Cortical progenitor cells in the developing human telencephalon

Radial glial (RG) cells have been demonstrated to be a major neural progenitor cell type, but in the human fetal brain, neither their molecular nor their spatiotemporal characteristics are well known. We used glial and neuronal-specific antibodies to determine the antigen characteristics and distribution of RG cells and other neuronal progenitors in the human brain during the first half of intrauterine development. Proliferating RG (4A4+) cells in the ventricular zone (VZ) showed clear caudorostral and ventrodorsal gradients, spreading from the spinal cord to the ventral rhombencephalon, at embryonic stages (4.5-5.5 gestational weeks [gw]). However, in the same embryo, other dividing cells expressed the neuronal marker SMI-31 and were present throughout the entire CNS, including the rostral prosencephalon. At the beginning of cortical neurogenesis (6 gw), proliferating VZ cells labeled either with neuronal markers (SMI-31, MAP2, beta-III-tubulin), double-labeled 4A4(+)/SMI-31+ cells, or cells not labeled with these antibodies, were in close proximity to each other. At midgestation (17-24 gw), RG divisions were less frequent, but were spread throughout the entire cerebral cortex, including the subventricular and intermediate zones and the subpial granular layer. Several subtypes of RG were co-labeled with vimentin and other glial markers (BLBP, GFAP, or GLAST) and quantified in vitro. In conclusion, the diversity of cortical progenitors in the human brain may, in part, explain the unique complexity of the human cerebral cortex.     

Distinctive population of Gfap‐expressing neural progenitors arising around the dentate notch migrate and form the granule cell layer in the developing hippocampus

In the adult hippocampus, granule cells continue to be generated from astrocyte‐like progenitors expressing glial fibrillary acidic protein (GFAP) that differ from embryonic neocortical progenitors. However, during the embryonic period, dentate granule neurons and neocortical pyramidal neurons are derived from the ventricular zone (VZ) of the pallium. Our question is when do GFAP+ progenitors of granule neurons appear in the developing hippocampus during the embryonic period, and how do they form the granule cell layer. The present analysis using Gfap‐GFP transgenic mice shows that the GFP+ distinct cell population first appears in the VZ of the medial pallium at the dorsal edge of the fimbria on embryonic day 13.5. During the perinatal period, they form a migratory stream from the VZ to the developing dentate gyrus, and establish the germinal zones in the migratory stream, and the marginal and hilar regions in the developing dentate gyrus. GFP+ cells in these regions were positive for Sox2 and Ki67, but negative for BLBP. GFP+ cells with Neurogenin2 expression were largely distributed in the VZ, whereas GFP+ cells with Tbr2 and NeuroD expressions were seen in the migratory stream and developing dentate gyrus. Prox1‐expressing GFP+ cells were restricted to the developing dentate gyrus. These results suggest that distinctive Gfap‐expressing progenitors arising around the dentate notch form germinal regions in the migratory stream and the developing dentate gyrus where they differentiate into granule neurons, indicating that distinct astrocyte‐like neural progenitors continue to generate granule neurons, from the beginning of dentate development and throughout life. J. Comp. Neurol. 522:261–283, 2014. © 2013 Wiley Periodicals, Inc.     

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ. © 2010 Wiley‐Liss, Inc.     

Selective apoptosis within the rat subependymal zone: a plausible mechanism for determining which lineages develop from neural stem cells

During development, the output of the subventricular zone (SZ) becomes increasingly restricted, yet it still harbors multipotential progenitors. The output of the SZ could be gated by selectively eliminating inappropriately specified progenitors. Using in situ end-labeling (ISEL) to identify apoptotic cells, nearly 60% of the ISEL(+) cells in the juvenile forebrain were localized to the SZ. Of these dying cells, at least 9% could be identified as neurons, 4% as astrocytes, and 12% as oligodendrocytes. The remainder were negative for the stem cell marker nestin, as well as other markers evaluated. To test the hypothesis that committed progenitors were under selective pressures, neural stem/progenitor cells were allowed to differentiate in vitro in the presence or absence of the caspase 3 inhibitor z-DEVD-fmk. DEVD increased neuronal production 10-fold over control cultures. By contrast, the development of astrocytes and oligodendrocytes was not affected. Altogether, these data support the hypothesis that selective forces within the postnatal rat forebrain control the types of precursors that emerge from the germinal matrix. Furthermore, they suggest that different mechanisms control neuronal versus glial cell numbers.     

