Consequences of pilocarpine-induced recurrent seizures in neonatal rats

Accumulated evidence have shown that a series of morphological alternations occur in patients with epilepsy and in different epileptic animal models. Given most of animal model studies have been focused on adulthood stage, the effect of recurrent seizures to immature brain in neonatal period has not been well established. This study was designed to observe the certain morphological changes following recurrent seizures occurred in the neonatal rats. For seizure induction, neonatal Wistar rats were intraperitoneally injected with pilocarpine on postnatal day 1 (P1), P4 and P7. Rat pups were grouped and sacrificed at 1d, 7d, 14d and 42d after the last pilocarpine injection respectively. Bromodeoxyuridine (BrdU) was intraperitoneally administered 36h before the rats were sacrificed. BrdU single and double labeling with neuronal markers were used to analyze cell proliferation and differentiation. Nissl and Timm staining were performed to evaluate cell loss and mossy fiber sprouting. Rats with neonatal seizures had a significant reduction in the number of Bromodeoxyuridine-(BrdU) labeled cells in the dentate gyrus compared with the control groups when the animals were killed either 1 or 7 days after the third seizure (P<0.05) but there was no difference between two groups on P21. On the contrary, BrdU-labeled cells significantly increased in the experimental group compared with control group on P49 (P<0.05). The majority of the BrdU-labeled cells colocalized with neuronal marker-NF200 (Neurofilament-200). Nissl staining showed that there was no obvious neuronal loss after seizure induction over all different time points. Rats with the survival time of 42 days after neonatal seizures developed to increased mossy fiber sprouting in both the CA3 region and supragranular zone of the dentate gyrus compared with the control groups (P<0.05). Taken together, the present findings suggest that synaptic reorganization only occurs at the later time point following recurrent seizures in neonatal rats, and neonatal recurrent seizures can modulate neurogenesis oppositely over different time window with a down-regulation at early time and up-regulation afterwards.     

新生期大鼠反复痫性发作的形态学及行为学结果与糖皮质激素水平升高有关(英文)

目的探讨新生期大鼠反复痫性发作后的形态学,行为学以及糖皮质激素水平的变化。方法64只出生后一天的Wistar大鼠随机分为惊厥组40只和对照组24只。惊厥组的新生鼠在出生后1天(P1)、4天(P4)、7天(P7)给予腹腔注射匹罗卡品,制备新生鼠癫痫模型;对照组的新生鼠腹腔注射生理盐水。惊厥组分别在第 3次致痫后在即刻(Ⅰ组)、第4 天(Ⅱ组)、第14 天(Ⅲ组)、第42天(Ⅳ组)四个时间点处死,各时间点设相应对照组,处死前36 h惊厥组和对照组的大鼠腹腔注射BrdU。所有大鼠处死前均取血检测糖皮质激素。第Ⅳ组从P40开始进行Morris水迷宫试验。结果新生鼠3次发作后即刻和第4天与相应日龄对照组相比,齿状回BrdU阳性细胞数明显减少(P<0.05),而癫痫发作后14天和42天BrdU阳性细胞数增加,但发作后14天差异无统计学意义(P>0.05)。在4天的Morris水迷宫试验中,匹罗卡品处理组大鼠到达平台的时间均长于对照组,但是只有第1天和第2天有统计学意义(P<0.05)。检测结果表明高水平的糖皮质激素一直持续到发作后第4天,糖皮质激素水平与BrdU阳性细胞数呈负相关。结论新生大鼠反复痫性发作会造成早期神经发生减少,而后期神经发生增加;造成大鼠成年后认知功能缺陷;造成糖皮质激素水平增高,这与痫性大鼠形态学和行为学方面的改变有关。     

