Cells In Subgranular Zone Research Articles

The study of neurogenesis contributed by the expression of survivin in hippocampus after traumatic brain injury

Objective To investigate the expression of survivin after traumatic brain injury(TBI);and the relationship between the up- regulation of survivin and endogenous neurogenesis. Further to demonstrate the role of increased survivin to promote the adult neurogenesis, and detect the possible mechanism in this process.Finally to find the approach that can promote restoration of nervous function after TBI. Methods A-dult male C57BL/6 mice, total n= 80, were randomly divided into sham- operation group and TBI group. Mice in TBI and sham- operation group were sacrificed at 12 hours, 1, 2, 5, 7 and 14 days post TBI and sham- operation(n= 6 for each point), and hippocampus tissues from the ipsilateral hemispheres were carried out for Western blotting.Double- label immunofluorescence staining was adopted to detect survivin expression in difference kinds of cells in the hippocampus(n= 4). Results The up- regulation of survivin protein starting at day 1(42 003.15, P<0.05)and lasting to day 7, peaking at day 2(90 403.34, P<0.01). The number of survivin+, Brd U+ and doublecortin(DCX+) cells in subgranular zone(SGZ) of ipsilateral hippocampus was significantly increased after TBI by immunofluorescence staining. The results also revealed that the majority of survivin+ cells were BrdU+ cells,and a part of survivin+ cells were DCX+ cells in SGZ of dentate gyrus(DG)in the hippocampus. Conclusion Survivin gene was activated follow brain injury. The expression of survivin protein were increased in the hippocampus after TBI. Increased survivin protein is expressed in neural stem cells and immature neurons in ipsilateral hippocampus after TBI, which correlate to the proliferation of neurogenic cell in SGZ of hippocampus. Key words: Survivin; Traumatic brain injury; Hippocampus; Adult neurogenesis

Vascular endothelial growth factor receptor 3 controls neural stem cell activation in mice and humans.

SUMMARYNeural stem cells (NSCs) continuously produce new neurons within the adult mammalian hippocampus. NSCs are typically quiescent but activated to self-renew or differentiate into neural progenitor cells. The molecular mechanisms of NSC activation remain poorly understood. Here, we show that adult hippocampal NSCs express vascular endothelial growth factor receptor (VEGFR) 3 and its ligand VEGF-C, which activates quiescent NSCs to enter the cell cycle and generate progenitor cells. Hippocampal NSC activation and neurogenesis are impaired by conditional deletion of Vegfr3 in NSCs. Functionally, this is associated with compromised NSC activation in response to VEGF-C and physical activity. In NSCs derived from human embryonic stem cells (hESCs), VEGF-C/VEGFR3 mediates intracellular activation of AKT and ERK pathways that control cell fate and proliferation. These findings identify VEGF-C/VEGFR3 signaling as a specific regulator of NSC activation and neurogenesis in mammals.

Growth hormone pathways signaling for cell proliferation and survival in hippocampal neural precursors from postnatal mice.

BackgroundAccumulating evidence suggests that growth hormone (GH) may play a major role in the regulation of postnatal neurogenesis, thus supporting the possibility that it may be also involved in promoting brain repair after brain injury. In order to gain further insight on this possibility, in this study we have investigated the pathways signaling the effect of GH treatment on the proliferation and survival of hippocampal subgranular zone (SGZ)-derived neurospheres.ResultsOur results demonstrate that GH treatment promotes both proliferation and survival of SGZ neurospheres. By using specific chemical inhibitors we have been also able to demonstrate that GH treatment promotes the activation of both Akt-mTOR and JNK signaling pathways, while blockade of these pathways either reduces or abolishes the GH effects. In contrast, no effect of GH on the activation of the Ras-ERK pathway was observed after GH treatment, despite blockade of this signaling path also resulted in a significant reduction of GH effects. Interestingly, SGZ cells were also capable of producing GH, and blockade of endogenous GH also resulted in a decrease in the proliferation and survival of SGZ neurospheres.ConclusionsAltogether, our findings suggest that GH treatment may promote the proliferation and survival of neural progenitors. This effect may be elicited by cooperating with locally-produced GH in order to increase the response of neural progenitors to adequate stimuli. On this view, the possibility of using GH treatment to promote neurogenesis and cell survival in some acquired neural injuries may be envisaged.

