Characterization of a unique cell population marked by transgene expression in the adult cochlea of nestin‐CreERT2/tdTomato‐reporter mice

Hair cells in the adult mammalian cochlea cannot spontaneously regenerate after damage, resulting in the permanency of hearing loss. Stem cells have been found to be present in the cochlea of young rodents; however, there has been little evidence for their existence into adulthood. We used nestin‐CreER^T2/tdTomato‐reporter mice to trace the lineage of putative nestin‐expressing cells and their progeny in the cochleae of adult mice. Nestin, an intermediate filament found in neural progenitor cells during early development and adulthood, is regarded as a multipotent and neural stem cell marker. Other investigators have reported its presence in postnatal and young adult rodents; however, there are discrepancies among these reports. Using lineage tracing, we documented a robust population of tdTomato‐expressing cells and evaluated these cells at a series of adult time points. Upon activation of the nestin promoter, tdTomato was observed just below and medial to the inner hair cell layer. All cells colocalized with the stem cell and cochlear‐supporting‐cell marker Sox2 as well as the supporting cell and Schwann cell marker Sox10; however, they did not colocalize with the Schwann cell marker Krox20, spiral ganglion marker NF200, nor glial fibrillary acidic acid (GFAP)‐expressing supporting cell marker. The cellular identity of this unique population of tdTomato‐expressing cells in the adult cochlea of nestin‐CreER^T2/tdTomato mice remains unclear; however, these cells may represent a type of supporting cell on the neural aspect of the inner hair cell layer. J. Comp. Neurol. 523:1474–1487, 2015. © 2015 Wiley Periodicals, Inc.     

Specificity and efficiency of reporter expression in adult neural progenitors vary substantially among nestin‐CreERT2 lines

Transgenic lines expressing a controllable form of Cre recombinase have become valuable tools for manipulating gene expression in adult neural progenitors and their progeny. Neural progenitors express several proteins that distinguish them from mature neurons, and the promoters for these genes have been co‐opted to produce selective transgene expression within this population. To date, nine CreER^T2 transgenic lines have been designed using the nestin promoter; however, only a subset are capable of eliciting expression within both neurogenic zones of the adult brain. Here we compare three such nestin‐CreER^T2 lines to evaluate specificity of expression and efficiency of recombination. Each line was examined by using three different Cre reporter strains that varied in sensitivity. We found that all three nestin‐CreER^T2 strains induced reporter expression within the main neurogenic areas, albeit to varying degrees depending on the reporter. Unexpectedly, we found that two of the three lines induced substantial reporter expression outside of neurogenic areas. These lines produced strong labeling in cerebellar granule neurons, with additional expression in the cortex, hippocampus, striatum, and thalamus. Reporter expression in the third nestin‐CreER^T2 line was considerably more specific, but was also less efficient, labeling a smaller percentage of the target population than the other two drivers. Our findings suggest that each nestin‐CreER^T2 line may best serve different experimental needs, depending on whether specificity or efficiency is of greatest concern. Our study further demonstrates that each new pair of driver and responder lines should be evaluated independently, as both components can significantly influence the resulting expression pattern. J. Comp. Neurol. 522:1191–1208, 2014. © 2013 Wiley Periodicals, Inc.     

Critical neuroprotective roles of heme oxygenase‐1 induction against axonal injury‐induced retinal ganglion cell death