新生期大鼠反复痫性发作的形态学及行为学结果与糖皮质激素水平升高有关

目的探讨新生期大鼠反复痫性发作后的形态学,行为学以及糖皮质激素水平的变化。方法64只出生后一天的Wistar大鼠随机分为惊厥组40只和对照组24只。惊厥组的新生鼠在出生后1天（P1）、4天（P4）、7天（P7）给予腹腔注射匹罗卡品,制备新生鼠癫痫模型;对照组的新生鼠腹腔注射生理盐水。惊厥组分别在第3次致痫后在即刻（Ⅰ组）、第4天（Ⅱ组）、第14天（Ⅲ组）、第42天（Ⅳ组）四个时间点处死,各时间点设相应对照组,处死前36h惊厥组和对照组的大鼠腹腔注射BrdU。所有大鼠处死前均取血检测糖皮质激素。第Ⅳ组从P40开始进行Morris水迷宫试验。结果新生鼠3次发作后即刻和第4天与相应日龄对照组相比,齿状回BrdU阳性细胞数明显减少（P〈0．05）,而癫痫发作后14天和42天BrdU阳性细胞数增加,但发作后14天差异无统计学意义（P〉0．05）。在4天的Morris水迷宫试验中,匹罗卡品处理组大鼠到达平台的时间均长于对照组,但是只有第1天和第2天有统计学意义（P〈0．05）。检测结果表明高水平的糖皮质激素一直持续到发作后第4天,糖皮质激素水平与BrdU阳性细胞数呈负相关。结论新生大鼠反复痫性发作会造成早期神经发生减少,而后期神经发生增加;造成大鼠成年后认知功能缺陷;造成糖皮质激素水平增高,这与痫性大鼠形态学和行为学方面的改变有关。     

Periodic maternal separation decreases hippocampal neurogenesis without affecting basal corticosterone during the stress hyporesponsive period, but alters HPA axis and coping behavior in adulthood

Although not directly evaluated, the early rise of glucocorticoid (GC) levels, as occur after exposure to adverse early life experience, are assumed to affect hippocampal ontogeny by altering the hippocampus negative feedback on adult HPA axis. To test whether hippocampal ontogeny is affected by early exposure to stress we estimated the survival of recently formed hippocampal granule cells in rat pups subjected to periodic maternal separation (180 min/day; MS180) from postnatal days (PND) 1 to 14. Accordingly, MS180 pups injected with bromodeoxyuridine (BrdU, 50 mg/kg, ip) at PND 5 showed decreased density of doublecortin (DCX) positive BrdU-labeled cells at PND 15. MS180 and AFR pups showed similar corticosterone (CORT) basal levels between PND 3 and 12, whereas adult MS180 rats presented with higher CORT levels than AFR adults. Nonetheless, both AFR and MS180 pups and adults showed similar transient increments of CORT levels in response to stress. In addition, MS180 had no effect on the adult anxiety-like behavior evaluated in the elevated plus maze, but evoked a passive coping strategy in the forced swimming test. The data show that the decrease in hippocampal neurogenesis is an early onset phenomenon, and suggests that adverse experiences alter hippocampal ontogeny without chronic elevation of GC levels.     

Differential inhibition of neurogenesis and angiogenesis by corticosterone in rats stimulated with electroconvulsive seizures

Antidepressant drugs and electroconvulsive seizure (ECS)-treatment, an animal model of electroconvulsive therapy, induce neurogenesis in adult rats. Stress and high levels of corticosterone (CORT) on the contrary inhibit neurogenesis. Hippocampal neurogenesis has been described to occur in an angiogenic niche where proliferation of neural progenitors takes place in an environment with active vascular growth. Here we investigate the effect of ECS-treatment on the proliferation of endothelial cells and neuronal precursors in hippocampus of CORT-treated rats. Bromodeoxyuridine (BrdU) was used to identify dividing cells. The number of newborn neuronal precursors and endothelial cells was quantified in the subgranular zone (SGZ) and the molecular layer (ML) of the dentate gyrus. The increase in neuronal precursor proliferation in the SGZ following ECS-treatment was not inhibited by elevated levels of CORT despite CORT strongly inhibiting ECS-induced endothelial cell proliferation. Also in the ML CORT-treatment inhibited the ECS-induced angiogenic response. We conclude that despite common factors regulating neurogenesis and angiogenesis, ECS-induced proliferation of neuronal precursors can take place even if the angiogenic response is blunted. Whether inhibition of angiogenesis affects other steps in the chain of events leading to the formation of fully integrated granule neurons remains to be elucidated.     

