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.     

Distinct stages of adult hippocampal neurogenesis are regulated by running and the running environment

Hippocampal neurogenesis continues into adulthood in mammalian vertebrates, and in experimental rodent models it is powerfully stimulated by exposure to a voluntary running wheel. In this study, we demonstrate that exposure to a running wheel environment, in the absence of running, is sufficient to regulate specific aspects of hippocampal neurogenesis. Adult mice were provided with standard housing, housing enriched with a running wheel or housing enriched with a locked wheel (i.e., an environment comparable to that of running animals, without the possibility of engaging in running). We found that mice in the running wheel and locked wheel groups exhibited equivalent increases in proliferation within the neurogenic niche of the dentate gyrus; this included comparable increases in the proliferation of radial glia‐like stem cells and the number of proliferating neuroblasts. However, only running animals displayed increased numbers of postmitotic neuroblasts and mature neurons. These results demonstrate that the running wheel environment itself is sufficient for promoting proliferation of early lineage hippocampal precursors, while running per se enables newly generated neuroblasts to survive and mature into functional hippocampal neurons. Thus, both running‐independent and running‐dependent stimuli are integral to running wheel‐induced hippocampal neurogenesis. © 2010 Wiley Periodicals, Inc.     

Functional recruitment of newborn hippocampal neurons after experimental stroke

The adult brain responds to diverse pathologies such as stroke with increased generation of neurons in the dentate gyrus of the hippocampus. However, only little is known regarding the functional integration of newborn neurons into pre-existing neuronal circuits. In this study, we investigated whether newborn neurons generated after experimental stroke are recruited for different behavioral tasks. Adult mice received photochemical cortical infarcts in the sensorimotor cortex and proliferating cells were labeled using the proliferation marker, bromodeoxyuridine. Eight weeks after stroke induction, the animals were trained to perform either a spatiotemporal task or a sensorimotor task. Immediate early gene expression (c-fos, Zif268) in newborn neurons was analyzed directly after the last session. Using this approach, we demonstrate that post-stroke generated neurons are recruited within the hippocampal networks. The sensorimotor task activates significantly more newborn neurons compared to the spatiotemporal task. Further experiments employing the two well-established stimulators of neurogenesis, enriched environment and voluntary wheel running, both significantly increase post-stroke neurogenesis in the dentate gyrus but do not affect the percentage of recruited neurons compared to controls. Significantly, the spatiotemporal task leads to a higher portion of activated newborn neurons in the granule cell layer, suggesting a specific spatial activation pattern of new neurons in the dentate gyrus.     

Human wild-type alpha-synuclein impairs neurogenesis

Neurodegenerative diseases classified as synucleinopathies are characterized by alpha-synuclein inclusions. In these disorders, alpha-synuclein accumulates within glial or neuronal cells in the brain including regions of adult neurogenesis. We hypothesized a pathophysiological role for alpha-synuclein in newly generated cells of the adult brain and in this study examined regions of neurogenesis in adult mice overexpressing human wild-type alpha-synuclein under the control of the platelet-derived growth factor promoter. The number of proliferating cells and the fate of newly generated cells were analyzed in the olfactory bulb system and in the hippocampal dentate gyrus. There were no effects on proliferation detectable; however, significantly less neurogenesis and fewer neurons were observed in the olfactory bulb as well as in the hippocampus of adult human alpha-synuclein mice compared to control littermates. This effect was almost exclusively due to diminished survival of neuronal precursors in the target regions of neurogenesis. Our data imply that the finely tuned equilibrium of neuronal cell birth and death in neurogenic regions may be altered in human alpha-synuclein-overexpressing mice. We hypothesize that reduced adult neurogenesis in the olfactory bulb may contribute to olfactory deficits in neurodegenerative disorders associated with alpha-synuclein inclusions.     