Survival of adult generated hippocampal neurons is altered in circadian arrhythmic mice.

The subgranular zone of the hippocampal formation gives rise to new neurons that populate the dentate gyrus throughout life. Cells in the hippocampus exhibit rhythmic clock gene expression and the circadian clock is known to regulate the cycle of cell division in other areas of the body. These facts suggest that the circadian clock may regulate adult neurogenesis in the hippocampus as well. In the present study, neurogenesis in the hippocampal subgranular zone was examined in arrhythmic Bmal1 knockout (-KO) mice and their rhythmic heterozygous and wildtype littermates. Proliferation and survival of newly generated subgranular zone cells were examined using bromodeoxyuridine labelling, while pyknosis (a measure of cell death) and hippocampal volume were examined in cresyl violet stained sections. There was no significant difference in cellular proliferation between any of the groups, yet survival of proliferating cells, 6 weeks after the bromodeoxyuridine injection, was significantly greater in the BMAL1-KO animals. The number of pyknotic cells was significantly decreased in Bmal1-KO animals, yet hippocampal volume remained the same across genotypes. These findings suggest that while a functional circadian clock is not necessary for normal proliferation of neuronal precursor cells, the normal pruning of newly generated neurons in the hippocampus may require a functional circadian clock.

The P7C3 class of neuroprotective compounds exerts antidepressant efficacy in mice by increasing hippocampal neurogenesis.

Augmenting hippocampal neurogenesis represents a potential new strategy for treating depression. Here we test this possibility by comparing hippocampal neurogenesis in depression-prone ghrelin receptor (Ghsr)-null mice to that in wild-type littermates and by determining the antidepressant efficacy of the P7C3 class of neuroprotective compounds. Exposure of Ghsr-null mice to chronic social defeat stress (CSDS) elicits more severe depressive-like behavior than in CSDS-exposed wild-type littermates, and exposure of Ghsr-null mice to 60% caloric restriction fails to elicit antidepressant-like behavior. CSDS resulted in more severely reduced cell proliferation and survival in the ventral dentate gyrus (DG) subgranular zone of Ghsr-null mice than in that of wild-type littermates. Also, caloric restriction increased apoptosis of DG subgranular zone cells in Ghsr-null mice, although it had the opposite effect in wild-type littermates. Systemic treatment with P7C3 during CSDS increased survival of proliferating DG cells, which ultimately developed into mature (NeuN+) neurons. Notably, P7C3 exerted a potent antidepressant-like effect in Ghsr-null mice exposed to either CSDS or caloric restriction, while the more highly active analog P7C3-A20 also exerted an antidepressant-like effect in wild-type littermates. Focal ablation of hippocampal stem cells with radiation eliminated this antidepressant effect, further attributing the P7C3 class antidepressant effect to its neuroprotective properties and resultant augmentation of hippocampal neurogenesis. Finally, P7C3-A20 demonstrated greater proneurogenic efficacy than a wide spectrum of currently marketed antidepressant drugs. Taken together, our data confirm the role of aberrant hippocampal neurogenesis in the etiology of depression and suggest that the neuroprotective P7C3-compounds represent a novel strategy for treating patients with this disease.

N-Methyl-N-nitrosourea during late gestation results in concomitant but reversible progenitor cell reduction and delayed neurogenesis in the hippocampus of rats