Although axonal damage induces significant retinal ganglion cell (RGC) death, small numbers of RGCs are able to survive up to 7 days after optic nerve crush (NC) injury. To develop new treatments, we set out to identify patterns of change in the gene expression of axonal damage‐resistant RGCs. To compensate for the low density of RGCs in the retina, we performed retrograde labeling of these cells with 4Di‐10ASP in adult mice and 7 days after NC purified the RGCs with fluorescence‐activated cell sorting. Gene expression in the cells was determined with a microarray, and the expression of Ho‐1 was determined with quantitative PCR (qPCR). Changes in protein expression were assessed with immunohistochemistry and immunoblotting. Additionally, the density of Fluoro‐gold‐labeled RGCs was counted in retinas from mice pretreated with CoPP, a potent HO‐1 inducer. The microarray and qPCR analyses showed increased expression of Ho‐1 in the post‐NC RGCs. Immunohistochemistry also showed that HO‐1‐positive cells were present in the ganglion cell layer (GCL), and cell counting showed that the proportion of HO‐1‐positive cells in the GCL rose significantly after NC. Seven days after NC, the number of RGCs in the CoPP‐treated mice was significantly higher than in the control mice. Combined pretreatment with SnPP, an HO‐1 inhibitor, suppressed the neuroprotective effect of CoPP. These results reflect changes in HO‐1 activity to RGCs that are a key part of RGC survival. Upregulation of HO‐1 signaling may therefore be a novel therapeutic strategy for glaucoma. © 2014 Wiley Periodicals, Inc.     

Deletion of TLX and social isolation impairs exercise‐induced neurogenesis in the adolescent hippocampus

Adolescence is a sensitive period of neurodevelopment during which life experiences can have profound effects on the brain. Hippocampal neurogenesis, the neurodevelopmental process of generating functional new neurons from neural stem cells, occurs throughout the lifespan and has been shown to play a role in learning, memory and in mood regulation. In adulthood it is influenced by extrinsic environmental factors such as exercise and stress. Intrinsic factors that regulate hippocampal neurogenesis include the orphan nuclear receptor TLX (Nr2e1) which is primarily expressed in the neurogenic niches of the brain. While mechanisms regulating adult hippocampal neurogenesis have been widely studied, less is known on how hippocampal neurogenesis is affected during adolescence. The aim of this study was to investigate the influence of both TLX and isolation stress on exercise‐induced increases in neurogenesis in running and sedentary conditions during adolescence. Single‐ (isolation stress) wild type and Nr2e1^‐/‐ mice or pair‐housed wild type mice were housed in sedentary conditions or allowed free access to running wheels for 3 weeks during adolescence. A reduction of neuronal survival was evident in mice lacking TLX, and exercise did not increase hippocampal neurogenesis in these Nr2e1^‐/‐ mice. This suggests that TLX is necessary for the pro‐neurogenic effects of exercise during adolescence. Interestingly, although social isolation during adolescence did not affect hippocampal neurogenesis, it prevented an exercise‐induced increase in neurogenesis in the ventral hippocampus. Together these data demonstrate the importance of intrinsic and extrinsic factors in promoting an exercise‐induced increase in neurogenesis at this key point in life.     

Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell‐autonomous fashion

Ca²⁺ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha‐1C subunit (i.e., Ca_v1.2) of the voltage‐dependent l ‐type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Ca_v1.2 channels located on astrocyte‐like stem cells and their descendants in the development of new granule neurons, we created Tg^{GLAST‐CreERT2}/Cacna1c^fl/fl/RCE:loxP mice, a transgenic tool that allows cell‐type‐specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type‐1 cells. FACS‐sorted Cacna1c‐deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c‐deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type‐1 cells decreased type‐1 cell proliferation and reduced the neuronal fate‐choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type‐1 cells, indicating that there must be Ca_v1.2‐independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L‐type 1.2 channels on type‐1 cells. Ca_v1.2 channels promote type‐1 cell proliferation and push the glia‐to‐neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Ca_v1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell‐autonomous fashion.     

In vivo bioluminescence imaging of neurogenesis – the role of the blood brain barrier in an experimental model of Parkinson's disease

