雌激素在脑缺血诱导的大鼠齿状回神经再生中的作用

为研究雌激素对成年动物局灶性脑缺血诱导成年动物海马齿状回神经元再生的影响,将雄性SD大鼠分为假手术 雌激素组(SE)、假手术 生理盐水替代组(SN)、缺血 雌激素组(ME)和缺血 生理盐水替代组(MN),右侧大脑中动脉闭塞(MCAO)建立脑缺血模型。在缺血90min后恢复供血再灌注,分别于再灌注后1、3、12、24和28h处死老鼠并检测各组大鼠脑梗死体积、细胞凋亡以及脑缺血诱导的成年动物海马齿状回神经元再生的情况。在5个时间点的检测中,ME组脑梗死体积显著小于SE组(P<0.05);在MCAO大鼠中,海马齿状回区域并未发现有神经元丢失及凋亡的现象。同时,MN组与SN组相比较,损伤侧齿状回新生神经元数目明显增多(P<0.05),说明这种缺血诱导的神经元再生并不依赖于齿状回区域神经细胞的死亡;ME组与MN组相比较,损伤侧新生神经元数目显著增多(P<0.05);SE与SN组相比较,手术侧和对侧的新生神经元数目都显著增加(P<0.05)。结果提示雌激素对局灶性脑缺血后海马齿状回神经元再生具有促进作用,且这种促进作用与海马缺血损伤程度无关。     

Unsupervised Learning and Adaptation in a Model of Adult Neurogenesis

Adult neurogenesis has long been documented in the vertebrate brain and recently even in humans. Although it has been conjectured for many years that its functional role is related to the renewing of memories, no clear mechanism as to how this can be achieved has been proposed. Using the mammalian olfactory bulb as a paradigm, we present a scheme in which incorporation of new neurons proceeds at a constant rate, while their survival is activity-dependent and thus contingent on new neurons establishing suitable connections. We show that a simple mathematical model following these rules organizes its activity so as to maximize the difference between its responses and can adapt to changing environmental conditions in unsupervised fashion, in agreement with current neurophysiological data.     

PlexinA2 Forward Signaling through Rap1 GTPases Regulates Dentate Gyrus Development and Schizophrenia-like Behaviors

1. Download high-res image (171KB)
2. Download full-size image

     

Not all water mazes are created equal: cyclin D2 knockout mice with constitutively suppressed adult hippocampal neurogenesis do show specific spatial learning deficits

Studies using the Morris water maze to assess hippocampal function in animals, in which adult hippocampal neurogenesis had been suppressed, have yielded seemingly contradictory results. Cyclin D2 knockout (Ccnd2^?/?) mice, for example, have constitutively suppressed adult hippocampal neurogenesis but had no overt phenotype in the water maze. In other paradigms, however, ablation of adult neurogenesis was associated with specific deficits in the water maze. Therefore, we hypothesized that the neurogenesis‐related phenotype might also become detectable in Ccnd2^?/? mice, if we used the exact setup and protocol that in our previous study had revealed deficits in mice with suppressed adult neurogenesis. Ccnd2^?/? mice indeed learned the task and developed a normal preference for the goal quadrant, but were significantly less precise for the exact goal position and were slower in acquiring efficient and spatially more precise search strategies. Upon goal reversal (when the hidden platform was moved to a new position) Ccnd2^?/? mice showed increased perseverance at the former platform location, implying that they were less flexible in updating the previously learned information. Both with respect to adult neurogenesis and behavioral performance, Ccnd2^+/? mice ranged between wild types and knockouts. Importantly, hippocampus‐dependent learning was not generally impaired by the mutation, but specifically functional aspects relying on precise and flexible encoding were affected. Whether ablation of adult neurogenesis causes a specific behavioral phenotype thus also depends on the actual task demands. The test parameters appear to be important variables influencing whether a task can pick up a contribution of adult neurogenesis to test performance.     

