In vivo contribution of nestin‐ and GLAST‐lineage cells to adult hippocampal neurogenesis

Radial glia‐like cells (RGCs) are the hypothesized source of adult hippocampal neurogenesis. However, the current model of hippocampal neurogenesis does not fully incorporate the in vivo heterogeneity of RGCs. In order to better understand the contribution of different RGC subtypes to adult hippocampal neurogenesis, we employed widely used transgenic lines (Nestin‐CreER^T2 and GLAST::CreER^T2 mice) to explore how RGCs contribute to neurogenesis under basal conditions and after stimulation and depletion of neural progenitor cells. We first used these inducible fate‐tracking transgenic lines to define the similarities and differences in the contribution of nestin‐ and GLAST‐lineage cells to basal long‐term hippocampal neurogenesis. We then explored the ability of nestin‐ and GLAST‐lineage RGCs to contribute to neurogenesis after experimental manipulations that either ablate neurogenesis (i.c.v. application of the anti‐mitotic AraC, cytosine‐β‐D‐arabinofuranoside) or stimulate neurogenesis (wheel running). Interestingly, in both ablation and stimulation experiments, labeled RGCs in GLAST::CreER^T2 mice appear to contribute to neurogenesis, whereas RGCs in Nestin‐CreER^T2 mice do not. Finally, using NestinGFP reporter mice, we expanded on previous research by showing that not all RGCs in the adult dentate gyrus subgranular zone express nestin, and therefore RGCs are antigenically heterogeneous. These findings are important for the field, as they allow appropriately conservative interpretation of existing and future data that emerge from these inducible transgenic lines. These findings also raise important questions about the differences between transgenic driver lines, the heterogeneity of RGCs, and the potential differences in progenitor cell behavior between transgenic lines. As these findings highlight the possible differences in the contribution of cells to long‐term neurogenesis in vivo, they indicate that the current models of hippocampal neurogenesis should be modified to include RGC lineage heterogeneity. © 2013 Wiley Periodicals, Inc.     

Transient Cnp expression by early progenitors causes Cre‐Lox‐based reporter lines to map profoundly different fates

NG2 expressing oligodendroglial precursor cells are ubiquitous in the central nervous system and the only cell type cycling throughout life. Previous fate mapping studies have remained inconsistent regarding the question whether NG2 cells are capable of generating certain types of neurons. Here, we use CNP‐Cre mice to map the fate of a sub‐population of NG2 cells assumed to be close to differentiation. When crossing these mice with the ROSA26/YFP Cre‐reporter line we discovered large numbers of reporter‐expressing pyramidal neurons in the piriform and dorsal cortex. In contrast, when using Z/EG reporter mice to track the fate of Cnp‐expressing NG2 cells only oligodendroglial cells were found reporter positive. Using BrdU‐based birth dating protocols and inducible NG2CreER:ROSA26/YFP mice we show that YFP positive neurons are generated from radial glial cells and that these radial glial cells display temporary and low level activity of certain oligodendroglial genes sufficient to recombine the Cre‐inducible reporter gene in ROSA26/YFP but not in Z/EG mice. Taken together, we did not obtain evidence for generation of neurons from NG2 cells. Our results suggest that with an appropriate reporter system Cnp activity can be used to define a proliferative subpopulation of NG2 cells committed to generate oligodendrocytes. However, the strikingly different results obtained from ROSA26/YFP versus Z/EG mice demonstrate that the choice of Cre‐reporter line can be of crucial importance for fate mapping studies and other applications of the Cre‐lox technology. GLIA 2017;65:342–359     

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.     

Relationship between post‐traumatic stress disorder‐like behavior and reduction of hippocampal 5‐bromo‐2′‐deoxyuridine‐positive cells after inescapable shock in rats

