110 years from the appearance of the first bacteriology treatise and other priorities of Victor Babe?

The mouse Engrailed-2 gene, En-2, appears to be involved in cerebellar pattern formation. Homozygous null mutants for En-2 have abnormal foliation patterns in the posterior half of the cerebellum and there are changes in Purkinje and granule cell gene expression in some posterior folia, possibly reflecting changes in cell identity. We have examined the distribution of spinocerebellar mossy fiber terminals in homozygous En-2hd null mutants to determine if En-2 is involved in regulating the pattern of afferent connectivity in the cerebellum. Spinocerebellar mossy fiber terminals were labeled following WGA-HRP injections in the lumbar region of 5 homozygous En-2hd mutants and 4 heterozygous controls. The distribution of spinocerebellar mossy fiber terminals was consistently altered in lobules VIII and IX of the En-2hd mutants. The principal changes were a reduction in the number of mossy fiber terminal fields in the dorsal aspect of lobule VIII and the dorsal midline field in lobule IX was fused into a single compartment. The results suggest that the deletion of En-2 expression does not transform lobule identity, at least with respect to afferent fiber positional information cues. However, the changes in foliation and afferent connectivity in the En-2 mutant support a broad role for the En-2 gene in cerebellar patterning.     

Synchronization of golgi and granule cell firing in a detailed network model of the cerebellar granule cell layer

The granular layer of the cerebellum has a disproportionately large number of excitatory (granule cells) versus inhibitory neurons (Golgi cells). Its synaptic organization is also unique with a dense reciprocal innervation between granule and Golgi cells but without synaptic contacts among the neurons of either population. Physiological recordings of granule or Golgi cell activity are scarce, and our current thinking about the way the granular layer functions is based almost exclusively on theoretical considerations. We computed the steady-state activity of a large-scale model of the granular layer of the rat cerebellum. Within a few tens of milliseconds after the start of random mossy fiber input, the populations of Golgi and granule cells became entrained in a single synchronous oscillation, the basic frequency of which ranged from 10 to 40 Hz depending on the average rate of firing in the mossy fiber population. The long parallel fibers ensured, through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-mediated synapses, a coherent excitation of Golgi cells, while the regular firing of each Golgi cell synchronized all granule cells within its axonal radius through transient activation of their gamma-aminobutyric acid-A (GABAA) receptor synapses. Individual granule cells often remained silent during a few successive oscillation cycles so that their average firing rates, which could be quite variable, reflected the average activities of their mossy fiber afferents. The synchronous, rhythmic firing pattern was robust over a broad range of biologically realistic parameter values and to parameter randomization. Three conditions, however, made the oscillations more transient and could desynchronize the entire network in the end: a very low mossy fiber activity, a very dominant excitation of Golgi cells through mossy fiber synapses (rather than through parallel fiber synapses), and a tonic activation of granule cell GABAA receptors (with an almost complete absence of synaptically induced inhibitory postsynaptic currents). These three conditions were associated with a reduction in the parallel fiber activity, and synchrony could be restored by increasing the mossy fiber firing rate. The model predicts that, under conditions of strong mossy fiber input to the cerebellum, Golgi cells do not only control the strength of parallel fiber activity but also the timing of the individual spikes. Provided that their parallel fiber synapses constitute an important source of excitation, Golgi cells fire rhythmically and synchronized with granule cells over large distances along the parallel fiber axis. According to the model, the granular layer of the cerebellum is desynchronized when the mossy fiber firing rate is low.     

Developmental changes in human cerebellum: expression of intracellular calcium receptors, calcium-binding proteins, and phosphorylated and nonphosphorylated neurofilament protein

