Enriched environment increases neurogenesis in the adult rat dentate gyrus and improves spatial memory

The fetal and even the young brain possesses a considerable degree of plasticity. The plasticity and rate of neurogenesis in the adult brain is much less pronounced. The present study was conducted to investigate whether housing conditions affect neurogenesis, learning, and memory in adult rats. Three‐month‐old rats housed either in isolation or in an enriched environment were injected intraperitoneally with bromodeoxyuridine (BrdU) to detect proliferation among progenitor cells and to follow their fate in the dentate gyrus. The rats were sacrificed either 1 day or 4 weeks after BrdU injections. This experimental paradigm allows for discrimination between proliferative effects and survival effects on the newborn progenitors elicited by different housing conditions. The number of newborn cells in the dentate gyrus was not altered 1 day after BrdU injections. In contrast, the number of surviving progenitors 1 month after BrdU injections was markedly increased in animals housed in an enriched environment. The relative ratio of neurogenesis and gliogenesis was not affected by environmental conditions, as estimated by double‐labeling immunofluorescence staining with antibodies against BrdU and either the neuronal marker calbindin D28k or the glial marker GFAp, resulting in a net increase in neurogenesis in animals housed in an enriched environment. Furthermore, we show that adult rats housed in an enriched environment show improved performance in a spatial learning test. The results suggest that environmental cues can enhance neurogenesis in the adult hippocampal region, which is associated with improved spatial memory. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 569–578, 1999     

The multi-herbal formula Chong-Myung-Tang improves spatial memory and increases cell genesis in the dentate gyrus of aged mice

Chong-Myung-Tang (CMT) is a multi-herbal formula that has been used to improve memory. However, the potential mechanism remains unknown. The present study investigated the effects of CMT (50, 100, and 200?mg/kg) on spatial memory of aged mice. The behavioral training tests indicated that 200?mg/kg CMT treatment can significantly improve spatial memory of aged mice in the Morris water maze. Moreover, cell survival was examined by injecting bromodeoxyuridine (BrdU) on the first three days. The result showed that 200?mg/kg CMT treatment significantly increased cell survival in the dentate gyrus. Cell proliferation was determined by injecting BrdU 2?h before the mice were killed. The result suggested that CMT treatments had no influence on cell proliferation in the dentate gyrus. Thus, an increase in cell survival in the dentate gyrus stimulated by CMT may be involved in the effect of CMT on spatial memory improvement.     

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。     

Stimulatory effects of prostaglandin E2 on neurogenesis in the dentate gyrus of the adult rat

Neurogenesis in the dentate gyrus of adult rodents is elicited by transient global ischemia. Cyclooxygenase (COX) -2, a rate-limiting enzyme for prostanoid synthesis, is also induced by ischemia. We recently found that the administration of a non-selective COX inhibitor to ischemic animals suppressed cell proliferation in the subgranular zone (SGZ) at the dentate gyrus of the hippocampus. To clarify whether prostaglandin E2 (PGE2) synthesis by COX's is involved in neurogenesis, sulprostone, an analogue of PGE2, was injected into the rat hippocampus. Sulprostone injection increased the number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the SGZ. BrdU-positive cells also expressed polysialylated isoforms of neural cell adhesion molecule and neuronal nuclear antigen. These results suggest that PGE2 plays an important role in the proliferation of cells in the SGZ.     

Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.     

胎次、性别对成年小鼠海马齿状回神经发生及学习记忆的影响

目的探讨胎次、性别是否对成年小鼠海马齿状回神经发生及学习记忆产生影响。方法运用Morris水迷宫系统检测第1～3胎成年小鼠的学习记忆能力,腹腔注射BrdU,标记神经干细胞,检测不同胎次、性别小鼠海马齿状回中的神经发生的差异。结果 （1）在同性别、不同胎次成年小鼠间,第2胎的学习记忆能力（LMA）均显著地高于第1、3胎的,其影响规律为LMA2〉LMA1〉LMA3,且P〈0.05;在同胎次、不同性别成年小鼠间,雌性小鼠的LMA均高于雄性小鼠的,但其差异无显著性（P〉0.05）。（2）在同性别、不同胎次成年小鼠间,第2胎海马DG新生神经细胞的数量（N）均高于第1、3胎的,其影响规律分别为NF2〉NF3〉NF1和NM2〉NM1〉NM3,但其差异无显著性（P〉0.05）;在同胎次、不同性别成年小鼠间,雌性小鼠的N均高于雄性小鼠的,但其差异无显著性（P〉0.05）。结论胎次、性别对实验动物神经发生及学习记忆等方面产生的影响是肯定的。因此,在使用实验动物时,应予以充分考虑,尽量使用胎次、性别相同的。     

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.     

