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R Hawkes

Publications and source records attributed to R Hawkes.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cerebellar abnormalities in the disabled (mdab1-1) mouse.

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Genomic sequences of aldolase C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic mice.

Aldolase C is regarded as the brain-specific form of fructose-1, 6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5' to the transcription start site to 0.5 kb 3' to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed beta-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.

Animals↗
PubMed1998

Pattern formation in the cerebellum of murine embryonic stem cell chimeras.

The cerebellar cortex is subdivided into an elaborate, stereotyped array of transverse zones and parasagittal stripes. It has been speculated that (i) all Purkinje cells derive from 10 to 20 precursors allocated early in embryogenesis and (ii) that pattern formation is based on cell lineage restriction in the founder pool. These hypotheses have been tested by clonal analysis of embryonic stem cell chimeras. Neither speculation is supported: the analysis suggests that Purkinje cells derive from a founder population of > 102 precursors, and that neither cerebellar transverse developmental boundaries nor parasagittal stripes have a clonal origin. We conclude that early lineage restriction plays no role in cerebellar pattern formation.

Animals↗
PubMed1998

Blebs in the mouse cerebellar granular layer as a sign of structural inhomogeneity. 2. Posterior lobe vermis.

When the adult mouse cerebellum is perfusion-fixed with 70% ethanol, paraffin-embedded, sectioned, and the sections are rehydrated, the granular layer wrinkles into an elaborate array of blebs. In the posterior lobe vermis these blebs are seen in both transverse and sagittal sections, are symmetrical about the midline, reproducible between neighboring sections and between individuals, and bear a constant relationship to the Purkinje cell bands as revealed by zebrin II immunocytochemistry. The data suggest that blebs represent individual cytoarchitectonic units and thus that the mouse cerebellum is subdivided into several thousand modules, each comprising >10(2) Purkinje cells and their associated interneurons and glial cells.

Animals↗
PubMed1997

A search for primitive Purkinje cells: zebrin II expression in sea lampreys (Petromyzon marinus).

Zebrin II/aldolase C is a 36 kDa polypeptide expressed by Purkinje cells in the cerebellum of elasmobranchs, teleosts, birds, and mammals, and by octavolateralis pyramidal cells in developing teleosts. To better understand the evolution of these two systems we determined if zebrin II is expressed (1) in previously described primitive Purkinje cells, and (2) in octavolateralis pyramidal cells of sea lampreys (Petromyzon marinus). Ammocete and adult stages were reacted with mab anti-zebrin II. In ammocetes the large pyramidal cells of the anterior octavomotor nucleus (AON) were mab anti-zebrin II immunoreactive, but immunoreactivity was not detected in the cerebellar plate. In adults there was no immunoreactivity in any portion of the brain, including the cerebellar plate and the AON. The data indicate that zebrin II immunoreactivity may prove valuable in studying the development of the octavolateralis system across vertebrates. Three explanations are proposed to account for the absence of zebrin II+ Purkinje cells: aldolase C is expressed in Purkinje cells but the zebrin II epitope has not yet evolved; the zebrin II epitope was present in ancestral lampreys but has since been lost; or sea lampreys do not have Purkinje cells. The evolutionary implications of these results are briefly reviewed.

Animals↗
PubMed1997

Neuronal position in the developing brain is regulated by mouse disabled-1.

During mammalian brain development, immature neurons migrate radially from the neuroectoderm to defined locations, giving rise to characteristic cell layers. Here we show that targeted disruption of the mouse disabled1 (mdab1) gene disturbs neuronal layering in the cerebral cortex, hippocampus and cerebellum. The gene encodes a cytoplasmic protein, mDab1 p80, which is expressed and tyrosine-phosphorylated in the developing nervous system. It is likely to be an adaptor protein, docking to others through its phosphotyrosine residues and protein-interacting domain. The mdab1 mutant phenotype is very similar to that of the reeler mouse. The product of the reeler gene, Reelin, is a secreted protein that has been proposed to act as an extracellular signpost for migrating neurons. Because mDab1 is expressed in wild-type cortical neurons, and Reelin expression is normal in mdab1 mutants, mDab1 may be part of a Reelin-regulated or parallel pathway that controls the final positioning of neurons.

Animals↗
PubMed1997

Reestablishment of the olivocerebellar projection map by compensatory transcommissural reinnervation following unilateral transection of the inferior cerebellar peduncle in the newborn rat.