Aim: Inescapable shocks (IS) have been reported to reduce the number of 5‐bromo‐2′‐deoxyuridine (BrdU)‐positive cells in hippocampus. Antidepressants prevent this reduction, and the role of neurogenesis in depression is now suggested. It has been reported, however, that the number of BrdU‐positive cells was not different between the rats that developed learned helplessness and those that did not. This suggests that reduction of neurogenesis does not constitute a primary etiology of depression. It has been previously shown that IS can cause various post‐traumatic stress disorder (PTSD)‐like behavioral changes in rats. The aim of the present was therefore to examined whether the reduction of BrdU‐positive cells relates to any PTSD‐like behavioral changes in this paradigm. Methods: Rats were given either inescapable foot‐shocks (IS) or not shocked (non‐S) treatment in a shuttle box on day 1 and received BrdU injections once daily during the first week after IS/non‐S treatment. On day 14, rats treated with IS and non‐S were given an avoidance/escape test in the shuttle box and dorsal hippocampal SGZ were analyzed by BrdU immunohistochemistry. Results: In accordance with previously reported results, IS loading resulted in fewer BrdU‐positive cells in the hippocampal subgranular zone (SGZ). Furthermore, in the IS‐treated group, the number of BrdU‐positive cells in the hippocampal SGZ was negatively correlated at a significant level with several hyperactive behavioral parameters but not with hypoactive behavioral parameters. Earlier findings had indicated that chronic selective serotonin re‐uptake inhibitor administration, which is known to increase hippocampal neurogenesis, restored the increase in hypervigilant/hyperarousal behavior but did not attenuate the increase in numbing/avoidance behavior. Conclusion: The regulatory mechanism responsible for the decreased proliferation and survival of cells in the hippocampus may be related to the pathogenic processes of hypervigilance/hyperarousal behaviors.     

Impact of melatonin receptor‐signaling on Zeitgeber time‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)‐mediated signaling. Adult melatonin‐proficient C3H/HeN mice and melatonin‐proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12‐h light, 12‐h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT‐dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin‐proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT‐dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24‐h light‐dark cycle. Apoptosis showed ZT‐dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT‐dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT‐dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2‐mediated signaling appears to be crucial for generation and timing of ZT‐dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.     

Expression of hairy/enhancer of split genes in neural progenitors and neurogenesis domains of the adult zebrafish brain

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.     

Prolonged voluntary wheel‐running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice

Voluntary wheel‐running induces a rapid increase in proliferation and neurogenesis by neural precursors present in the adult rodent hippocampus. In contrast, the responses of hippocampal and other central nervous system neural precursors following longer periods of voluntary physical activity are unclear and are an issue of potential relevance to physical rehabilitation programs. We investigated the effects of a prolonged, 6‐week voluntary wheel‐running paradigm on neural precursors of the CD1 mouse hippocampus and forebrain. Examination of the hippocampus following 6 weeks of running revealed two to three times as many newly born neurons and 60% more proliferating cells when compared with standard‐housed control mice. Among running mice, the number of newly born neurons correlated with the total running distance. To establish the effects of wheel‐running on hippocampal precursors dividing during later stages of the prolonged running regime, BrdU was administered after 3 weeks of running and the BrdU‐retaining cells were analyzed 18 days later. Quantifications revealed that the effects of wheel‐running were maintained in late‐stage proliferating cells, as running mice had two to three times as many BrdU‐retaining cells within the hippocampal dentate gyrus, and these yielded greater proportions of both mature neurons and proliferative cells. The effects of prolonged wheel‐running were also detected beyond the hippocampus. Unlike short‐term wheel‐running, prolonged wheel‐running was associated with higher numbers of proliferating cells within the ventral forebrain subventricular region, a site of age‐associated decreases in neural precursor proliferation and neurogenesis. Collectively, these findings indicate that (i) prolonged voluntary wheel‐running maintains an increased level of hippocampal neurogenesis whose magnitude is linked to total running performance, and (ii) that it influences multiple neural precursor populations of the adult mouse brain. © 2009 Wiley‐Liss, Inc.     

Seizure severity‐dependent selective vulnerability of the granule cell layer and aberrant neurogenesis in the rat hippocampus

The pilocarpine‐induced status epilepticus rodent model has been commonly used to analyze the mechanisms of human temporal lobe epilepsy. Recent studies using this model have demonstrated that epileptic seizures lead to increased adult neurogenesis of the dentate granule cells, and cause abnormal cellular organization in dentate neuronal circuits. In this study, we examined these structural changes in rats with seizures of varying severity. In rats with frequent severe seizures, we found a clear loss of Prox1 and NeuN expression in the dentate granule cell layer (GCL), which was confined mainly to the suprapyramidal blade of the GCL at the septal and middle regions of the septotemporal axis of the hippocampus. In the damaged suprapyramidal region, the number of immature neurons in the subgranular zone was markedly reduced. In contrast, in rats with less frequent severe seizures, there was almost no loss of Prox1 and NeuN expression, and the number of immature neurons was increased. In rats with no or slight loss of Prox1 expression in the GCL, ectopic immature neurons were detected in the molecular layer of the suprapyramidal blade in addition to the hilus, and formed chainlike aggregated structures along the blood vessels up to the hippocampal fissure, suggesting that newly generated neurons migrate at least partially along blood vessels to the hippocampal fissure. These results suggest that seizures of different severity cause different effects on GCL damage, neurogenesis, and the migration of new neurons, and that these structural changes are selective to subdivisions of the GCL and the septotemporal axis of the hippocampus.     