N-Methyl-N-nitrosourea (MNU) is an alkylating agent having genotoxic potential to cause gene mutations and antiproliferative cytotoxic activity on developing brains to cause microcephaly by mid-gestational exposure in rodents. This study investigated the transient genotoxic and cytocidal effect of MNU at the beginning of the subgranular zone (SGZ) formation in the hippocampal dentate gyrus on neurogenesis in later life using rats. Pregnant rats were injected with MNU at 0 (vehicle controls), 1 or 3mg/kg body weight intraperitoneally from gestational day (GD) 18 to GD 20 once a day. In offspring, effects were observed at 3mg/kg. Fetal brains on GD 21 after the last MNU injection increased TUNEL+ apoptotic cells in the tertiary germinal matrices. At postnatal day (PND) 21 on weaning, offspring displayed decrease of doublecortin (Dcx)+ cells and cell proliferation in the SGZ and increase of calbindin (Calb1)+ interneurons in the dentate hilus. Postnatal single bromodeoxyuridine (BrdU) injection on PND 3 resulted in an increase of BrdU+/Dcx+ cells. On PND 77, Dcx+ cells recovered in number, but cell proliferation increased in the SGZ. Thus, late-gestational maternal MNU exposure may induce reversible reductions of type-3 progenitor and/or immature granule cells and cell proliferation on weaning in response to progenitor cell apoptosis, as well as delayed neurogenesis due to cell cycle arrest. Increases of Calb1+ interneurons on weaning and SGZ cell proliferation later on may reflect compensatory mechanism for MNU-induced aberrant neurogenesis. Considering the lack of effects on PND 77, MNU may mainly target transient populations of highly proliferative progenitor cells without affecting their stem cells to undergo progenitor production. Protective and plasticity mechanism may be operated against genotoxic agents on hippocampal neurogenesis.

Knockout of CXCR5 increases the population of immature neural cells and decreases proliferation in the hippocampal dentate gyrus

BackgroundThe process of neurogenesis in which new neurons are generated by proliferation and differentiation of neural stem/progenitor cells (NSCs/NPCs) has been a topic of intensive recent investigation. Investigations of the factors which regulate this process have recently begun to include immune factors including immune cells and cytokines, however the class of immune proteins designated as chemokines have been relatively neglected. Increasing evidence for novel brain-specific mechanisms of chemokines beyond their classical chemotactic functions has suggested that they may play a role in the regulation of NSC/NPC biology.MethodsWe have investigated the role of the chemokine receptor CXCR5 (ligand is CXCL13) in the activity of these cells through neurobiological and behavioural analysis of CXCR5-deficient mice (CXCR5-/-). These investigations included: immunohistochemistry for the markers Ki67, nestin, doublecortin, and IBA-1, neurosphere assays, and the baseline behavioural tests: open field test and sucrose preference test.ResultsWe observed a significant increase in doublecortin and nestin staining in the hippocampal dentate gyrus (P = 0.02 and P = 0.0008, respectively) of CXCR5-/- animals as compared to wild-type controls. This was accompanied by a decrease in Ki67 staining subgranular zone (P = 0.009). Behavioural correlates included a significant increase in baseline locomotor activity in an open field test (P <0.00018) and a decrease in stress reactivity in that test (P = 0.015). Deficiency in CXCR5 was not associated with alterations in hippocampal microglial density, microglial activation or systemic cytokine levels, nor with loss of NSC/NPC populations in the neurosphere assay.ConclusionsThese findings are the first to describe a brain-specific function of CXCR5 under physiological conditions. CXCR5 reduces maintenance of immature neural cell populations and enhances proliferation of subgranular zone cells in the hippocampal dentate gyrus, however the mechanism of these effects remains unclear. Further research into the regulation of NSC/NPC activity should consider investigation of CXCR5 and other chemokines which may be relevant to the pathophysiology of psychiatric disorders including depression, anxiety and cognitive impairment/dementia.

Ectopic hippocampal neurogenesis in adolescent male rats following alcohol dependence

