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Roles of the mammalian subventricular zone in cell replacement after brain injury.
The subventricular zones (SVZs) are essential sources of new cells in the developing brain and remnants of these germinal zones persist into adulthood. As these cells have the capacity to replenish neurons and glia that are turning over, many investigators have assessed the SVZ's role in replacing neural cells eliminated by brain injuries. A review of the literature reveals that the progenitors within the SVZs are vulnerable to chemical, radiation and ischemia-induced damage, whereas the neural stem cells are resilient. With moderate insults, the SVZ can recover, but it cannot recover after more severe injury. Thus, the vulnerability of these cells has important ramifications when considering therapeutic interventions for the treatment of brain tumors and for the prospect of recovery after ischemia. The cells of the perinatal and adult SVZ not only have the capacity to replenish their own numbers, but they also have the capacity to replace neurons and glia after ischemic and traumatic brain injuries. Moreover, the mechanisms underlying these regenerative responses are beginning to be revealed. By reviewing, comparing and contrasting the responses of the SVZs to different injuries, our goal is to provide a foundation from which current and future studies on the potential of the SVZs for cell replacement can be evaluated.
Corticotropin-releasing factor: a marked circadian rhythm in primate cerebrospinal fluid peaks in the evening and is inversely related to the cortisol circadian rhythm.
Continuous sampling of cerebrospinal fluid (CSF) over 24-h periods in 10 rhesus monkeys revealed a 2-fold, highly reproducible circadian rhythm in CRF concentrations. Peak CRF values of 77.9 +/- 6.4 pg/ml occurred in the evening at 1930 h, while the CRF nadir (38.4 +/- 4.2 pg/ml) occurred at 0745 h. Simultaneously sampled CSF cortisol peaked at 0913 h, with a nadir at 2226 h. Both CRF and cortisol rhythms closely fit sinusoidal circadian models, with r2 values of 0.94 and 0.92, respectively. While hypothalamic CRF is regarded as a major physiological regulator of pituitary ACTH secretion and, thereby, of the circadian and stress-related release of cortisol from the adrenal gland, CRF and CRF receptors are also widely distributed in other brain areas of primates and rodents. The marked difference in the circadian rhythm of CRF vs. that of cortisol suggests that CRF in CSF reflects or mediates some nonhypophysiotropic brain functions of this peptide.
Differential activation of microglia in neurogenic versus non-neurogenic regions of the forebrain.
Proliferation decreases in the neurogenic subventricular zone (SVZ) of mice after aspiration lesions of the cerebral cortex. We hypothesized that microglial activation may contribute to this given microglial activation attenuates neurogenesis in the hippocampus. Using CD45, CD11b, IB4, and IL-6 immunohistochemistry (IHC), BrdU IHC, and fluorescent bead tracking of peripheral monocytes into the brain, we compared microglial activation in the SVZ to non-neurogenic forebrain regions. SVZ microglia exhibited greater constitutive activation and proliferation than did microglia in non-neurogenic regions. In contrast to the SVZ, the dentate gyrus (DG) contained relatively few CD45(+) cells. After aspiration cerebral cortex lesions, microglia became activated in the cerebral cortex, corpus callosum, and striatum. SVZ microglial activation did not increase, and similarly, microglia in the DG were less activated after injury than in adjacent non-neurogenic regions. We next showed that SVZ microglia are not categorically refractory to activation, since deep cortical contusion injuries increased SVZ microglial activation. Macrophages migrate into the brain during development, but it is unclear if this is recapitulated after injury. Infiltration of microbead-labeled macrophages into the brain did not change after injury, but resident SVZ microglia were induced to migrate toward the injury. Our data show that both constitutive and postlesion levels of microglial activation differ between neurogenic and non-neurogenic regions.
Dopamine stimulation of postnatal murine subventricular zone neurogenesis via the D3 receptor.
