaptic remodeling in the hippocampus of epileptic rats

Seizures set in motion complex molecular and morphological changes in vulnerable structures, such as the hippocampal complex. A number of these changes are responsible for neuronal death of CA3 and hilar cells, which involves necrotic and apoptotic mechanisms. In surviving dentate granule cells seizures induce an increased expression of tubulin subunits and microtubule-associated proteins, suggesting that an overproduction of tubulin polymers would lead to a remodeling of mossy fibers (the axons of granule cells). In fact, these fibers sprout in the dentate gyrus to innervate granule cell dendrites, creating recurrent excitatory circuits. In contrast, terminal mossy fibers do not sprout in the CA3 field. Navigation of mossy fiber's growth cones may be facilitated by astrocytes, which would exert differential effects by producing and excreting cell adhesion and substrate molecules. In the light of the results discussed here, we suggest that in adult brain activated-resident astrocytes (nonproliferating, tenascin-negative, neuronal cell-adhesion molecule-positive astrocytes) could contribute to the process of axonal outgrowth and synaptogenesis in the dentate gyrus, while proliferating astrocytes, tenascin-positive, could impede any axonal rearrangement in CA3. © 1995 John Wiley & Sons, Inc.     

Alterations of Glial Cells in the Mouse Hippocampus During Postnatal Development

We investigated the postnatal alterations of neurons, astrocyte, oligodendrocyte, and microglia in the mouse hippocampal CA1 sector and dentate gyrus under the same conditions using immunohistochemistry. Neuronal nuclei (NeuN), Glial fibrillary acidic protein (GFAP), 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), and ionized calcium binding adaptor molecule 1 (Iba 1) immunoreactivity were measured in 1-, 2-, 4-, and 8-week-old mice. Total number of NeuN-positive neurons was unchanged in the mouse hippocampal CA1 sector and dentate gyrus from 1 to 8 weeks of birth. In contrast, a significant increase in the number of GFAP-positive astrocytes was observed only in the hippocampal CA1 sector of 1-week-old mice when compared with 8-week-old animals. Thereafter, total number of GFAP-positive astrocytes was unchanged in the hippocampal CA1 sector and dentate gyrus from 2 to 8 weeks of birth. For microglia, a significant increase in the number of Iba 1-positive microglia was observed in the hippocampal CA1 sector and dentate gyrus of 1-, 2-, and 4-week-old mice as compared with 8-week-old animals. On the other hand, a significant decrease in the area of expression of CNPase-positive fibers was observed in the hippocampal CA1 sector of 1- and 2-week-old mice as compared with 8-week-old animals. In dentate gyrus, a significant decrease in the area of expression of CNPase-positive fibers was found in 1-, 2-, and 4-week-old mice. Furthermore, our double-labeled immunostaining showed that brain-derived neurotrophic factor (BDNF) immunoreactivity was observed in GFAP-positive astrocytes and Iba 1-positive microglia in the hippocampal CA1 sector and dentate gyrus of 1- and 2-week-old mice. These results show that glial cells may play some role in the maintenance and neuronal functions of hippocampal CA1 pyramidal neurons and granule cells of dentate gyrus during postnatal development. Furthermore, our results demonstrate that glial BDNF may play an important role in the maturation of oligodendrocyte in the hippocampal CA1 sector and dentate gyrus during postnatal development. Thus, our findings provide valuable information on the developmental processes.     

The chemokine SDF1 regulates migration of dentate granule cells

The dentate gyrus is the primary afferent pathway into the hippocampus, but there is little information concerning the molecular influences that govern its formation. In particular, the control of migration and cell positioning of dentate granule cells is not clear. We have characterized more fully the timing and route of granule cell migration during embryogenesis using in utero retroviral injections. Using this information, we developed an in vitro assay that faithfully recapitulates important events in dentate gyrus morphogenesis. In searching for candidate ligands that may regulate dentate granule cell migration, we found that SDF1, a chemokine that regulates cerebellar and leukocyte migration, and its receptor CXCR4 are expressed in patterns that suggest a role in dentate granule cell migration. Furthermore, CXCR4 mutant mice have a defect in granule cell position. Ectopic expression of SDF1 in our explant assay showed that it directly regulates dentate granule cell migration. Our study shows that a chemokine is necessary for the normal development of the dentate gyrus, a forebrain structure crucial for learning and memory.     

Gas7在大鼠海马和齿状回发育过程中的动态表达

目的研究生长休止蛋白7（Gas7）在大鼠海马和齿状回不同发育阶段的表达。方法采用免疫组织化学方法观察Gas7在SD大鼠胚胎第18d（E18）、新生（P0）、生后第7d（P7）、P14、P21和成年海马和齿状回中的表达和分布。结果在大鼠脑海马和齿状回部位的冠状切片上,Gas7免疫反应阳性产物主要表达在海马的锥体细胞、齿状回的颗粒细胞和门区的多形层细胞。随着发育的进程,在海马,Gas7较早表达在CA3区,其次是CA2和CA1区;在齿状回,Gas7在外臂的表达早于内臂,在颗粒细胞层的表达是按先外层后内层的顺序。在围生期,Gas7在海马和齿状回各区的表达逐渐增强,至P14达到高峰,后逐渐降低,至P21其表达强度和分布趋于恒定至成年水平。结论 Gas7在大鼠海马和齿状回发育过程中的动态表达具有时间和空间上的特异性,提示Gas7可能参与了海马和齿状回形态形成和功能成熟的调控。     

Linking Macroscopic with Microscopic Neuroanatomy Using Synthetic Neuronal Populations

Dendritic morphology has been shown to have a dramatic impact on neuronal function. However, population features such as the inherent variability in dendritic morphology between cells belonging to the same neuronal type are often overlooked when studying computation in neural networks. While detailed models for morphology and electrophysiology exist for many types of single neurons, the role of detailed single cell morphology in the population has not been studied quantitatively or computationally. Here we use the structural context of the neural tissue in which dendritic trees exist to drive their generation in silico. We synthesize the entire population of dentate gyrus granule cells, the most numerous cell type in the hippocampus, by growing their dendritic trees within their characteristic dendritic fields bounded by the realistic structural context of (1) the granule cell layer that contains all somata and (2) the molecular layer that contains the dendritic forest. This process enables branching statistics to be linked to larger scale neuroanatomical features. We find large differences in dendritic total length and individual path length measures as a function of location in the dentate gyrus and of somatic depth in the granule cell layer. We also predict the number of unique granule cell dendrites invading a given volume in the molecular layer. This work enables the complete population-level study of morphological properties and provides a framework to develop complex and realistic neural network models.     

Prox1 Is Required for Granule Cell Maturation and Intermediate Progenitor Maintenance During Brain Neurogenesis

The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.