Enhanced neurogenesis in the rodent hippocampus following traumatic brain injury

Recent studies have shown that neurogenesis in the dentate gyrus of the rodent hippocampus continues throughout life. Several physiological and pathological conditions have been reported to alter the rate of progenitor cell division resulting in the increased production of mature granule neurons. Excitotoxic and mechanical lesions of the granule cell layer also stimulate the proliferation of precursor cells suggesting that the death of pre-existing granule neurons may act as a trigger for enhanced neurogenesis. Hippocampal pyramidal neurons, and to a lesser extent granule neurons, have been reported to die as a result of traumatic brain injury in rodents. To determine if the proliferation of precursor cells is enhanced as a result of brain injury in rodents, newly divided cells were labeled with the thymidine analog, bromodeoxyuridine (BrdU). Traumatic brain injury increased the production of BrdU-labeled cells in the dentate gyrus with a maximal rate observed at 3 days post-injury. These cells, a proportion of which co-localize with the immature neuronal marker TOAD-64, implanted themselves into the granule cell layer where they accumulated over time. When examined 1 month post-injury, the majority of BrdU-labeled cells co-labeled with the mature neuronal marker calbindin. These findings show that traumatic brain injury increases neurogenesis in the granule cell layer and suggests that these new cells may contribute to the function of the hippocampus.     

Hippocampal adult neurogenesis occurs in a microenvironment provided by PSA-NCAM-expressing immature neurons

Neurons continue to be generated in the adult hippocampus. In the present study, the early developmental processes of newly generated neurons in the adult rat hippocampus were examined by confocal laser scanning microscopy using a combination of bromodeoxyuridine (BrdU) labeling and immunohistochemistry for highly polysialylated neural cell adhesion molecule (PSA-NCAM) and NeuroD, which are markers for immature neurons, and glial fibrillary acidic protein (GFAP). Rats were injected with BrdU and 2 hours, 1, 3, and 7 days after the injection, the hippocampus was processed for immunohistochemistry. One day after the injection, BrdU-labeled cells were found frequently in clusters consisting of dividing cells, putative undifferentiated cells, NeuroD-positive differentiated neurons, and GFAP-positive astrocytes. Three days later, BrdU-labeled cells were loosely aggregated and BrdU-positive fragmented nuclei were sometimes observed, suggesting that apoptosis occurred in the clusters. These BrdU-labeled nuclei were frequently associated in various ways with the processes of immature PSA-positive granule cells. They are positioned along PSA-positive apical and basal dendrites or surrounded by these processes. By 7 days after the injection, the number of the clusters was reduced and the BrdU-labeled cells had developed dendrites. These cell-to-cell associations support the hypothesis that the clustering and a microenvironment provided by the PSA-expressing immature neurons contribute to the early developmental events of adult neurogenesis, such as proliferation, differentiation, apoptosis, and neurophilic migration in the adult hippocampus.     

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.     

Sleep deprivation suppresses neurogenesis in the adult hippocampus of rats

We reported previously that 96 h of sleep deprivation (SD) reduced cell proliferation in the dentate gyrus (DG) of the hippocampus in adult rats. We now report that SD reduces the number of new cells expressing a mature neuronal marker, neuronal nuclear antigen (NeuN). Rats were sleep-deprived for 96 h, using an intermittent treadmill system. Total sleep time was reduced to 6.9% by this method in SD animals, but total treadmill movement was equated in SD and treadmill control (CT) groups. Rats were allowed to survive for 3 weeks after 5-bromo-2-deoxyuridine (BrdU) injection. The phenotype of BrdU-positive cells in the DG was assessed by immunofluorescence and confocal microscopy. After 3 weeks the number of BrdU-positive cells was reduced by 39.6% in the SD group compared with the CT. The percentage of cells that co-localized BrdU and NeuN was also lower in the SD group (SD: 46.6 +/- 1.8% vs. CT: 71.9 +/- 2.1, P < 0.001). The percentages of BrdU-labeled cells co-expressing markers of immature neuronal (DCX) or glial (S100-beta) cells were not different in SD and CT groups. Thus, SD reduces neurogenesis in the DG by affecting both total proliferation and the percentage of cells expressing a mature neuronal phenotype. We hypothesize that sleep provides anabolic or signaling support for proliferation and cell fate determination.     