Bioluminescence imaging in transgenic mice expressing firefly luciferase in Doublecortin⁺ (Dcx) neuroblasts might serve as a powerful tool to study the role of neurogenesis in models of brain injury and neurodegeneration using non‐invasive, longitudinal in vivo imaging. Therefore, we aimed to use BLI in B6(Cg)‐Tyrc‐2J/J Dcx‐Luc (Doublecortin‐Luciferase, Dcx‐Luc) mice to investigate its suitability to assess neurogenesis in a unilateral injection model of Parkinson's disease. We further aimed to assess the blood brain barrier leakage associated with the intranigral 6‐OHDA injection to evaluate its impact on substrate delivery and bioluminescence signal intensity. Two weeks after lesion, we observed an increase in bioluminescence signal in the ipsilateral hippocampal region in both, 6‐OHDA and vehicle injected Dcx‐Luc mice. At the same time, no corresponding increase in Dcx⁺ neuroblast numbers could be observed in the dentate gyrus of C57Bl6 mice. Blood brain barrier leakage was observed in the hippocampal region and in the degenerating substantia nigra of C57Bl6 mice in vivo using T1 weighted Magnetic Resonance Imaging with Gadovist^® and ex vivo using Evans Blue Fluorescence Reflectance Imaging and mouse Immunoglobulin G staining. Our data suggests a BLI signal dependency on blood brain barrier permeability, underlining a major pitfall of substrate/tracer dependent imaging in invasive disease models.     

Running promotes spatial bias independently of adult neurogenesis

Different memory systems offer distinct advantages to navigational behavior. The hippocampus forms complex associations between environmental stimuli, enabling flexible navigation through space. In contrast, the dorsal striatum associates discrete cues and favorable behavioral responses, enabling habit‐like, automated navigation. While these two systems often complement one another, there are instances where striatal‐dependent responses (e.g. approach a cue) conflict with hippocampal representations of spatial goals. In conflict situations, preference for spatial vs. response strategies varies across individuals and depends on previous experience, plasticity and the integrity of these two memory systems. Here, we investigated the role of adult hippocampal neurogenesis and exercise on mouse search strategies in a water maze task that can be solved with either a hippocampal‐dependent place strategy or a striatal‐dependent cue‐response strategy. We predicted that inhibiting adult neurogenesis would impair hippocampal function and shift behavior towards striatal‐dependent cue responses. However, blocking neurogenesis in a transgenic nestin‐TK mouse did not affect strategy choice. We then investigated whether a pro‐neurogenic stimulus, running, would bias mice towards hippocampal‐dependent spatial strategies. While running indeed promoted spatial strategies, it did so even when neurogenesis was inhibited in nestin‐TK mice. These findings indicate that exercise‐induced increases in neurogenesis are not always required for enhanced cognitive function. Furthermore, our data identify exercise as a potentially useful strategy for promoting flexible, cognitive forms of memory in habit‐related disorders that are characterized by excessive responding to discrete cues.     

Focal cerebral ischemia induces a multilineage cytogenic response from adult subventricular zone that is predominantly gliogenic

The purpose of this study was to ascertain the relative contribution of neural stem/progenitor cells (NSPCs) of the subventricular zone (SVZ) to lineages that repopulate the injured striatum following focal ischemia. We utilized a tamoxifen‐inducible Cre/loxP system under control of the nestin promoter, which provides permanent YFP labeling of multipotent nestin⁺ SVZ‐NSPCs prior to ischemic injury and continued YFP expression in all subsequent progeny following stroke. YFP reporter expression was induced in adult male nestin‐CreER^T2:R26R‐YFP mice by tamoxifen administration (180 mg kg⁻¹, daily for 5 days). Fourteen days later, mice were subjected to 60‐min transient middle cerebral artery occlusion (MCAO) and sacrificed at 2 days, 2 weeks, or 6 weeks post‐MCAO for phenotypic fate mapping of YFP⁺ cells using lineage‐specific markers. Migration of YFP⁺ cells from SVZ into the injured striatal parenchyma was apparent at 2 and 6 weeks, but not 2 days, post‐MCAO. At 2 weeks post‐MCAO, the average percent distribution of YFP⁺ cells within the injured striatal parenchyma was as follows: 10% Dcx⁺ neuroblasts, 15–20% oligodendrocyte progenitors, 59% GFAP⁺ astrocytes, and only rare NeuN⁺ postmitotic neurons. A similar phenotypic distribution was observed at 6 weeks, except for an increased average percentage of YFP⁺ cells that expressed Dcx⁺ (20%) or NeuN (5%). YFP⁺ cells did not express endothelial markers, but displayed unique anatomical relationships with striatal vasculature. These results indicate that nestin⁺ NSPCs within the SVZ mount a multilineage response to stroke that includes a gliogenic component more predominant than previously appreciated. © 2010 Wiley‐Liss, Inc.     

