Three-dimensional ultrastructural and immunohistochemical study of immature neurons in the subgranular zone of the rat dentate gyrus

The present study is devoted to three-dimensional ultrastructural organization of mitotically dividing immature neurons in dentate gyrus using biophysical approaches. In adult vertebrate brain, cell proliferation persists throughout life mainly in dentate gyrus of the hippocampus (DG) and olfactory bulb. Neurogenesis has been demonstrated using tagged thymidine analogues incorporated into the S phase of the cell cycle, but these may also detect repaired DNA in postmitotic neurons. Recent retroviral labelling has shown that neuronal progenitors/neuroblasts divide and produce functional neurons. Providing ultrastructural evidence of mitotically active cells has proven problematical, not only because of technical issues of identifying dividing cells at electron microscope level, but also because it is difficult to demonstrate unequivocally that neurons identified in the electron microscope are really post mitotic. However by characterising post mitotic cells labelled with BrdU and doublecortin and comparing these with post mitotic cells reconstructed in 3-dimensions from ultrathin serial sections, we have been able to illustrate individual mitotic elements and phases of cells within the GC layer of adult rat dentate gyrus. Here we show dividing cells in metaphase within clusters of immature GCs in subgranular zone (SGZ). These reconstructions provide ultrastructural confirmation that cells expressing doublecortin (DCX), a microtubule-associated protein expressed in migrating neurons, localize as clusters in the subgranular zone (SGZ) of dentate gyrus (DG) in the hippocampus during all animal life. Such DG cells with clear synaptic specializations, somatic spines and basal dendrites are exclusive to immature GC that appear to re-enter the cell cycle, suggesting the possibility that newly generated neurons within the DG might arise not only from precursors, but also from clusters of immature GC.     

Mitogen‐ and stress‐activated kinases regulate progenitor cell proliferation and neuron development in the adult dentate gyrus

The neurogenic niche within the subgranular zone (SGZ) of the dentate gyrus is a source of new neurons throughout life. Interestingly, SGZ proliferative capacity is regulated by both physiological and pathophysiological conditions. One outstanding question involves the molecular mechanisms that regulate both basal and inducible adult neurogenesis. Here, we examined the role of the MAPK‐regulated kinases, mitogen‐ and stress‐activated kinase (MSK)1 and MSK2. as regulators of dentate gyrus SGZ progenitor cell proliferation and neurogenesis. Under basal conditions, MSK1/2 null mice exhibited significantly reduced progenitor cell proliferation capacity and a corollary reduction in the number of doublecortin (DCX)‐positive immature neurons. Strikingly, seizure‐induced progenitor proliferation was totally blocked in MSK1/2 null mice. This blunting of cell proliferation in MSK1/2 null mice was partially reversed by forskolin infusion, indicating that the inducible proliferative capacity of the progenitor cell population was intact. Furthermore, in MSK1/2 null mice, DCX‐positive immature neurons exhibited reduced neurite arborization. Together, these data reveal a critical role for MSK1/2 as regulators of both basal and activity‐dependent progenitor cell proliferation and morphological maturation in the SGZ.     

Kinship and Descent: Redefining the Stem Cell Compartment in the Adult Hippocampus

Identifying multipotent, self-renewing neural stem cells (NSCs) within the adult hippocampus in vivo has been somewhat elusive. In this issue of Cell Stem Cell, Suh et al. (2007) show that Sox2-expressing cells in the subgranular zone (SGZ) of the dentate gyrus not only have NSC characteristics but also display an unexpected degree of heterogeneity.     

Estradiol initially enhances but subsequently suppresses (via adrenal steroids) granule cell proliferation in the dentate gyrus of adult female rats