Multipotential and lineage restricted precursors coexist in the mammalian perinatal subventricular zone

Developmental studies have shown that both neurons and glia arise from the subventricular zone (SVZ) but there have been no clonal analyses to determine whether a single progenitor can produce both. Therefore, we used replication deficient retroviral vectors to analyze the clonal progeny of single rat SVZ cells that were maintained in culture media permissive or non-permissive for neuronal differentiation. When maintained in medium supplemented with 5% fetal bovine serum, all surviving progenitors generated glial cell clones. Within these glial clones we often observed both type 1 astrocytes and O-2A lineage cells. When SVZ cells were maintained in medium permissive for neurogenesis approximately 50% of the total clones contained at least one antigenically defined neuron. Of those clones that contained neurons, 60% contained neurons and glia. The other 50% of the total clones were either comprised of only astrocytes, astrocytes and oligodendrocytes, or were unidentifiable. Since the culture environment permitted multilineage clone formation, yet many homogeneous neuronal or astrocytic clones were obtained, some progenitors must become developmentally restricted while they are in the germinal zone. Therefore, we conclude that the perinatal SVZ is a mosaic of multipotential, bipotential, and lineage restricted precursors, and that the lack of postnatal neocortical neurogenesis is not due to the absence of potential neuroblasts. J. Neurosci. Res. 48:83–94, 1997. © 1997 Wiley-Liss, Inc.     

Clonal mixing, clonal restriction, and specification of cell types in the developing rat olfactory bulb

To understand the clonal relationship of various olfactory bulb (OB) cell types, OB progenitor cells were infected at embryonic day (E) 14, E15, and E17 with retroviral libraries encoding alkaline phosphatase or beta-galactosidase. After survival to postnatal day 10-15, sibling relationships were identified by polymerase chain reaction DNA amplification of distinct sequences in the retroviral constructs. Within the OB, clonal progeny dispersed widely in all directions. In sharp contrast, however, clonal dispersion between the OB and neocortex was not observed, although occasional clonal dispersion between the OB and pyriform and hippocampal regions could not be excluded. Most clones (84%) contained a single cell type, especially after E17 injections, suggesting the existence of either restricted precursors, or multipotential progenitors instructed by a restricted cellular environment. Mixed OB clones (16%) contained multiple cell types in the OB, or occasionally glial or neuronal cells outside the OB, demonstrating the existence of multipotential OB progenitors, likely at a stage before formation of the olfactory rostral migratory stream. Surprisingly, OB glial cells were not labeled, suggesting distinct lineages or perhaps distinct migratory paths for glia and neurons into the OB. A hierarchical cell lineage is proposed that involves a multipotential progenitor that gives rise to potentially more limited progenitors.     

Noggin is a negative regulator of neuronal differentiation in developing neocortex

Bone morphogenetic proteins (BMPs) trigger neuronal differentiation of neocortical precursors within the ventricular zone (VZ) [Li et al. (1998): J Neurosci 18:8853-8862]. BMP-2/4 protein is concentrated at the VZ surface and BMPs rapidly promote the differentiation of neocortical precursors in both dissociated cell and explant cultures. Noggin binds to BMP-2/4 with high affinity, and prevents binding to cell surface receptors. In the present study, we used human recombinant noggin protein to determine whether endogenous BMP-2/4 triggers neuronal differentiation in dissociated cell culture. We find that noggin inhibits the differentiation of neocortical neurons: noggin decreases the number of MAP-2- and TUJ1-positive cells after 24 h of treatment, yet has no effect on either proliferation or cell survival. Noggin also significantly decreases neurite growth of MAP-2-positive cells. In addition, using Western blot analysis we show that noggin protein is present in developing cortex at E15. These results are consistent with previous results showing that endogenous BMPs trigger neuronal differentiation in the neocortical VZ and also indicate that a balance of noggin and BMP may regulate the differentiation of neocortical neurons in vivo.     

Doublecortin-like, a microtubule-associated protein expressed in radial glia, is crucial for neuronal precursor division and radial process stability