The adolescent hippocampus is highly vulnerable to alcohol-induced damage, which could contribute to their increased susceptibility to alcohol use disorder. Altered adult hippocampal neurogenesis represents one potential mechanism by which alcohol (ethanol) affects hippocampal function. Based on the vulnerability of the adolescent hippocampus to alcohol-induced damage, and prior reports of long-term alcohol-induced effects on adult neurogenesis, we predicted adverse effects on adult neurogenesis in the adolescent brain following abstinence from alcohol dependence. Thus, we examined neurogenesis in adolescent male rats during abstinence following a 4-day binge model of alcohol dependence. Bromodeoxyuridine and Ki67 immunohistochemistry revealed a 2.2-fold increase in subgranular zone cell proliferation after 7 days of abstinence. Increased proliferation was followed by a 75% increase in doublecortin expression and a 56% increase in surviving bromodeoxyuridine-labeled cells 14 and 35 days post-ethanol exposure, respectively. The majority of newborn cells in ethanol and control groups co-localized with NeuN, indicating a neuronal phenotype and therefore a 1.6-fold increase in hippocampal neurogenesis during abstinence. Although these results mirror the magnitude of reactive neurogenesis described in adult rat studies, ectopic bromodeoxyuridine and doublecortin positive cells were detected in the molecular layer and hilus of adolescent rats displaying severe withdrawal symptoms, an effect that has not been described in adults. The presence of ectopic neuroblasts suggests that a potential defect exists in the functional incorporation of new neurons into the existing hippocampal circuitry for a subset of rats. Age-related differences in functional incorporation could contribute to the increased vulnerability of the adolescent hippocampus to ethanol.

Secreted Frizzled-Related Protein 3 Regulates Activity-Dependent Adult Hippocampal Neurogenesis

Adult neurogenesis, the process of generating mature neurons from adult neural stem cells, proceeds concurrently with ongoing neuronal circuit activity and is modulated by various physiological and pathological stimuli. The niche mechanism underlying the activity-dependent regulation of the sequential steps of adult neurogenesis remains largely unknown. Here, we report that neuronal activity decreases the expression of secreted frizzled-related protein 3 (sFRP3), a naturally secreted Wnt inhibitor highly expressed by adult dentate gyrus granule neurons. Sfrp3 deletion activates quiescent radial neural stem cells and promotes newborn neuron maturation, dendritic growth, and dendritic spine formation in the adult mouse hippocampus. Furthermore, sfrp3 reduction is essential for activity-induced adult neural progenitor proliferation and the acceleration of new neuron development. Our study identifies sFRP3 as an inhibitory niche factor from local mature dentate granule neurons that regulates multiple phases of adult hippocampal neurogenesis and suggests an interesting activity-dependent mechanism governing adult neurogenesis via the acute release of tonic inhibition.

Long-Term Upregulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model

The long-term consequences of traumatic brain injury (TBI), specifically the detrimental effects of inflammation on the neurogenic niches, are not very well understood. In the present in vivo study, we examined the prolonged pathological outcomes of experimental TBI in different parts of the rat brain with special emphasis on inflammation and neurogenesis. Sixty days after moderate controlled cortical impact injury, adult Sprague-Dawley male rats were euthanized and brain tissues harvested. Antibodies against the activated microglial marker, OX6, the cell cycle-regulating protein marker, Ki67, and the immature neuronal marker, doublecortin, DCX, were used to estimate microglial activation, cell proliferation, and neuronal differentiation, respectively, in the subventricular zone (SVZ), subgranular zone (SGZ), striatum, thalamus, and cerebral peduncle. Stereology-based analyses revealed significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle. In parallel, significant decrements in Ki67-positive proliferating cells in SVZ and SGZ, but only trends of reduced DCX-positive immature neuronal cells in SVZ and SGZ were detected relative to sham control group. These results indicate a progressive deterioration of the TBI brain over time characterized by elevated inflammation and suppressed neurogenesis. Therapeutic intervention at the chronic stage of TBI may confer abrogation of these deleterious cell death processes.

Preliminary morphological and immunohistochemical changes in rat hippocampus following postnatal exposure to sodium arsenite

