Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.     

Cerebellar granule cell differentiation in mutant and X-irradiated rodents revealed by the neural adhesion molecule TAG-1

In the external granular layer of the cerebellum, the granule cell precursors express the transient axonal glycoprotein TAG-1, a molecule involved in adhesion and neurite outgrowth. Granule cells express TAG-1 transiently, just as they extend neurites before migrating over the radial glia. The present study aims to investigate whether the expression pattern of TAG-1 is altered when granule cells develop abnormally. We studied in vivo models in which Purkinje and/or granule cell defects occur during postnatal development. These include the cerebellar mutant mice staggerer and lurcher as well as rats irradiated during postnatal development. Neither alterations in Purkinje cell differentiation nor the related granule cell loss in the mouse mutants impairs the ability of the surviving granule cell precursors to express TAG-1. Also, early granule cell loss in the X-irradiated rats do not disturb the TAG-1 expression phase in the patches of surviving granule cell precursors. Ectopic granule cells found in the adult cerebellum of X-irradiated rats do not bear the molecule, although they are located in the most superficial part of the molecular layer, occupied by the immunopositive cells a few days earlier. Thus, TAG-1 marks a very precise stage of granule cell differentiation, and the inward migration process itself is not required for the cessation of the expression. We postulate that TAG-1 may be involved in local differentiation steps restricted to the deep external granular layer such as parallel migratory routes or synchrony of axonal growth.     

Localization and developmental changes of the expression of two inward rectifying K(+)-channel proteins in the rat brain

We have raised affinity-purified polyclonal antibodies specific for the inward rectifying K+ channel (IRK1/Kir2.1) and the G protein-activated inward rectifying K+ channel (GIRK1/Kir3.1) examined their distributions in the rat brain immunohistochemically. The regional expression pattern of the IRK1 and GIRK1 proteins were similar to those of mRNA of the previous in situ hybridization study. The subcellular distribution was studied in the cerebellum; cerebral cortex and hippocampus. In the cerebellum, the IRK1 protein was clearly detected in the somata and proximal dendrites of Purkinje cells, while the GIRK1 protein was present in the somata and clustered dendrites of granule cells. In the cerebral cortex and hippocampus, both IRK1- and GIRK1-immunoreactivities were detected in the somata and apical dendrites of the pyramidal cells. The presence of IRK1 or GIRK1 proteins in the axons could not proved by the present study. The developmental changes of the expression pattern of the GIRK1 protein were also investigated in the hippocampus and in the cerebellum of postnatal day (P) 7 to P17 rats. The GIRK1 protein was detected neither in the subgranular zone of the dentate gyrus nor in the proliferative zone of the external granule cell layer of the cerebellum, in which granule cell precursors are reported to proliferate, while it was clearly detected in the adjacent layer in which postmitotic but immature cells exist. These results imply that the expression of the GIRK1 protein starts just after the neuronal precursors finished the last mitotic cell division.     

Divergent responses to thyroid hormone treatment of the different secondary germinal layers in the postnatal rat brain

The effect of daily triiodothyronine (T3) treatment (first injection on the day of birth) was studied on the postnatal development of various parts of the rat brain. It was found that the T3 treatment resulted in an increase of the cell multiplication in the external grannular layer of the cerebellum but decreased the cell division or had no significant effect in the periventricular germinal layer and in the polymorph layer of the dentate gyrus. From the 10th day the T3 treatment resulted in a decrease of the cell division in all secondary germinal layers examined. As a reason for this different effect is can be suggested that the triidothyronine acts differently upon the various neuronal and glial precursors or upon the germinal layers producing them.     

