Age‐dependent effects of hippocampal neurogenesis suppression on spatial learning

Reducing hippocampal neurogenesis sometimes, but not always, disrupts hippocampus‐dependent learning and memory. Here, we tested whether animal age, which regulates rate of hippocampal neurogenesis, is a factor that influences whether deficits in spatial learning are observed after reduction of neurogenesis. We found that suppressing the generation of new hippocampal neurons via treatment with temozolomide, an antiproliferation agent, impaired learning the location of a hidden platform in the water maze in juvenile mice (1–2 months old) but not in adult mice (2–3 months old) or middle‐aged mice (11–12 months old). These findings suggest that during juvenility, suppression of neurogenesis may alter hippocampal development, whereas during adulthood and aging, pre‐existing neurons may compensate for the lack of new hippocampal neurons. © 2012 Wiley Periodicals, Inc.     

Exogenous neuropeptide Y promotes in vivo hippocampal neurogenesis

Adult neurogenesis mainly occurs in two brain regions, the subventricular zone and the dentate gyrus (DG) of the hippocampus. Neuropeptide Y (NPY) is widely expressed throughout the brain and is known to enhance in vitro hippocampal cell proliferation. Mice lacking either NPY or the Y1 receptor display lower levels of cell proliferation, thereby suggesting a role for NPY in basal in vivo neurogenesis. Here, we investigated whether exogenous NPY stimulates DG progenitors proliferation in vivo. We show that intracerebroventricular administration of NPY increases DG cell proliferation and promotes neuronal differentiation in C57BL/6 adult mice. In these mice, the proliferative effect of NPY is mediated by the Y1 and not the Y2 receptor, as a Y1 ([Leu³¹,Pro³⁴]), but not a Y2 (NPY_3–36), receptor agonist enhanced proliferation. In addition, no NPY‐induced DG cellular proliferation is observed following NPY injection when coadministered with a Y1 antagonist or in the Y1 receptor knockout mouse. These results are in line with data obtained in Y1^?/? mice, demonstrating that NPY regulates in vivo hippocampal neurogenesis. © 2010 Wiley‐Liss, Inc.     

Effects of exercise on neurogenesis in the dentate gyrus and ability of learning and memory after hippocampus lesion in adult rats

Objective To explore the effects of exercise on dentate gyrus (DG) neurogenesis and the ability of learning and memory in hippocampus-lesioned adult rats. Methods Hippocampus lesion was produced by intrahippocampal microinjection of kainic acid (KA). Bromodeoxyuridine (BrdU) was used to label dividing cells. Y maze test was used to evaluate the ability of learning and memory. Exercise was conducted in the form of forced running in a motor-driven running wheel. The speed of wheel revolution was regulated at 3 kinds of intensity: lightly running, moderately running, or heavily running. Results Hippocampus lesion could increase the number of BrdU-labeled DG cells, moderately running after lesion could further enhance the number of BrdU-labeled cells and decrease the error number (EN) in Y maze test, while neither lightly running, nor heavily running had such effects. There was a negative correlation between the number of DG BrdU-labeled cells and the EN in the Y maze test after running. Conclusion Moderate exercise could enhance the DG neurogenesis and ameliorate the ability of learning and memory in hippocampus-lesioned rats.     

Effects of exercise on neurogenesis in the dentate gyrus and ability of learning and memory after hippocampus lesion in adult rats

Objective To explore the effects of exercise on dentate gyrus （DG） neurogenesis and the ability of learning and memory in hippocampus-lesioned adult rats. Methods Hippocampus lesion was produced by intrabippocampal microinjection of kainic acid （KA）. Bromodeoxyuridine （BrdU） was used to label dividing cells. Y maze test was used to evaluate the ability of learning and memory. Exercise was conducted in the form of forced running in a motor-driven running wheel. The speed of wheel revolution was regulated at 3 kinds of intensity： lightly running, moderately running, or heavily running. Results Hippocampus lesion could increase the number of BrdU-labeled DG cells, moderately running after lesion could further enhance the number of BrdU-labeled cells and decrease the error number （EN） in Y maze test, while neither lightly running, nor heavily running had such effects. There was a negative correlation between the number of DG BrdU-labeled cells and the EN in the Y maze test after running. Conclusion Moderate exercise could enhance the DG neurogenesis and ameliorate the ability of learning and memory in hippocampus-lesioned rats.     