We investigated the expression and role of the dopamine receptor 3 (D3R) in postnatal mouse subventricular zone (SVZ). In situ hybridization detected selective D3R mRNA expression in the SVZ. Fluorescence activated cell sorting (FACS) of adult SVZ subtypes using hGFAP-GFP and Dcx-GFP mice showed that transit amplifying progenitor cells and niche astrocytes expressed D3R whereas stem cell-like astrocytes and neuroblasts did not. To determine D3R's role in SVZ neurogenesis, we administered U-99194A, a D3R preferential antagonist, and bromodeoxyuridine in postnatal mice. In vivo D3R antagonism decreased the numbers of newborn neurons reaching the core and the periglomerular layer of the olfactory bulb. Moreover, it decreased progenitor cell proliferation but did not change the number of label-retaining (stem) cells, commensurate with its expression on transit amplifying progenitor cells but not SVZ stem cell-like astrocytes. Collectively, this study suggests that dopaminergic stimulation of D3R drives proliferation via rapidly amplifying progenitor cells to promote murine SVZ neurogenesis.
Proliferation but not migration is associated with blood vessels during development of the rostral migratory stream.
Blood vessels play a critical role in regulating neural stem cell proliferation and migration. We show here that blood vessels became progressively aligned in the direction of the rostral migratory stream (RMS) from embryonic day 14 to postnatal day 4. Dividing cells revealed by phosphohistone H3+ immunoreactivity were statistically closer to isolectin B4+ blood vessels than predicted by chance in the emerging RMS. The close proximity of blood vessels and H3+ cells was consistent regardless of the age of the RMS and was strikingly similar to the embryonic cerebral cortex. In contrast to the adult RMS, we found no evidence for preferential juxtaposition of migratory doublecortin-positive neuroblasts and vasculature in the neonatal RMS. Our work provides an important framework for understanding the precise mechanism behind regulation of proliferation.
Early induction of mRNA for calbindin-D28k and BDNF but not NT-3 in rat hippocampus after kainic acid treatment.
The influence of kainic acid (KA), which induces acute seizures, on expression of mRNA for the calcium-binding protein, calbindin-D28k, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and early-response genes [c-fos, zif268 (NGFI-A), nur77 (NGFI-B)] was examined in rat hippocampus by Northern blot analysis. A significant increase (3.2-fold) in BDNF mRNA was observed 1 h after KA injection (12 mg/kg i.p.) and peak expression (9.4-fold) occurred 3 h after KA. The induction of BDNF mRNA was preceded by the induction of c-fos, mRNA (30 min after KA) and was followed by the induction of calbindin-D28k mRNA (3.5-fold 3 h after KA; a maximal response was at 3-6 h after KA). Region-specific changes, analyzed by immunocytochemistry and in situ hybridization, indicated that the most dramatic increases in calbindin protein and mRNA after KA treatment were in the dentate gyrus. Although calbindin-D28k and BDNF mRNAs were induced, a 3.4-3.8-fold decrease in NT-3 mRNA was observed by Northern analysis 3-24 h after KA treatment. Calbindin-D28k gene expression was also examined in rats with a chronic epileptic state characterized by recurrent seizures established with an episode of electrical stimulation-induced status epilepticus (SE). When these animals were examined 30 days post-SE, no changes in hippocampal calbindin-D28k mRNA were observed. Our findings suggest that the induction of calbindin-D28k mRNA (which may be interrelated to the induction of BDNF mRNA) is an early response which may not be related to enhanced neuronal activity or seizures per se, but rather to maintaining neuronal viability.
Techniques and strategies to analyze neural progenitor cell migration.
One of the most surprising aspects of neural development is that cells do not remain in their birthplace but actively migrate along a variety of routes to their final destinations. This review traces past, present, and future techniques used to analyze progenitor cell migration in the brain, and also discusses their relevant strengths and weaknesses. The large majority of information regarding cell migration is from studies where migratory cells have been labeled, but in which the actual movements are not observed, ie., from static experiments. More recently, dynamic imaging of cell migration in living slices and, even in vivo, has provided a glimpse of how complex these phenomena truly are. A variety of new techniques, such as 2-photon videomicroscopy, are emerging that will continue to add to our body of knowledge concerning the migration of cells in the central nervous system.
Expression of molecules associated with neuronal plasticity in the striatum after aspiration and thermocoagulatory lesions of the cerebral cortex in adult rats.