Pituitary adenylate cyclase-activating polypeptide stimulates both c-fos gene expression and cell survival in rat cerebellar granule neurons through activation of the protein kinase A pathway

A high density of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors coupled to both adenylyl cyclase and phospholipase C is found in the external granule cell layer of the rat cerebellum during postnatal development. It has recently been reported that synthetic PACAP promotes cell survival and neurite outgrowth in immature granule cells. In the present study, we have investigated the transduction pathways that mediate the neurotrophic activity of PACAP in cultured granule cells from eight-day-old rat cerebellum. The effect of PACAP on cell survival was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate suggesting that only the adenylyl cyclase pathway is involved in the neurotrophic activity of PACAP. PACAP also induced a transient increase in c-fos messenger RNA level. The ability of PACAP to stimulate c-fos gene expression was mimicked by dibutyryladenosine 3',5'-cyclic-monophosphate but not phorbol 12-myristate 13-acetate. Similar effects of PACAP on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The protein kinase A inhibitor H89 significantly reduced the effect of PACAP on c-fos messenger RNA level whereas the specific protein kinase C inhibitor chelerythrine did not modify c-fos gene expression. These data indicate that the action of PACAP on cerebellar granule cell survival and c-fos gene expression are both mediated through the adenylyl cyclase/protein kinase A pathway. The observation that a short-term stimulation by PACAP can be converted into a long-lasting response indicates that the effect of the peptide on cell survival must involve immediate-early gene activation. The fact that a brief exposure to PACAP causes both c-fos gene expression and promotes cell survival strongly suggests that c-fos is involved in the trophic effect of PACAP on immature cerebellar granule cells.     

Re-examination of the ontogeny in the gene expression of DARPP-32 in the rat brain

By in situ hybridization histochemistry, we have re-examined the ontogeny of the gene expression of mRNA encoding the dopamine- and cyclic AMP-regulated phosphoprotein with a molecular weight of 32,000, termed DARPP-32. On E13 and E15, weak expression signals were detected in the mantle zones and ventricular germinal zones of the fore-, mid-, hind-brain, and spinal cord. In the caudate putamen, the expression signals were first visible at its lateral margin on E15. The ventrolateral region of the caudate putamen expressed the gene intensely, while its ventricular germinal zone expressed it weakly on E18-20. Thereafter, the mRNA for DARPP-32 were expressed over the entire caudate putamen in patchy patterns. After birth, the expression levels in the caudate putamen increased markedly, with the majority of the neurons in the caudate putamen expressing the gene intensely on P7 and thereafter. In addition to the caudate putamen, expression signals were detected, albeit faintly, in the olfactory bulb, cortical plate, hippocampal pyramidal cell layer, and their ventricular zones on E18-20. The olfactory tubercle and medial habenular nucleus expressed the gene at slightly higher levels. In the cerebellum, the Purkinje cells showed progressively increasing gene expression from E20 to P7, whereas the external granule cell layer expressed the gene weakly. The ontogeny of the gene expression is largely consistent with previous immunohistochemical findings by other authors. Furthermore, the present finding suggests that DARPP-32 is involved in the regulation of the mitosis-related dephosphorylation by protein phosphatase 1 in the neuroepithelium.     

Ectopic overexpression of engrailed-2 in cerebellar Purkinje cells causes restricted cell loss and retarded external germinal layer development at lobule junctions

Members of the En and Wnt gene families seem to play a key role in the early specification of the brain territory that gives rise to the cerebellum, the midhindbrain junction. To analyze the possible continuous role of the En and Wnt signaling pathway in later cerebellar patterning and function, we expressed En-2 ectopically in Purkinje cells during late embryonic and postnatal cerebellar development. As a result of this expression, the cerebellum is greatly reduced in size, and Purkinje cell numbers throughout the cerebellum are reduced by more than one-third relative to normal animals. Detailed analysis of both adult and developing cerebella reveals a pattern of selectivity to the loss of Purkinje cells and other cerebellar neurons. This is observed as a general loss of prominence of cerebellar fissures that is highlighted by a total loss of sublobular fissures. In contrast, mediolateral patterning is generally only subtly affected. That En-2 overexpression selectively affects Purkinje cells in the transition zone between lobules is evidenced by direct observation of selective Purkinje cell loss in certain fissures and by the observation that growth and migration of the external germinal layer (EGL) is selectively retarded in the deep fissures during early postnatal development. Thus, in addition to demonstrating the critical role of Purkinje cells in the generation and migration of granule cells, the heterogeneous distribution of cellular effects induced by ectopic En expression suggests a relatively late morphogenetic role for this and other segment polarity proteins, mainly oriented at lobule junctions.     

