Notch1信号系统在氟西汀上调大鼠海马神经再生中的作用

目的 了解慢性氟西汀干预正常大鼠所导致的海马神经再生上调与Notch1信号系统功能改变的关系.方法 应用大鼠腹腔注射氟西汀建立在体模型,分为对照组、14 d干预组、28 d干预组(n=12),采用免疫组化、real time PCR和Western blot,测定大鼠海马神经干细胞的增殖、存活和分化以及Notch1信号通路各个因子(NICD、Hes1、Hes5、Jag1)的基因及蛋白表达水平的改变.结果 ①与对照组(2919.50±188.80)比较,14 d氟西汀干预组(3706.50±228.04)、28 d氟西汀干预组(4334.33±217.48)海马齿状回神经干细胞增殖明显增加(P<0.001);与对照组(2404.50±148.77)相比,Flu干预28 d组(3273.16±156.68)海马齿状回神经干细胞存活明显增加(P<0.001);与对照组比较,氟西汀干预组NeuN/BrdU、GFAP/BrdU比例无明显差异(P＞0.05).②与对照组[NICDmRNA (0.30±0.03),Hes1mRNA (0.53±0.03),Hes5mRNA (0.21±0.02),Jag1mRNA(1.04±0.07)]比较,氟西汀(Flu)干预14d组[NICDmRNA (0.45±0.05),Hes1mRNA (0.65±0.06),Hes5mRNA (0.31±0.06),Jag1mRNA(2.46±0.39)]和Flu干预28 d组[NICDmRNA (0.42±0.03),Hes1mRNA (0.85±0.06),Hes5mRNA (0.39±0.02),Jag1mRNA(3.21±0.34)]Notch1信号通路各因子基因水平均明显升高(P<0.01或P<0.001).③与对照组[NICD(2.36±0.17),Hes1(1.09±0.25),Jag1(2.33±0.31)]比较,Flu干预14 d组[NICD(3.20±0.25),Jag1(2.86±0.25)]和Flu干预28 d组[NICD(3.40±0.19),Hes1(1.43±0.13),Jag1(3.35±0.14)]NICD、Hes1、Jag1蛋白水平明显升高,差异有统计学意义(P<0.01或P<0.001).与对照组Hes5比较,Flu干预14 d组Hes5和Flu干预28 d Hes5蛋白水平无改变,差异无统计学意义(P＞0.05).结论 氟西汀促进大鼠海马齿状回神经干细胞的增殖和存活,但对分化无影响;同时,海马Notch信号功能激活,提示Notch1信号系统可能参与氟西汀介导的大鼠海马神经再生上调.     

Developmental and degenerative modulation of GABAergic transmission in the mouse hippocampus

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter involved in synaptic plasticity. GABAergic transmission is also implicated in developmental and degenerative processes in the brain. The goal of the present study was to understand the developmental and degenerative regulation of GABAergic transmission in the mouse hippocampus by examining changes in GABA receptor subunit mRNA levels and GABA-related protein expression during postnatal development of the hippocampus and trimethyltin (TMT)-induced neurodegeneration in the juvenile (postnatal day [PD] 24) and adult hippocampus (PD 56). During postnatal development, the mRNA levels of GABA A receptor (GABA_AR) subunits, including α1, α4, β1, β2, and δ; GABA B receptor (GABA_BR) subunit 2; and the expression of GABA-related proteins, including glutamic acid decarboxylase, vesicular GABA transporter (VGAT), and potassium chloride cotransporter 2 increased gradually in the mouse hippocampus. The results of seizure scoring and histopathological findings in the hippocampus revealed a more pronounced response to the same administered TMT dose in juvenile mice, compared with that in adult mice. The mRNA levels of most GABA receptor subunits in the juvenile hippocampus, excluding GABA_AR subunit β3, were dynamically altered after TMT treatment. The mRNA levels of GABA_AR subunits γ2 and δ decreased significantly in the adult hippocampus following TMT treatment, whereas the level of GABA_BR subunit 1 mRNA increased significantly. Among the GABA-related proteins, only VGAT decreased significantly in the juvenile and adult mouse hippocampus after TMT treatment. In conclusion, regulation of GABAergic signaling in the mouse hippocampus may be related to maturation of the central nervous system and the degree of neurodegeneration during postnatal development and TMT-induced neurodegeneration in the experimental animals.     