Few recent data are available on the development of the precise projection maps of the cerebellar cortex in humans. To address this topic, we studied temporal and spatial distribution of several antigens involved in calcium (Ca)-dependent processes: the intracellular Ca receptors, inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR); the Ca-binding proteins, calbindin D-28k (CB), parvalbumin (PV), and synaptophysin; and phosphorylated (SMI 31) and nonphosphorylated (SMI 32) forms of neurofilament protein. All antigens were studied in the human cerebellum during intrauterine development. The results of this study show that immunocytochemical markers appeared in the following sequence: CB and both forms ofneurofilament protein were observed at 4-5 gestational weeks (g.w.), PV appeared in the external granular layer and in a few Purkinje cells at 11 g.w., a diffuse immunostaining for IP3R1 and synaptophysin were observed at 13 g.w., whereas RyR was observed at 17-18 g.w. From 24 g.w. on, Purkinje cells expressed all four examined markers of intracellular Ca signaling as well as two forms of neurofilament protein. At the same time, compartmentation of the Purkinje cell layer was detected with three intracellular Ca-signaling molecules (IP3R1, CB, and PV) and with SMI 32. These results indicate that the developmentally regulated expression of antigens studied here may play a role in establishing a highly regular organization of terminal fields in the human cerebellar cortex. Moreover, the initial expression of these antigens is correlated temporally with other developmental processes in the cerebellum, such as cellular maturation, revealed by the immunoreaction to cytoskeletal protein, and synaptogenesis, revealed by immunoreaction to synaptophysin.     

Control of spine formation by electrical activity in the adult rat cerebellum

Dendritic spines are a key structure in neuronal plasticity. Enhanced activity is commonly associated with an increase in spine size and density. Purkinje cell dendrites are characterized by a proximal and a distal compartment on which climbing fibers and parallel fibers, respectively, impinge. The proximal region has a very low spine density, whereas the distal region has a high density. Previous experiments showed that after climbing fiber deletion, Purkinje cells become hyperactive, and a large number of spines develop on the proximal dendrites. Here we show that the same hyperspiny transformation occurs in the proximal dendrites of adult Purkinje cells by depressing electrical activity with tetrodotoxin. Thus, spines in different dendritic compartments are created or maintained independently from the level of Purkinje cell-firing rate and when the afferent activity is blocked. This conclusion supports the view that spinogenesis is the expression of an intrinsic program and the two regions of the dendritic tree respond differently to activity block because of differences in the inputs that they receive. On tetrodotoxin treatment, climbing fibers become atrophic and may sprout thin collateral ramifications directed mainly toward the granular layer. All changes are reversible on tetrodotoxin removal. Therefore, Purkinje cells provide a model where spines in different compartments of the same neuron are differently regulated by the activity of their local afferents. In addition, electrical activity is also essential to maintain the full climbing fiber innervation.     

Membrane-fusions and cytoplasmic bridges in the cells of the developing cerebellum

In the developing cerebellum of the neonate rats membrane-fusions and cytoplasmic bridges between cells were observed. These membrane-fusions were characterized by the presence of loops of membrane and cytoplasmic bridges between the two limits of the membrane-fusions. They were found between Purkinje cells, Purkinje cells and the migratory cells, mitotically potent cells of the external granular layer, and differentiating granule cells of the internal granular layer. The membrane-fusions were found to be a transient developmental phenomenon. Issues pertaining to the universality of membrane-fusions, their significance in the induction for cell differentiation, and the problem of fixation artifacts are discussed.     

Patches of synchronized activity in the cerebellar cortex evoked by mossy-fiber stimulation: questioning the role of parallel fibers

The discrepancy between the structural longitudinal organization of the parallel-fiber system in the cerebellar cortex and the functional mosaic-like organization of the cortex has provoked controversial theories about the flow of information in the cerebellum. We address this issue by characterizing the spatiotemporal organization of neuronal activity in the cerebellar cortex by using optical imaging of voltage-sensitive dyes in isolated guinea-pig cerebellum. Parallel-fiber stimulation evoked a narrow beam of activity, which propagated along the parallel fibers. Stimulation of the mossy fibers elicited a circular, nonpropagating patch of synchronized activity. These results strongly support the hypothesis that a beam of parallel fibers, activated by a focal group of granule cells, fails to activate the Purkinje cells along most of its length. It is thus the ascending axon of the granule cell, and not its parallel branches, that activates and defines the basic functional modules of the cerebellar cortex.     