Sevoflurane Induces Hippocampal Neuronal Apoptosis by Altering the Level of Neuropeptide Y in Neonatal Rats

Numerous studies have shown that the inhaled general anesthetic sevoflurane imposes toxicity on the central nervous system during the developmental period but the underlying mechanisms remain unclear. Neuropeptide Y (NPY) was reported to have important neuroprotective effects, which can attenuate neuronal loss under pathological conditions. However, the effects of NPY on sevoflurane-induced hippocampal neuronal apoptosis have not been investigated. In this study, postnatal day 7 (PND7) Sprague–Dawley rats and primary cultured cells separated from hippocampi were exposed to sevoflurane (2.4% for 4 h) and the NPY expression levels after treatment were analyzed. Furthermore, neuronal apoptosis assay was conducted via immunofluorescence staining of cleaved caspase-3 and flow cytometry after exogenous NPY administration to PND7 rats as well as cultured hippocampal neurons to elucidate the role of NPY in sevoflurane-induced neurotoxicity. Our results showed the level of NPY gradually decreased within 24 h after sevoflurane exposure in both the hippocampus of PND7 rats and cultured hippocampal neurons, but not in cultured astrocytes. In the exogenous NPY pretreatment study, the proportion of cleaved caspase-3 positive cells in the CA1 region of the hippocampus was increased significantly at 24 h after sevoflurane treatment, while NPY pretreatment could reduce it. Similarly, NPY could also reverse the apoptogenic effect of sevoflurane on cultured neurons. Herein, our results showed that sevoflurane caused a significant decrease in NPY expression, whereas exogenous NPY supplementation could reduce sevoflurane-induced hippocampal neuronal apoptosis both in vivo and in vitro.

     

Pulse labeling and long-term tracing of newborn neurons in the adult subgranular zone

Research over the past decades has demonstrated that adult brain produces neural progenitor cells which proliferate and differentiate to newborn neurons that integrate into the existing circuit. However, detailed differentiation processes and underlying mechanisms of newly generated neurons are largely unknown due to the limitation of available methods for labeling and manipulating neural progenitor cells and newborn neurons. In this study, we designed a tightly controlled, noninvasive system based on Cre/loxP recombination to achieve long-term tracing and genetic manipulation of adult neurons in vivo. In this system, tamoxifen-inducible recombinase, CreER(T2), was driven by BAC-based promoter of doublecortin (DCX, a marker of newborn neurons). By crossing this Cre line with reporter mouse, we found that newborn neurons in the dentate gyrus (DG) could be selectively pulse-labeled by tamoxifen-induced expression of yellow fluorescent protein (YFP). YFP-positive neurons were identified by coimmunostaining with cell type-specific markers and characterized by electrophysiological recording. Furthermore, analysis of the migration of these neurons showed that the majority of these labeled neurons migrated to the inner part of granule cell layer. Moreover, spine growth of inner molecular layer of newborn granule neurons takes a dynamic pattern of invert U-shape, in contrast to the wedge-shaped change in the outer molecular layer. Our transgenic tool provides an efficient way to selectively label and manipulate newborn neuron in adult mouse DG.     

Structural Remodeling of the Neuronal Network in the Dentate Gyrus of the Epileptically Transformed Murine Hippocampus

Immunohistochemical analysis of the hippocampus of a transgene mutant line of the mice (genetic model of temporal epilepsy) demonstrated a progressive increase in the level of brain-derived neurotrophic factor (BDNF) in granular cells of the dentate gyrus and their axons; this increase correlated with an enhancement of the level of epileptic activity. Epileptogenic reprojection of mossy fibers toward an internal zone of the molecular layer of the hippocampus was also observed. In addition, we observed excessive spreading of the zone of axon branching of GABA-ergic basket cells, as compared with the norm; their maximum collateralization was found within an internal zone of the molecular layer of the dentate gyrus. This allows us to hypothesize that the processes of sprouting of the mossy fibers and recovery of recurrent inhibition of the granular cells are interrelated.     