Seizures induce proliferation and dispersion of doublecortin-positive hippocampal progenitor cells

One neuropathological hallmark of temporal lobe epilepsy is granule cell dispersion, a widening of the hippocampal granule cell layer (GCL) with abnormally positioned excitatory neurons. The finding that seizure activity also induces adult hippocampal neurogenesis was taken largely as indicative of a regenerative attempt, not as part of the pathology. The aim of our study was to characterize a potential relationship between granule cell dispersion and seizure-induced neurogenesis. Kainic acid (KA)-induced seizures in mice led to increased cell proliferation and new neurons persisted for months after the seizures. We show that the proliferative stimulus did not affect nestin-expressing early precursor cells that primarily respond to physiologic mitogenic stimuli, but stimulated the division of late type-3 progenitor cells, which express doublecortin (DCX), a protein associated with cell migration. This delayed proliferation presumably interfered with migration, leading to a significant dispersion of DCX-positive progenitors and early postmitotic neurons within the dentate gyrus granule cell layer. We propose that initial seizures induce ectopic precursor cell proliferation resulting in the dispersion of immature neurons within the adult granule cell layer. Thus, seizure-generated neurons might contribute to the disease process of epilepsy.     

Granule Cell Neurogenesis After Status Epilepticus in the Immature Rat Brain

Summary: Purpose : Several experimental paradigms of seizure induction that produce epilepsy as a consequence have been shown to be associated with the proliferation of dentate granule cells. In developing animals, the acute sequela of hilar damage and the chronic sequelae of spontaneous seizures and mossy fiber synaptic reorganization, in response to status epilepticus, occur in an age-dependent manner. We investigated seizure-induced granule cell neurogenesis in developing rat pups to study the association between hilar injury, granule cell neurogenesis, and epilepsy.
Methods : Rat pups of 2 and 3 weeks postnatal age were subjected to lithium-pilocarpine status epilepticus (LiPC SE). Rats were given bromodeoxyuridine (BrdU; 50 mg/kg intra-peritoneal) twice daily for 4 days beginning 3 days after SE to label dividing cells. Routine immunocytochemistry and quantification of BrdU labeling by image analysis were performed. Results were compared with previously reported data on cellular injury, mossy fiber sprouting, and spontaneous seizures in rat pups of these ages after LiPC SE.
Results : In 3-week-old pups, which demonstrate SE-induced hilar damage and develop spontaneous seizures accompanied by mossy fiber sprouting, the BrdU-immunoreactive area (percent) in the subgranular proliferative zone increased to 10·6 ± 2·5 compared with 1·4 ± 0·5 in the control animals (p < 0·05). The 2-week-old animals, which show neither hilar damage nor sprouting and rarely develop spontaneous seizures, also showed a comparable extent of SE-induced neurogenesis [8·0 ± 1·4 (LiPC SE) versus 0·4 ± 0·2 (control), p < 0·05].
Conclusions : Seizure-induced granule cell neurogenesis does not appear to be a function of seizure-induced hilar cellular damage. Granule cell neurogenesis induced by SE does not determine epileptogenesis in the developing rat.     