Cerebellar nitric oxide synthase is expressed within granule cell patches innervated by specific mossy fiber terminals: a developmental profile

The nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining technique was utilized as a marker of nitric oxide synthase (NOS) to map NOS expression in developing and adult rat cerebellum. NADPH-d-positive cells were first visualized in the cerebellar cortex at postnatal day 5 (PND5) which increased to peak levels by PND30 when they began to exhibit a patch-like organization. In order to determine the relationship of the NADPH-d staining pattern with mossy fiber innervation, mossy fiber projections were traced using cholera toxin B subunit or biocytin injected into the lateral reticular nuclei (LRtN) or pontine nuclei (PtN), respectively. Double staining revealed that the clustered mossy fiber terminals projecting from the ventrorostral LRtN and caudal PtN were well matched with NADPH-d-stained patches. This patch-like localization of NOS matched with specific mossy fiber terminals in adult cerebellum implicates these NOS patches as defining distinct anatomical zones.     

Parallin, a cerebellar granule cell protein the expression of which is developmentally regulated by Purkinje cells: evidence from mutant mice

In this paper we report on monoclonal antibody 3H6 with unique specificities for development of the cerebellum. Immunohistochemical studies on normal and mutant mice suggest that it is primarily located in or on granule cell parallel fibers in the cerebellum. The only other region showing immunoreactivity is a small region of the hippocampus. The antigen is detected immunohistochemically as early as postnatal day 11 in the molecular layer of the cerebellum. In adult wild-type mice parallin expression is seen in the molecular layer and to a lesser degree in the internal granular layer. In the cerebella of two neurological granule cell-deficient mutants, weaver (wv) and staggerer (sg), parallin is not detected. However, in two Purkinje cell-deficient mutants, Purkinje cell degeneration (pcd) and nervous (nr), a more complex and interesting pattern is observed. These two mutants do have granule cells and parallel fibers and 3H6 immunoreactivity is observed. However, in both of these Purkinje cell-deficient mutants the 3H6 immunoreactivity is drastically reduced in regions where Purkinje cells have degenerated. Furthermore, in nr mutants, the antigen appears to be concentrated in regions of the parallel fiber that are in close proximity to Purkinje cells, suggesting its possible association with synapses. Taken together these results suggest that parallin is a marker of granule cells and their parallel fibers, its onset correlates with the formation of granule cell synapses on developing Purkinje cells, and it requires Purkinje cells for the maintenance of expression.     

Profiles in discouragement: two studies of variability in the time course of smoking withdrawal symptoms

A mouse homolog of the Drosophila Disabled (dab) gene, disabled-1 (mdab1), encodes an adaptor molecule that functions in neural development. Targeted disruption of the mdab1 gene (mdab1-1 mice) leads to anomalies in the development of the cerebrum, hippocampus, and cerebellum. Here we describe a number of histologic abnormalities in the cerebellum of the mdab1-1 mouse. There is a complete absence of foliation, and most Purkinje cells are clumped in central clusters. However, lamination appears to develop normally in areas where the Purkinje cells and external granular layer are closely apposed. The granular layer forms a thin rind over most of the cerebellar surface, but is subdivided by both transverse and parasagittal boundaries. The Purkinje cells, identified by anti-zebrin II in the adult or anti-calbindin in the new born mdab1-1 mutant cerebellum, form a parasagittal banding pattern, similar to but distorted compared with the wild-type design. The data suggest that the development of the mdab1-1 cerebellum parallels the development of reeler. The reeler gene encodes an extracellular protein (Reelin) that is secreted by the external granular layer. Because Reelin expression is retained in the mdab1-1 mutant mouse, mDab1 p80 may act in a parallel pathway or downstream of Reelin, leading to the transformation of embryonic Purkinje cell clusters into the adult parasagittal bands.     

From zebra stripes to postal zones: deciphering patterns of gene expression in the cerebellum

The analysis of patterned gene expression has been an important tool for dissecting the molecular and developmental bases of functional compartmentalization in the mammalian cerebellum. In particular, sagittally-oriented cellular aggregates arranged along the mediolateral axis are the patterning element most commonly invoked to illustrate cerebellar compartmentalization, and these are revealed both by patterns of afferent projection and by a number of classical biochemical markers that are distributed in a pattern of'zebra stripes'. Compartmentation along both the mediolateral and rostrocaudal axes might be linked mechanistically to segmentation in the fruit fly, since early cerebellar development is especially dependent upon the expression of mammalian homologs of Drosophila segmentation genes. In addition, as has been demonstrated in the retinotectal system, some of these genes are likely to control positional information required for the sagittal organization of cerebellar afferent projections. However, in contrast to these global or macro zones, the cerebellum is also compartmentalized at the subcellular or micro level. This can be visualized by differential patterns of mRNA distribution within the sole cerebellar efferent system, the Purkinje cell, defining within such cells a number of distinct subcellular domains or 'postal zones'. The global versus subcellular levels of cerebellar compartmentalization are related since they both appear to be linked to patterns of afferent innervation.A major goal of cerebellar research will be to unravel the true nature of such a relationship, and its relevance to function and behavior.     