The effects of arsenic exposure during rapid brain growth period (RBGP) (postnatal period 4-11) on pyramidal neurons of cornu ammonis (specifically CA1 and CA3 regions) and granule cells of dentate gyrus (DG) of rat hippocampus were studied. Wistar rat pups, subdivided into the control (group I) and the experimental groups (group II, III, and IV), received distilled water and sodium arsenite (aqueous solution of 1.0, 1.5, and 2.0 mg/kg body weight, respectively) by intraperitoneal (i.p.) route. On postnatal day (PND) 12, the animals were sacrificed and brain tissue obtained. Paraffin sections (8 μm thick) stained with Cresyl Violet (CV) were observed for morphological and morphometric parameters. Arsenic induced programmed cell death (apoptosis) was studied using Terminal deoxyribonucleotidyl transferase mediated dUTP biotin Nick End Labeling (TUNEL) technique on the paraffin sections. Microscopy revealed decreased number and isolation of pyramidal neurons in superficial layers, misalignments of pyramidal cells in stratum pyramidale (SP) of CA1 and CA3 in experimental group III and IV, and presence of polymorphic cells in subgranular zone of ectal limb of dentate gyrus (suggestive of arsenic induced proliferation and migration of granule cells in the dentate gyrus). Morphometric assessments quantified and confirmed the microscopic findings. The mean nuclear area of pyramidal cells was increased and cell density was decreased in the CA1, CA3, and DG of experimental groups in comparison to the control group. Increase in the TUNEL positive cells in DG was observed in the experimental group IV, suggestive of increased apoptosis. These observations confirm vulnerability of pyramidal (CA1, CA3) and granule cells (DG) of hippocampus during RBGP.

Lhx8 promote differentiation of hippocampal neural stem/progenitor cells into cholinergic neurons in vitro

Lhx8, also named L3, is a recently identified member of the LIM homeobox gene family. Previously, we found acetylcholinesterase (AChE)-positive cells in fimbria-fornix (FF) transected rat hippocampal subgranular zone (SGZ). In the present study, we detected choline acetyltransferase (ChAT)-positive cholinergic cells in hippocampal SGZ after FF transaction, and these ChAT-positive cells were double labeled by Lhx8. Then we overexpressed Lhx8 during neural differentiation of hippocampal neural stem/progenitor cells on adherent conditions using lentivirus Lenti6.3-Lhx8. The result indicated that overexpression of Lhx8 did not affect the proportion of MAP2-positive neurons, but increased the proportion of ChAT-positive cells in vitro. These results suggested that FF-transected hippocampal niche promoted the ChAT/Lhx8-positive cholinergic neurons generation in rodent hippocampus, and Lhx8 was not associated with the MAP2-positive neurons differentiation on adherent conditions, but played a role in the specification of cholinergic neurons derived from hippocampal neural stem/progenitor cells in vitro.

Effects of Chinese herbal medicine Shenxiong Yujing Granule on regeneration of neural cells in rats with diabetes-associated cerebral ischemia

To establish a rat model of diabetes-associated cerebral ischemia due to qi and yin deficiency and blood stasis, and to investigate the effects of Radix Ginseng, Rhizoma Chuanxiong, Rhizoma Polygonati Odorati and Rhizoma Polygonati Sibirici Granule (Shenxiong Yujing Granule), which has the function of strengthening qi, nourishing yin, and activating blood, on proliferation, differentiation and survival of neural cells in rats with diabetes-associated cerebral ischemia. Rats were divided into sham-operation, diabetes plus ischemia reperfusion injury model, Shenxiong Yujing Granule and Radix Ginseng and Rhizoma Chuanxiong Granule (Shenxiong Granule) groups with 20 rats in each. The 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and immunohistochemical method were used to investigate the proliferation, differentiation and survival of neural cells in dentate gyrus of rats with diabetes-associated cerebral ischemia. The number of newly proliferating cells in subgranular zone of dentate gyrus was increased in the model group, but there was no significant difference compared with 7 day treatment with Shenxiong Yujing Granule. Shenxiong Yujing Granule significantly increased the survival rate and promoted the differentiation of newly proliferating neurons after 21-day treatment (P<0.01). In addition, the beneficial effect of Shenxiong Yujing Granule was considerably greater than that of the Shenxiong Granule (P<0.01). Shenxiong Yujing Granule can increase the survival rate and promote the differentiation of newly proliferating neurons in rats with diabetes-associated cerebral ischemia of dual deficiency of qi and yin and blood stasis obstructing the collaterals. The effect is greater than that of Shenxiong Granule.