S-100 proteins in trimethyltin-induced neurodegeneration in the rat hippocampus

After acute trimethyltin (TMT) intoxication (21 d after a single ip injection at a dose of 8 mg/kg) the histological, immunohistochemical, and immunochemical investigation of adult rat hippocampus showed a distinct pattern of neuronal loss, and an increase in both glial fibrillary acidic protein- (GFAP) immunoreactive cells and GFAP concentration, as expected. S-100-immunoreactive cells also increased markedly, whereas the concentration of S-100 increased even more than that of GFAP. The data show that S-100 is an index of glial reaction to damage after TMT intoxication and suggest the potential usefulness of exploring the possibility that it may play a role in induced neurodegenerative processes.     

A decrease of cell proliferation by hypothermia in the hippocampus of the neonatal rat

Hypothermia is a potential therapy for cerebral hypoxic ischemic injury of not only adults but also neonates. However, the side effects of hypothermia in the developing brain, where a massive amount of neurogenesis occurs, remain unclear. We investigated the proliferation of neural progenitor cells by systemic application of the thymidine analog 5-bromodeoxyuridine (BrdU) in neonatal rats in a severe hypothermic environment. The rat pups were divided into two groups, a hypothermia group (30 degrees C: n=10) and a normothermia group (37 degrees C: n=10). After the pups were placed for 21 h in each environment, 100 mg/kg/day of BrdU was injected intraperitoneally to label dividing cells, and then the pups were sacrificed at 24 h. We examined the number of BrdU-labeled cells in the subventricular zone of the periventricle and the subgranular zone of the dentate gyrus. In the hypothermic environment, BrdU-labeled cells significantly decreased in number in the dentate gyrus, but not in the periventricular region. Thus, the severe hypothermic environment induced a decrease of neurogenesis in the neonatal rat. These observations are noteworthy regarding clinical hypothermia therapy following cerebral hypoxic ischemic injury during the perinatal period.     

Postnatal effect of embryonic neurogenesis disturbance on reelin level in organotypic cultures of rat hippocampus

Despite a delayed emergence of the symptoms, schizophrenia is thought to be a late consequence of early disturbances during development. Several reports have found decreased levels of reelin in the cortex and the hippocampus of postmortem brains of schizophrenic patients. In the rat, intraperitoneal injection of the anti-mitotic agent methylazoxymethanol (MAM) during intra-uterine development (embryonic day 17) induces cytoarchitectural abnormalities in the hippocampus and the cortex and behavioural changes reminiscent of positive, negative and cognitive symptoms of schizophrenia. We aimed to examine whether a transient prenatal disturbance of neurogenesis induces postnatal changes in the expression of reelin in the hippocampus. Cellular modifications were explored using hippocampal organotypic slice cultures, which allow for conservation of the in vivo cytoarchitecture. MAM effect on hippocampal neurogenesis was confirmed by birthdating experiments. After 3 weeks in vitro, reelin was expressed by calretinin-negative cells. The number of reelin-positive neurons was increased whereas the total neuron number was decreased in the stratum oriens in the E17 MAM-exposed animals as compared to the control group. Not only an increase in the number of cells expressing reelin was observed, but there was also a slight increase in reelin mRNA levels in hippocampal pyramidal cells of MAM-exposed animals. In contrast, there was no significant change in the dentate gyrus. These results show that transient prenatal disturbance of neurogenesis induces long-term modifications in specific areas of the hippocampus and in particular in the number of neurons expressing reelin. They also confirm the value of organotypic slices to study postnatal maturation in the hippocampus.     

卡马西平对匹罗卡品诱导成年癫癎大鼠海马齿状回神经发生影响的研究

目的研究卡马西平对成年癫癎大鼠海马齿状回神经发生的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫癎模型,于干预后第6d腹腔注射5-溴脱氧尿核苷嘧啶标记海马齿状回内源性神经前体细胞的增殖情况;用免疫组织化学方法及免疫荧光双标方法观察海马齿状回新生细胞的增殖、存活、分化及迁移情况。结果 （1）卡马西平可明显抑制癫癎大鼠海马齿状回新生细胞增殖;（2）卡马西平可明显促进癫癎大鼠海马齿状回新生细胞的存活;（3）卡马西平可增加癫癎大鼠海马齿状回新生神经元的数量,但不增加新生细胞分化为成熟神经元的比例;（4）卡马西平对新生神经细胞的异位迁移无抑制作用。结论卡马西平对癫癎大鼠海马齿状回神经发生的影响是其控制癫癎临床症状的可能机制之一。     

