Expression pattern of alpha CaMKII in the mouse main olfactory bulb.

Multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is highly enriched at synapses and has been implicated in regulating the formation and function of several sensory systems, including the visual and the somatosensory systems. Although there is evidence for CaMKII expression in the olfactory system, the cellular localization of CaMKII has not been well studied and its function remains unknown. In this study, we examined the normal expression patterns of the predominant alpha CaMKII in the mouse main olfactory bulb. We showed that alpha CaMKII expression levels were high in the olfactory bulb and were developmentally regulated. Immunoreactivity to alpha CaMKII was heavy in the external plexiform layer and the granule cell layer but minimal in the olfactory nerve layer and the glomerular layer. At the cellular level, alpha CaMKII was selectively expressed in the gamma-aminobutyric acid (GABA)ergic granule cells but not in the GABAergic periglomerular cells. Unexpectedly, alpha CaMKII was not detected in the glutamatergic mitral/tufted cells. At the ultrastructural level, alpha CaMKII immunoreactivity was positive in granule cell spines and dendrites, but negative in mitral/tufted cell dendrites. In contrast, in the piriform cortex, as in the majority of cortical regions, alpha CaMKII was expressed in the glutamatergic neurons but not in the GABAergic neurons. Our results set the stage for ongoing investigations of the roles of CaMKII in the formation and function of the olfactory system.     

Distribution of the protein kinase C substrates MARCKS and MRP in the postnatal developing rat brain

The myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are both membrane-associated phosphoproteins that interact with calmodulin and filamentous actin in a protein kinase C phosphorylation-dependent manner. In the present study, we examined MARCKS and MRP gene expression in the postnatal (P) rat brain (1, 7, 14, 21, and 90 days after birth) by using quantitative in situ hybridization. At P1, MRP expression was high in neocortex, striatum, thalamus, cerebellar cortex, and hippocampus (CA1–CA3, hilus, and granule cell layer) but low in brainstem and, between P7 and P14, exhibited a dramatic decline in each of these regions except hippocampal CA1 and granule cell layers. Between P14 and P21, MRP expression increased in white matter regions including the corpus callosum, fimbria/fornix, and cerebellar deep white matter. At P90 (adult), MRP remained strongly expressed in the olfactory bulb, medial habenula, hippocampal CA1, and the inner two-thirds of granule cell layer, temporal, and entorhinal cortices, the corpus callosum and fimbria/fornix, and cerebellar white matter. At P1, MARCKS was strongly expressed in the majority of brain regions except the brainstem, which subsequently declined gradually to approximate adult levels by P14. Between P14 and P21, MARCKS expression declined gradually in the hilus, remained elevated in hippocampal CA1, CA3, and granule cell layers, and increased dramatically in the corpus callosum and fimbria/fornix. At P90, MARCKS expression declined in hippocampal CA3 and hilus and remained strongly expressed in hippocampal CA1 and granule cell layers, regions of the olfactory bulb, the medial habenula, temporal cortex, and cerebellar granule and Purkinje cells. Expression of both MARCKS and MRP in regions undergoing neuronal proliferation, migration, and neurite outgrowth suggest a common role in these developmental events, whereas differences in expression during development and in the adult brain provide evidence of differential regulation. J. Comp. Neurol. 397:337–356, 1998. © 1998 Wiley-Liss, Inc.     

Regional and cellular localization of the CXCl12/SDF-1 chemokine receptor CXCR7 in the developing and adult rat brain