During corticogenesis, progenitors divide within the ventricular zone where they rely on radial process extensions, formed by radial glial cell (RG) scaffolds, along which they migrate to the proper layers of the cerebral cortex. Although the microtubule-associated proteins doublecortin (DCX) and doublecortin-like kinase (DCLK) are critically involved in dynamic rearrangement of the cytoskeletal machinery that allow migration, little is known about their role in early corticogenesis. Here we have functionally characterized a mouse splice-variant of DCLK, doublecortin-like (DCL), exhibiting 73% amino acid sequence identity with DCX over its entire length. Unlike DCX, DCL is expressed from embryonic day 8 onwards throughout the early neuroepithelium. It is localized in mitotic cells, RGs and radial processes. DCL knockdown using siRNA in vitro induces spindle collapse in dividing neuroblastoma cells, whereas overexpression results in elongated and asymmetrical mitotic spindles. In vivo knockdown of the DCLK gene by in utero electroporation significantly reduced cell numbers in the inner proliferative zones and dramatically disrupted most radial processes. Our data emphasize the unique role of the DCLK gene in mitotic spindle integrity during early neurogenesis. In addition, they indicate crucial involvement of DCLK in RG proliferation and their radial process stability, a finding that has thus far not been attributed to DCX or DCLK.     

Laminin regulates postnatal oligodendrocyte production by promoting oligodendrocyte progenitor survival in the subventricular zone

The laminin family of extracellular matrix proteins are expressed broadly during embryonic brain development, but are enriched at ventricular and pial surfaces where laminins mediate radial glial attachment during corticogenesis. In the adult brain, however, laminin distribution is restricted, yet is found within the vascular basal lamina and associated fractones of the ventricular zone (VZ)‐subventricular zone (SVZ) stem cell niche, where laminins regulate adult neural progenitor cell proliferation. It remains unknown, however, if laminins regulate the wave of oligodendrogenesis that occurs in the neonatal/early postnatal VZ‐SVZ. Here we report that Lama2, the gene that encodes the laminin α2‐subunit, regulates postnatal oligodendrogenesis. At birth, Lama2?/? mice had significantly higher levels of dying oligodendrocyte progenitor cells (OPCs) in the OPC germinal zone of the dorsal SVZ. This translated into fewer OPCs, both in the dorsal SVZ well as in an adjacent developing white matter tract, the corpus callosum. In addition, intermediate progenitor cells that give rise to OPCs in the Lama2?/? VZ‐SVZ were mislocalized and proliferated nearer to the ventricle surface. Later, delays in oligodendrocyte maturation (with accompanying OPC accumulation), were observed in the Lama2?/? corpus callosum, leading to dysmyelination by postnatal day 21. Together these data suggest that prosurvival laminin interactions in the developing postnatal VZ‐SVZ germinal zone regulate the ability, or timing, of oligodendrocyte production to occur appropriately. © 2012 Wiley Periodicals, Inc.     

Glial Cell Lineages in the Rat Cerebral Cortex

I have traced the fates of glial cell progenitors in the rat cerebral cortex marked with a recombinant retrovirus throughout most of the period of corticogenesis, from embryonic (E) day 14 to postnatal (P) day 14. Discrete clusters of clonally related glia were examined in serially cut sections, and their phenotypes identified using reliable light and electron microscopic criteria. Analysis of a large number of clones marked with retrovirus at various stages of embryonic life contained, with very few exceptions, either all astrocytes or all oligodendrocytes. This observation suggests that the ventricular zone contains separate progenitor cells for the two glial cell types. Oligodendrocyte clones were rarely seen in the cortices injected with retrovirus at the early stages of corticogenesis (E14-E16), suggesting that there is a very small number of oligodendrocyte progenitors in the ventricular zone at these early stages. Their frequency increased significantly at later embryonic ages. At these later stages, ventricular zone cells also give rise to progenitor cells that make up the subventricular zone in early postnatal life. Injections of retrovirus in this proliferative zone shortly after birth resulted in the generation of labeled astrocyte and oligodendrocyte clones in the cortical gray and white matter, with the astrocyte clones being in the majority. Injections at increasingly later stages resulted in the presence, predominantly in the white matter of both hemispheres and in the corpus callosum, of progressively more oligodendrocyte clones and fewer astrocyte clones. Injections at P14 generated only oligodendrocyte clones in the white matter of both hemispheres. A small number of clusters (<10%) generated after subventricular zone injections contained both astrocytes and oligodendrocytes, suggesting that single subventricular zone cells can differentiate into both glial cell types.     