Adult neurogenesis in the four-striped mice （Rhabdomys pumilio）

In this study, we investigated non-captive four-striped mice （Rhabdomys pumilio） for evidence that adult neurogenesis occurs in the adult brain of animal models in natural environment. Ki-67 （a marker for cell proliferation） and doublecortin （a marker for immature neurons） immunos-taining conifrmed that adult neurogenesis occurs in the active sites of subventricular zone of the lateral ventricle with the migratory stream to the olfactory bulb, and the subgranular zone of the dentate gyrus of the hippocampus. No Ki-67 proliferating cells were observed in the striatum substantia nigra, amygdala, cerebral cortex or dorsal vagal complex. Doublecortin-immunore-active cells were observed in the striatum, third ventricle, cerebral cortex, amygdala, olfactory bulb and along the rostral migratory stream but absent in the substantia nigra and dorsal vagal complex. The potential neurogenic sites in the four-striped mouse species could invariably lead to increased neural plasticity.     

Impaired fear memory and decreased hippocampal neurogenesis following olfactory bulbectomy in rats

It has been proposed that a decrease in adult hippocampal neurogenesis provides a biological and cellular basis for major depression. The olfactory bulbectomy (OB) in rats is widely accepted as an animal model of depression. In the present study, we investigated the effect of OB on memory formation in the memory tasks related to the hippocampal function and adult hippocampal neurogenesis. OB induced a behavioural syndrome, which was characterized by an increased activity in the open-field test and impairment in passive avoidance behaviour and contextual fear conditioning. The behavioural changes, following OB, were accompanied by a decrease in the number of proliferating cells in the dentate gyrus. Furthermore, the differentiation of the newly born cells, into mature calbindin-positive neurons, was also retarded. Stereological analysis revealed a decrease in the total granule neuron numbers within the granule cell layer of the dentate gyrus, without a significant decrease in volume of the dentate gyrus. Although a relationship between altered neurogenesis and behavioural syndrome, induced by OB, is not established yet, our results suggest that decreased neurogenesis might at least partly contribute for behavioural deficits following OB.     

Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.     

Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice

Neurons are generated from neural progenitor cells not only during development but also in the mature brain. To develop an in vivo system for analyzing neurogenesis, we generated transgenic mice expressing green fluorescent protein (GFP) under the control of regulatory regions of the nestin gene. GFP fluorescence was observed in areas and during periods connected with neurogenesis, including embryonic neuroepithelium, neonatal cerebellum, and hippocampal dentate gyrus and rostral migratory pathway from the subventricular zone to the olfactory bulb in the adult. GFP-positive cells in the adult brain included immature neuronal cells expressing polysialylated NCAM. BrdU labeling experiments revealed that newly generated interneurons which migrated rostrally from the subventricular zone expressed GFP until they reached the olfactory bulb. These results indicate that nestin promoter-GFP transgenic mice can be utilized to visualize the regions of neurogenesis throughout the life of the animals and to follow the migration and differentiation of newly generated neurons.     

Widespread deficits in adult neurogenesis precede plaque and tangle formation in the 3xTg mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) affects cognitive modalities that are known to be regulated by adult neurogenesis, such as hippocampal‐ and olfactory‐dependent learning and memory. However, the relationship between AD‐associated pathologies and alterations in adult neurogenesis has remained contentious. In the present study, we performed a detailed investigation of adult neurogenesis in the triple transgenic (3xTg) mouse model of AD, a unique model that generates both amyloid plaques and neurofibrillary tangles, the hallmark pathologies of AD. In both neurogenic niches of the brain, the hippocampal dentate gyrus and forebrain subventricular zone, we found that 3xTg mice had decreased numbers of (i) proliferating cells, (ii) early lineage neural progenitors, and (iii) neuroblasts at middle age (11 months old) and old age (18 months old). These decreases correlated with major reductions in the addition of new neurons to the respective target areas, the dentate granule cell layer and olfactory bulb. Within the subventricular zone niche, cytological alterations were observed that included a selective loss of subependymal cells and the development of large lipid droplets within the ependyma of 3xTg mice, indicative of metabolic changes. Temporally, there was a marked acceleration of age‐related decreases in 3xTg mice, which affected multiple stages of neurogenesis and was clearly apparent prior to the development of amyloid plaques or neurofibrillary tangles. Our findings indicate that AD‐associated mutations suppress neurogenesis early during disease development. This suggests that deficits in adult neurogenesis may mediate premature cognitive decline in AD.     