It is unclear whether reparative processes in the injured mammalian brain are able to restore the topographic organisation of neuronal connections. To address this question, we have investigated the plasticity of the olivocerebellar system. This pathway has a precise topographic arrangement, in which subsets of inferior olivary neurons project to parasagittally oriented Purkinje cell compartments. Following unilateral transection of the inferior cerebellar peduncle in newborn rats, axons from the contralateral projection cross the cerebellar midline and reinnervate the deafferented hemicerebellum. By this experimental approach, we first analysed the behaviour of calcitonin gene-related peptide (CGRP)-immunoreactive climbing fibres. This marker is transiently expressed by a subset of developing inferior olivary axons, which terminate in the cerebellar cortex into several parasagittal strips. We show that transcommissural axons reestablish the original pattern of climbing fibre bands within a few days after lesion. Then, in adult animals injured at birth, we assessed whether the newly formed climbing fibre bands align with zebrin II+/- Purkinje cell compartments, as in normal conditions. The newly formed projection is organised in parasagittally oriented strips which mirror the distribution of their counterparts on the intact side and are precisely aligned to the heterogeneous Purkinje cell compartments. In addition, the patchy distribution of olivo-nuclear fibres suggests that specific reinnervation is also achieved in the deep nuclei. Thus, transcommissural olivocerebellar reinnervation is not random, but it is regulated by selective interactions between distinct subsets of olivocerebellar axons and target neurons aimed at reestablishing the correct projection map.

Animals↗
PubMed1997

Blebs in the mouse cerebellar granular layer as a sign of structural inhomogeneity. 1. Anterior lobe vermis.

The cerebellum is a modular structure. However, the size of the fundamental compartments is uncertain, with anatomical methods showing a parasagittal band arrangement but electrophysiological mapping suggesting a finer subdivision into microzones and patches. A new anatomical way to demonstrate compartmentation is described. The cerebellum is fixed by perfusion with 70% ethanol, paraffin-embedded and sectioned. When the sections are rehydrated the granular layer pleats into an elaborate array of blebs. These blebs are seen in both transverse and sagittal sections, found in all lobules of both the vermis and the hemispheres, symmetrical about the midline, reproducible between neighboring sections and between individuals, and bear a constant relationship to the Purkinje cell bands as revealed by zebrin II immunocytochemistry. The data suggest that the granular layer of the adult mouse cerebellum is divided into several thousand modules. These modules may reflect the mossy fiber topography, and may be the anatomical equivalents of the tactile receptive field patches. Such a profound compartmentation has important implication for theories of cerebellar structure and development.

Animals↗
PubMed1997

Compartmentation of the granular layer of the cerebellum.

Numerous studies have demonstrated that the cerebellum is highly compartmentalized. In most cases, compartmentation involves the Purkinje cells and the molecular layer, but there is also substantial evidence that the granular layer is subdivided into a large number of highly reproducible modules. We first review the evidence for a modular granular layer. Compartmentation of the granular layer has been revealed both functionally and structurally. First, tactile receptive field mapping has revealed numerous discrete functional modules within the granular layer. The molecular correlates of the receptive fields may be the compartments revealed by histological staining of the cerebellum for several enzymes and antigens. The structural substrate of the receptive fields is the mossy fiber afferent projection map, and anterograde tracing of various mossy fiber projections shows afferent terminals in parasagittal bands within the granular layer that are topographically aligned with the Purkinje cell compartments. Based on this evidence we argue that the cerebellum consists of many hundreds of reproducible structural/functional modules, and that a modular organization is a prerequisite for the efficient parallel processing of information during motor control. The complex organization of the adult granular layer implies elaborate developmental mechanisms. In the second part of the review we consider five developmental models to generate the modular organization of the adult granular layer: 1) the external granular layer is heterogeneous, and its topography translates directly into a modular granular layer; 2) granular layer modules are clones, derived from single external granular layer precursors; 3) modules in the granular layers are a secondary epigenetic response to the compartmentation of the Purkinje cells; 4) modules are secondary to the compartmentation of the afferent terminal fields; 5) modules are sculpted by activity-dependent processes.

Adult↗
PubMed1997

Stripes and zones: the origins of regionalization of the adult cerebellum.

The mammalian cerebellum is subdivided into an elaborate, reproducible array of parasagittal stripes and transverse zones. Stripes and zones are most clearly revealed by the patterns of expression of numerous genes and by the consequences of several naturally-occurring mutations. Because the stripe and zone boundaries are orthogonal, they subdivide the cerebellum into a patchwork grid. How is this elaborate topography created during cerebellar development? This article reviews the evidence for cerebellar regionalization and considers various mechanisms by which it might arise during embryogenesis.