Like the hippocampus, the striatum receives excitatory afferents from the cerebral cortex but, in the case of the striatum, very little is known about the molecular events associated with plasticity after lesions of this pathway. Using immunohistochemical techniques, we have examined the effects of cortical lesions induced either by aspiration of the frontoparietal cortex or by thermocoagulation of pial blood vessels on axonal and glial molecules associated with neuronal plasticity in the striatum. The growth associated protein GAP-43, a molecule present in axons and growth cones, decreased in the dorsolateral striatum after aspiration but not after thermocoagulatory lesions. In contrast, synaptophysin, a marker of synaptic vesicles, remained unchanged in the denervated striatum after both types of lesions. Immunostaining for basic fibroblast growth factor (bFGF) markedly decreased in striatal astrocytes after both lesions, despite an increased staining for glial fibrillary acidic protein (GFAP). The adhesion molecules tenascin, chondroitin sulfate proteoglycans, highly polysialylated neural cell adhesion molecule (PSA-NCAM), and laminin did not change significantly in the gray matter of the dorsolateral striatum after either type of lesion. These effects differed from those observed after partial denervation of the hippocampus and spinal cord, revealing marked regional differences in the response of axonal and glial proteins to afferent lesions. In addition, the results further indicate that cortical lesions have both similar and distinct consequences, depending on the procedure by which the lesions are induced, suggesting that cortical lesions associated with different types of pathology may differentially affect subcortical structures.
High affinity agonist binding to cloned 5-hydroxytryptamine2 receptors is not sensitive to GTP analogs.
Agonists for GTP-binding protein (G protein)-coupled receptors are thought to bind with high affinity to the complex of receptor and G protein. Nonhydrolyzable GTP analogs have been shown to disrupt this complex and reduce the binding affinity for many agonists. Antagonists are thought to bind to the receptor whether or not it is coupled to the G protein, and therefore binding remains unchanged in the presence of GTP analogs. The binding of the serotonin 5-hydroxytryptamine (5-HT)2 receptor agonists serotonin (5-HT) and 4-bromo-2,5-dimethoxyphenylisopropylamine is not affected by the presence of GTP analogs when the cloned 5-HT2 receptor is expressed in the 293 human embryonic kidney cell line. The same receptor expressed in mouse NIH3T3 cells is partially sensitive to GTP analogs. Both cell lines have similar proportions of agonist and antagonist binding sites, and agonist stimulation of both cell lines leads to a robust increase in phosphoinositide hydrolysis. Differences in GTP metabolism in 293 cells is not likely to be the cause of the observed difference in inhibition of agonist binding, because the cloned 5-HT1A serotonin receptor expressed in these cells is sensitive to GTP analogs. The GTP-insensitive agonist binding is best explained by the existence of a G protein-receptor complex in 293 cells that is not sensitive to GTP analogs. Such a G protein-receptor complex may explain the fraction of agonist binding in the brain that is not sensitive to GTP analogs.
Pattern of expression of highly polysialylated neural cell adhesion molecule in the developing and adult rat striatum.
In rats, morphological and synaptic maturation of the striatum, a brain area involved in the control of movement and in cognitive behaviour, proceeds for several weeks postnatally. Little is known, however, about the molecular events associated with the final maturation of the striatum. In particular, there is little information on molecules playing a role in cell adhesion, a phenomenon of particular importance for neuronal development. We have examined the time course and topography of expression of the highly polysialylated form of the neural cell adhesion molecule in the rat striatum during postnatal development and in the adult, and compared it to growth-associated protein-43, a marker of axonal growth. As earlier during development [Aaron L. I. and Chesselet M.-F. (1989) Neuroscience 28, 701-710], immunolabelling for polysialylated neural cell adhesion molecule was very intense in the entire striatum at postnatal days 17-19. At postnatal days 21 and 22, loss of polysialylated neural cell adhesion molecule immunoreactivity in the caudal part of the striatum contrasted with the persistence of immunoreactivity at more rostral levels. Most of the striatum was devoid of polysialylated neural cell adhesion molecule immunoreactivity by postnatal day 25. At this age, as well as in the striatum of adult rats, immunolabelling was only observed along the ventricular edge of the striatum. In contrast to polysialylated neural cell adhesion molecule immunoreactivity, immunolabelling for growth-associated protein-43 had reached its adult pattern by postnatal day 17, indicating that polysialylated neural cell adhesion molecule persists beyond the period of major axonal growth. In the adult, an area of stronger growth associated protein-43 immunoreactivity overlapped with the region which retained immunoreactivity to polysialylated neural cell adhesion molecule. The results indicate that, in the developing rat striatum, the neural cell adhesion molecule remains highly sialylated not only during the ingrowth of cortical and nigral inputs but also during the formation of dendritic spine and synaptogenesis. Loss of polysialyated neural cell adhesion molecule occurs at the time of emerging spontaneous activity in cerebral cortex, and precedes the development of mature responses to cortical stimulation and adult membrane properties in a majority of striatal neurons.