Cell death in regenerating populations of neurons in BDNF mutant mice

There are two populations of neurons which are continually renewed in the adult, the dentate gyrus granule neurons and the olfactory bulb granule and periglomerular neurons. In the dentate gyrus, a secondary proliferative zone termed the subgranular zone is established along the interface between the dentate gyrus and the hilus where granule cells are born throughout life. Olfactory bulb neurons are generated in the anterior subventricular zone of the lateral ventricle and migrate via the rostral migratory stream to the olfactory bulb. We examined animals lacking brain-derived neurotrophic factor (BDNF) in order to establish whether this neurotrophin could be involved in the generation and/or survival of these neurons in vivo. We find that cells in nestin-positive regions of both the subgranular layer of the dentate gyrus and the subventricular zone of the olfactory bulb undergo apoptosis starting 2 weeks after birth in the absence of BDNF. However, increased apoptosis was not limited to precursors, as apoptotic cells were also found in the granule cell layer of the dentate gyrus and in the granule and periglomerular layers of the olfactory bulb. The excessive cell death was limited to these populations of neurons as no excessive cell death was detected in other forebrain areas. We conclude that BDNF is essential for the survival of neurons specifically in populations which are continuously being regenerated in the brain.     

Voluntary exercise-induced neurogenesis in the postischemic dentate gyrus is associated with spatial memory recovery from stroke

Spatial cognitive impairment is common after stroke insults. Voluntary exercise could improve the impaired spatial memory. Newly generated neurons in the dentate gyrus are necessary for the acquisition of new hippocampus-dependent memories. However, it is not well known whether voluntary exercise after stroke promotes neurogenesis in the adult dentate gyrus, thereby promoting spatial memory recovery. Here, we examined in mice subjected to focal cerebral ischemia the effect of voluntary or forced exercise on neurogenesis in the ischemic dentate gyrus and spatial memory. Exposure to voluntary wheel running after stroke enhanced newborn cell survival and up-regulated the phosphorylation of cAMP response element binding protein (CREB) in the dentate gyrus and reversed ischemia-induced spatial memory impairment. However, the enhanced newborn cell survival and CREB phosphorylation in the dentate gyrus and improved spatial memory were not observed in the mice exposed to forced swimming. Moreover, there was a significant correlation between the total number of surviving newborn cells in the dentate gyrus and the ability of mice to locate the platform in the Morris water maze. These results suggest that, in the adult mice, exposure to voluntary exercise after ischemic stroke may promote newborn cells survival in the dentate gyrus by up-regulating CREB phosphorylation and consequently restore impaired hippocampus-dependent memory.     

Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics,migration and transformation of cells incoorporating tritiated thymidine in neonate rats,with special reference to postnatal neurogenesis in some brain regions

Neonate rats were injected systemically with thymidine-H³ and killed after different periods of survival. Cell proliferation, migration and transformation in the brain were studied autoradiographically. It was established that cells multiplying in the ependymal and subependymal walls of the olfactory ventricle migrate outward into the olfactory bulb, where they become differentiated into granule cells. These postnatally formed granule cells contribute to the formation of the granular and several other layers of the olfactory bulb. Cells multiplying at a high rate in the wall of the lateral ventricle migrate to the hippocampus and contribute to the formation of the granule cells in the granular layer of the dentate gyrus. Cells multiplying at a high rate in the external and internal granular layers of the cerebellum become differentiated into granule cells, and, to a lesser extent, other types of nerve cells of the cerebellar cortex. Evidence was also obtained of the postnatal origin of many of the granule cells of the cochlear nucleus. Postnatal neurogenesis is restricted to these short-axoned granule cells or microneurons; the long-axoned nerve cells or macroneurons of the brain are formed prenatally.     