The chemokine stromal cell-derived factor-1 (SDF-1) regulates neuronal development via the chemokine receptor CXCR4. In the adult brain the SDF-1/CXCR4 system was implicated in neurogenesis, neuromodulation, brain inflammation, tumor growth, and HIV encephalopathy. Until the recent identification of RDC1/CXCR7 as the second SDF-1 receptor, CXCR4 was considered to be the only receptor for SDF-1. Here we provide the first map of CXCR7 mRNA expression in the embryonic and adult rat brain. At embryonic stages, CXCR7 and CXCR4 were codistributed in the germinative zone of the ganglionic eminences, caudate putamen, and along the routes of GABAergic precursors migrating toward the cortex. In the cortex, CXCR7 was identified in GABAergic precursors and in some reelin-expressing Cajal-Retzius cells. Unlike CXCR4, CXCR7 was abundant in neurons forming the cortical plate and sparse in the developing dentate gyrus and cerebellar external germinal layer. In the adult brain, CXCR7 was expressed by blood vessels, pyramidal cells in CA3, and mature dentate gyrus granule cells, which is reminiscent of the SDF-1 pattern. CXCR7 and CXCR4 overlapped in the wall of the four ventricles. Further neuronal structures expressing CXCR7 comprised the olfactory bulb, accumbens shell, supraoptic and ventromedial hypothalamic nuclei, medial thalamus, and brain stem motor nuclei. Also, GLAST-expressing astrocytes showed signals for CXCR7. Thus, CXCR4 and CXCR7 may cooperate or act independently in SDF-1-dependent neuronal development. In mature neurons and blood vessels CXCR7 appears to be the preponderant SDF-1-receptor.     

细胞趋化因子SDF-1α对小胶质细胞趋化作用的研究

目的观察细胞趋化因子SDF-1对小胶质细胞的趋化作用以及机制探讨。方法体外培养BV2细胞系,给予不同浓度SDF-1α,进行迁移实验以确定SDF-1α对小胶质细胞趋化作用的浓度;将C6小鼠分为对照组和SDF-1α干预组,分别予以侧脑室注射PBS和SDF-1α各8周,采用免疫荧光组织化学方法观察注射后小鼠脑内小胶质细胞的数量,进一步采用Western Blot检测小胶质细胞标志物Iba-1的表达水平。结果予以SDF-1干预6 h后BV2细胞的迁移指数增加,SDF-1的受体CXCR4拮抗剂AMD3100能抑制SDF-1所致的小胶质细胞迁移;长程的SDF-1α侧脑室注射能够增加C6小鼠皮层和海马小胶质细胞的数量;Western Blot检测发现SDF-1α干预组脑组织中Iba-1表达量也明显增加。结论 SDF-1在离体和在体实验中均能趋化小胶质细胞的迁移。     

Dynamics of granule cells migration into the internal granular layer in developing human cerebellum

The aim of these investigations was to find out the dynamics of migration of granule cells into the internal granular layer in three different parts of cerebellar cortex. The dynamics of changes was followed in vermis, in lateral hemispheres and in flocculus. For that purpose, 25 fetal brains of different postovulatory age (12.5, 15, 17.5, 19.5, 21, 31 week and one brain of 6 days old newborn) were fixed in formalin, embedded in paraffin, than stained with Cresyl violet and cut in 6, 15 and 30 microm. Each fifth slice was analyzed using the light microscope and numerical density of granule cell nuclei was established by point counting method according to Weibel. Results show a steady increase of the granule cell population in the internal granular layer of all regions investigated, being still greater in vermis and in hemispheres. This increase in the cell number lasted during the whole period of intrauterine development. Moreover, according to the trend of the curves, the increase of the cell number continues even after the birth, suggesting that at least some synaptic connections of granule cells are not established yet. For each region the adequate mathematical model of migration dynamics was calculated.     

Role of the alpha-chemokine stromal cell-derived factor (SDF-1) in the developing and mature central nervous system

alpha-chemokines, which control the activation and directed migration of leukocytes, participate in the inflammatory processes in host defense response. One of the alpha-chemokines, CXCL12 or stromal cell-derived factor 1 (SDF-1), not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development as we discuss here. SDF-1 indeed activates the CXCR4 receptor expressed in a variety of neural cells, and this signaling results in diverse biological effects. It enhances migration and proliferation of cerebellar granule cells, chemoattracts microglia, and stimulates cytokine production and glutamate release by astrocytes. Moreover, it elicits postsynaptic currents in Purkinje cells, triggers migration of cortical neuron progenitors, and produces pain by directly exciting nociceptive neurons. By modulating cell signaling and survival during neuroinflammation, SDF-1 may also play a role in the pathogenesis of brain tumors, experimental allergic encephalitis, and the nervous system dysfunction associated with acquired immunodeficiency syndrome.     

