Protective and regenerative response endogenously induced in the ischemic brain

Neuronal cells are highly vulnerable to ischemic insult. Because adult neurons are highly differentiated and cannot self-propagate, loss of neurons often results in functional deficits in mammalian brains. However, it has recently been shown that neurons and neuronal circuits exhibit protective and regenerative responses in a rodent model of experimental ischemia. At first, neurons respond by producing several protective proteins such as heat shock proteins (HSPs) after sublethal ischemia and then acquire tolerance against a subsequent ischemic insult (ischemic tolerance). Once neurons suffer irreversible injury, two repair processes, neurogenesis and synaptogenesis, are endogenously induced. Neuronal stem and (or) progenitor cells can proliferate in two brain areas in adult animals: the subventricular zone and the subgranular zone in the dentate gyrus. After ischemic insult, these stem (progenitor) cells proliferate and differentiate into neurons in the dentate gyrus of the hippocampus. Reactive synaptogenesis has been also observed in the injured brain following a period of long-term infarction, but it is unclear if it can compensate for disconnected circuits. Understanding the molecular mechanism underlying these protective and regenerative responses will be important in developing a new strategy for aimed at the augmentation of resistance against ischemic insult and the replacement of injured neurons and neuronal circuits.     

成年哺乳动物皮质区神经发生的研究进展

神经发生是神经干细胞在适当的条件下分化成功能性整合神经元的过程,主要包括细胞的增殖、迁移、分化和存活。成年神经发生区以前脑室管膜下区(Subventricular zones,SVZ)和海马齿状回颗粒层下区(Subgranular zones,SGZ)为主,但皮质作为神经元和神经胶质细胞数量最多、分布最广,同时也是哺乳动物高度发展的脑区,是否有成年神经元新生,已成为近年来神经科学领域的研究热点[1,2]。现本文就未成熟神经元在皮质区的研究方法、分布、来源与转归、病理生理功能影响等方面探讨成年哺乳动物皮质神经发生现象。     

Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal

Seasonal changes in vertebrate brain function are pervasive, but annual cycles in the rates of neuronal incorporation are established only in songbirds. Although cell division continues in the subependymal and hippocampal subgranular zones of adult rodents, there exists no parallel evidence that seasonal plasticity in mammals extends to changes in neuronal or glial number. We examined the effect of photoperiod on incorporation of new neurons in the brain of the adult golden hamster, a long-day breeder. We administered the cell birth marker 5′-bromode-oxyuridine (BrdU) to males which had either been maintained in long days, transferred to short days for 10 weeks, or moved acutely from long to short or short to long days. The number of cells in specific brain regions immunoreactive (ir) for this thymidine analog was determined 7 weeks later. The number of BrdU-ir cells in the dentate gyrus and subependymal zone increased twofold in short days. Transfer between photoperiods 10 days before the BrdU injections produced intermediate numbers of BrdU-labeled cells in the dentate gyrus, but was as effective as long-term photoperiodic exposure in the subependymal zone. Photoperiod also had similar effects in the hypothalamus and cingulate/retrosplenial cortex, but not in the central gray or preoptic area. Double-label immunocytochemistry indicated that very few of the BrdU-ir cells were glia, but that a majority had neuronal phenotype. In the subependymal zone, short days significantly increased the number of BrdU-labeled neurons. We did not detect significant effects of photoperiod on the volume of either the granule cell layer of the hippocampus or the dentate gyrus as a whole. We conclude that short day lengths increase neuronal birth and/or survival in several brain regions of adult hamsters. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 410–420, 1998     

Region- and neuronal phenotype-specific expression of NELL2 in the adult rat brain

NELL2, a neural tissue-enriched protein, is produced in the embryo, and postembryonically in the mammalian brain, with a broad distribution. Although its synthesis is required for neuronal differentiation in chicks, not much is known about its function in the adult mammalian brain. We investigated the distribution of NELL2 in various regions of the adult rat brain to study its potential functions in brain physiology. Consistent with previous reports, NELL2-immunoreactivity (ir) was found in the cytoplasm of neurons, but not in glial fibrillary acidic protein (GFAP)-positive glial cells. The highest levels of NELL2 were detected in the hippocampus and the cerebellum. Interestingly, in the cerebellar cortex NELL2 was observed only in the GABAergic Purkinje cells not in the excitatory granular cells. In contrast, it was found mainly in the hippocampal dentate gyrus and pyramidal cell layer that contains mainly glutamatergic neurons. In the dentate gyrus, NELL2 was not detected in the GFAP-positive neural precursor cells, but was generally present in mature neurons of the subgranular zone, suggesting a role in this region restricted to mature neurons.     