Brain-derived neurotrophic factor (BDNF) is required for the enhancement of hippocampal neurogenesis following environmental enrichment

Neurogenesis continues to occur in the adult mammalian hippocampus and is regulated by both genetic and environmental factors. It is known that exposure to an enriched environment enhances the number of newly generated neurons in the dentate gyrus. However, the mechanisms by which enriched housing produces these effects are poorly understood. To test a role for neurotrophins, we used heterozygous knockout mice for brain-derived neurotrophic factor (BDNF+/-) and mice lacking neurotrophin-4 (NT-4-/-) together with their wild-type littermates. Mice were either reared in standard laboratory conditions or placed in an enriched environment for 8 weeks. Animals received injections of the mitotic marker bromodeoxyuridine (BrdU) to label newborn cells. Enriched wild-type and enriched NT-4-/- mice showed a two-fold increase in hippocampal neurogenesis as assessed by stereological counting of BrdU-positive cells in the dentate gyrus and double labelling for BrdU and the neuronal marker NeuN. Remarkably, this enhancement of hippocampal neurogenesis was not seen in enriched BDNF+/- mice. Failure to up-regulate BDNF accompanied the lack of a neurogenic response in enriched BDNF heterozygous mice. We conclude that BDNF but not NT-4 is required for the environmental induction of neurogenesis.     

Experience‐dependent regulation of adult hippocampal neurogenesis: Effects of long‐term stimulation and stimulus withdrawal

Exposure to an enriched environment has been shown to cause an increase in neurogenesis in the dentate gyrus of adult mice. In this study we examined how this experience‐dependent response in adult hippocampal neurogenesis of C57BL/6 mice is modulated under the conditions of long‐term stimulation and of withdrawal from the enriched environment. We found that a group which experienced withdrawal from the enriched environment 3 months earlier, had more than twice as many proliferating cells in the subgranular zone as controls and mice experiencing long‐term stimulation. We propose that the greater number of proliferating cells after withdrawal reflects a survival‐promoting effect on the dividing neuronal stem and progenitor cells during the earlier period of stimulation. No differences between the groups were observed in the number of surviving progeny or their phenotypes. Therefore, the existence of more dividing cells in the withdrawal group did not translate into a significant net increase in neurogenesis in the absence of continued stimulation. Similarly, the finding in the group experiencing long‐term stimulation showing no clear benefit over controls could be interpreted as a diminished efficiency of continued environmental stimuli to elicit a neurogenic response. Thus, we propose as a working hypothesis that: 1) stimulation early in life may preserve the neurogenic potential in the dentate gyrus, and 2) the novelty of complex stimuli rather than simply continued exposure to complex stimuli elicits the environmental effects on adult hippocampal neurogenesis. Hippocampus 1999;9:321–332. © 1999 Wiley‐Liss, Inc.     

Monday July 3, 200612:00–13:30Hall 3DPlatform SessionBasic Science I: Molecules and Networks for Epileptogenesis

¹ M. Kokaia (   ¹ Wallenberg Neuroscience Center, Lund University Hospital, Sweden )
Purpose: Neural stem cells in the adult mammalian brain (including humans) continue to produce new functional granule cells in the dentate gyrus subgranular zone and new olfactory bulb neurons in the subventricular zone during an entire life. In the hippocampus, neurogenesis has been proposed to play a role in learning and memory and mood regulation. The new cells develop electrophysiological characteristics and synaptic inputs very similar to those of the rest of the cell population. The purpose of the study was to explore whether tissue environment in an epileptic brain influences properties of afferent synapses formed on newborn granule cells.
Method: Rats were exposed to either a physiological stimulus, i.e., running, or status epilepticus, which gives rise to neuronal death, inflammation, increased network excitability and recurrent spontaneous seizures. Both treatments increase neurogenesis in the dentate gyrus. We labelled newborn cells by GFP-retroviral vector injections right after these treatments to identify the cells and apply whole-cell patch-clamp recordings in live hippocampal slices.
Results: Granule cells formed after running and status epilepticus exhibited similar intrinsic membrane properties. However, new neurons born into the epileptic environment differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. The new granule cells formed after status epilepticus exhibited functional connectivity consistent with reduced synaptic network excitability of the dentate gyrus, i.e., decreased excitatory and increased inhibitory input activity.
Conclusion: We demonstrate for the first time a high degree of plasticity in synaptic inputs to the new neurons, which could mitigate pathological activity in the epileptic brain.     