Cerebellar histogenesis in the lurcher (Lc) mutant mouse

The postnatal development of the cerebellar cortex of normal and lurcher (Lc) mutant mice was studied by neurohistological and autoradiographic techniques at ages ranging from 2 days through 18 days after birth. Lurcher shows severe defects in the granule cells and Purkinje cells soon after birth. A decrease in the generative layers of the external granular layer is soon as early as two days in the lobulus simplex and by six days of age in the uvula. Granule cell death is common before and during granule cell migration, from 2 to 18 days of age. Loss of granule cells is reflected in reduced growth of the molecular and granular layers. Purkinje cell abnormalities appear at three to four days after birth in the form of crowding, failure of nuclear growth, and condensed or lessened cytoplasm. Purkinje cell death is apparent at four to six days of age depending on the region of the cerebellum.     

Cellular migration in the postnatal rat cerebellar cortex: confocal-infrared microscopy and the rapid Golgi method

Confocal laser microscopy of DiI-labeled slices of postnatal rat cerebellum (postnatal Day 4-10; P4-10) was compared to infrared microscopy and the rapid Golgi method (P0-14) to investigate postnatal migration of granule neurons. Vertical migration of the granule neurons occurred already at birth (P0). Surprisingly, mossy fibers often reached the external granule cell layer and were in close contact with the external granule cells. These mossy fibers may play a role in initiating granule cell migration. At this age, cell bodies of the immature neurons were attached to the external basal lamina by a process and extended down toward the presumptive internal granule cell layer. At P14, some granule cells remained attached to the surface, although their cell bodies exhibited the typical morphology of mature granule neurons and were located deep in the internal granule cell layer. These cells extended their endfeet-like processes all the way to the surface of the brain. These results indicate that the vertical pathways of granule cell migration form early and persist throughout the period of granule cell migration. Confocal infrared microscopy of DiI-labeled sections and the rapid Golgi method also allowed demonstration of tangentially migrating neurons that made one or more turns on the way to the internal granule cell layer. The rapid Golgi method confirmed that many Bergmann glial processes end at the level of the tangentially migrating granule cells whereas others project to the surface. These observations show that migratory granule cells take several different routes to their final destination, which cannot be explained by so-called radial glial guidance. The only mode of migration in evidence is consistent with process elongation and translocation of the nucleus within the preformed processes.     

Expression of Rho GTPases Rho-A and Rac1 in the adult and developing gerbil cerebellum

Rho GTPases proteins are essential for cytoskeletal reorganization and play important roles in the development of neuronal dendrites and axons. Several studies have implicated two members of the Rho GTPase family Rho-A and Rac1 activities in the neuronal polarization and the formation of axons and dendrites. In order to correlate cellular expressions of Rho-A and Rac1 with neuronal polarity (axons versus dendrite formation) in the central nervous system, the cerebellum and immunochemical techniques have been chosen. In the adult cerebellar cortex differential pattern of distribution between Rho-A and Rac1 was observed. While Rac1 expression was restricted to Purkinje cell (somata, dendrites and axons), Rho-A was ubiquitously distributed within the cerebellar cortex. Rac1 was localized in the Purkinje cell dendritic arborization (largest and tiny dendrites) and in their axons. This pattern of distribution was also observed during the postnatal development and followed the dendritic morphogenesis of Purkinje cell. Rho-A was highly expressed in the adult Purkinje cells somata, in cells of the granular layer, in glia within the white matter and in axons. Intense staining was observed in Bergmann glia cell bodies and processes. In the developing cerebellum, Rho-A was highly present in cells of the external and internal granule layers and in the Purkinje cell layer. Bergmann glia cell bodies and processes had the most intense staining during the development. The present study reveals a high expression of Rac1 and Rho-A during Purkinje cell neurites outgrowth period which occurred after birth in the cerebellum. In addition Rho-A is highly expressed in granule cell progenitor cells present in the external granular layer and therefore may play an important role in granule cell progenitor migration.     