Novel control by the CA3 region of the hippocampus on neurogenesis in the dentate gyrus of the adult rat

The dentate gyrus is a site of continued neurogenesis in the adult brain. The CA3 region of the hippocampus is the major projection area from the dentate gyrus. CA3 sends reciprocal projections back to the dentate gyrus. Does this imply that CA3 exerts some control over neurogenesis? We studied the effects of lesions of CA3 on neurogenesis in the dentate gyrus, and on the ability of fluoxetine to stimulate mitotic activity in the progenitor cells. Unilateral ibotenic-acid generated lesions were made in CA3. Four days later there was no change on the number of either BrdU or Ki67-positive progenitor cells in the dentate gyrus. However, after 15 or 28 days, there was a marked reduction in surviving BrdU-labelled cells on the lesioned side (but no change in Ki-67+ cells). pCREB or Wnt3a did not co-localise with Ki-67 but with NeuN, a marker of mature neurons. Lesions had no effect on the basal expression of either pCREB or Wnt3a. Subcutaneous fluoxetine (10 mg/kg/day) for 14 days increased the number of Ki67+ cells as expected on the control (non-lesioned) side but not on that with a CA3 lesion. Nevertheless, the expected increase in BDNF, pCREB and Wnt3a still occurred on the lesioned side following fluoxetine treatment. Fluoxetine has been reported to decrease the number of “mature” calbindin-positive cells in the dentate gyrus; we found this still occurred on the side of a CA3 lesion. We then showed that the expression GAP-43 was reduced in the dentate gyrus on the lesioned side, confirming the existence of a synaptic connection between CA3 and the dentate gyrus. These results show that CA3 has a hitherto unsuspected role in regulating neurogenesis in the dentate gyrus of the adult rat.     

Vitamin E affects cell death in adult rat dentate gyrus

We have previously reported the presence of dying cells in the granule cell layer (GCL) of adult rat dentate gyrus (DG), where neurogenesis occurs. In particular, we found that cell death in the GCL increased in vitamin E deficiency and decreased in vitamin E supplementation. These findings were regarded as related to changes in neurogenesis rate, which in turn was influenced by vitamin E availability; a neuroprotective effect of vitamin E on cell death was also proposed. In order to verify this latter hypothesis, we have studied cell death in all layers of DG in vitamin E-deficient and vitamin E-supplemented rats and in control rats at different ages, using TUNEL and nick translation techniques. The phenotype of TUNEL-positive cells was characterized and the existence of dying BrdU-positive cells was investigated. Dying cells with neuronal phenotype were observed throughout the DG in all experimental groups. The number of TUNEL-positive cells decreased from juvenile to adult age. A higher number of TUNEL-positive cells in vitamin E-deficient rats and a lower number in vitamin E-supplemented rats, with respect to age-matched controls, were found; moreover, in these groups, TUNEL-positive cells had a different percentage distribution in the different layers of the DG. Our results confirm the occurrence of cell death in DG, demonstrate that cell death affects neuronal cells and support the hypothesis that the effect of vitamin E on cell death is not related to neurogenesis.     

The neuronal primary cilium: Driver of neurogenesis and memory formation in the hippocampal dentate gyrus?

Considerable attention has recently been focused on the postnatal persistence of neurogenesis in the dentate gyrus of the hippocampus and the roles of signals from the primary cilium in the different functions of an increasing number of tissues and their malfunctions. Here we summarize the evidence that ties sonic hedgehog-triggered proliferogenic signaling from the primary cilia on granule cell progenitors in the adult dentate subgranular zone to maintain a pool of new “blank slate” dentate granule cells. These can be recruited to bundle and encode novel inputs flowing from various regions of the brain into the dentate gyrus via the entorhinal cortex without altering and erasing the synaptic patterns from previous inputs inscribed on older granule cells.     

Regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia

Neurogenesis in the dentate gyrus occurs throughout life. We observed regional differences in neurogenesis in the dentate gyrus of adult rats following transient forebrain ischemia. Nine days after ischemic-reperfusion or sham manipulation, rats were given 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU), a marker for dividing cells. They were killed 1 or 28 days later to distinguish between cell proliferation and survival. Neurogenesis was evaluated by BrdU incorporation as well by identifying neuronal and glial markers in six regions of the dentate gyrus: rostral, middle and caudal along the rostrocaudal axis, each further divided into suprapyramidal and infrapyramidal blade subregions. In control rats BrdU-positive cells in the rostral subregions were significantly lower in the suprapyramidal than in the infrapyramidal blades at both 1 and 28 days after BrdU injection. One day after injection, BrdU-positive cells had increased more in five of the subregions in the ischemic rats than in the controls, the exception being the suprapyramidal blade of the rostral subregion. At 28 days after BrdU injection, numbers of BrdU-positive cells were higher in four subregions in the ischemic group, the exceptions being the rostral suprapyramidal and middle infrapyramidal blades. At 28 days after BrdU injection, the percentages of BrdU positive cells that expressed a neuronal marker (NeuN) were the same in the dentate granule cell layers of ischemic and control rats. Our data thus demonstrate regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia.     