Further characterization of embryonic stem cell-derived radial glial cells

Previously, we showed that radial glia-like (RG) cells differentiated from embryonic stem (ES) cells after retinoic acid induction (Liour and Yu, 2003: Glia 42:109-117). In the present study, we demonstrate that the production of RG cells from ES cells is independent of the neural differentiation protocol used. These ES cell-derived RG (ES-RG) cells are similar in morphology to RG cells in vivo and express several characteristic RG cell markers. The processes of these ES-RG cells are organized into radial arrays similar to the RG scaffold in developing CNS. Expression of Pax6, along with other circumstantial data, suggests that at least some of these ES-RG cells are neural progenitors. The progression of neurogenesis into gliogenesis during the in vitro neural differentiation of ES cells recapitulates the in vivo developmental process. The identification of two cell surface markers, SSEA-1 and GM1, on both the native embryonic RG cells and ES-RG cells, may facilitate purification of radial glial cells for future studies and cell therapy. Overall, our study suggests that differentiation of radial glial cells is a common pathway during the neural differentiation of ES cells.     

BM88 is an early marker of proliferating precursor cells that will differentiate into the neuronal lineage

Progression of progenitor cells towards neuronal differentiation is tightly linked with cell cycle control and the switch from proliferative to neuron-generating divisions. We have previously shown that the neuronal protein BM88 drives neuroblastoma cells towards exit from the cell cycle and differentiation into a neuronal phenotype in vitro. Here, we explored the role of BM88 during neuronal birth, cell cycle exit and the initiation of differentiation in vivo. By double- and triple-labelling with the S-phase marker BrdU or the late G2 and M-phase marker cyclin B1, antibodies to BM88 and markers of the neuronal or glial cell lineages, we demonstrate that in the rodent forebrain, BM88 is expressed in multipotential progenitor cells before terminal mitosis and in their neuronal progeny during the neurogenic interval, as well as in the adult. Further, we defined at E16 a cohort of proliferative progenitors that exit S phase in synchrony, and by following their fate for 24 h we show that BM88 is associated with the dynamics of neuron-generating divisions. Expression of BM88 was also evident in cycling cortical radial glial cells, which constitute the main neurogenic population in the cerebral cortex. In agreement, BM88 expression was markedly reduced and restricted to a smaller percentage of cells in the cerebral cortex of the Small eye mutant mice, which lack functional Pax6 and exhibit severe neurogenesis defects. Our data show an interesting correlation between BM88 expression and the progression of progenitor cells towards neuronal differentiation during the neurogenic interval.     

Contributions of cortical subventricular zone to the development of the human cerebral cortex

The cortical subventricular zone (SVZ), a proliferative compartment in the forebrain, has a uniquely important role during the second half of intrauterine development in human. This is best observed in numerous neonatal pathologies that result from prenatal SVZ damage. These conditions highlight a need to better understand the contribution of the SVZ to the development of the human cerebral cortex. With this goal in mind, we analyzed histological organization, cell proliferation, and molecular diversity in the human fetal SVZ from 7-27 gestational weeks (gw) using light and electron microscopy, immunohistochemistry, and in vitro methods. Complex histological organization distinguishes human cortical SVZ from that of other mammals. In vitro quantification showed that approximately 50% of cells in the VZ/SVZ region are neurons, 30% are astroglia, 15% are nestin+ cells, with other cell types representing smaller fractions. Immunolabeling with BrdU showed that a considerable number of cells ( approximately 10%) are generated in the human cortical SVZ during midgestation (18-24 gw) under in vitro conditions. Immunofluorescence with cell type-specific markers and BrdU revealed that all major cell types, neural precursors (nestin+), astroglia including radial glia (GFAP+, vimentin+), and oligodendrocyte progenitors (PDGFR-alpha+) were proliferating. An increase in the ratio of the size of the SVZ to VZ, protracted period of cell proliferation, as well as cellular and histological complexity of the human fetal SVZ are directly related to the evolutionary expansion of the human cerebral cortex.     

Visualization of embryonic neural stem cells using Hes promoters in transgenic mice

In the central nervous system, neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner, suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist, we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice, GFP expression was restricted to undifferentiated cells in the VZ, which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture, indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development.     

Evidence for astrocyte heterogeneity: a distinct subpopulation of protoplasmic-like glial cells is detected in transgenic mice expressing Lmo1-lacZ

The adult mammalian central nervous system (CNS) contains a large number of different cell types, which arise from the ventricular (VZ) and subventricular zones during embryonic development. In this study, we used a transgenic mouse expressing Lmo1-LacZ from a randomly inserted promoter/reporter gene construct to identify a glial subpopulation. LMO1 is an LIM domain-containing protein, thought to act in protein-protein interactions. We found first that in the adult transgenic CNS, beta-galactosidase (beta-gal) was expressed in a specific subpopulation of protoplasmic-like cells, which did not express detectable levels of glial fibrilary acidic protein unless a lesion was produced. Secondly, during development, beta-gal(+) cells were found arising from discrete regions of the VZ. Taken together, these results identify a subpopulation of protoplasmic glial cells in the adult CNS and suggest that they arise from a restricted VZ region during CNS development.     