Developmental expression of GABA and subunits of the GABAA receptor complex in an inhibitory synaptic circuit in the rat cerebellum

The temporal relationship between the expression of a transmitter and its corresponding receptor may provide important insights into the development of synaptic circuits in the central nervous system. Here we examined the emergence of the inhibitory transmitter GABA, and subunits of the GABAA/benzodiazepine receptor complex in a well-characterized cerebellar circuit formed by granule cells and the synapses they make with Golgi II neurons in the cerebella of rats ranging in age from birth to 21 days. The presence of GABA was determined immunocytochemically. The presence of the GABAA receptor was demonstrated by localizing the alpha 1 subunit of the receptor using in situ hybridization and immunochemical localization of a 50 kDa benzodiazepine-binding subunit using monoclonal antibodies. Germinal cells of the external granular layer which give rise to granule cells did not express the GABAA receptor at any age. Similarly, receptor labeling could not be detected in granule cells during their postmitotic migratory period. In the internal granular layer, immature postmigratory granule cells are unlabeled. The expression of GABAA receptor subunits was first observed on the fifth postnatal day (P5) and then only in the more mature granule cells which have well elaborated dendrites in contact with presynaptic elements. The number of labeled neurons increased over the subsequent ages examined. Presynaptic elements in association with the dendrites of labeled granule cells had ultrastructural features characteristic of Golgi II cell axon terminals. These elements demonstrate GABA transmitter as early as P3, preceded by 2-3 days receptor labeling in the granular layer. Therefore, granule cells express GABAA receptor subunits only after they have completed migration and their dendrites have become involved in specific synaptic circuits, including innervation by GABAergic afferents.     

PSA-NCAM immunocytochemistry in the cerebral cortex and other telencephalic areas of the lizard Podarcis hispanica: differential expression during medial cortex neuronal regeneration

The lizard medial cortex, a region homologous to the mammalian dentate gyrus, shows postnatal neurogenesis and the surprising ability to replace its neurons after being lesioned specifically with the neurotoxin 3-acetylpyridine. As the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is expressed during neuronal migration and differentiation, we have studied its distribution in adult lizards and also during the lesion-regeneration process. In the medial cortex of control animals, many labeled fusiform somata, presumably corresponding to migratory neuroblasts, appeared in the inner plexiform layer. There were also scattered immunoreactive granule neurons in the cell layer. Double immunocytochemistry with 5'-bromodeoxyuridine revealed that some of the PSA-NCAM-expressing cells in the inner plexiform and cell layers were generated recently. PSA-NCAM immunoreactivity was also present in the dorsomedial, dorsal, and lateral cortices, as well as in the dorsal ventricular ridge, the nucleus accumbens, and the nucleus sphericus. Twelve hours after the injection of 3-acetylpyridine, some medial cortex granule neurons appeared degenerated, although some of them still expressed PSA-NCAM. One to 2 days after the injection, most granule neurons appeared degenerated and no PSA-NCAM immunoreactivity was detected in the medial cortex cell layer. Four to 7 days after treatment, abundant labeled fusiform cells populated the inner plexiform layer and some immunoreactive somata were seen in the cell layer. Fifteen to 30 days after the neurotoxin injection, the number of PSA-NCAM expressing granule neurons augmented considerably and the level was still above control levels in lizards that survived 42 days. Our results show for the first time the expression of PSA-NCAM in a reptile brain, where it appears to participate in the migration and differentiation of granule neurons during adult neurogenesis and regeneration.     

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.     

Expression and function of ganglioside 9-O-acetyl GD3 in postmitotic granule cell development

We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-O-acetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dose-dependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.