In the dentate gyrus of adult female meadow voles, a high dose of estradiol benzoate (EB) increases (within 4 h) then decreases (within 48) the number of dividing progenitor cells (Ormerod BK, Galea LAM. 2001. Reproductive status regulates cell proliferation within the dentate gyrus of the adult female meadow vole: A possible regulatory role for estradiol. Neurosci 2:169-179). We investigated whether time-dependent EB exposure differentially influences the number of new granule cells produced in the adult female rat dentate gyrus and whether EB-stimulated adrenal activity mediates the decrease in cell proliferation. Ovariectomized rats received either an EB (10 microg in 0.1 mL) or vehicle (0.1 mL) injection either 4 or 48 h (Experiment 1) before a BrdU injection (200 mg/kg) and were perfused 24 h later to assess the number of new cells. Relative to vehicle, the number of new cells increased following a 4 h exposure (p < or = 0.04) but decreased following a 48 h exposure (p < or = 0.006) to EB. In Experiment 2, the number of new cells within the dentate gyrus of ovariectomized and adrenalectomized females did not significantly differ between groups exposed to EB versus vehicle for 48 h prior to BrdU administration, suggesting the decreased number of new cells observed within the dentate gyrus of adrenal-intact adult female rats is mediated by EB-stimulated adrenal activity. We conclude that estradiol dynamically regulates cell proliferation within the dentate gyrus of adult female rats in the time-dependent manner observed previously in voles and suppresses cell proliferation by influencing adrenal steroids. Investigating how estradiol dynamically regulates neurogenesis could provide insight into the mechanisms by which the proliferation of progenitor cells is controlled within the adult rodent hippocampus.     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.     

牦牛海马的形态特征及成体神经发生的初探

采用传统H.E 染色和Golgi-Cox 染色方法观察成年牦牛海马结构的形态和细胞构筑,并通过DCX - DAB免疫组化染色和DCX/ NeuN、GFAP / NeuN 双重免疫荧光标记等技术观察齿状回颗粒下层中的新生神经元和放射状胶质细胞。结果表明,牦牛海马结构主要包括齿状回和海马本部,二者分层清晰。海马的主要细胞为颗粒细胞、苔藓细胞和锥体细胞。CA3 区的锥体细胞胞体较CA1 区的大,但其顶树突的平均长度较短。CA1 区的锥体细胞明显分为两层,而CA3 区的则为一层。DCX 阳性细胞的胞体主要集中在齿状回颗粒下层靠近门区处,沿颗粒层内侧单个或少数聚集分布。沿齿状回颗粒下层分布着一层GFAP 阳性的放射状胶质细胞样细胞,其胞质和单极性的细长突起均呈GFAP 阳性,而胞核为阴性。在整个海马结构中均有大量星形GFAP 阳性细胞散在分布,特别是海马分子层和门区内靠近颗粒层部分的密度较其它部位大。牦牛海马的形态结构与绵羊的相似,而与大鼠、小鼠、家猫、兔子等小型哺乳动物有一定差别。两种DCX 免疫组化实验结果表明在牦牛海马中存在着新生神经元。GFAP 免疫荧光标记表明,牦牛海马结构中分布有星形胶质细胞;特别是放射状胶质细胞。     

Treadmill exercise suppresses ischemia-induced increment in apoptosis and cell proliferation in hippocampal dentate gyrus of gerbils

Cerebral ischemia resulting from transient or permanent occlusion of cerebral arteries leads to neuronal cell death and eventually causes neurological impairments. In the present study, the effects of treadmill exercise on apoptosis and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils were investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspase-3 and 5-bromo-2'-deoxyuridine (BrdU). It was shown that apoptotic cell death and cell proliferation in the hippocampal dentate gyrus were significantly increased following transient global ischemia in gerbils and that treadmill exercise suppressed the ischemia-induced increase in apoptosis and cell proliferation in the dentate gyrus. The present results suggest that treadmill exercise may protect cells from apoptotic death and aid in recovery from the central nervous system sequelae following stroke.     

Cellular organization of adult neurogenesis in the Common Marmoset

Adult neurogenesis within the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricle (LV) has been most intensely studied within the brains of rodents such as mice and rats. However, little is known about the cell types and processes involved in adult neurogenesis within primates such as the common marmoset (Callithrix jacchus). Moreover, substantial differences seem to exist between the neurogenic niche of the LV between rodents and humans. Here, we set out to use immunohistochemical and autogradiographic analysis to characterize the anatomy of the neurogenic niches and the expression of cell type-specific markers in those niches in the adult common marmoset brain. Moreover, we demonstrate significant differences in the activity of neurogenesis in the adult marmoset brain compared to the adult mouse brain. Finally, we provide evidence for ongoing proliferation of neuroblasts within both the SGZ and SVZ of the adult brain and further show that the age-dependent decline of neurogenesis in the hippocampus is associated with a decrease in neuroblast cells.     