Influence of superior cervical ganglionectomy on hippocampal neurogenesis and learning and memory in adult rats

BACKGROUND： Studies have shown that neurogenesis in the dentate gyrus plays an important role in learning and memory. However, studies have not determined whether the superior cervical ganglion or the sympathetic nerve system influences hippocampal neurogenesis or learning and memory in adult rats. OBJECTIVE： To observe differences in dentate gyrus neurogenesis, as well as learning and memory, in adult rats following superior cervical ganglionectomy. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Immunohistochemistry Laboratory of the School of Life Sciences in Lanzhou University from July 2006 to July 2007. MATERIALS： Doublecortin polyclonal antibody was provided by Santa Cruz Biotechnology, USA; avidin-biotin-peroxidase complex was purchased from Zhongshan Goldenbride Biotechnology, China; Morris water maze was bought from Taimeng Technology, China. METHODS： A total of 20 adult, male, Wistar rats were randomly divided into surgery and control groups, with 10 rats in each group. In the surgery group, the bilateral superior cervical ganglions were transected. In the control group, the superior cervical ganglions were only exposed, but no ganglionectomy was performed. MAIN OUTCOME MEASURES： To examine distribution, morphology, and number of newborn neurons in the dentate gyrus using doublecortin immunohistochemistry at 36 days following surgical procedures. To examine ability of learning and memory in adult rats using the Morris water maze at 30 days following surgical procedures. RESULTS： Doublecortin immunohistochemical results showed that a reduction in the number of doublecortin-positive neurons in the surgery group compared to the control group （P 〈 0.05）, while the distribution of doublecortin-positive neurons was identical in the two groups. The surgery group exhibited significantly worse performance in learning and spatial memory tasks compared to the control group （P 〈 0.05）. CONCLUSION： Superior cervical ganglienectomy inhibited neurogenesis in the dentate gyrus and decreased learning and memory abilities in adult rats.     

冬南瓜醇提物干预雄性大鼠齿状回细胞增殖和空间学习功能的变化

Previous studies reported that some plants,including butternut squash,exert positive effects on the brain.However,few studies have examined the effects of butternut squash on learning,memory,and neurogenesis.This study studied the effects of butternut squash extract on spatial learning and cell proliferation in the dentate gyrus of healthy male rats.Thirty-five male Wistar rats were intrap-eritoneally injected with 0,50,100,200 and 400 mg/kg butternut squash extract once daily for 2 months.After the last administration,rat’s spatial memory was studied using the Morris water maze.Finally,rats were sacrificed and hippocampal sections were prepared for light microscopy and bromodeoxyuridine immunohistochemistry studies.The results revealed that escape latency and swim distance decreased in all treatment groups compared with the control rats,and that the number of bromodeoxyuridine-positive cells in the dentate gyrus was significantly increased in the treatment groups compared with the controls.These findings suggest that butternut squash extract improves the learning and memory abilities of male rats,and increases the proliferation of dentate gyrus cells.     