Regionalization defects in the weaver mouse cerebellum

The mammalian cerebellum consists of parasagittal bands and transverse zones that are laid down early in development. When the adult cerebellum is immunostained for the Purkinje cell-specific antigen zebrin II (i.e., aldolase C), compartmentation is reflected in alternating zebrin II+ (P+) and zebrin II- bands (P ). The zebrin II phenotype is Purkinje cell autonomous; thus, disruptions in the zebrin pattern may reflect early problems in pattern formation. Zebrin II expression has been examined in the weaver (wv) mouse cerebellum. Both zebrin II- and zebrin II Purkinje cells are present in the homozygous weaver (wv/wv) mouse, but they are not distributed normally. In the posterior vermis, although the zebrin II+ bands are wider and multilaminate, the standard compartmentation is present. However, a large zebrin II+ cell mass is absent from the central vermis, and analysis of the anterior lobe reveals several missing zebrin II- bands. The cytoarchitectonic defects in wv mice are not simply related to the Purkinje cell abnormalities. Instead, serial reconstruction reveals two transverse boundaries-one rostrally in lobule VI and the other caudally in lobule IX-that delineate cytoarchitectonic transverse zones important in cerebellar development. The abnormal zebrin expression pattern in wv/wv mice may be secondary to the deletion of a transverse zone. This is the first demonstration that Purkinje cell compartmentation can be altered by mutation; therefore, the wv mutation should prove valuable in understanding cerebellar regionalization.     

Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum

In the developing chicken cerebellar cortex, three cadherins (Cad6B, Cad7, and R-cadherin) are expressed in distinct parasagittal segments that are separated from each other by ribbons of migrating interneurons and granule cells which express R-cadherin and Cad7, respectively. The segment/ribbon pattern is respected by the expression of other types of molecules, such as engrailed-2 and SC1/BEN/DM-GRASP. The cadherin-defined segments contain young Purkinje cells which are connected to underlying nuclear zones expressing the same cadherin, thereby forming parasagittal cortico-nuclear zones of topographically organized connections. In addition, R-cadherin-positive mossy fiber terminals display a periodic pattern in the internal granular layer. In this layer, Cad7 and R-cadherin are associated with synaptic complexes. These results suggest that cadherins play a pivotal role in the formation of functional cerebellar architecture by providing a three-dimensional scaffold of adhesive information.     

Organization and histochemical phenotype of human fetal cerebellar cells following transplantation into the cerebellum of nude mice

Previous rodent studies have demonstrated the capacity of cerebellar transplants to organize into trilaminar cell layers typically observed in the normal cerebellum. In Purkinje Cell (PC)-deficient animals, PCs will migrate into the host and form synaptic connections. Recently, fetal cerebellar grafts transplanted into the Purkinje cell degeneration (pcd) mutant mouse were shown to result in an improvement of motor behaviors. These studies indicate the potential therapeutic use of neural transplantation in patients with cerebellar degeneration. In the present study, human fetal cerebellar tissue (8.5 wk postconception) was dissociated and transplanted into the normal cerebellum of nude mice. Six months following transplantation, histological analysis revealed donor cells in recipient mice. Immunostaining for the 28 kDa calcium-binding protein (calbindin) revealed the presence of donor PCs that were organized in discrete cellular layers within the transplant neuropil. In most cases the dendritic processes were oriented in a planar fashion perpendicular to the transplant cell layer. Human neurofilament immunostaining revealed bundles of donor fibers within the core of the transplant and/or at the periphery. These bundles were found to be calbindin positive (PC fibers). Three animals provided evidence of donor PC axon growth ventrally into host white matter, and in one case, this ventral migration reached the deep cerebellar nuclei. Most notable was the development of a pronounced folia-like organization by the implanted cell suspensions. Glial processes within the grafts were aligned perpendicular to the long axis of the transplant folia. These results demonstrate the capacity of human fetal cerebellar cell suspension to reorganize into cell layers typical of the normal cerebellum following transplantation into the rodent cerebellum, and develop an organotypic folia-like organization.     