Maternal experience inhibits the production of immature neurons in the hippocampus during the postpartum period through elevations in adrenal steroids

Motherhood is accompanied by alterations in numerous nonreproductive behaviors, including learning and memory, as well as anxiety and stress regulation. These functions have been linked to adult neurogenesis in the hippocampus, but the effect of maternal experience on this brain region has not been completely explored. To determine whether the production of new hippocampal granule cells is altered during the postpartum period, we examined the number of proliferating cells and their progeny in the dentate gyrus of primiparous female rats at various time points during the postpartum period while they were caring for their offspring, as well as after weaning. Additionally, we investigated whether cell proliferation in the postpartum female is affected by the presence of offspring and nursing-induced increases in glucocorticoids. Analysis of the number of BrdU-labeled cells revealed that cell proliferation in the dentate gyrus was suppressed in lactating postpartum females until the time of weaning. This effect was temporary; a difference was detectable at 1 week after BrdU-labeling, when the majority of cells expressed a marker of immature and mature granule neurons (TuJ1) but not at 2 weeks, when most cells expressed a marker of mature neurons (NeuN). The decrease in cell proliferation was dependent on elevated basal glucocorticoid levels associated with lactation; removal of nursing pups reduced basal corticosterone levels and prevented the decrease in the number of BrdU-labeled cells. Moreover, preventing increased basal corticosterone levels by means of adrenalectomy and low-dose corticosterone replacement eliminated the reduction in cell proliferation. These findings indicate that offspring interactions inhibit adult neurogenesis through changes in adrenal steroids, and further suggest a potential mechanism for alterations in hippocampal function during the postpartum period.     

Lamotrigine increases the number of BrdU-labeled cells in the rat hippocampus

Antidepressant medication and electroconvulsive therapy stabilize mood symptoms and increase hippocampal neurogenesis. We examined whether lamotrigine, suggested to give rise to mood-stabilizing and antidepressant effects in addition to its antiepileptic properties, also increases the number of newborn cells in rat hippocampus. Rats (on day P21) received lamotrigine, valproate, or saline intraperitoneally once daily for 7 days. All animals received four intraperitoneal injections of bromodeoxyuridine (BrdU) on day P28 and were sacrificed the next day. Quantification of BrdU-labeled cells in the granule cell layer of the dentate gyrus showed an increased number of newborn cells in rats receiving lamotrigine (42.6 ± 3.5 cells/slice) compared with valproate (31.6 ± 2.8) and controls (32.2 ± 3.1; P<0.05). The increased number of BrdU-labeled cells suggests increased neurogenesis, possibly explaining the mood-stabilizing and antidepressant effects of lamotrigine.     

Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production

Neurogenesis continues throughout life in the hippocampus. To study postnatal neurogenesis in vitro, hippocampal slices from rats on postnatal day 5 (P5) were cultured on a porous membrane for 14 or 21 days. In the initial experiments, precursor cells were labeled with bromodeoxyuridine (BrdU) after 7 days in culture because hippocampal slices are generally used in experiments after 1-2 weeks in culture. Fourteen days after labeling, however, only about 10% of BrdU-labeled cells expressed neuronal markers, although in living rats, about 80% of cells labeled with BrdU on P5 had become neurons by P19. Next, rats were injected with BrdU 30 min before culture, after which hippocampal slices were cultured for 14 days to examine the capacity of in vivo-labeled neural precursors to differentiate into neurons in vitro. In this case, more than two-thirds of BrdU-labeled cells expressed neuronal markers, such as Hu, NeuN, and PSA-NCAM. Furthermore, precursor cells underwent early in vitro labeling by incubation with BrdU or a modified retrovirus vector carrying EGFP for 30 min from the beginning of the culture. This procedure resulted in a similar high rate of neuronal differentiation and normal development into granule cells. In addition, time-lapse imaging with retrovirus-EGFP revealed migration of neural precursors from the hilus to the granule cell layer. These results indicate that in vivo- and early in vitro-labeled cultures are readily available ex vivo models for studying postnatal neurogenesis and suggest that the capacity of neural precursors to differentiate into neurons is reduced during the culture period.     

Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice

Neurogenesis in the adult mammalian CNS occurs in the subventricular zone (SVZ) and dentate gyrus. The receptor for ciliary neurotrophic factor (CNTF), CNTFRalpha, is expressed in the adult subventricular zone. Because the in vitro effects of CNTF on neural precursors have been varied, including proliferation and differentiation into neurons or glia, we investigated its role in vivo. Injection of CNTF in the adult C57BL/6 mice forebrain increased the number of cells labeled with ip BrdU in both neurogenic regions. In the dentate gyrus, CNTF also appeared to enhance differentiation of precursors into neurons, i.e., increased the proportion of NeuN+/BrdU+ cells from approximately 14 to approximately 29%, but did not affect differentiation into astrocytes (GFAP+) or oligodendrocytes (CNPase+). In the SVZ, CNTF increased the proportion of GFAP+/BrdU+ cells from approximately 1 to approximately 2%. CNTF enhanced the distance of migration of new neurons into the granule cell layer. Intraventricular injection of neutralizing anti-CNTF antibodies reduced the number of BrdU-labeled cells in the SVZ. These results suggest that endogenous CNTF regulates adult neurogenesis by increasing proliferation of neural stem cells and/or precursors. Alternatively, CNTF could maintain cells longer in the S-phase, resulting in increased BrdU labeling. In the neurogenic region of the SVZ, CNTFRalpha was exclusively present in GFAP-positive process-bearing cells, suggesting that CNTF affects neurogenesis indirectly via neighboring astroglia. Alternatively, these cells may be part of the neural precursor lineage. The restricted expression of CNTF within the nervous system makes it a potential selective drug target for cell replacement strategies.     

Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus

Summary. Male gerbils were bred and reared grouped under enriched semi-natural environmental conditions. The objective of the present study was to examine the influence of an acute treatment with the neuroleptic haloperidol on adult granule cell neurogenesis in the hippocampus. For that purpose, at the age of postnatal day 90 adult animals received 4 challenges of either haloperidol (5 mg/kg, i.p.) or saline. Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2′-desoxyuridine (BrdU) which was applied 1 hour after the final dose of haloperidol. BrdU-labeled granule cell nuclei were identified in consecutive horizontal slices along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 7 days after the BrdU-labeling. It was found that in both saline- and haloperidol-treated animals there was a highly significant spatial septotemporal gradient in granular cell proliferation with numbers of BrdU-labeled cells gradually declining from the septal towards the temporal pole. The acute treatment with haloperidol stimulated granule cell proliferation by about 75% and the septotemporal gradient of mitotic activity became significantly enhanced. The present results are discussed with regard to known factors regulating cell proliferation in the hippocampus and other cell systems. Accepted January 27, 1998; received December 12, 1997     

Dentate granule cell neurogenesis after seizures induced by pentylenetrazol in rats

Epileptic seizures originating from the limbic system have been shown to stimulate the proliferation rate of granule cell precursors in the adult brain, but it is not clear if other type(s) of seizures have the similar effects. This study examined the effects of pentylenetrazol (PTZ)-induced generalized clonic seizures on dentate granule cell neurogenesis in adult rats. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we studied the proliferation rate of neural precursor cells in the dentate gyrus at various time points after PTZ-induced seizures. The double-label immunofluorescence with confocal microscopy was used to determine the newborn cell phenotypes. Quantitative analysis of BrdU labeling revealed a significant increase in the proliferation rate of neural precursor cells in the dentate gyrus 3, 7, and 14 days after seizures. The number of BrdU-labeled cells in the dentate gyrus returned to baseline levels by 28 days after the initial seizures. Most of newborn cells migrated into the granule cell layer from the subgranular zone, displayed the neuronal phenotype, and developed morphological characteristics of differentiated dentate granule cells. These results indicated that neuron proliferation in the dentate gyrus was enhanced during a time window (3-14 days) after PTZ-induced seizures. Its underlying mechanism is discussed.     