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF‐κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF‐κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell‐autonomous and noncell‐autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC–MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin‐2 (LCN‐2) was recognized as a novel astroglial‐derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN‐2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN‐2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin‐1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC‐astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169–181     

Transient expression of the conserved zinc finger gene INSM1 in progenitors and nascent neurons throughout embryonic and adult neurogenesis

INSM1 is a zinc-finger protein expressed in the developing nervous system and pancreas as well as in medulloblastomas and neuroendocrine tumors. With in situ hybridization combined with immunohistochemistry, we detected INSM1 mRNA in all embryonic to adult neuroproliferative areas examined: embryonic neocortex, ganglionic eminence, midbrain, retina, hindbrain, and spinal cord; autonomic, dorsal root, trigeminal and spiral ganglia; olfactory and vomeronasal organ epithelia; postnatal cerebellum; and juvenile to adult subgranular zone of dentate gyrus, subventricular zone, and rostral migratory stream leading to olfactory bulb. In most of these neurogenic areas, subsets of neuronal progenitors and nascent, but not mature, neurons express INSM1. For example, in developing cerebellum, INSM1 is present in proliferating progenitors of the outer external granule layer (EGL) and in postmitotic cells of the inner EGL, but not in mature granule cell neurons. Also, lining the neural tube from spinal cord to neocortex in mouse as well as human embryos, cells undergoing mitosis apically do not express INSM1. By contrast, nonsurface progenitors located in the basal ventricular and/or subventricular zones express INSM1. Whereas apical progenitors are proliferative and generate one or two additional progenitors, basal progenitors are thought to divide terminally and symmetrically to produce two neurons. The nematode ortholog of INSM1, EGL-46, is expressed during terminal symmetric neurogenic divisions and regulates the termination of proliferation. We propose that, in mice and humans, INSM1 is likewise expressed transiently during terminal neurogenic divisions, from late progenitors to nascent neurons, and particularly during symmetric neuronogenic divisions.     

Ras‐GRF2 mediates long‐term potentiation,survival, and response to an enriched environment of newborn neurons in the hippocampus

Hippocampal adult neurogenesis contributes to key functions of the dentate gyrus (DG), including contextual discrimination. This is due, at least in part, to the unique form of plasticity that new neurons display at a specific stage of their development when compared with the surrounding principal neurons. In addition, the contribution that newborn neurons make to dentate function can be enhanced by an increase in their numbers induced by a stimulating environment. However, signaling mechanisms that regulate these properties of newborn neurons are poorly understood. Here, we show that Ras‐GRF2 (GRF2), a calcium‐regulated exchange factor that can activate Ras and Rac GTPases, contributes to both of these properties of newborn neurons. Using Ras‐GRF2 knockout mice and wild‐type mice stereotactically injected with retrovirus containing shRNA against the exchange factor, we demonstrate that GRF2 promotes the survival of newborn neurons of the DG at approximately 1–2 weeks after their birth. GRF2 also controls the distinct form of long‐term potentiation that is characteristic of new neurons of the hippocampus through its effector Erk MAP kinase. Moreover, the enhancement of neuron survival that occurs after mice are exposed to an enriched environment also involves GRF2 function. Consistent with these observations, GRF2 knockout mice display defective contextual discrimination. Overall, these findings indicate that GRF2 regulates both the basal level and environmentally induced increase of newborn neuron survival, as well as in the induction of a distinct form of synaptic plasticity of newborn neurons that contributes to distinct features of hippocampus‐derived learning and memory. © 2014 Wiley Periodicals, Inc.     