Pyridoxine Enhances Cell Proliferation and Neuroblast Differentiation by Upregulating the GABAergic System in the Mouse Dentate Gyrus

We investigated the effects of pyridoxine (vitamin B₆) on cell death, cell proliferation, neuroblast differentiation, and the GABAergic system in the mouse dentate gyrus. We administered pyridoxine (350 mg/kg intraperitoneally) to 8 week old mice twice a day for 14 days and sacrificed them at 10 weeks of age. Pyridoxine treatment did not induce neuronal death or activate microglia in the dentate gyrus, while glial fibrillary acidic protein (GFAP)-positive cells were significantly increased in the subgranular zone of the dentate gyrus. The increase in GFAP-positive cells was confirmed to be due to proliferating cells based on double immunofluorescence staining. GFAP-positive cells, which were also labeled with Ki67, a marker for cell proliferation, and doublecortin, a marker for neuroblast differentiation, were significantly increased in the pyridoxine-treated group compared to those in the vehicle-treated group. Pyridoxine treatment also increased the protein levels of glutamic acid decarboxylase (GAD) 67, an enzyme for GABA synthesis, and pyridoxal 5′-phosphate (PNP) oxidase, an enzyme for pyridoxal phosphate synthesis, in the dentate gyrus. These results suggest that pyridoxine treatment distinctly increases cell proliferation, neuroblast differentiation, and upregulated the GABAergic system, as revealed by the increases of GAD67 and PNP oxidase in the mouse dentate gyrus.     

Estradiol and GPER Activation Differentially Affect Cell Proliferation but Not GPER Expression in the Hippocampus of Adult Female Rats

Estradiol increases cell proliferation in the dentate gyrus of the female rodent but it is not known whether the G protein-coupled estrogen receptor (GPER), a membrane receptor, is involved in this process, nor whether there are regional differences in estradiol’s effects on cell proliferation. Thus, we investigated whether estradiol exerts its effects on cell proliferation in the dorsal and ventral dentate gyrus through GPER, using the GPER agonist, G1, and antagonist, G15. Ovariectomized adult female rats received a single injection of either: 17β-estradiol (10 μg), G1 (0.1, 5, 10 μg), G15 (40 μg), G15 and estradiol, or vehicle (oil, DMSO, or oil+DMSO). After 30 min, animals received an injection of bromodeoxyuridine (BrdU) and were perfused 24 h later. Acute treatment with estradiol increased, while the GPER agonist G1 (5 μg) decreased, the number of BrdU+ cells in the dentate gyrus relative to controls. The GPER antagonist, G15 increased the number of BrdU+ cells relative to control in the dorsal region and decreased the number of BrdU+ cells in the ventral region. However, G15 treatment in conjunction with estradiol partially eliminated the estradiol-induced increase in cell proliferation in the dorsal dentate gyrus. Furthermore, G1 decreased the expression of GPER in the dentate gyrus but not the CA1 and CA3 regions of the hippocampus. In summary, we found that activation of GPER decreased cell proliferation and GPER expression in the dentate gyrus of young female rats, presenting a potential and novel estrogen-independent role for this receptor in the adult hippocampus.     

Antidepressant-induced vascular dynamics in the hippocampus of adult mouse brain

New neurons are continuously added to hippocampal circuitry involved with spatial learning and memory throughout life. These new neurons originate from neural stem/progenitor cells (NSPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG). Recent studies indicate that vascular reconstruction is closely connected with neurogenesis, but little is known about its mechanism. We have examined vascular reconstruction in the hippocampus of adult mouse brain after the administration of the antidepressant fluoxetine, a potent inducer of hippocampal neurogenesis. The immunohistochemistry of laminin and CD31 showed that filopodia of endothelial cells sprouted from existing thick microvessels and often formed a bridge between two thick microvessels. These filopodia were frequently seen at the molecular layer and dentate hilus of the DG, the stratum lacunosum-moleculare of the CA1, and the stratum oriens of the CA3. The filopodia were exclusively localized along cellular processes of astrocytes, but such intimate association was not seen with cell bodies and processes of NSPCs. The administration of fluoxetine significantly increased vascular density by enlarging the luminal size of microvessels and eliminating the filopodia of endothelial cells in the molecular layer and dentate hilus. Treatment with fluoxetine increased the number of proliferating NSPCs in the granule cell layer and dentate hilus, and that of endothelial cells in the granule cell layer. Thus, antidepressant-induced vascular dynamics in the DG are possibly attributable to the alteration of the luminal size of microvessels rather than to proliferation of endothelial cells.     

Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis

In the adult hippocampus, neuroprogenitor cells in the subgranular zone (SGZ) of the dentate gyrus give rise to newborn neuroblasts. However, only a small subset of these cells integrates into the hippocampal circuitry as mature neurons at the end of a 4 week period. Here, we show that the majority of the newborn cells undergo death by apoptosis in the first 1 to 4 days of their life, during the transition from amplifying neuroprogenitors to neuroblasts. These apoptotic newborn cells are rapidly cleared out through phagocytosis by unchallenged microglia present in the adult SGZ niche. Phagocytosis by the microglia is efficient and undeterred by increased age or inflammatory challenge. Our results suggest that the main critical period of newborn cell survival occurs within a few days of birth and reveal a new role for microglia in maintaining the homeostasis of the baseline neurogenic cascade.     

A circuit‐based gatekeeper for adult neural stem cell proliferation

Newborn neurons are generated in the adult hippocampus from a pool of self‐renewing stem cells located in the subgranular zone (SGZ) of the dentate gyrus. Their activation, proliferation, and maturation depend on a host of environmental and cellular factors but, until recently, the contribution of local neuronal circuitry to this process was relatively unknown. In their recent publication, Song and colleagues have uncovered a novel circuit‐based mechanism by which release of the neurotransmitter, γ‐aminobutyric acid (GABA), from parvalbumin‐expressing (PV) interneurons, can hold radial glia‐like (RGL) stem cells of the adult SGZ in a quiescent state. This tonic GABAergic signal, dependent upon the activation of γ₂ subunit‐containing GABA_A receptors of RGL stem cells, can thus prevent their proliferation and subsequent maturation or return them to quiescence if previously activated. PV interneurons are thus capable of suppressing neurogenesis during periods of high network activity and facilitating neurogenesis when network activity is low.     

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.     

Destruction of Dopaminergic Neurons in the Midbrain by 6-Hydroxydopamine Decreases Hippocampal Cell Proliferation in Rats: Reversal by Fluoxetine

Background

Non-motor symptoms (e.g., depression, anxiety, and cognitive deficits) in patients with Parkinson disease (PD) precede the onset of the motor symptoms. Although these symptoms do not respond to pharmacological dopamine replacement therapy, their precise pathological mechanisms are currently unclear. The present study was undertaken to examine whether the unilateral 6-hydroxydopamine (6-OHDA) lesion to the substantia nigra pars compacta (SNc), which represents a model of long-term dopaminergic neurotoxicity, could affect cell proliferation in the adult rat brain. Furthermore, we examined the effects of the selective serotonin reuptake inhibitor (SSRI) fluoxetine and the selective noradrenaline reuptake inhibitor maprotiline on the reduction in cell proliferation in the subgranular zone (SGZ) by the unilateral 6-OHDA lesion.

Methodology/Principal Findings

A single unilateral injection of 6-OHDA into the rat SNc resulted in an almost complete loss of tyrosine hydroxylase (TH) immunoreactivity in the striatum and SNc, as well as in reductions of TH-positive cells and fibers in the ventral tegmental area (VTA). On the other hand, an injection of vehicle alone showed no overt change in TH immunoreactivity. A unilateral 6-OHDA lesion to SNc significantly decreased cell proliferation in the SGZ ipsilateral to the 6-OHDA lesion, but not in the contralateral SGZ or the subventricular zone (SVZ), of rats. Furthermore, subchronic (14 days) administration of fluoxetine (5 mg/kg/day), but not maprotiline significantly attenuated the reduction in cell proliferation in the SGZ by unilateral 6-OHDA lesion.

Conclusions/Significance

The present study suggests that cell proliferation in the SGZ of the dentate gyrus might be, in part, under dopaminergic control by SNc and VTA, and that subchronic administration of fluoxetine reversed the reduction in cell proliferation in the SGZ by 6-OHDA. Therefore, SSRIs such as fluoxetine might be potential therapeutic drugs for non-motor symptoms as well as motor symptoms in patients with PD, which might be associated with the reduction in cell proliferation in the SGZ.     