Adult treatment with methamphetamine transiently decreases dentate granule cell proliferation in the gerbil hippocampus

Summary. The objective of the present study was to examine whether acute treatment with the recreational drug methamphetamine influences adult granule cell proliferation in the dentate gyrus of the hippocampus. For that purpose, at the age of postnatal day 90 adult male gerbils (Meriones unguiculatus) received a single dose of either methamphetamine (25 mg/kg; i.p.) or saline. Proliferation of granule cells was identified by in-vivo labeling with 5-bromo-2'-desoxyuridine (BrdU) which was applied either simultaneously with methamphetamine or 36 h after administration of the drug. BrdU-labeled granule cell nuclei were identified in consecutive horizontal slices along the mid-septotemporal axis of the hippocampus and light-microscopically quantified 7 days after the BrdU-labeling. It was found that in both saline- and methamphetamine-treated animals there was a highly significant spatial septotemporal gradient in granule cell proliferation with numbers of BrdU-labeled cells gradually declining from the septal towards the temporal pole. The acute treatment with methamphetamine suppressed granule cell proliferation by about 28% and the septotemporal gradient of mitotic activity became significantly attenuated. It was further found that 36 h after the drug challenge granule cell proliferation rates had been restored almost to the control values along the whole septotemporal axis of the hippocampus. The present results are discussed with regard to (1) pharmacological regulation of neurogenesis in the hippocampus and (2) probable clues they may provide for both understanding the biological correlates of psychotic disorders and evolution of future concepts in neuropharmacological intervention. Received January 8, 1999; accepted July 12, 1999     

Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice

We here show that the early postmitotic stage of granule cell development during adult hippocampal neurogenesis is characterized by the transient expression of calretinin (CR). CR expression was detected as early as 1 day after labeling dividing cells with bromodeoxyuridine (BrdU), but not before. Staining for Ki-67 confirmed that no CR-expressing cells were in cell cycle. Early after BrdU, CR colocalized with immature neuronal marker doublecortin; and later with persisting neuronal marker NeuN. BrdU/CR-labeled cells were negative for GABA and GABAA1 receptor, but early on expressed granule cell marker Prox-1. After 6 weeks, no new neurons expressed CR, but all contained calbindin. Stimuli inducing adult neurogenesis have limited (enriched environment), strong (voluntary wheel running), and very strong effects on cell proliferation (kainate-induced seizures). In these models the induction of cell proliferation was paralleled by an increase of CR-positive cells, indicating the stimulus-dependent progression from cell division to a postmitotic stage.     

NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus.

The effect of N-methyl-D-aspartate (NMDA) and 2-(aminomethyl)phenylacetic acid/kainate (AMPA/kainate) glutamate receptors on dentate cell proliferation and hippocampal synapsin-I induction was examined after global ischemia. Cell proliferation was assessed using BrdU labeling, and synaptic responses were assessed using synapsin-I expression. Systemic glutamate receptor antagonists (MK-801 and NBQX) increased BrdU-labeled cells in the dentate subgranular zone (SGZ) of control adult gerbils (30% to 90%, P < 0.05). After global ischemia (at 15 days after 10 minutes of ischemia), most CA1 pyramidal neurons died, whereas the numbers of BrdU-labeled cells in the SGZ increased dramatically (>1000%, P < 0.0001). Systemic injections of MK801 or NBQX, as well as intrahippocampal injections of either drug, when given at the time of ischemia completely blocked the birth of cells in the SGZ and the death of CA1 pyramidal neurons at 15 days after ischemia. Glutamate receptor antagonists had little effect on cell birth and death when administered 7 days after ischemia. The induction of synapsin-I protein in stratum moleculare of CA3 at 7 and 15 days after global ischemia was blocked by pretreatment with systemic or intrahippocampal MK-801 or NBQX. It is proposed that decreased dentate glutamate receptor activation--produced by glutamate receptor antagonists in normal animals and by chronic ischemic hippocampal injury--may trigger dentate neurogenesis and synaptogenesis. The synapsin-I induction in mossy fiber terminals most likely represents re-modeling of dentate granule cell neuron presynaptic elements in CA3 in response to the ischemia. The dentate neurogenesis and synaptogenesis that occur after ischemia may contribute to memory recovery after hippocampal injury caused by global ischemia.