Sox-9 and cDachsund-2 expression in the developing chick telencephalon.
The expression patterns of the transcription factor, Sox-9, and of the nuclear factor, cDachsund-2, were examined in the developing chick telencephalon. Both genes were expressed in the ventricular zone and in the subventricular zone of the telencephalon during the period of neurogenesis. Whereas Sox-9 was not expressed in postmitotic tissues, cDachsund-2 was specifically expressed in the neostriatum and in subdivisions of the hyperstriatum embryonically and in posthatch chicks.
Rostral migratory stream neuroblasts turn and change directions in stereotypic patterns.
Neuroblasts generated in the adult subventricular zone (SVZ) migrate through the rostral migratory stream (RMS) to the olfactory bulb (OB). Previous work uncovered motility ranging from straight to complex, but it was unclear if directional changes were stochastic or exhibited stereotypical patterns. Here, we provide the first in-depth two-photon time-lapse microscopy study of morphological and dynamic features that accompany turning and direction reversals in the RMS. We identified three specific kinds of turning (30-90 degrees): bending of the leading process proximal to the cell body (P-bending 47% of cases), bending of the distal leading process (D-bending 30%) or branching of the leading process or lamellipodium (23%). Bending and branching angles were remarkably constrained and were significantly different from one another. Cells reversed direction (> 90 degrees) through D-bendings (54%), branching (11%) or de novo growth of processes from the soma (23%), but not P-bending. Direction reversal was often composed of several iterations of D-bending or branching as opposed to novel modalities. Individual neuroblasts could turn or change direction in multiple patterns suggesting that the patterns are not specific for different lineages. These findings show that neuroblasts in the RMS use a limited number of distinct and constrained modalities to turn or reverse direction.
Activation of subventricular zone stem cells after neuronal injury.
The mammalian subventricular zone (SVZ) has garnered a tremendous amount of attention as a potential source of replacement cells for neuronal injury. This zone is highly neurogenic, harbours stem cells and supports long-distance migration. The general pattern of activation includes increased proliferation, neurogenesis and emigration towards the injury. Intrinsic transcription factors and environmental signalling molecules are rapidly being discovered that may facilitate the induction of these cells to mount appropriate therapeutic responses. The extent of SVZ neurogenesis in humans is controversial. However, tantalizing new data suggest that humans are capable of generating increased numbers of neurons after a variety of diseases.
Radial glia-like cells at the base of the lateral ventricles in adult mice.
During development radial glia (RG) are neurogenic, provide a substrate for migration, and transform into astrocytes. Cells in the RG lineage are functionally and biochemically heterogeneous in subregions of the brain. In the subventricular zone (SVZ) of the adult, astrocyte-like cells exhibit stem cell properties. During examination of the response of SVZ astrocytes to brain injury in adult mice, we serendipitously found a population of cells in the walls of the ventral lateral ventricle (LV) that were morphologically similar to RG. The cells expressed vimentin, glial fibrillary acidic protein (GFAP), intermediate filament proteins expressed by neural progenitor cells, RG and astrocytes. These RG-like cells had long processes extending ventrally into the nucleus accumbens, ventromedial striatum, ventrolateral septum, and the bed nucleus of the stria terminalis. The RG-like cell processes were associated with a high density of doublecortin-positive cells. Lesioning the cerebral cortex did not change the expression of vimentin and GFAP in RG-like cells, nor did it alter their morphology. To study the ontogeny of these cells, we examined the expression of molecules associated with RG during development: vimentin, astrocyte-specific glutamate transporter (GLAST), and brain lipid-binding protein (BLBP). As expected, vimentin was expressed in RG in the ventral LV embryonically (E16, E19) and during the first postnatal week (P0, P7). At P14, P21, P28 as well as in the adult (8-12 weeks), the ventral portion of the LV retained vimentin immunopositive RG-like cells, whereas RG largely disappeared in the dorsal two-thirds of the LV. GLAST and BLBP were expressed in RG of the ventral LV embryonically and through P7. In contrast to vimentin, at later stages BLBP and GLAST were found in RG-like cell somata but not in their processes. Our results show that cells expressing vimentin and GFAP (in the radial glia-astrocyte lineage) are heterogeneous dorsoventrally in the walls of the LV. The results suggest that not all RG in the ventral LV complete the transformation into astrocytes and that the ventral SVZ may be functionally dissimilar from the rest of the SVZ.