For the long run: maintaining germinal niches in the adult brain

The adult mammalian brain retains neural stem cells that continually generate new neurons within two restricted regions: the subventricular zone (SVZ) of the lateral ventricle and the dentate gyrus subgranular zone (SGZ) of the hippocampus. Though these cellular populations are spatially isolated and subserve different brain systems, common themes begin to define adult neurogenic niches: (1) astrocytes serve as both stem cell and niche cell, (2) a basal lamina and concomitant vasculogenesis may be essential components of the niche, and (3) "embryonic" molecular morphogens and signals persist in these niches and play critical roles for adult neurogenesis. The adult neurogenic niches can be viewed as "displaced" neuroepithelium, pockets of cells and local signals that preserve enough embryonic character to maintain neurogenesis for life.     

NADPH oxidase NOX5-S mediates acid-induced cyclooxygenase-2 expression via activation of NF-kappaB in Barrett's esophageal adenocarcinoma cells

We have shown that the NADPH oxidase NOX5-S may play an important role in the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) by increasing cell proliferation and decreasing apoptosis. However, the mechanism of the acid-induced NOX5-S-mediated increase in cell proliferation is not known. We found that, in SEG1 EA cells, the acid-induced increase in prostaglandin E2 (PGE2) production was mediated by activation of cyclooxygenase-2 (COX2) but not by COX1. Acid treatment increased intracellular Ca2+, and a blockade of intracellular Ca2+ increase inhibited the acid-induced increase in COX2 expression and PGE2 production. Knockdown of NOX5-S or NF-kappaB1 p50 by their small interfering RNA significantly inhibited acid-induced COX2 expression and PGE2 production in SEG1 cells. Acid treatment significantly decreased IkappaBalpha and increased luciferase activity when SEG1 cells were transfected with an NF-kappaB in vivo activation reporter plasmid, pNF-kappaB-Luc. In a novel Barrett's cell line overexpressing NOX5-S, IkappaBalpha was significantly reduced, and luciferase activity increased when these Barrett's cells were transfected with pNF-kappaB-Luc. Overexpression of NOX5-S in Barrett's cells significantly increased H2O2 production, COX2 expression, PGE2 production, and thymidine incorporation. The increase in thymidine incorporation occurring in NOX5-S-overexpressing Barrett's cells or induced by acid treatment in SEG1 EA cells was significantly decreased by COX2 inhibitors or small interfering RNA. We conclude that acid-induced COX2 expression and PGE2 production depend on an increase in cytosolic Ca2+ and sequential activation of NOX5-S and NF-kappaB in SEG1 cells. COX2-derived PGE2 production may contribute to NOX5-S-mediated cell proliferation in SEG1 cells.     

Hippocampal enlargement in <Emphasis Type="Italic">Bassoon</Emphasis>-mutant mice is associated with enhanced neurogenesis,reduced apoptosis,and abnormal BDNF levels

Mice mutant for the presynaptic protein Bassoon develop epileptic seizures and an altered pattern of neuronal activity that is accompanied by abnormal enlargement of several brain structures, with the strongest size increase in hippocampus and cortex. Using manganese-enhanced magnetic resonance imaging, an abnormal brain enlargement was found, which is first detected in the hippocampus 1 month after birth and amounts to an almost 40% size increase of this structure after 3 months. Stereological quantification of cell numbers revealed that enlargement of the dentate gyrus and the hippocampus proper is associated with larger numbers of principal neurons and of astrocytes. In search for the underlying mechanisms, an approximately 3-fold higher proportion of proliferation and survival of new-born cells in the dentate gyrus was found to go hand in hand with similarly larger numbers of doublecortin-positive cells and reduced numbers of apoptotic cells in the dentate gyrus and the hippocampus proper. Enlargement of the hippocampus and of other forebrain structures was accompanied by increased levels of brain-derived neurotrophic factor (BDNF). These data show that hippocampal overgrowth in Bassoon-mutant mice arises from a dysregulation of neurogenesis and apoptosis that might be associated with unbalanced BDNF levels.