Increased adult hippocampal neurogenesis is not necessary for wheel running to abolish conditioned place preference for cocaine in mice

Recent evidence suggests that wheel running can abolish conditioned place preference (CPP) for cocaine in mice. Running significantly increases the number of new neurons in the hippocampus, and new neurons have been hypothesised to enhance plasticity and behavioral flexibility. Therefore, we tested the hypothesis that increased neurogenesis was necessary for exercise to abolish cocaine CPP. Male nestin–thymidine kinase transgenic mice were conditioned with cocaine, and then housed with or without running wheels for 32 days. Half of the mice were fed chow containing valganciclovir to induce apoptosis in newly divided neurons, and the other half were fed standard chow. For the first 10 days, mice received daily injections of bromodeoxyuridine (BrdU) to label dividing cells. On the last 4 days, mice were tested for CPP, and then euthanized for measurement of adult hippocampal neurogenesis by counting the number of BrdU‐positive neurons in the dentate gyrus. Levels of running were similar in mice fed valganciclovir‐containing chow and normal chow. Valganciclovir significantly reduced the numbers of neurons (BrdU‐positive/NeuN‐positive) in the dentate gyrus of both sedentary mice and runner mice. Valganciclovir‐fed runner mice showed similar levels of neurogenesis as sedentary, normal‐fed controls. However, valganciclovir‐fed runner mice showed the same abolishment of CPP as runner mice with intact neurogenesis. The results demonstrate that elevated adult hippocampal neurogenesis resulting from running is not necessary for running to abolish cocaine CPP in mice.     

Age‐dependent role for Ras‐GRF1 in the late stages of adult neurogenesis in the dentate gyrus

The dentate gyrus of the hippocampus plays a pivotal role in pattern separation, a process required for the behavioral task of contextual discrimination. One unique feature of the dentate gyrus that contributes to pattern separation is adult neurogenesis, where newly born neurons play a distinct role in neuronal circuitry. Moreover, the function of neurogenesis in this brain region differs in adolescent and adult mice. The signaling mechanisms that differentially regulate the distinct steps of adult neurogenesis in adolescence and adulthood remain poorly understood. We used mice lacking RAS‐GRF1 (GRF1), a calcium‐dependent exchange factor that regulates synaptic plasticity and participates in contextual discrimination performed by mice, to test whether GRF1 plays a role in adult neurogenesis. We show Grf1 knockout mice begin to display a defect in neurogenesis at the onset of adulthood (～2 months of age), when wild‐type mice first acquire the ability to distinguish between closely related contexts. At this age, young hippocampal neurons in Grf1 knockout mice display severely reduced dendritic arborization. By 3 months of age, new neuron survival is also impaired. BrdU labeling of new neurons in 2‐month‐old Grf1 knockout mice shows they begin to display reduced survival between 2 and 3 weeks after birth, just as new neurons begin to develop complex dendritic morphology and transition into using glutamatergic excitatory input. Interestingly, GRF1 expression appears in new neurons at the developmental stage when GRF1 loss begins to effect neuronal function. In addition, we induced a similar loss of new hippocampal neurons by knocking down expression of GRF1 solely in new neurons by injecting retrovirus that express shRNA against GRF1 into the dentate gyrus. Together, these findings show that GRF1 expressed in new neurons promotes late stages of adult neurogenesis. Overall our findings show GRF1 to be an age‐dependent regulator of adult hippocampal neurogenesis, which contributes to ability of mice to distinguish closely related contexts. © 2013 Wiley Periodicals, Inc.     