Fast,Potent Pharmacological Expansion of Endogenous Hes3+/Sox2+ Cells in the Adult Mouse and Rat Hippocampus

The adult hippocampus is involved in learning and memory. As a consequence, it is a brain region of remarkable plasticity. This plasticity exhibits itself both as cellular changes and neurogenesis. For neurogenesis to occur, a population of local stem cells and progenitor cells is maintained in the adult brain and these are able to proliferate and differentiate into neurons which contribute to the hippocampal circuitry. There is much interest in understanding the role of immature cells in the hippocampus, in relation to learning and memory. Methods and mechanisms that increase the numbers of these cells will be valuable in this research field. We show here that single injections of soluble factors into the lateral ventricle of adult rats and mice induces the rapid (within one week) increase in the number of putative stem cells/progenitor cells in the hippocampus. The established progenitor marker Sox2 together with the more recently established marker Hes3, were used to quantify the manipulation of the Sox2/Hes3 double-positive cell population. We report that in both adult rodent species, Sox2+/Hes3+ cell numbers can be increased within one week. The most prominent increase was observed in the hilus of the dentate gyrus. This study presents a fast, pharmacological method to manipulate the numbers of endogenous putative stem cells/progenitor cells. This method may be easily modified to alter the degree of activation (e.g. by the use of osmotic pumps for delivery, or by repeat injections through implanted cannulas), in order to be best adapted to different paradigms of research (neurodegenerative disease, neuroprotection, learning, memory, plasticity, etc).     

Time-of-Day-Dependent Enhancement of Adult Neurogenesis in the Hippocampus

Background

Adult neurogenesis occurs in specific regions of the mammalian brain such as the dentate gyrus of the hippocampus. In the neurogenic region, neural progenitor cells continuously divide and give birth to new neurons. Although biological properties of neurons and glia in the hippocampus have been demonstrated to fluctuate depending on specific times of the day, it is unclear if neural progenitors and neurogenesis in the adult brain are temporally controlled within the day.

Methodology/Principal Findings

Here we demonstrate that in the dentate gyrus of the adult mouse hippocampus, the number of M-phase cells shows a day/night variation throughout the day, with a significant increase during the nighttime. The M-phase cell number is constant throughout the day in the subventricular zone of the forebrain, another site of adult neurogenesis, indicating the daily rhythm of progenitor mitosis is region-specific. Importantly, the nighttime enhancement of hippocampal progenitor mitosis is accompanied by a nighttime increase of newborn neurons.

Conclusions/Significance

These results indicate that neurogenesis in the adult hippocampus occurs in a time-of-day-dependent fashion, which may dictate daily modifications of dentate gyrus physiology.     

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.     

Temporal Changes in Prosaposin Expression in the Rat Dentate Gyrus after Birth

Neurogenesis in the hippocampal dentate gyrus occurs constitutively throughout postnatal life. Adult neurogenesis includes a multistep process that ends with the formation of a postmitotic and functionally integrated new neuron. During adult neurogenesis, various markers are expressed, including GFAP, nestin, Pax6, polysialic acid-neural cell adhesion molecule (PSA-NCAM), neuronal nuclei (NeuN), doublecortin, TUC-4, Tuj-1, and calretinin. Prosaposin is the precursor of saposins A–D; it is found in various organs and can be excreted. Strong prosaposin expression has been demonstrated in the developing brain including the hippocampus, and its neurotrophic activity has been proposed. This study investigated changes in prosaposin in the dentate gyrus of young and adult rats using double immunohistochemistry with antibodies to prosaposin, PSA-NCAM, and NeuN. Prosaposin immunoreactivity was intense in the dentate gyrus at postnatal day 3 (P3) and P7, but decreased gradually after P14. In the dentate gyrus at P28, immature PSA-NCAM-positive neurons localized exclusively in the subgranular zone were prosaposin-negative, whereas mature Neu-N-positive neurons were positive for prosaposin. Furthermore, these prosaposin-negative immature neurons were saposin B-positive, suggesting that the neurons take up and degrade prosaposin. In situ hybridization assays showed that prosaposin in the adult dentate gyrus is dominantly the Pro+9 type, a secreted type of prosaposin. These results imply that prosaposin secreted from mature neurons stimulates proliferation and maturation of immature neurons in the dentate gyrus.     