Temporal proteomic profile of memory consolidation in the rat hippocampal dentate gyrus

Information storage in the brain depends on the ability of neurons to alter synaptic connectivity within key circuitries such as the hippocampus. Memory-associated synaptic plasticity is mediated by a temporal cascade of de novo protein synthesis and altered protein processing. Here, we have used two-dimensional difference in gel electrophoresis (2-D DIGE) to investigate memory-specific protein changes in the hippocampal dentate gyrus at increasing times following spatial learning. We identified 42 proteins that were significantly regulated in the first 24 h of spatial memory consolidation. Two distinct waves of protein expression regulation were evident, at 3 and 12 h post-learning and this is in agreement with studies employing inhibitors of global translation. Functional classification of the memory-associated proteins revealed that the majority of regulated proteins contributed either to cellular structure or cellular metabolism. For example, actins, tubulins and intermediate filament proteins, core proteins of the three major cytoskeletal components, were dynamically regulated at times that suggest a role in memory-associated synaptic reorganization. Increased proteasome-mediated protein degradation was evident in the early post-training period including the down-regulation of phosphoprotein enriched in astrocytes 15 kDa, a key inhibitor of extracellular signal-regulated kinase signaling. Some of the most substantial protein expression changes were observed for secreted carrier proteins including transthyretin and serum albumin at 6-12 h post-learning, regulations that could serve an important role in increasing the supply of retinoic acid and thyroid hormone, key synaptic plasticity-promoting signals in the adult brain. Together these observations provide further insight into protein level regulations occurring in the hippocampus during spatial memory consolidation.     

Impaired adult neurogenesis in the dentate gyrus of a triple transgenic mouse model of Alzheimer's disease

It has become generally accepted that new neurones are added and integrated mainly in two areas of the mammalian CNS, the subventricular zone and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus, which is of central importance in learning and memory. The newly generated cells display neuronal morphology, are able to generate action potentials and receive functional synaptic inputs, i.e. their properties are similar to those found in mature neurones. Alzheimer's disease (AD) is the primary and widespread cause of dementia and is an age-related, progressive and irreversible neurodegenerative disease that deteriorates cognitive functions. Here, we have used male and female triple transgenic mice (3xTg-AD) harbouring three mutant genes (beta-amyloid precursor protein, presenilin-1 and tau) and their respective non-transgenic (non-Tg) controls at 2, 3, 4, 6, 9 and 12 months of age to establish the link between AD and neurogenesis. Using immunohistochemistry we determined the area density of proliferating cells within the SGZ of the DG, measured by the presence of phosphorylated Histone H3 (HH3), and their possible co-localisation with GFAP to exclude a glial phenotype. Less than 1% of the HH3 labeled cells co-localised with GFAP. Both non-Tg and 3xTg-AD showed an age-dependent decrease in neurogenesis. However, male 3xTg-AD mice demonstrated a further reduction in the production of new neurones from 9 months of age (73% decrease) and a complete depletion at 12 months, when compared to controls. In addition, female 3xTg-AD mice showed an earlier but equivalent decrease in neurogenesis at 4 months (reduction of 63%) with an almost inexistent rate at 12 months (88% decrease) compared to controls. This reduction in neurogenesis was directly associated with the presence of beta-amyloid plaques and an increase in the number of beta-amyloid containing neurones in the hippocampus; which in the case of 3xgTg females was directly correlated. These results suggest that 3xTg-AD mice have an impaired ability to generate new neurones in the DG of the hippocampus, the severity of which increases with age and might be directly associated with the known cognitive impairment observed from 6 months of age onwards . The earlier reduction of neurogenesis in females, from 4 months, is in agreement with the higher prevalence of AD in women than in men. Thus it is conceivable to speculate that a recovery in neurogenesis rates in AD could help to rescue cognitive impairment.     

Multipotent progenitor cells in the adult dentate gyrus

Neurogenesis persists in the adult dentate gyrus of rodents throughout the life of the organism. The factors regulating proliferation, survival, migration, and differentiation of neuronal progenitors are now being elucidated. Cells from the adult hippocampus can be propagated, cloned in vitro, and induced to differentiate into neurons and glial cells. Cells cultured from the adult rodent hippocampus can be genetically marked and transplanted back to the adult brain, where they survive and differentiate into mature neurons and glial cells. Although multipotent stem cells exist in the adult rodent dentate gyrus, their biological significance remains elusive. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 249–266, 1998.     