Genetic labeling of steroidogenic factor‐1 (SF‐1) neurons in mice reveals ventromedial nucleus of the hypothalamus (VMH) circuitry beginning at neurogenesis and development of a separate non‐SF‐1 neuronal cluster in the ventrolateral VMH

The ventromedial nucleus of the hypothalamus (VMH) influences a wide variety of physiological responses. Here, using two distinct but complementary genetic tracing approaches in mice, we describe the development of VMH efferent projections, as marked by steroidogenic factor‐1 (SF‐1; NR5A1). SF‐1 neurons were visualized by Tau‐green fluorescent protein (GFP) expressed from the endogenous Sf‐1 locus (Sf‐1^TauGFP) or by crossing the transgenic Sf1:Cre driver to a GFP reporter strain (Z/EG^Sf1:Cre). Strikingly, VMH projections were visible early, at embryonic (E) 10.5, when few postmitotic SF1 neurons have been born, suggesting that formation of VMH circuitry begins at the onset of neurogenesis. At E14.5, comparison of these two reporter lines revealed that SF1‐positive neurons in the ventrolateral VMH (VMH_vl) persist in Z/EG^Sf1:Cre embryos but are virtually absent in Sf‐1^TauGFP. Therefore, although the entire VMH including the VMH_vl shares a common lineage, the VMH_vl further differentiates into a neuronal cluster devoid of SF‐1. At birth, extensive VMH projections to broad regions of the brain were observed in both mouse reporter lines, matching well with those previously discovered by injection of axonal anterograde tracers in adult rats. In summary, our genetic tracing studies show that VMH efferent projections are highly conserved in rodents and are established far earlier than previously appreciated. Moreover, our results imply that neurons in the VMH_vl adopt a distinct fate early in development, which might underlie the unique physiological functions associated with this VMH subregion. J. Comp. Neurol., 521:1268–1288, 2013. © 2012 Wiley Periodicals, Inc.     

The TIR‐domain‐containing adapter inducing interferon‐β‐dependent signaling cascade plays a crucial role in ischemia–reperfusion‐induced retinal injury,whereas the contribution of the myeloid differentiation primary response 88‐dependent signaling cascade is not as pivotal

Toll‐like receptor 4 (Tlr4) plays an important role in ischemia–reperfusion (IR)‐induced retinal inflammation and damage. However, the role of two Tlr4‐dependent signaling cascades, myeloid differentiation primary response 88 (Myd88) and TIR‐domain‐containing adapter inducing interferon‐β (Trif), in retinal IR injury is poorly understood. In this study, we investigated the contribution of the Myd88‐dependent and Trif‐dependent signaling cascades in retinal damage and inflammation triggered by IR, by using Myd88 knockout (Myd88KO) and Trif knockout (TrifKO) mice. Retinal IR injury was induced by unilateral elevation of intraocular pressure for 45 min by direct corneal cannulation. To study IR‐induced retinal ganglion cell (RGC) death in vitro, we used an oxygen and glucose deprivation (OGD) model. Our data suggested that Myd88 was present in many retinal layers of sham‐operated and ischemic mice, whereas Trif was mainly present in the ganglion cell layer (GCL). The level of Myd88 was increased in the retina after IR. We found that retinas of TrifKO mice had a significantly reduced neurotoxic pro‐inflammatory response and significantly increased survival of the GCL neurons after IR. Although Myd88KO mice had relatively low levels of inflammation in ischemic retinas, their levels of IR‐induced retinal damage were notably higher than those of TrifKO mice. We also found that Trif‐deficient RGCs were more resistant to death induced by OGD than were RGCs isolated from Myd88KO mice. These data suggested that, as compared with the Myd88‐dependent signaling cascade, Trif signaling contributes significantly to retinal damage after IR.     