Pre- and Posttreatment With Edaravone Protects CA1 Hippocampus and Enhances Neurogenesis in the Subgranular Zone of Dentate Gyrus After Transient Global Cerebral Ischemia in Rats

Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs) in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15), control (n = 15), and edaravone-treated (n = 15) groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α) and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p < .05). Treatment with edaravone also decreased apoptosis of NSPCs (p < .01). Furthermore, treatment with edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia.     

Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats

Past research suggested that androgens may play a role in the regulation of adult neurogenesis within the dentate gyrus. We tested this hypothesis by manipulating androgen levels in male rats. Castrated or sham castrated male rats were injected with 5-Bromo-2'deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or glial) using immunohistochemistry. Castrated males showed a significant decrease in 30-day cell survival within the dentate gyrus but there was no significant change in cell proliferation relative to control males, indicating that androgens positively affect cell survival, but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were injected with testosterone s.c. for 30 days starting the day after BrdU injection. Higher doses (0.5 and 1.0 mg/kg) but not a lower dose (0.25 mg/kg) of testosterone resulted in a significant increase in neurogenesis relative to controls. We next tested the role of testosterone's two major metabolites, dihydrotestosterone (DHT), and estradiol, upon neurogenesis. Thirty days of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a significant increase in hippocampal neurogenesis. These results suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism.     

Polysialic acid regulates the clustering, migration, and neuronal differentiation of progenitor cells in the adult hippocampus

Newborn cells of the adult dentate gyrus in the hippocampus are characterized by their abundant expression of polysialic acid (PSA), a carbohydrate attached to the neural cell adhesion molecule (NCAM). PSA+ newborn cells of the dentate gyrus form clusters with proliferating neural progenitor cells, migrate away from these clusters, and terminally differentiate. To identify the roles of PSA in the development of adult progenitors of the dentate gyrus, we injected endoneuraminidase N (endoN) into the hippocampus of adult rats to specifically cleave PSA from NCAM. Two days later, we administered the mitotic marker, 5-bromo-2'-deoxyuridine (BrdU). Three days after BrdU injection, BrdU+ cells were found inside and outside the clusters of newborn cells. In endoN-treated animals, the total number of BrdU+ cells was not changed but significantly more BrdU+ cells were present within clusters, suggesting that PSA normally facilitates the migration of progenitors away from the clusters. Seven days post-BrdU injection, endoN-treated animals had significantly more BrdU+ cells which were also positive for the mature neuronal nuclear marker NeuN compared with controls, indicating that the loss of PSA from progenitor cells increases neuronal differentiation. This report is the first demonstration that PSA is involved in controlling the spatio-temporal neuronal maturation of adult hippocampal progenitors in the normal brain. In vitro, the removal of PSA from adult-derived neural progenitors significantly enhanced neuronal differentiation, strengthening our in vivo findings and indicating that PSA removal on isolated progenitor cells, apart from a complex in vivo environment, induces neuronal maturation.     

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.     

强制运动促进成年大鼠海马齿状回神经发生并提高学习能力

为了探讨强制运动对成年大鼠海马齿状回（dentate gyrus,DG）神经发生的影响,强制大鼠在马达驱动的转轮中跑步,用5-溴-2-脱氧尿苷（5-bromo-2-deoxyuridine,BrdU）标记增殖细胞,巢蛋白（neuroepthelial stem cell protein,nestin）标记神经干细胞／前体细胞,然后用免疫细胞化学技术检测大鼠DG中BrdU及nestin阳性细胞。为了解强制运动后DG增殖细胞的功能意义,采用Y-迷宫检测大鼠的学习能力。结果表明,强制运动组DG中BrdU及nestin阳性细胞数均日月显多于对照组（P〈0．05）：强制运动对DG神经发生的效应有强度依赖性。Y-迷宫检测结果显示,强制运动能明显改善大鼠的学习能力。结果提示,在转轮中进行强制跑步能促进成年火鼠DG的神经发生,并改善学习能力。     

An inducible transgenic Cre mouse line for the study of hippocampal development and adult neurogenesis