Hypoxic ischaemic hypothermia promotes neuronal differentiation and inhibits glial differentiation from newly generated cells in the SGZ of the neonatal rat brain

Hypothermia is a potential therapy for cerebral hypoxic ischaemic injury in adults and neonates. The mechanism of the neuroprotective effects of hypothermia after hypoxia–ischaemia (HI) in the developing rat brain remains unclear. In this research, 7-day-old rats underwent left carotid artery ligation followed by the administration of 8% oxygen for 2h. These rats were divided into hypothermic (rectal temperature, 32–33°C for 24h) and normothermic (36–37°C for 24h) groups immediately after HI. All rats were given 50mg/kg/day 5-bromodeoxyuridine (BrdU) intraperitoneally at 4–6 days and sacrificed at 1 or 2 weeks after HI. We found a significant decrease in infarct volume and the neuron loss were also detected in the subgranular zone (SGZ) in the hypothermic group at 7 and 14 days after HI compared with the normothermic group. BrdU immunopositive cells were reduced greatly in the hypothermic group compared with the normothermic group. Hypothermia did not change the number of nestin-labelled cells in the ipsilateral SGZ at 1 and 2 weeks after HI. The differentiation of newly generated cells was assessed by double immunolabelling of BrdU with glial fibrillary acidic protein (GFAP), O4 or Neuronal Nuclei (NeuN). The ratio of BrdU+–GFAP+ or BrdU+–O4+ to total BrdU+ staining decreased dramatically, but the ratio of BrdU+–NeuN+ to total BrdU+ staining increased significantly in the hypothermic group compared to the normothermic group at 2 and 6 weeks after HI. These results suggest that the reduction in neuron loss observed after mild hypothermia may be associated with enhanced neuronal differentiation and decreased glial differentiation in the SGZ after HI. These observations are noteworthy for clinical hypothermia therapy following cerebral HI injury during the perinatal period.

A dual function of Bcl11b/Ctip2 in hippocampal neurogenesis

The development of the dentate gyrus is characterized by distinct phases establishing a durable stem-cell pool required for postnatal and adult neurogenesis. Here, we report that Bcl11b/Ctip2, a zinc finger transcription factor expressed in postmitotic neurons, plays a critical role during postnatal development of the dentate gyrus. Forebrain-specific ablation of Bcl11b uncovers dual phase-specific functions of Bcl11b demonstrated by feedback control of the progenitor cell compartment as well as regulation of granule cell differentiation, leading to impaired spatial learning and memory in mutants. Surprisingly, we identified Desmoplakin as a direct transcriptional target of Bcl11b. Similarly to Bcl11b, postnatal neurogenesis and granule cell differentiation are impaired in Desmoplakin mutants. Re-expression of Desmoplakin in Bcl11b mutants rescues impaired neurogenesis, suggesting Desmoplakin to be an essential downstream effector of Bcl11b in hippocampal development. Together, our data define an important novel regulatory pathway in hippocampal development, by linking transcriptional functions of Bcl11b to Desmoplakin, a molecule known to act on cell adhesion.

Reversible aberration of neurogenesis targeting late-stage progenitor cells in the hippocampal dentate gyrus of rat offspring after maternal exposure to acrylamide

We have recently shown that maternal exposure to acrylamide (AA) impaired neurogenesis in rat offspring measured by the increase in interneurons producing reelin, a molecule regulating migration and correct positioning of developing neurons, in the hippocampal dentate gyrus. To clarify the cellular target of AA on hippocampal neurogenesis and its reversibility after maternal exposure, pregnant Sprague-Dawley rats were given drinking water containing AA at 0, 4, 20, 100 ppm on day 10 of pregnancy through day 21 after delivery on weaning. Male offspring were examined immunohistochemically on postnatal day (PND) 21 and PND 77. For comparison, male pups of direct AA-injection control during lactation (50 mg/kg body weight, intraperitoneally, 3 times/week) were also examined. On PND 21, maternal AA-exposure decreased progenitor cell proliferation in the subgranular zone (SGZ) from 20 ppm accompanied with increased density of reelin-producing interneurons and NeuN-expressing mature neurons within the hilus at 100 ppm, similar to the direct AA-injection control. In the SGZ examined at 100 ppm, cellular populations immunoexpressing doublecortin or dihydropyrimidinase-like 3, suggesting postmitotic immature granule cells, were decreased. On PND 77, the SGZ cell proliferation and reelin-producing interneuron density recovered, while the hilar mature neurons sustained to increase from 20 ppm, similar to the direct AA-injection control. Thus, developmental exposure to AA reversibly affects hippocampal neurogenesis targeting the proliferation of type-3 progenitor cells resulting in a decrease in immature granule cells in rats. A sustained increase in hilar mature neurons could be the signature of the developmental effect of AA.