Effects of adult-generated granule cells on coordinated network activity in the dentate gyrus

Throughout the adult life of most mammals, new neurons are continuously generated in the dentate gyrus of the hippocampal formation. Recent work has documented specific cognitive deficits after elimination of adult hippocampal neurogenesis in rodents, suggesting that these neurons may contribute to information processing in hippocampal circuits. Young adult-born neurons exhibit enhanced excitability and have altered capacity for synaptic plasticity in hippocampal slice preparations in vitro. Still, little is known about the effect of adult-born granule cells on hippocampal activity in vivo. To assess the impact of these new neurons on neural circuits in the dentate, we recorded perforant-path evoked responses and spontaneous network activity from the dentate gyrus of urethane-anesthetized mice whose hippocampus had been focally X-irradiated to eliminate the population of young adult-born granule cells. After X-irradiation, perforant-path responses were reduced in magnitude. In contrast, there was a marked increase in the amplitude of spontaneous γ-frequency bursts in the dentate gyrus and hilus, as well as increased synchronization of dentate neuron firing to these bursts. A similar increase in gamma burst amplitude was also found in animals in which adult neurogenesis was eliminated using the GFAP:TK pharmacogenetic ablation technique. These data suggest that young neurons may inhibit or destabilize recurrent network activity in the dentate and hilus. This unexpected result yields a new perspective on how a modest number of young adult-generated granule cells may modulate activity in the larger population of mature granule cells, rather than acting solely as independent encoding units.     

Granule cell dispersion and aberrant neurogenesis in the adult hippocampus of an LIS1 mutant mouse

Human type 1 lissencephaly is a severe brain malformation associated with cognitive dysfunction and intractable epilepsy. Mutant mice with a heterozygous deletion of LIS1 show varying degrees of hippocampal abnormality and enhanced excitability. Whether a reduction of LIS1 function affects adult hippocampal neurogenesis, and if so, whether aberrant neurogenesis contributes to the generation of a disorganized hippocampus remain unknown. Previous reports indicate the presence of multiple pyramidal cell layers and granule cell dispersion in LIS1 mutant mice. Here we observed disruption of the subgranular zone and glial fibrillary acidic protein-immunoreactive radial astrocytes in the dentate gyrus of adult LIS1 mice. Using pulse-chase bromodeoxyuridine (BrdU) labeling combined with neuronal and glial antibody staining we provide evidence for ectopic adult neurogenesis in LIS1 mice. A gradually decreased survival rate for these newborn granule cells was also demonstrated in LIS1 mice 7 days after BrdU injection. This reduced survival rate was associated with impaired neuronal differentiation 28 days after BrdU administration. Thus, LIS1 haploinsufficiency can lead to abnormal cell proliferation, migration and differentiation in the adult dentate gyrus.     

Mechanisms and functional significance of aberrant seizure-induced hippocampal neurogenesis

Studies of experimental mesial temporal lobe epilepsy (mTLE) indicate that prolonged seizures in the adult not only damage the hippocampal formation but also dramatically stimulate neurogenesis. Endogenous neural progenitor cells (NPCs) located in the adult rodent dentate gyrus and striatal subventricular zone are stimulated by experimental status epilepticus (SE) to generate increased numbers of dentate granule cells (DGCs) and olfactory interneurons, respectively ( Bengzon et al., 1997 ; Parent et al., 1997, 2002 ; Scott et al., 1998 ). In this review, we discuss current knowledge regarding the consequences of seizure activity on NPC proliferation, focusing on the hippocampus, and on the migration and integration of adult-born hippocampal neurons. We also describe the effects of seizure-induced neurogenesis on hippocampal network function and the potential relevance of aberrant neurogenesis to human mTLE.