Impairment of hippocampal neurogenesis in streptozotocin-treated diabetic rats

Objectives –  Adult neurogenesis in dentate gyrus (DG) is an evolutionarily preserved trait in most mammals examined thus far. Neuronal proliferation and subsequent integration of new neurons into the hippocampal circuit are regulated processes that can have profound effects on an animal's behaviour. A streptozotocin model of type I diabetes, characterized by low insulin and high plasma glucose levels, affects not only body's overall metabolism but also brain activity.
Materials and methods –  Neurogenesis was measured within the DG of the hippocampus using immunohistochemical markers Ki67, Doublecortin, Calbindin (CaBP) and bromodeoxyuridine (BrdU).
Results –  Cell proliferation, measured with the endogenous marker Ki67, was reduced by 45%, and cell survival, measured with BrdU, was reduced by 64% of the control. Combined effects on proliferation and survival produced dramatically lower neuronal production. Among the surviving cells only 33% matured normally as judged by the co-labelling of BrdU and CaBP.
Conclusion –  Such a reduction lowered the number of surviving cells with neuronal phenotype by over 80% of the control values and this is expected to cause a significant functional impairment of learning and memory in diabetic animals. These results may shed light on causes of diabetic neuropathology and provide an explanation for the memory deficiencies seen in some diabetic patients.     

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.     

Decreased neurogenesis after cholinergic forebrain lesion in the adult rat

Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival-promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.     

Spatial selectivity of unit activity in the hippocampal granular layer

Single neuron activity was recorded in the granular layer of the fascia dentata in freely moving rats, while the animals performed a spatial “working” memory task on an eight-arm maze. Using recording methods that facilitate detection of units with low discharge rates, it was found that the majority (88%) of cells in this layer have mean rates below 0.5 Hz, with a minimum of 0.01 Hz or less. The remaining recorded cells exhibited characteristics typical of the theta interneurons found throughout the hippocampus. Based on several criteria including relative proportion and the relation of their evoked discharges to the population spike elicited by perforant path stimulation, it was concluded that the low-rate cells correspond to granule cells. Granule cells exhibited clear spatially and directionally selective discharge that was at least as selective as that of a sample of CA3 pyramidal cells recorded under the same conditions. Granule cells had significantly smaller place fields than pyramidal cells, and tended to have more discontiguous subfields. There was no spatial correlation among simultaneously recorded adjacent granule cells. Granule cells also exhibited burst discharges reminiscent of complex spikes from pyramidal cells while the animals sat quietly; however, the spike duration of granule cells was significantly shorter than CA3 pyramidal cell spike durations. Under conditions of environmental stability, granule cell place fields were stable for at least several days. Following occasional maze rotations relative to the (somewhat impoverished) visual stimuli of the recording room, granule cell place fields were maintained relative to the distal spatial cues; however, frequent rotations of the maze sometimes resulted in a shift in the reference frame to the maze itself. These observations indicate that granule cells of the fascia dentata provide their CA3 targets with a high degree of spatial information, in the form of a sparsely coded, distributed representation.     

Thymidine analog methods for studies of adult neurogenesis are not equally sensitive

Adult neurogenesis is often studied by labeling new cells with the thymidine analog bromodeoxyuridine (BrdU) and using immunohistochemical methods for their visualization. Using this approach, considerable variability has been reported in the number of new cells produced in the dentate gyrus of adult rodents. We examined whether immunohistochemical methods, including BrdU antibodies from different vendors (Vector, BD, Roche, Dako, Novocastra, and Accurate) and DNA denaturation pretreatments alter the quantitative and qualitative patterns of BrdU labeling. We also compared the sensitivity and specificity of BrdU with two other thymidine analogs, iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU). We found that the number of BrdU‐labeled cells in the dentate gyrus of adult rats was dependent on the BrdU antibody used but was unrelated to differences in antibody penetration. Even at a higher concentration, some antibodies (Vector and Novocastra) stained fewer cells. A sensitive BrdU antibody (BD) was specific for dividing cells; all BrdU‐labeled cells stained for Ki67, an endogenous marker of cell proliferation. We also observed that DNA denaturation pretreatments affected the number of BrdU‐labeled cells and staining intensity for a marker of neuronal differentiation, NeuN. Finally, we found that IdU and CldU, when used at molarities comparable to those that label the maximal number of cells with BrdU, are less sensitive. These data suggest that antibody and thymidine analog selection, as well as the staining procedure employed, can affect the number of newly generated neurons detected in the adult brain, thus providing a potential explanation for some of the variability in the adult neurogenesis literature. J. Comp. Neurol. 517:123–133, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the role of adult neurogenesis in touch‐screen discrimination learning