The hippocampus is crucial for higher brain functions, such as learning, memory, and emotion. Many diseases like epilepsy and Down's syndrome are associated with abnormalities in early hippocampal development. In addition, adult dentate neurogenesis is thought to be defective in several classes of psychiatric disorders. However, the mechanisms regulating hippocampal development and adult neurogenesis remain unclear. One of the limitations to studying these processes is the scarcity of available specific mouse tools. Here, we report an inducible transgenic Cre mouse line, Frizzled 9‐CreER?, in which tamoxifen administration induces Cre recombinant. Our data show that Cre is expressed in the developing hippocampal primordium, confined to the granule cell layer at P20 and further limited to the subgranular zone in the adult dentate gyrus. Cre recombinase shows very high activity in all of these regions. Thus, this transgenic line will be a powerful tool in understanding the mechanisms of hippocampal development, adult neurogenesis, and associated diseases. genesis 49:919–926, 2011. © 2011 Wiley Periodicals, Inc.     

Lithium enhances long-term potentiation independently of hippocampal neurogenesis in the rat dentate gyrus

We measured the temporal and spatial profiles of neural precursor cells, hippocampal long-term potentiation (LTP), and signaling molecules in neurogenesis-induced adult rats. Chronic lithium treatment produced a significant 54% and 40% increase in the numbers of bromodeoxyuridine [BrdU(+)] cells after 12 h and 28 days, respectively, after treatment completion in the dentate gyrus (DG). Both LTP obtained from slices perfused with artificial cerebrospinal fluid (ACSF-LTP) and LTP recorded in the presence of bicuculline (bicuculline-LTP) were significantly greater in the lithium group than in the saline controls. Although the number of BrdU(+) cells, approximately 90% of which were double-labeled with a neural marker neuronal nuclear protein, were markedly increased in the granule cell layer (GCL) 28 days after the completion of the 28-day lithium treatment, the magnitude of LTP observed at this time was similar to that observed 12 h after completing the 28-day lithium treatment. However, protein levels of calcium and calmodulin-dependent protein kinase II, p-Elk and TrkB were highly elevated until 28 days after the 28-day lithium treatment. Acute lithium treatment for 2 days also enhanced LTP, which was accompanied by the elevated expression of p-CREB, but not by neurogenesis. Our results suggest that the enhancement of LTP is independent of the increased number of neurons per se and it is more closely associated with key molecules, which are probably involved in neurogenesis.     

Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal

Seasonal changes in vertebrate brain function are pervasive, but annual cycles in the rates of neuronal incorporation are established only in songbirds. Although cell division continues in the subependymal and hippocampal subgranular zones of adult rodents, there exists no parallel evidence that seasonal plasticity in mammals extends to changes in neuronal or glial number. We examined the effect of photoperiod on incorporation of new neurons in the brain of the adult golden hamster, a long-day breeder. We administered the cell birth marker 5′-bromode-oxyuridine (BrdU) to males which had either been maintained in long days, transferred to short days for 10 weeks, or moved acutely from long to short or short to long days. The number of cells in specific brain regions immunoreactive (ir) for this thymidine analog was determined 7 weeks later. The number of BrdU-ir cells in the dentate gyrus and subependymal zone increased twofold in short days. Transfer between photoperiods 10 days before the BrdU injections produced intermediate numbers of BrdU-labeled cells in the dentate gyrus, but was as effective as long-term photoperiodic exposure in the subependymal zone. Photoperiod also had similar effects in the hypothalamus and cingulate/retrosplenial cortex, but not in the central gray or preoptic area. Double-label immunocytochemistry indicated that very few of the BrdU-ir cells were glia, but that a majority had neuronal phenotype. In the subependymal zone, short days significantly increased the number of BrdU-labeled neurons. We did not detect significant effects of photoperiod on the volume of either the granule cell layer of the hippocampus or the dentate gyrus as a whole. We conclude that short day lengths increase neuronal birth and/or survival in several brain regions of adult hamsters. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 410–420, 1998