Mouse Zic1 is involved in cerebellar development

Zic genes encode zinc finger proteins, the expression of which is highly restricted to cerebellar granule cells and their precursors. These genes are homologs of the Drosophila pair-rule gene odd-paired. To clarify the role of the Zic1 gene, we have generated mice deficient in Zic1. Homozygous mice showed remarkable ataxia during postnatal development. Nearly all of the mice died within 1 month. Their cerebella were hypoplastic and missing a lobule in the anterior lobe. A bromodeoxyuridine labeling study indicated a reduction both in the proliferating cell fraction in the external germinal layer (EGL), from 14 d postcoitum, and in forward movement of the EGL. These findings suggest that Zic1 may determine the cerebellar folial pattern principally via regulation of cell proliferation in the EGL.     

Quantitative analysis of cerebellar lobulation in normal and agranular rats

Cerebellar pattern formation was investigated in rats treated with DNA modifying agents. Animals were subjected to combinations of daily injections of methylazoxymethanol acetate (MAM) for the last 6 days gestation and/or localised X-irradiation of the hindbrain on postnatal days 1 and 5 (P1 and P5). Animals were analysed on embryonic day 18 (E18), P0, P3, P7, and P14. Five parameters of the cerebellum were recorded from midsagittal sections: the number of primary lobules; the thickness of the external germinal layer (EGL); the density of cells in the internal granule cell layer (IGL) region; and the midsagittal area and perimeter. In addition, the laterolateral cerebellar distance was calculated. The data demonstrate that pre- and postnatal reduction of the EGL results in reduced cerebellar growth and folding. Cessation of the treatment at birth results in a recovery and eventual overproduction of EGL, but cerebellar growth and the development of fissures lags behind that of normal rats. Pre- and postnatal destruction of the EGL severely limited cerebellar growth and fissuration, and the cerebella contained only five primary lobules at P14. Rats subjected to postnatal X-irradiation alone had a similar low density of granule cells relative to those treated with a combination of prenatal MAM injections and postnatal X-irradiation, and yet the cerebella contained deeper fissures and more lobules (nine at P14). The data indicate that there are two phases of cerebellar folding: the establishment of five lobules that arise independent of granule cell production, and the granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development. We propose that the lack of correlation between the severity of the granule cell loss and degree of lobulation in agranular rats indicates that granule cells exert an inductive influence over lobulation that is in part independent of the forces generated by their production and differentiation.     

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.     

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.     

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.     

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.     

Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice

The chemokine stromal cell-derived factor 1, SDF-1, is an important regulator of leukocyte and hematopoietic precursor migration and pre-B cell proliferation. The receptor for SDF-1, CXCR4, also functions as a coreceptor for T-tropic HIV-1 entry. We find that mice deficient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-deficient mice have severely reduced B-lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-lymphopoiesis is unaffected. Furthermore, the cerebellum develops abnormally with an irregular external granule cell layer, ectopically located Purkinje cells, and numerous chromophilic cell clumps of abnormally migrated granule cells within the cerebellar anlage. Identical defects are observed in mice lacking SDF-1, suggesting a monogamous relationship between CXCR4 and SDF-1. This receptor-ligand selectivity is unusual among chemokines and their receptors, as is the function in migration of nonhematopoietic cells.     