Cocaine causes deficits in radial migration and alters the distribution of glutamate and GABA neurons in the developing rat cerebral cortex

Prenatal cocaine exposure induces cytoarchitectural changes in the embryonic neocortex; however, the biological mechanisms and type of cortical neurons involved in these changes are not known. Previously, we found that neural progenitor proliferation in the neocortical ventricular zone (VZ) is inhibited by cocaine; here, we examine the changes in cortical neurogenesis and migration of glutamate and GABA neurons induced by prenatal cocaine exposure. Pregnant rats received 20 mg/kg of cocaine intraperitoneally twice at an interval of 12 h during three periods of neocortical neurogenesis. Neocortical area and distribution of developing neurons were examined by counting Tuj1+, glutamate+, or GABA+ cells in different areas of the cerebral cortex. Cocaine decreased neocortical area by reducing the size of the Tuj1+ layer, but only when administered during early periods of neocortical neurogenesis. The number of glutamatergic neurons was increased in the VZ but was decreased in the outer cortical laminae. Although the number of GABA+ neurons in the VZ of both the neocortex and ganglionic eminences was unchanged, GABA+ cells decreased in all other neocortical laminae. Tangential migration of GABA+ cells was also disrupted by cocaine. These findings suggest that in utero cocaine exposure disturbs radial migration of neocortical neurons, possibly because of decreased radial glia guiding support through enhanced differentiation of neocortical VZ progenitors. Cocaine interrupts radial migration of both glutamatergic and GABAergic neurons within the neocortex, in addition to the tangential migration of GABAergic neurons from the subcortical telecephalon. This may result in abnormal neocortical cytoarchitecture and concomitant adverse functional effects. Synapse 65:21–34, 2011. © 2010 Wiley‐Liss, Inc.     

Tcf7L2 is essential for neurogenesis in the developing mouse neocortex

Generation of neurons in the embryonic neocortex is a balanced process of proliferation and differentiation of neuronal progenitor cells. Canonical Wnt signalling is crucial for expansion of radial glial cells in the ventricular zone and for differentiation of intermediate progenitors in the subventricular zone. We detected abundant expression of two transcrtiption factors mediating canonical Wnt signalling, Tcf7L1 and Tcf7L2, in the ventricular zone of the embryonic neocortex. Conditional knock-out analysis showed that Tcf7L2, but not Tcf7L1, is the principal Wnt mediator important for maintenance of progenitor cell identity in the ventricular zone. In the absence of Tcf7L2, the Wnt activity is reduced, ventricular zone markers Pax6 and Sox2 are downregulated and the neuroepithelial structure is severed due to the loss of apical adherens junctions. This results in decreased proliferation of radial glial cells, the reduced number of intermediate progenitors in the subventricular zone and hypoplastic forebrain. Our data show that canonical Wnt signalling, which is essential for determining the neuroepithelial character of the neocortical ventricular zone, is mediated by Tcf7L2.     

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.     

QKI expression is regulated during neuron-glial cell fate decisions

QKI proteins are expressed by differentiated glia and have been implicated as regulators of myelination, but are also thought to function during early neural development. This study shows that QKI proteins are expressed in neural progenitors of the ventricular zone (vz) during murine CNS development, but that their expression is down-regulated during neuronal differentiation. By contrast, neural progenitors located in specific subdomains of the vz maintain expression of QKI proteins as they differentiate and migrate away into the emerging nervous system. These QKI⁺ cells have characteristics consistent with the acquisition of a glial rather than neuronal fate; they express nestin, incorporate BrdU, fail to express neuronal markers, and similar QKI⁺ cells are found in the postnatal subventricular zone, a known area of gliogenesis. In vitro, neural progenitor cells also down-regulate QKI expression as they differentiate into neurons, but not if they differentiate into glia. Furthermore, neural progenitors in strictly delineated subdomains of the vz dramatically up-regulate expression of the QKI-5 isoform prior to the emergence of QKI⁺ cells from these regions. Taken together, these data indicate that (1) glia are generated from subsets of neural progenitors found in specific, identifiable subdomains of the vz (2) QKI expression is regulated as neural progenitors undergo the neuron-glial cell fate decision and (3) QKI expression is a characteristic of glial progenitors. J. Neurosci. Res. 54:46–57, 1998. © 1998 Wiley-Liss, Inc.