Cerebral cortex lesions decrease the number of bromodeoxyuridine-positive subventricular zone cells in mice.
We previously showed that cortical lesions in rats increase the number of subventricular zone (SVZ) cells. Here, we examined the response of the SVZ to cortical lesions in mice from 6 h to 35 days later. Whereas the total number of cells did not change, the number of cells in S-phase (bromodeoxyuridine-positive) decreased in a biphasic manner (from 6 h to day 3, and again at days 25-35). In addition, there was a delayed (days 25-35) increase in immunoreactivity for polysialylated neural cell adhesion molecule, a marker of neuroblasts. The results suggest that in mice there are rapid as well as delayed responses in the SVZ to injury of the overlying cerebral cortex. They also show that the SVZ of different mammalian species can exhibit widely divergent responses to the same brain injury.
Subventricular zone cells remain stable in vitro after brain injury.
Subventricular zone (SVZ) cells emigrate toward brain injury but relatively few survive. Thus, if they are to be used for repair, ex vivo expansion and autologous transplantation of SVZ cells may be necessary. Since it is unclear how brain injury alters SVZ cell culture, we studied neurosphere formation, differentiation, and migration, after cortical lesions. The number of neurosphere forming cells from lesioned mice was comparable to controls. Also, the proportion of astrocytes and neurons generated in vitro remained unchanged after cortical lesions. Cell emigration from neurospheres was characterized by increased cell-cell contact after injury in adults and neonates. However, neither molecules implicated in SVZ migration nor the extent of migration changed after injury. Thus, neurospheres can be successfully cultured after extensive brain damage, and they are remarkably stable in vitro, suggesting suitability for ex vivo expansion and autologous transplantation.
Migration patterns of subventricular zone cells in adult mice change after cerebral cortex injury.
The subventricular zone (SVZ) generates the largest number of migratory cells in the adult brain. SVZ neuroblasts migrate to the olfactory bulbs (OB) in the adult, whereas during development, SVZ cells migrate into many adjacent nuclei. Previously, we showed that cerebral cortex injury in the adult causes molecular and cellular changes which may recapitulate the developmental migratory directions. Consistent with this, growth factors, as well as models of illness or injury can cause adult SVZ cells to migrate into non-olfactory bulb nuclei. Here, we tested the hypothesis that cerebral cortex injury in the adult mouse induces changes in migration, by labeling adult SVZ cells with a retroviral vector and examining the distribution of cells 4 days and 3 weeks later. Four days after cortical lesions, disproportionately fewer retrovirally-labeled cells had migrated to the olfactory bulb in lesioned mice than in controls. Conversely, the number of cells found in non-olfactory bulb regions (primarily the area of the lesion and the corpus callosum) was increased in lesioned mice. The morphology of these emigrated cells suggested that they were differentiating into glial cells. Three weeks after cortical injury, the majority of retrovirally-labeled cells in both groups of mice had migrated into the granule and periglomerular layers of the olfactory bulb. At 3 weeks, we still observed retrovirally-labeled glial cells in the corpus callosum and in the area of the injury in lesioned mice. These results suggest that cortical lesions cause a transient change in migration patterns of SVZ progeny, which is characterized by decreases in migration to the olfactory bulb but increased migration towards the injury. Our studies also suggest that cortical lesions induce the production of new glial cells which survive for at least 3 weeks after injury. The data support the concept that in the adult, SVZ cells can generate progeny that migrate towards injured areas and thus potentially be harnessed for neural repair.