Amyloid precursor protein expression in the rat hippocampal dentate gyrus modulates during memory consolidation

Despite advances in our understanding of the basic biology of amyloid precursor protein (APP), the normal physiological function(s) of APP in learning and memory remains unclear. Here we show increased APP degradation in the hippocampus to be associated with the consolidation of a passive avoidance response. Neurone-specific APP695 expression became transiently reduced 2-4 h post-training through association with endosomal adaptin proteins and enhanced internalization. By contrast, internalization of glial-associated APP containing a Kunitz protease inhibitor-like domain (APP-KPI) was dependent on the low-density lipoprotein receptor-related protein (LRP). In addition, LRP expression and association with apolipoprotein E increased in the 2-4 h post-training period. The LRP antagonist receptor-associated protein prevented the APP-KPI internalization and LRP-apolipoprotein E association and this resulted in amnesia. Degradation of APP695 and APP-KPI did not appear to be related to alpha-secretase activity, as no learning-associated increase of secreted APP was observed in the CSF. Moreover, as internalization of APP isoforms was observed only in dentate gyrus, it probably relates to the learning-associated restructuring of the perforant path terminals. Memory-associated APP processing in both neuronal and glial compartments points to a role for glial unsheathing of synaptic connections, an event required for the synaptic restructuring that accompanies memory consolidation. These observations may have a direct relevance to understanding the pathophysiology of Alzheimer's disease as beta/gamma-secretase-derived beta-amyloid is formed following internalization of cell surface APP into the endosomal compartment.     

Resolving new memories: a critical look at the dentate gyrus, adult neurogenesis, and pattern separation

Recently, investigation of new neurons in memory formation has focused on a specific function-pattern separation. However, it has been difficult to reconcile the form of separation tested in behavioral tasks with how it is conceptualized according to computational and electrophysiology perspectives. Here, we propose a memory resolution hypothesis that considers the unique information contributions of broadly tuned young neurons and highly specific mature neurons and describe how the fidelity of memories can relate to spatial and contextual discrimination. See the related Perspective from Sahay, Wilson, and Hen, "Pattern Separation: A?Common Function for New Neurons in Hippocampus and Olfactory Bulb," in this issue of Neuron.     

Chronic lithium enhances hippocampal long-term potentiation,but not neurogenesis,in the aged rat dentate gyrus

We investigated the hippocampal long-term potentiation (LTP), neurogenesis, and the activation of signaling molecules in the 20-month-old aged rats following chronic lithium treatment. Chronic lithium treatment produced a significant 79% increase in the numbers of BrdU(+) cells 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. Our results show that as with young rats, chronic lithium can substantially increase LTP and the number of BrdU(+) cells in the aged rats. However, neurogenesis, assessed by colocalization of NeuN and BrdU, was not detected in the aged rat DG subjected to chronic lithium treatment. Therefore, it is concluded that the increase in LTP and the number of BrdU(+) cells might not be associated with increases in neurogenesis in the granule cell layer of the DG. Lithium might has a beneficial effects through other signaling pathways in the aged brain.     

Synaptically-silent immature neurons show gaba and glutamate receptor-mediated currents in adult rat dentate gyrus

The fate of adult-generated neurons in dentate gyrus is mainly determined early, before they receive synapses. In developing brain, classical neurotransmitters such as GABA and glutamate exert trophic effects before synaptogenesis. In order for this to occur in adult brain as well, immature non-contacted cells must express functional receptors to GABA and glutamate. In this investigation, patch-clamp recordings were used in adult rat dentate gyrus slices to assess the presence and analyze the characteristics of GABA- and glutamate-evoked currents in highly immature, synaptically-silent granule cells. Whole-cell patch-clamp recordings showed that all the analyzed cells responded to puff application of GABA and most of them responded to glutamate. Currents evoked by GABA were mediated exclusively by GABAA receptors and those elicited by glutamate were mediated by NMDA and AMPA/Kainate receptors. GABAA receptor-mediated currents were reduced by furosemide, which suggests that synaptically-silent immature neurons express high-affinity, alpha4-subunit-containing GABAA receptors. Gramicidin-perforated-patch recordings showed that GABAA receptor-mediated currents exerted a depolarizing effect due to high intracellular chloride concentration. Synaptically-silent immature cells shared morphological and electrophysiological properties with GFP-expressing, 7-day-old adult-generated granule layer cells, indicating that they could be in the first week of life, the period of maximal newborn cell death. Moreover, the presence of functional GABA and glutamate receptors was confirmed in these GFP-expressing cells. Present findings are mostly consistent with previous data obtained in female mice undergoing spontaneous activity and in transgenic mice, except for some inconsistencies about the presence of functional glutamatergic receptors. We speculate that adult-generated, non-contacted granule cells may be able to sense activity-related variations of GABA and glutamate extracellular levels. This condition is necessary, even if not sufficient, for these neurotransmitters to have a direct role in addressing cell survival.