Recent theories posit that adult neurogenesis supports dentate gyrus pattern separation and hence is necessary for some types of discrimination learning. Using an inducible transgenic mouse model, we investigated the contribution of adult‐born neurons to spatial and nonspatial touch‐screen discriminations of varying levels of difficulty. Arresting neurogenesis caused a modest but statistically significant impairment in a position discrimination task. However, the effect was present only on trials after a learned discrimination was reversed, suggesting that neurogenesis supports cognitive flexibility rather than spatial discrimination per se. The deficit was present 4–10 weeks after the arrest of neurogenesis but not immediately after, consistent with previous evidence that the behavioral effects of arresting neurogenesis arise because of the depletion of adult‐born neurons at least 1 month old. The arrest of neurogenesis failed to affect a nonspatial brightness discrimination task that was equal in difficulty to the spatial task. The data suggest that adult neurogenesis is not strictly necessary for spatial or perceptual discrimination learning and instead implicate adult neurogenesis in factors related to reversal learning, such as cognitive flexibility or proactive interference. © 2014 Wiley Periodicals, Inc.     

Sexual experience restores age‐related decline in adult neurogenesis and hippocampal function

Aging is associated with compromised hippocampal function and reduced adult neurogenesis in the dentate gyrus. As new neurons have been linked to hippocampal functions, such as cognition, age‐related decline in new neuron formation may contribute to impaired hippocampal function. We investigated whether a rewarding experience known to stimulate neurogenesis in young adult rats, namely sexual experience, would restore new neuron production and hippocampal function in middle‐aged rats. Sexual experience enhanced the number of newly generated neurons in the dentate gyrus with both single and repeated exposures in middle‐aged rats. Following continuous long‐term exposure to sexual experience, cognitive function was improved. However, when a prolonged withdrawal period was introduced between the final mating experience and behavioral testing, the improvements in cognitive function were lost despite the presence of more new neurons. Taken together, these results suggest that repeated sexual experience can stimulate adult neurogenesis and restore cognitive function in the middle‐aged rat as long as the experience persists throughout the testing period. The extent to which changes in adult neurogenesis underlie those in cognition remain unknown. © 2013 Wiley Periodicals, Inc.     

Associative learning increases adult neurogenesis during a critical period

Learning increases the number of immature neurons that survive and mature in the adult hippocampus. One-week-old cells are more likely to survive in response to learning than cells in animals that are exposed to training but do not learn. Because neurogenesis is an ongoing and overlapping process, it is possible that learning differentially affects new cells as a function of their maturity. To address this issue, we examined the effects of associative learning on the survival of cells at different stages of development. Training did not alter the number of cells that were produced during the training experience. Cells that were 1-2 weeks of age at the time of training survived after learning but cells that were younger or older did not. In contrast, cells that were produced during training were less likely to survive than cells in untrained animals. Additionally, the number of cells that were generated after learning in trained animals was not different from the number in untrained animals. Finally, survival was not increased if the memory was expressed when the cells were about 1-week-old. Together, these results indicate that new neurons are rescued from death by initial acquisition, not the expression or reacquisition, of an associative memory and only during a critical period. Overall, these results suggest the presence of a feedback system, which controls how many new neurons become incorporated into the adult brain in response to learning.     

Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience

After a spatial behavioral experience, hippocampal CA1 pyramidal cells express the activity-regulated, immediate early gene Arc in an environment-specific manner, and in similar proportions ( 40%) to cells exhibiting electrophysiologically recorded place fields under similar conditions. Theoretical accounts of the function of the fascia dentata suggest that it plays a role in pattern separation during encoding. The hypothesis that the dentate gyrus (DG) uses a sparse, and thus more orthogonal, coding scheme has been supported by the observation that, while granule cells do exhibit place fields, most are silent in a given environment. To quantify the degree of sparsity of DG coding and its corresponding ability to generate distinct environmental representations, behaviorally induced Arc expression was assessed using in situ hybridization coupled with confocal microscopy. The proportion of Arc(+) cells in the "upper blade" of the fascia dentata (i.e., the portion that abuts CA1) increased in an environment-specific fashion, approximately 4-fold above cage-control activity, after behavioral exploration. Surprisingly, cells in the lower blade of the fascia dentata, which are capable of expressing Arc following electrical stimulation, exhibited virtually no behaviorally-induced Arc expression. This difference was confirmed using "line scan" analyses, which also revealed no patterns or gradients of activity along the upper blade of the DG. The expression of Arc in the upper blade was quantitatively similar after exploring familiar or novel environments. When animals explored two different environments, separated by 20 min, a new group of cells responded to the second environment, whereas two separated experiences in the same environment did not activate a new set of granular cells. Thus, granule cells generate distinct codes for different environments. These findings suggest differential contribution of upper and lower blade neurons to plastic networks and confirm the hypothesis that the DG uses sparse coding that may facilitate orthogonalization of information.     

PACAP promotes neural stem cell proliferation in adult mouse brain

In recent years, it has become evident that neural stem cells in the adult mammalian brain continuously generate new neurons, mainly in the hippocampus and olfactory bulb. Although different growth factors have been shown to stimulate neurogenesis in the adult brain, very little is known about the role of neuropeptides in this process. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic effects acting through three receptors to which it has high affinity, namely, PACAP receptor 1 (PAC1), vasoactive intestinal peptide (VIP) receptor 1, and VIP receptor 2. We show that PAC1 is expressed in the neurogenic regions of the adult mouse brain, namely the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus. Cultured neural stem cells isolated from the lateral ventricle wall of adult mice express PAC1 and proliferate in vitro in response to two PAC1 agonists, PACAP and Maxadilan, but not VIP at physiologic concentrations, indicating PAC1 as a mediator of neural stem cell proliferation. Pharmacologic and biochemical characterization of PACAP-induced neural stem cell proliferation revealed the protein kinase C pathway as the principal signaling pathway, whereas addition of epidermal growth factor synergistically enhanced the proliferating effect of PACAP. Further in vitro characterization of the effect of PACAP on neural stem cells showed PACAP capable of stimulating ex novo in vitro formation of multipotent neurospheres with the capacity to generate both neuronal and glial cells. Finally, intracerebroventricular infusion of PACAP increases cell proliferation in the ventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus. We conclude that PACAP, through PAC1, is a potent mediator of adult neural stem cell proliferation.     

The effects of running and of inhibiting adult neurogenesis on learning and memory in rats

The presence of ongoing adult neurogenesis within the highly plastic hippocampal circuitry poses questions as to the relevance of new neurons to learning and memory. Correlational and causal evidence suggests that some, but not all, hippocampal tasks involve the new neurons. The evidence with regard to spatial learning in the water maze, one of the most commonly used hippocampal tasks, is contradictory. In this study we examined the effects of irradiation-induced reduction in neurogenesis on spatial learning and another standard hippocampal task, contextual fear conditioning, in rats that experienced normal cage conditions or voluntary running. The results indicate that reduced neurogenesis had little effect on spatial learning but severely impaired contextual fear conditioning. It was suggested that compensatory mechanisms within the hippocampus may have contributed selectively to sparing of spatial function. Performance on the fear conditioning task was weakly related to enhanced neurogenesis or running. The results improve our understanding of the functional role of adult neurogenesis in behaving animals.