Analysis of gene action in the meander tail mutant mouse: examination of cerebellar phenotype and mitotic activity of granule cell neuroblasts

The meander tail (mea) gene results in a stereotypic pattern of cerebellar abnormalities, most notably the virtual depletion of granule cells in the anterior lobe of the cerebellum. The causal basis of this mutation is unknown. In this paper we have taken a three-part approach to the analysis of mea gene action. First, we quantitatively determined the effect of the mea gene on granule cell and Purkinje cell number. We found, in addition to the marked depletion of anterior lobe granule cells ( > 90%), there were also significantly fewer granule cells in the posterior lobe (20-30%) without a concomitant loss of Purkinje cells. Second, we explored the relationship between granule cell depletion caused by the mea gene and by the mitotic poison, 5-fluoro-2'-deoxyuridine (FdU). Prenatal and postnatal ICR mice were treated with FdU to ascertain the regimen that best produces a meander tail-like cerebellar phenotype. The similarity of the effects of the mea gene and injections of FdU at E17 and PO suggests the hypothesis that the mea gene acts to disrupt the cell cycle of cerebellar granule cell precursors. Thus, the third part of this study was to test this hypothesis by using injections of either BrdU (5-bromo-2'-deoxyuridine) or 3H-thymidine into homozygous and heterozygous meander tail littermates at E17 or PO. After processing the tissue for BrdU immunocytochemistry or 3H-thymidine autoradiography, counts were made of the number of labeled and unlabeled external granule layer (EGL) cells to determine the percentage that had incorporated the mitotic label (labeling index). No difference in the labeling index was found between homozygous meander tail mice and normal, heterozygous littermate controls. Therefore, the mitotic activity of the EGL neuroblasts is not disrupted by the mea gene. Furthermore, while a mitotic poison can produce a phenotype similar to the action of the mea gene, mea is phenomenologically different from FdU treatment.     

N-Acetylglucosaminyl transferase regulates the expression of the sulfoglucuronyl glycolipids in specific cell types in cerebellum during development

In the adult cerebellum, sulfoglucuronyl glycolipids (SGGLs) are specifically localized in Purkinje cells and their dendrites in the molecular layer. Other major cell types such as granule neurons and glial cells lack SGGLs. To explain the cell specific localization and the known biphasic expression of SGGLs, enzymic activities of four glycosyltransferases involved in the biosynthesis of SGGLs were studied in murine cerebellar mutants, in distinct cellular layers of rat cerebellum, and in isolated granule neurons during development. The enzymes studied were lactosylceramide: N-acetylglucosaminyl transferase (GlcNAc-Tr), lactotriaosylceramide:galactosyltransferase, neolactotetraosylceramide:glucuronyltransferase, and glucuronylglycolipid:sulfotransferase. In the cerebellum of Purkinje cell-deficient mutants, such as (pcd/pcd) and lurcher (Lc/+) where Purkinje cells are lost, GlcNAc-Tr was absent, but the other three glycosyltransferase were not severely affected. This indicated that the latter three enzymes were localized in other cell types, such as in mature granule neurons and glial cells, in addition to that in Purkinje cells, and the lack of SGGLs in these mutants was due to absence of GlcNAc-Tr. Analyses of the enzymes in the specific micro-dissected cellular layers also showed that Purkinje cell layer and molecular layer (where Purkinje cell dendrites are localized) contained all four enzymes. However, granule neurons and glial cells in the white matter lacked GlcNAc-Tr, but expressed the other three enzymes. It was concluded that the absence of SGGLs in adult granule neurons and glial cells was due to specific deficiency of the GlcNAc-Tr. Although adult granule neurons lacked GlcNAc-Tr and therefore SGGLs, isolated granule neurons from the neonatal cerebellum contained all four enzymes necessary for the synthesis of SGGLs. With development, the activity of GlcNAc-Tr in the isolated granule neurons declined but the other enzymes were not as affected, indicating that immature granule neurons were capable of synthesizing SGGLs and with maturation the synthesis was down-regulated. This also explains the biphasic expression of SGGLs in the developing cerebellum.