Collagen peptides enhance hippocampal neurogenesis and reduce anxietyrelated behavior in mice

The present study examined the effects of enzymatically hydrolyzed collagen peptides on the level of hippocampal neurogenesis and emotional behavior in adult mice. For this purpose, two kinds of enzymatically hydrolyzed collagen peptides, the lower or higher molecular weight peptides (LP: below 2,000, HP: about 30,000) were administered orally to C57BL/6 mice for 4 weeks. As a result, the density of proliferating cells in subgranular zone of hippocampus showed a 1.2-fold increase in LP mice as compared with HP mice. Additionally, LP mice spent less time in closed arms than HP mice in elevated plus maze test to examine anxiety-related behavior. These results suggest that oral administration of the lower molecular weight peptides derived from collagen enhanced the hippocampal neurogenesis and exerted emotional behavior in adult mice.

Expression of Wnt 3a, β-Catenin, Cyclin D1 and PCNA in Mouse Dentate Gyrus Subgranular Zone (SGZ): a Possible Role of Wnt Pathway in SGZ Neural Stem Cell Proliferation

In mammalian dentate gyrus subgranular zone, the addition of new neurons throughout adult-hood is a remarkable form of structural plasticity. Yet, the molecular controls over subgranular zone neural stem cell proliferation, survival, and differentiation are poorly understood. In this study we analysed the expression of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in mouse subgranular zone to elucidate the involvement of Wnt pathway in subgranular zone neural stem cell proliferation. We performed immunohistochemistry and RT-PCR for the above molecules on adult and postnatal developing hippocampal tissues of mice, respectively. RT-PCR analysis showed a gradual increase in expression of mRNA of Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen as the postnatal hippocampus developed, and immunohistochemical analysis showed a highly positive immunoreactive expression for Wnt 3a, β-catenin, cyclin D1 and proliferating cell nuclear antigen in the subgranular zone cells. Together, our data suggested that the Wnt pathway is activated in subgranular zone and could play an important role in regulating subgranular zone neural stem cell proliferation in mouse hippocampus.

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates.

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.

Minor neuronal damage and recovered cellular proliferation in the hippocampus after continuous unilateral forelimb restraint in normal rats

Constraint-induced movement therapy (CIMT) involves the restraint of an intact limb to force the dominant use of an affected limb, in an attempt to enhance use-dependent plasticity and reduce dysfunction. To investigate whether forced disuse of an intact forelimb with CIMT causes a loss of limb function and degenerative damage in the brain, a staircase test and a horizontal ladder test were carried out in control rats and forelimb-restrained rats, and then Argyrophil III silver staining, which is capable of detecting subtle neuronal damage, was used to examine histological alterations associated with restraint. No significant changes in forelimb function were observed in restrained rats. However, atypical weak argyrophilic neurons, an indicator of minor neural damage, were found in the bilateral hippocampus of restrained rats. This damage was not found in the cortex, striatum, or spinal cord. Investigation of neurogenesis in the subventricular zone (SVZ) and subgranular zone (SGZ) revealed a clear reduction in the number of bromodeoxyuridine-positive cells in bilateral SGZ, but not in the SVZ, in restrained rats compared with controls. This reduction was accompanied by reduced mRNA expression of vascular endothelial growth factor and glial-derived neurotrophic factor. However, reduced cellular proliferation and decreased gene expression were recovered after the removal of the restraint. Our results suggest that forced disuse of the intact forelimb has no significant effect on skilled forelimb function but has a minor effect on neurogenesis in SGZ, suggesting that mild stress may be caused by the restraint.