All‐trans retinoic acid improved impaired proliferation of neural stem cells and suppressed microglial activation in the hippocampus in an Alzheimer's mouse model

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by cognitive impairment with neuronal loss. The number of patients suffering from AD has increased, but none of the present therapies stops the progressive symptoms in patients with AD. It has been reported that the activation of microglial cells induces harmful chronic inflammation, leading to neuronal death. Furthermore, the impairment of adult neurogenesis in the hippocampus has been observed earlier than amyloid plaque formation. Inflammatory response may lead to impaired adult neurogenesis in patients with AD. This study examines the relationship between adult neurogenesis and neuroinflammation using APPswe/PS1M146V/tauP301L (3 × Tg) mice. We observed a decline in the proliferation of neural stem cells and the occurrence of severe inflammation in the hippocampus of 3 × Tg mouse brains at 12 months of age. Previously, our research had shown an anti‐inflammatory effect of all‐trans retinoic acid (ATRA) in the 3 × Tg mouse brain. We found that ATRA has effects on the recovery of proliferative cells along with suppression of activated microglia in the hippocampus. These results suggest that the inhibition of microglial activation by ATRA leads to recovery of adult neurogenesis in the hippocampus in an AD mouse model. © 2016 Wiley Periodicals, Inc.     

Site‐specific regulation of adult neurogenesis by dietary fatty acid content,vitamin E and flight exercise in European starlings

Exercise is known to have a strong effect on neuroproliferation in mammals ranging from rodents to humans. Recent studies have also shown that fatty acids and other dietary supplements can cause an upregulation of neurogenesis. It is not known, however, how exercise and diet interact in their effects on adult neurogenesis. We examined neuronal recruitment in multiple telencephalic sites in adult male European starlings (Sturnus vulgaris) exposed to a factorial combination of flight exercise, dietary fatty acids and antioxidants. Experimental birds were flown in a wind tunnel following a training regime that mimicked the bird's natural flight behaviour. In addition to flight exercise, we manipulated the composition of dietary fatty acids and the level of enrichment with vitamin E, an antioxidant reported to enhance neuronal recruitment. We found that all three factors – flight exercise, fatty acid composition and vitamin E enrichment – regulate neuronal recruitment in a site‐specific manner. We also found a robust interaction between flight training and vitamin E enrichment at multiple sites of neuronal recruitment. Specifically, flight training was found to enhance neuronal recruitment across the telencephalon, but only in birds fed a diet with a low level of vitamin E. Conversely, dietary enrichment with vitamin E upregulated neuronal recruitment, but only in birds not flown in the wind tunnel. These findings indicate conserved modulation of adult neurogenesis by exercise and diet across vertebrate taxa and indicate possible therapeutic interventions in disorders characterized by reduced adult neurogenesis.     

Transplanted human embryonic neural stem cells survive,migrate, differentiate and increase endogenous nestin expression in adult rat cortical peri‐infarction zone

Transplantation of stem cells is a potential therapeutic strategy for stroke damage. The survival, migration, and differentiation of transplanted human embryonic neural stem cells in the acute post‐ischemic environment were characterized and endogenous nestin expression after transplantation was investigated. Human embryonic neural stem cells obtained from the temporal lobe cortex were cultured and labeled with fluorescent 1,1′‐dioctadecy‐6,6′‐di (4‐sulfopheyl)‐3,3,3′,3′‐tetramethylindocarbocyanin (DiI) in vitro. Labeled cells were transplanted into cortical peri‐infarction zones of adult rats 24 h after permanent middle cerebral artery occlusion. Survival, migration, and differentiation of grafted cells were quantified in immunofluorescence‐stained sections from rats sacrificed at 7, 14, and 28 days after transplantation. Endogenous nestin‐positive cells in the cortical peri‐infarction zone were counted at serial time points. The cells transplanted into the cortical peri‐infarction zone displayed the morphology of living cells and became widely located around the ischemic area. Moreover, some of the transplanted cells expressed nestin, GFAP, or NeuN in the peri‐infarction zone. Furthermore, compared with the control group, endogenous nestin‐positive cells in the peri‐infarction zone had increased significantly 7 days after cell transplantation. These results confirm the survival, migration, and differentiation of transplanted cells in the acute post‐ischemic environment and enhanced endogenous nestin expression within a brief time window. These findings indicate that transplantation of neural stem cells into the peri‐infarction zone may be performed as early as 24 h after ischemia.