Localized deposition of M-cadherin in the glomeruli of the granular layer during the postnatal development of mouse cerebellum

M-cadherin is a Ca2+-dependent cell adhesion molecule of the cadherin family, initially localized at the areas of contact between myotubes during myogenesis, but also detected in the peripheral nerve and at the adult neuromuscular junction. In this study, searching for the expression of M-cadherin in the adult mouse brain, we observed a restricted expression of M-cadherin in one of the three layers of the cerebellar cortex: the granular layer. M-cadherin was accumulated in structures rich in synapses and other intercellular junctions where mossy fibers connect granule cell dendrites, the glomeruli. This molecule was not expressed in the cerebellum during the first steps of postnatal cerebellar neurogenesis: granule cell proliferation and migration and Purkinje cell alignment. M-cadherin expression was first detected at postnatal day (P) 11, after the establishment of the synaptic connections between mossy fibers and granule cell dendrites. It then accumulated in glomeruli during their phase of maturation which is characterized by the formation of puncta adherentia between granule cell dendrites. M-cadherin was undetectable in the cerebella of the weaver and staggerer mutants, lacking granule cells, and therefore mature glomeruli and puncta adherentia. Furthermore, other components classically associated with intercellular junctions, i.e., alpha-caterin, beta-catenin and actin filaments, closely paralleled M-cadherin appearance and colocalized with M-cadherin in the mature glomeruli. M-cadherin, which appears as a molecular marker of glomerulus maturation, might be implicated in the formation, and be the ligand, of adherens junctions encountered in this structure.     

Localization of metallothionein-I and -III expression in the CNS of transgenic mice with astrocyte-targeted expression of interleukin 6

The effect of interleukin-6 (IL-6) on metallothionein-I (MT-I) and MT-III expression in the brain has been studied in transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), which develop chronic progressive neurodegenerative disease. In situ hybridization analysis revealed that GFAP-IL6 (G16-low expressor line, and G36-high expressor line) mice had strongly increased MT-I mRNA levels in the cerebellum (Purkinje and granular layers of the cerebellar cortex and basal nuclei) and, to a lesser degree, in thalamus (only G36 line) and hypothalamus, whereas no significant alterations were observed in other brain areas studied. Microautoradiography and immunocytochemistry studies suggest that the MT-I expression is predominantly localized to astrocytes throughout the cerebrum and especially in Bergman glia in the cerebellum. However, a significant expression was also observed in microglia of the GFAP-IL6 mice. MT-III expression was significantly increased in the Purkinje cell layer and basal nuclei of the cerebellum, which was confirmed by Northern blot analysis of poly(A)+ mRNA and by ELISA of the MT-III protein. In contrast, in the G36 but not G16 mice, transgene expression of IL-6 was associated with significantly decreased MT-III RNA levels in the dentate gyrus and CA3 pyramidal neuron layer of the hippocampus and, in both G36 and G16 mice, in the occipital but not frontal cortex and in ependymal cells. Thus, both the widely expressed MT-I isoform and the CNS specific MT-III isoform are significantly affected in a MT isoform- and CNS area-specific manner in the GFAP-IL6 mice, a chronic model of brain damage.     

Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: a possible mechanism underlying GABA's excitatory role in immature brain

An inhibitory neurotransmitter in mature brain, gamma-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl- accumulation in immature neurons and oligodendrocytes.     

The identification of mossy fibres and their cells of origin in the normal and Lurcher mutant mouse

The behavioural mutant mouse Lurcher survives to adult life as the heterozygote (Lc/+) and shows a disorder of gait. The neurological lesion has been shown to involve degeneration of Purkinje cells and inferior olivary neurones (Caddy and Biscoe 1976). It follows that the climbing fibre input is reduced and we wished to know if the mossy fibre input was also affected. Heterozygote Lurcher mutants were compared with the wild type in all experiments. Mossy fibre glomeruli were identified in the cerebellum of the mutant mouse by electron microscopy. Horseradish peroxidase (HRP) was injected into the cerebellum and was found in the cells of Clarke's column in the spinal cord. Electrophysiological experiments showed that following stimulation of the sciatic nerve evoked responses could be recorded in the cerebellum. It is concluded that the mossy fibre input to the cerebellum is intact in the Lurcher mutant mouse.     