Phosphatidylglucoside: a novel marker for adult neural stem cells

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.     

Immunohistological markers for staging neurogenesis in adult hippocampus

Neurogenesis in the adult dentate gyrus (DG) of the hippocampus occurs constitutively throughout postnatal life, and the rate of neurogenesis within the DG can be altered under various physiological and pathophysiological conditions. Adult neurogenesis includes the process in which the division of a precursor cell takes place and the multi-step process (proliferation, differentiation, migration, targeting, and synaptic integration) that ends with the formation of a postmitotic functionally integrated new neuron. During specific time-frames of adult neurogenesis, various markers are expressed that correlate with the differentiation steps along the pathway from early progenitor cells to newly generated postmitotic neurons within the DG. Markers that are currently widely used for the investigation of adult hippocampal neurogenesis are: glial fibrillary acidic protein, nestin, Pax6, NeuroD, PSA-NCAM, doublecortin, TUC-4, Tuj-1, and calretinin. The discovery and development of specific markers that allow the time-course and fate of neurons to be followed during adult neurogenesis in a detailed and precise fashion are not only helpful for gaining further insights into the genesis of new neurons in the hippocampus, but also might be applicable to the development of strategies for therapeutic interventions. This study was supported by the DFG (SFB 636/A5).     

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.     

Decreased Glucagon-Like Peptide-1 Receptor Immunoreactivity in the Dentate Granule Cell Layer from Adult in the Gerbil Hippocampus

In this study, we investigated age-related changes in glucagon-like peptide-1 receptor (GLP-1R) immunoreactivity and its protein levels in the gerbil hippocampus during normal aging. In the postnatal month 3 (PM 3) group, GLP-1R immunoreaction was well observed in neurons, especially pyramidal and non-pyramidal cells in the hippocampus proper, and granule and polymorphic cells in the dentate gyrus. In the hippocampus proper, GLP-1R immunoreactivity in neurons was maintained until PM 24. In the dentate gyrus, however, GLP-1R immunoreactivity in granule cells, not polymorphic cells, was hardly detected from PM 6. Western blot analysis also showed that age-dependent change patterns in GLP-1R protein levels in the gerbil hippocampus were similar to the immunohistochemical changes. These results indicate that GLP-1R immunoreactivity was markedly decreased in dentate granule cells from PM 6, showing that GLP-1R immunoreactivity and its protein levels were decreased in the adult and aged gerbil hippocampus.     

Differences in Doublecortin Immunoreactivity and Protein Levels in the Hippocampal Dentate Gyrus Between Adult and Aged Dogs

Doublecortin (DCX), a microtubule-associated protein, specifically expresses in neuronal precursors. This protein has been used as a marker for neuronal precursors and neurogenesis. In the present study, we observed differences in DCX immunoreactivity and its protein levels in the hippocampal dentate gyrus between adult and aged dogs. In the adult dog, DCX immunoreactive cells with well-stained processes were detected in the subgranular zone of the dentate gyrus. Numbers of DCX immunoreactive cells in the dentate gyrus of the aged dog were significantly decreased compared to those in the adult dog. DCX immunoreactive cells in both adult and aged dog did not show NeuN (a marker for mature neurons) immunoreactivity. NeuN immunoreactivity in the aged dog was poor compared to that in the adult dog. DCX protein level in the aged dentate gyrus was decreased by 80% compared to that in the adult dog. These results suggest that the reduction of DCX in the aged hippocampal dentate gyrus may be involved in some neural deficits related to the hippocampus.     

Functional convergence of neurons generated in the developing and adult hippocampus

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function.     