Radiation of the rat brain suppresses seizure-induced neurogenesis and transiently enhances excitability during kindling acquisition

PURPOSE: Adult hippocampal neurogenesis is enhanced in several models for temporal lobe epilepsy (TLE). In this study, we used low-dose whole brain radiation to suppress hippocampal neurogenesis and then studied the effect of this treatment on epileptogenesis in a kindling model for TLE. METHODS: Half of the rats were exposed to a radiation dose of 8 Gy one day before the initiation of a rapid kindling protocol. Afterdischarge threshold (ADT), afterdischarge duration (ADD), clinical seizure severity, and inflammation were compared between groups. On the first and third day after radiation, rats were injected with 5'-bromo-2'-deoxyuridine (BrdU) to evaluate neurogenesis. Seven and 21 days after radiation, numbers of doublecortin (DCX) positive neuroblasts in subgranular zone and granule cell layer were compared between groups. RESULTS: We showed that radiation significantly suppressed neurogenesis and neuroblast production during kindling acquisition. Radiation prevented an increase in ADT that became significantly lower in radiated rats. On the third and fourth kindling acquisition day radiated rats developed more severe seizures more rapidly, which resulted in a significantly higher mean severity score on these days. Differences in ADD could not be demonstrated. DISCUSSION: Our results demonstrate that brain radiation with a relatively low dose effectively suppressed the generation of new granule cells and transiently enhanced excitability during kindling acquisition. Although seizure-induced neurogenesis was lower in the radiated rats we could not detect a strong effect on the final establishment of the permanent fully kindled state, which argues against a prominent role of seizure-induced neurogenesis in epileptogenesis.     

Developmental profile of neurogenesis in prenatal human hippocampus: An immunohistochemical study

Hippocampus has attracted the attention of the neuroscientists for its involvement in a wide spectrum of higher-order brain functions and pathological conditions, especially its persistent neurogenesis in subgranular zone (SGZ). The development of hippocampus was intensively investigated on animals such as rodents. However, in prenatal human hippocampus, little information on the distribution of neural stem/progenitor cells, newly generated neurons and mature neurons is available and the timetable of a series of neurogenesis event is even more obscure. So in the present study, we aim at immunohistochemically providing more information on neurogenesis in prenatal human hippocampus from 9 weeks to 32 weeks of gestation. We found that the ki67-positive cells were always detected in hippocampus from 9 weeks to 32 weeks, with a peak at 9 weeks in cornu ammonis (CA) or 14 weeks in dentate gyrus (DG). At 9 weeks the nestin-expressing cells were distributed throughout the hippocampus, with concentrated immunoreactivity in intermediate zone (IZ), marginal zone (MZ), fimbria, and relatively sparse immunoreactivity in the ventricular zone (VZ) and hippocampal plate (HP). With development, the optical density (OD) and the number of nestin-positive cells decreased gradually. At 32 weeks, there were relatively more nestin-positive cells in DG than that in CA. About DCX-positive cells, they displayed a similar distribution as nestin-positive cells (immunoreactivity concentrated in IZ, MZ, fimbria and HP) and a dramatic decrease of OD or cell number density from 9 weeks on. NeuN-positive cells, with small nuclei, were firstly found in MZ and subplate of hippocampus at 9 weeks. After 14 weeks, many NeuN-positive cells extended from subplate into HP and the density of NeuN-positive cells peaked at 22 weeks. That the immunoreactivity for NeuN was the strongest and the nuclei were the biggest at 32 weeks suggests that the neurons reach maturity gradually. Therefore this study provides an important timetable of neurogenesis in prenatal human hippocampus for the clinicians in neuroscience or pediatrics.     

Nitric oxide synthase-containing neurons in the hippocampus are preserved in trimethyltin intoxication

We studied the effects of trimethyltin (TMT) (9 mg/kg, p.o.) on the nitric oxide synthase (NOS)-containing neurons in the rat hippocampus by NADPH-diaphorase histochemistry and a biochemical assay of NOS activity. TMT exposure caused the typical behavioral changes and a loss of the CA3/4 pyramidal cells, which were NADPH diaphorase-negative. The scattered interneurons and the CA1 pyramidal cells, which were NADPH diaphorase-positive, were spared. Hippocampal NOS activity showed no reduction in the TMT-treated rats compared with the controls. These results provide evidence of the preservation of the NOS-containing neurons in TMT intoxication.     