Modular organization of occipito-temporal pathways: cortical connections between visual area 4 and visual area 2 and posterior inferotemporal ventral area in macaque monkeys

The modular organization of cortical pathways linking visual area 4 (V4) with occipital visual area 2 (V2) and inferotemporal posterior inferotemporal ventral area (PITv) was investigated through an analysis of the patterns of retrogradely labeled cell bodies after injections of tracers into V4 and PITv. Although cytochrome oxidase or other stains have failed to yield reliable independent anatomical markers for cortical modules beyond V1 and V2, V4 and PITv seem to have modular compartments with specific patterns of cortico-cortical connectivity. Tracer injections of V4 labeled cells in V2 (1) thin stripes exclusively, (2) interstripes exclusively, or (3) specific combinations of interstripe and thin stripe subcompartments. These labeling patterns suggest (1) that there is a complicated organization of inputs to V4, (2) that projections from V2 to V4 display a submodular selectivity, and (3) that projections from V2 to V4 display some degree of cross-stream convergence. Consistent with this framework, extensive regions of PITv provide feedback projections to interstripe-recipient portions of V4, whereas more restricted portions of PITv provide feedback to thin stripe-recipient portions of V4. Similarly, the feedforward projection from V4 to PITv often arose from multiple cell clusters across a wide expanse of V4. When distinguishable fluorescent tracers were injected into two PITv sites separated by 3-5 mm, a variety of projection patterns was observed in V4. In most cases, labeled cells were found in multiple, interdigitating, nonoverlapping clusters of 1-3 mm width, whereas in other cases the two labeled fields were highly intermixed. These results suggest that V4 and PITv contain functional modules that can be characterized by the specific patterns of segregated and convergent projections they receive from lower cortical areas. These specific patterns of intercortical input, in conjunction with intrinsic cortical circuitry, may endow extrastriate cortical neurons with new and more complex receptive field properties.     

Distribution of a reeler gene-related antigen in the developing cerebellum: an immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice

We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.     

Role of arachidonic acid and its metabolites in the priming of NADPH oxidase in human polymorphonuclear leukocytes by peritoneal dialysis effluent

Immunohistochemical techniques were used to examine the distribution of prostaglandin H synthase (PGHS)-2 and neuronal nitric oxide synthase (nNOS) in piglet brain. Samples from parietal cortex, hippocampus, and cerebellum were immersion fixed in 10% formalin, sectioned at 50 microm, and immunostained using specific antibodies against PGHS-2 and nNOS. Immunoreactivity for PGHS-2 was extensive throughout the areas examined. For example, PGHS-2 immunoreactive cells were present in all layers of the cortex, but were particularly dense among neurons in layers II/II, V, and VI. In addition, glial cells associated with microvessels in white matter showed PGHS-2 immunoreactivity. In contrast, nNOS immunoreactive neurons were limited in number and widely dispersed across all layers of the cortex and thus did not form a definable pattern. In the hippocampus, heavy PGHS-2 immunoreactivity was present in neurons and glial cells in the subgranular region, stratum radiatum, adjacent to the hippocampal sulcus, and in CA1 and CA3 pyramidal cells. Immunostaining for nNOS displayed a different pattern from PGHS-2 in the hippocampus, and was mainly localized to the granule cell layer of the dentate gyrus and the mossy fiber layer. In the cerebellum, PGHS-2 immunoreactivity was heavily represented in the Bergmann glia and to a lesser extent in cells of the granular layer, whereas nNOS was detected only in Basket cells. There are four conclusions from this study. First, PGHS-2 immunoreactivity is widely represented in the cerebral cortex, hippocampus, and cerebellum of neonatal pigs. Second, glia cells as well as neurons can show immunoreactivity for PGHS-2. And third, the distribution of nNOS is different from PGHS-2 immunoreactivity in the cerebral cortex, hippocampus, and cerebellum.