NSCs promote hippocampal neurogenesis,metabolic changes and synaptogenesis in APP/PS1 transgenic mice

Adult neurogenesis and synaptic remodeling persist as a unique form of structural and functional plasticity in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) of the lateral ventricles due to the existence of neural stem cells (NSCs). Transplantation of NSCs may represent a promising approach for the recovery of neural circuits. Here, we aimed to examine effects of highly neuronal differentiation of NSCs transplantation on hippocampal neurogenesis, metabolic changes and synaptic formation in APP/PS1 mice. 12‐month‐old APP/PS1 mice were used for behavioral tests, immunohistochemistry, western blot, transmission electron microscopy and proton magnetic resonance spectroscopy (1H‐MRS). The results showed that N‐acetylaspartate (NAA) and Glutamate (Glu) levels were increased in the Tg‐NSC mice compared with the Tg‐PBS and Tg‐AD mice 10 weeks after NSCs transplantation. NSC‐induced an increase in expression of synaptophysin and postsynaptic protein‐95, and the number of neurons with normal synapses was significantly increased in Tg‐NSC mice. More doublecortin‐, BrdU/NeuN‐ and Nestin‐positive neurons were observed in the hippocampal DG and SVZ of the Tg‐NSC mice. This is the first demonstration that engrafted NSCs with a high differentiation rate to neurons can enhance neurogenesis in a mouse model of AD and can be detected by 1H‐MRS in vivo. It is suggested that engraft of NSCs can restore memory and promote endogenous neurogenesis and synaptic remodeling, moreover, 1H‐MRS can detect metabolite changes in AD mice in vivo. The observed changes in NAA/creatine (Cr) and glutamate (Glu)/Cr may be correlated with newborn neurons and new synapse formation.     

Pathological characterization of the glioblastoma border as shown during surgery using 5‐aminolevulinic acid‐induced fluorescence

Thirty consecutive surgical patients with glioblastoma, were operated upon using fluorescence induced by 5‐aminolevulinic acid as guidance. The fluorescent quality of the tissue was used to take biopsies from the tumor center, from the invasive area around it and from adjacent normal‐looking tissue. These samples were analyzed with HE, Ki‐67 and nestin. Nestin expression in tissue surrounding glioblastoma cases was compared to tissue surrounding vascular lesions, metastasis and hippocampal sclerosis. The rate of gross total resection assessed by volumetric MRI was 83%. Using HE examination as the gold standard, fluorescence identified solid tumor with 100% positive predictive value, invasive areas with 97%, and normal tissue with 67% negative predictive value. Ki67 stained some cells in 69% of the non‐fluorescent samples around the tumor. There was always strong nestin expression around the tumor but it was similar to control cases in non‐glioma lesions with subacute expansion. 5‐aminolevulinic acid fluorescence guidance is very reliable and can help to study the tumor–brain interface. Nestin expression is strong and constant in the tissue around the tumor, but is mostly an acute glial reaction, not specific of the neoplasm. Nestin staining is not recommended as a tumor stem cell marker.     

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.     

Nestin‐expressing cell types in the temporal lobe and hippocampus: Morphology,differentiation, and proliferative capacity

Nestin is expressed in immature neuroepithelial and progenitor cell types and transiently upregulated in proliferative neuroglial cells responding to acute brain injury, including following seizures. In 36 temporal lobe (TLobe) specimens from patients with TLobe epilepsy (age range 8–60 years) we studied the number, distribution and morphology of nestin‐expressing cells (NEC) in the pes, hippocampus body, parahippocampal gyrus, amygdala, temporal cortex and pole compared with post mortem control tissues from 26 cases (age range 12 gestational weeks to 76 years). The proliferative fraction of NEC was evaluated in selected regions, including recognized niches, using MCM2. Their differentiation was explored with neuronal (DCX, mushashi, βIII tubulin, NeuN) and glial (GFAP, GFAPdelta, glutamine synthetase, aquaporin4, EAAT1) markers, both in sections or following culture. Findings were correlated with clinical parameters. A stereotypical pattern in the distribution and morphologies of NEC was observed, reminiscent of patterns in the developing brain, with increased densities in epilepsy than adult controls (p < .001). Findings included MCM2‐positive radial glial‐like cells in the periventricular white matter and rows of NEC in the hippocampal fimbria and sulcus. Nestin cells represented 29% of the hippocampal proliferative fraction in epilepsy cases; 20% co‐expressed βIII tubulin in culture compared with 28% with GFAP. Significant correlations were noted between age at surgery, memory deficits and nestin populations. TLobe NEC with ongoing proliferative capacity likely represent vestiges of developmental migratory streams and resident reactive cell populations of potential relevance to hippocampal epileptogenesis, TLobe pathology, and co‐morbidities, including memory decline.     