Trimethyltin intoxication induces the migration of ventricular/subventricular zone cells to the injured murine hippocampus

Following the postnatal decline of cell proliferation in the mammalian central nervous system, the adult brain retains progenitor cells with stem cell-like properties in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampus. Brain injury can stimulate proliferation and redirect the migration pattern of SVZ precursor cells to the injury site. Sublethal exposure to the neurotoxicant trimethyltin (TMT) causes dose-dependent necrosis and apoptosis in the hippocampus dentate gyrus and increases SGZ stem cell proliferation to generate new granule cells. To determine whether SVZ cells also contribute to the repopulation of the TMT-damaged dentate gyrus, 6-8 week old male C3H mice were injected with the carbocyanine dye spDiI and bromodeoxyuridine (80 mg/kg; ip.) to label ventricular cells prior to TMT exposure. The presence of labeled cells in hippocampus was determined 7 and 28 days after TMT exposure. No significant change in the number of BrdU⁺ and spDiI⁺ cells was observed in the dentate gyrus 7 days after TMT treatment. However, 28 days after TMT treatment there was a 3–4 fold increase in the number of spDiI-labeled cells in the hippocampal hilus and dentate gyrus. Few spDiI⁺ cells stained positive for the mature phenotypic markers NeuN or GFAP, suggesting they may represent undifferentiated cells. A small percentage of migrating cells were BrdU⁺/spDiI⁺, indicating some newly produced, SVZ- derived precursors migrated to the hippocampus. Taken together, these data suggest that TMT-induced injury of the hippocampus can stimulate the migration of ventricular zone-derived cells to injured dentate gyrus.     

侧脑室注射Noggin对AD小鼠海马神经发生和学习记忆功能的影响

目的观察侧脑室给予Noggin对AD小鼠海马神经发生和学习记忆功能的影响。方法选取12月龄APP/PS1双转基因AD小鼠,实验组连续7d侧脑室注射Noggin,对照组向侧脑室注射等量生理盐水,Morris水迷宫检测其认知功能,免疫荧光检测海马齿回BrdU的表达。结果和生理盐水对照组相比,侧脑室注射Noggin组小鼠海马齿回BrdU阳性细胞数显著增加(P0.01),行为学检测显示逃逸潜伏期缩短、穿过原平台位置次数和在原平台象限探索时间百分率增加(P0.05)。结论侧脑室注射Noggin可促进AD小鼠海马神经发生并改善其学习记忆能力。     

The presenilin-1 familial Alzheimer disease mutant P117L impairs neurogenesis in the hippocampus of adult mice

The functions of presenilin 1 (PS1) and how PS1 mutations cause familial Alzheimer's disease (FAD) are incompletely understood. PS1 expression is essential for neurogenesis during embryonic development and may also influence neurogenesis in adult brain. We examined how increasing PS1 expression or expressing an FAD mutant would affect neurogenesis in the adult hippocampus. A neuron-specific enolase (NSE) promoter was used to drive neuronal overexpression of either wild-type human PS1 or the FAD mutant P117L in transgenic mice, and the animals were studied under standard-housing conditions or after environmental enrichment. As judged by bromodeoxyuridine (BrdU) labeling, neural progenitor proliferation rate was mostly unaffected by increasing expression of either wild-type or FAD mutant PS1. However, in both housing conditions, the FAD mutant impaired the survival of BrdU-labeled neural progenitor cells leading to fewer new beta-III-tubulin-immunoreactive neurons being generated in FAD mutant animals during the 4-week postlabeling period. The effect was FAD mutant specific in that neural progenitor survival and differentiation in mice overexpressing wild-type human PS1 were similar to nontransgenic controls. Two additional lines of PS1 wild-type and FAD mutant transgenic mice showed similar changes indicating that the effects were not integration site-dependent. These studies demonstrate that a PS1 FAD mutant impairs new neuron production in adult hippocampus by decreasing neural progenitor survival. They also identify a new mechanism whereby PS1 FAD mutants may impair normal neuronal function and may have implications for the physiological functioning of the hippocampus in FAD.     