Neural Precursor Cells from Adult Mouse Cerebral Cortex Differentiate into Both Neurons and Oligodendrocytes

Recent findings concerning adult neurogenesis in two selected structures of the mammalian brain, the olfactory bulb and dentate gyrus of the hippocampus, present the possibility that this mechanism of neurogenesis applies for all brain regions, including the cerebral neocortex. In this way, a small number of potential neural precursor cells may exist in the cerebral neocortex, but they do not normally differentiate into cortical neurons in vivo. It has, however, been reported recently that cycling cells isolated from non-neurogenic areas of adult rat cerebral cortex could generate neurons in vitro. In this study, we analyzed the lineage potential of cycling cells from the adult mouse neocortex. For the dissection of the cerebral cortex from the adult mouse, which is significantly smaller than that of the adult rat, we have modified the previous dissection protocol developed for the rat neocortex. As a result, cycling cells from adult mouse neocortex gave rise to neurons and oligodendrocytes, but not to astrocytes, whereas when the previous dissection method was used, cycling cells gave rise to neurons, oligodendrocytes and astrocytes. This discrepancy might stem from slight contamination of the dissected mouse neocortical tissue in the previous protocol used for the dissection of rat neocortex by cells from the surrounding subependymal zone, where typical adult neural stem cells exist. The results presented here will contribute to our understanding of the nature of cycling cells in the adult mammalian neocortex, and for which future stem cell research will provide new possibilities for cell replacement therapy to be used in the treatment of neurodegenerative conditions.     

Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family

Cells expressing mRNA for hippocampus-derived neurotrophic factor (HDNF/NT-3) or brain-derived neurotrophic factor (BDNF) were identified by in situ hybridization. In the rat brain, HDNF mRNA was predominantly found in pyramidal neurons in CA1 and CA2 of the hippocampus. Lower levels of HDNF mRNA were found in granular neurons of the dentate gyrus and in neurons of the taenia tecta and induseum griseum. BDNF mRNA-expressing cells were more widely distributed in the rat brain, with high levels in neurons of CA2, CA3, and the hilar region of the dentate gyrus, in the external and internal pyramidal layers of the cerebral cortex, in the claustrum, and in one brainstem structure. Lower levels were seen in CA1 and in the granular layer of the hippocampus, in the taenia tecta, and in the mammillary complex. In peripheral tissues, HDNF mRNA was found in glomerular cells in the kidney, secretory cells in the male rat submandibular gland, and epithelial cells in secondary and tertiary follicles in the ovary. Cells expressing BDNF mRNA were found in the dorsal root ganglia, where neurons of various sizes were labeled.     

Congruence of vascular network remodeling and neuronal dispersion in the hippocampus of reelin-deficient mice

In the hippocampus, neurons and fiber projections are strictly organized in layers and supplied with oxygen via a vascular network that also develops layer-specific characteristics in wild-type mice, as shown in the present study for the first time in a quantitative manner. By contrast, in the reeler mutant, well known for its neuronal migration defects due to the lack of the extracellular matrix protein reelin, emerging layer-specific characteristics of the vascular pattern were found to be remodeled during development of the dentate gyrus. Remarkably, in the first postnatal week, when a granule cell layer was still discernable in the reeler dentate gyrus, also the reeler vascular pattern resembled wild type. Thus, at postnatal day 6, unbranched microvessels traversed the granule cell layer and bifurcated when reaching the subgranular zone. Only after the first postnatal week vascular network remodeling in the reeler dentate gyrus became apparent, when the proportion of dispersed granule cells increased. Hence, vessel bifurcation frequency decreased in the maturing reeler dentate gyrus, but increased in wild type, resulting in significant differences (approx. 100%; p < 0.01) between adult wild type and reeler. Moreover, layer-specific vessel bifurcation frequencies disappeared in the maturing reeler dentate gyrus. Finally, a wild type-like vascular pattern was also found in the dentate gyrus of mice deficient for the reelin receptor very low density lipoprotein receptor (VLDLR), precluding a requirement of VLDLR for normal vascular pattern formation in the dentate gyrus. In sum, our findings show that vascular network remodeling in the reeler dentate gyrus is closely linked to the progression of granule cell dispersion.