Gfap‐positive radial glial cells are an essential progenitor population for later‐born neurons and glia in the zebrafish spinal cord

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189     

Time‐dependent enhancement of hippocampus‐dependent memory after treatment with memantine: Implications for enhanced hippocampal adult neurogenesis

Adult hippocampal neurogenesis has been suggested to play modulatory roles in learning and memory. Importantly, previous studies have shown that newborn neurons in the adult hippocampus are integrated into the dentate gyrus circuit and are recruited more efficiently into the hippocampal memory trace of mice when they become 3 weeks old. Interestingly, a single high‐dose treatment with the N‐methyl‐d ‐aspartate receptor antagonist memantine (MEM) has been shown to increase hippocampal neurogenesis dramatically by promoting cell proliferation. In the present study, to understand the impact of increased adult neurogenesis on memory performance, we examined the effects of a single treatment of MEM on hippocampus‐dependent memory in mice. Interestingly, mice treated with MEM showed an improvement of hippocampus‐dependent spatial and social recognition memories when they were trained and tested at 3–6 weeks, but not at 3 days or 4 months, after treatment with MEM. Importantly, we observed a significant positive correlation between the scores for spatial memory (probe trial in the Morris water maze task) and the number of young mature neurons (3 weeks old) in MEM‐treated mice, but not saline‐treated mice. We also observed that the young mature neurons generated by treatment with MEM were recruited into the trace of spatial memory similarly to those generated through endogenous neurogenesis. Taken together, our observations suggest that treatment with MEM temporally improves hippocampus‐dependent memory formation and that the newborn neurons increased by treatment with MEM contribute to this improvement when they become 3 weeks old. © 2014 The Authors. Hippocampus Published by Wiley Periodicals, Inc.     

Neuroprotective Effect of PACAP Against NMDA-Induced Retinal Damage in the Mouse

Retinal excitotoxicity is one of the major causes of retinal ganglion cell (RGC) death in glaucoma. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with potent neuroprotective activity; however, whether it exerts such an effect in the retina and the mechanism by which RGCs are protected is still not well understood. In this study, we examined the effect of exogenous and endogenous PACAP on RGC death induced by N-methyl-d-aspartate acid (NMDA). The vitreous body of anesthetized adult male mice (C57/BL6J) was injected with NMDA (40?nmol in a 2???L saline solution). The number of RGCs decreased from days?1 to 7 after NMDA injection, and the number of dUTP end-labeling (TUNEL)-positive cells, an indicator of cell death, peaked at day?3. However, when PACAP38 (10^?8, 10^?10, 10^?12, 10^?14, or 10^?16M) was co-administered with NMDA, the 10^?10M dose resulted in significantly increased RGC survival at day?7, and a decrease in the number of TUNEL-positive RGCs at day?3. We next investigated the neuroprotective effect of endogenous PACAP using PACAP heterozygote(+/?) mice. Under normal circumstances, there was no significant difference in the number of RGCs in the PACAP(+/?) mice compared with their wild-type counterparts. However, the number of RGCs significantly decreased in the PACAP(+/?) mice 7?days after NMDA injection, relative to their wild-type counterparts. The number of TUNEL-positive RGCs peaked at day?1 in the PACAP(+/?) mice. These effects in the PACAP(+/?) mice were reversed by intravitreous injection of 10^?10M PACAP38. This suggests that exogenous PACAP is able to counteract NMDA-induced toxicity, and that endogenous PACAP exerts a neuroprotective effect in the retina.