Electroconvulsive seizures protect against methamphetamine-induced inhibition of neurogenesis in the rat hippocampus

Following methamphetamine consumption and during abstinence many behavioral consequences emerge (i.e., cognitive deficits, ongoing episodes of psychosis, depression, severe cravings, brain neurotoxicity), which are likely linked to propensity to relapse. In this line of thought, we recently showed that binge methamphetamine administration enhanced negative affect and voluntary drug consumption in rats, while it induced persistent neurotoxic effects (i.e., impaired hippocampal neurogenesis), effects that emerged long after drug removal. To date, no pharmacological strategies have been proven to be effective for the treatment of methamphetamine toxicity. A few studies have evaluated the impact of combining methamphetamine pretreatment with electroconvulsive seizures (ECS) post-treatment, an alternative non-pharmacological option used in psychiatry for resistant depression that offers a safe and really potent therapeutic response. Against this background, the present study aimed at testing whether repeated ECS treatment could ameliorate some of the long-term neurotoxicity effects induced by adolescent methamphetamine exposure in rats and emerging after drug removal. At the behavioral level, the main results showed that methamphetamine administration did not alter negative affect immediate during adolescence or later on in adulthood. Interestingly, repeated ECS improved the negative impact of methamphetamine administration on reducing hippocampal neurogenesis, demonstrating that ECS can attenuate certain degree of methamphetamine-induced neurotoxicity in rats, and suggesting ECS as a good therapeutical candidate that deserves further studies.     

戊四氮点燃过程中大鼠海马 BMP4表达与神经增殖关系

目的探讨戊四氮(PTZ)点燃过程中大鼠海马骨形成蛋白4(bone morphogenetic protein-4,BMP4)的表达变化与神经增殖的关系。方法将成年大鼠分为对照组与模型组,模型组大鼠又根据在点燃中的不同时相点分为9组。用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA与BrdU阳性细胞数的变化。结果正常成年大鼠BMP4阳性细胞主要分布于齿状回的门区、颗粒下层、CA3、CA1区。BrdU阳性细胞主要分布在齿状回颗粒下层。BMP4阳性细胞与BrdU阳性细胞在点燃过程中均明显增加,呈明显正相关,点燃后2月降至基线水平。结论BMP4可能通过影响成年大鼠海马神经发生在PTZ点燃过程中起重要作用。     

Environmental enrichment promotes neurogenesis and changes the extracellular concentrations of glutamate and GABA in the hippocampus of aged rats

The aim of the present study was to investigate the effects of environmental enrichment on the neurogenesis and the extracellular concentrations of glutamate and GABA in the hippocampus of freely moving young and aged rats. Male Wistar rats of 2 (young) and 25 (old) months of age were housed during 8 weeks in an enriched environment; control rats were kept in individual plastic cages during that same period of time. Rats were injected intraperitoneally with bromodeoxyuridine (BrdU; 40 mg/kg; 7 days) during the fourth week of the housing period to detect neurogenesis in the dentate gyrus (DG) of the hippocampus. Rats were sacrified 6 weeks after the last injection of BrdU. During the last week of housing, rats were tested in the water maze for the evaluation of spatial learning. After the housing period, rats were stereotaxically implanted with guide-cannulas to accommodate microdialysis probes in the CA3 area of the hippocampus and the extracellular concentrations of glutamate and GABA were determined. Aged rats showed a decrease in the number of BrdU positive cells in the dentate gyrus compared to young rats. However, neurogenesis in the dentate gyrus of both young and old rats was increased in animals housed in an enriched environment. Microdialysis experiments in the CA3 area of the hippocampus showed that enriched housing conditions increased basal extracellular concentrations of glutamate in aged rats. Perfusion of KCl 100 mM produced a higher increase of extracellular glutamate and GABA in aged rats but not in young rats housed in an enriched environment compared to control rats. These results suggest that enriched housing conditions change both neurogenesis in the dentate gyrus and glutamate and GABA levels in the CA3 area of the hippocampus of aged rats.     

The prototypic mineralocorticoid receptor agonist aldosterone influences neurogenesis in the dentate gyrus of the adrenalectomized rat

Glucocorticoid receptor activation inhibits granule cell proliferation in the hippocampus, but little is known about the role of mineralocorticoid receptors in this process. Here we administered aldosterone to adrenalectomized (ADX) rats, and monitored neurogenesis by BrdU immunohistochemistry. ADX significantly increased the number of BrdU-positive cells and aldosterone replacement further augmented BrdU-positivity. Our results indicate that aldosterone, most probably acting through mineralocorticoid receptors, may positively influence the proliferation and survival of newly-generated granule cells.