Animals↗
PubMed1996

Absence epilepsy in tottering mutant mice is associated with calcium channel defects.

Mutations at the mouse tottering (tg) locus cause a delayed-onset, recessive neurological disorder resulting in ataxia, motor seizures, and behavioral absence seizures resembling petit mal epilepsy in humans. A more severe allele, leaner (tg(la)), also shows a slow, selective degeneration of cerebellar neurons. By positional cloning, we have identified an alpha1A voltage-sensitive calcium channel gene that is mutated in tg and tg(la) mice. The alpha1A gene is widely expressed in the central nervous system with prominent, uniform expression in the cerebellum. alpha1A expression does not mirror the localized pattern of cerebellar degeneration observed in tg(la) mice, providing evidence for regional differences in biological function of alpha1A channels. These studies define the first mutations in a mammalian central nervous system-specific voltage-sensitive calcium channel and identify the first gene involved in absence epilepsy.

Amino Acid Sequence↗
PubMed1996

Partial ablation of the neonatal external granular layer disrupts mossy fiber topography in the adult rat cerebellum.

The spinocerebellar projection in the rat is compartmentalized in an array of parasagittal bands of mossy fiber terminals. These bands align reproducibly with bands of Purkinje cells that differentially express zebrin II. To investigate whether this alignment is obligatory, Purkinje cell and mossy fiber compartmentation has been compared in the rat cerebellum where the cytoarchitecture was contorted by neonatal administration of methylazoxymethanol. Methylazoxymethanol ablates many granule cell precursors, leaving a much reduced external granular layer, and adult rats that received a single methylazoxymethanol injection at birth showed varying degrees of abnormal cerebellar foliation. Zebrin II immunocytochemistry nevertheless revealed no fundamental abnormality in the Purkinje cell compartments. However, despite the normal Purkinje cell compartmentation being retained, the spinocerebellar mossy fiber-Purkinje cell topography is disrupted by methylazoxymethanol treatment. The normal precise array of parasagittal mossy fiber terminal fields becomes blurred across the lobule, and the normal clear banding is difficult to follow. These data suggest that, despite the early topography being dependent on the Purkinje cells, the granule cell-mossy fiber interactions also regulate the topography of the spinocerebellar projection.

Animals↗
PubMed1996

Differential distribution of MAP1a and aldolase c in adult mouse cerebellum.

MAP1a is a microtubule-associated protein with an apparent molecular weight of 360 kDa that is found in the axonal and dendritic processes of neurons. Two monoclonal anti-MAP1a antibodies anti-A and anti-BW6, revealed different epitope distributions in the adult mouse cerebellum. Anti-A stained Purkinje and granule cells uniformly throughout the cerebellum. In contrast, anti-BW6 selectively stained the dendriites of a subset of Purkinje cells, revealing parasagittal bands of immunoreactivity in the molecular layer. The compartmentation of the BW6 epitope was compared to the Purkine cells as revealed by immunostaining with anti-zebrin II, a well known antigen expressed selectively by bands of Purkinje cells. The anti-BW6 staining pattern was complementary to the zebrin II bands, the zebrin II- Purkinjke cells having BW6+ dendrites. These results demonstrate that MAP1a is present in two forms in the mouse cerebellum, one of which is segregated into parasagittal bands. This may indicate a unique MAP1a isoform or may reflect differences in the metabolic states of Purkinje cell classes, and regional differences in their functions.

Animals↗
PubMed1996

Topographically organized climbing fibre sprouting in the adult rat cerebellum.

Adaptive recovery following brain injury requires the topography of projection maps to be restored. In the adult mammalian brain, the regeneration of severed axons does not normally occur and repair mainly relies on collateral reinnervation from uninjured neurons. Although reinnervation can be target specific at the single cell level, it is not known if the new connections are organized correctly. The normal olivocerebellar projection had precise topography in which subnuclei of the inferior olive terminate as climbing fibres on chemically defined bands of cerebellar Purkinje cells. This precision has been exploited to determine the topography of climbing fibre sprouting following an inferior olive lesion in the adult rat. Collateral reinnervation was found to respect the boundaries between the Purkinje cell compartments. Thus, topographical cues are available in the adult during post-lesion plasticity to guide the restoration of the olivocerebellar projection map.

Animals↗
PubMed1995

Developing mossy fiber terminal fields in the rat cerebellar cortex may segregate because of Purkinje cell compartmentation and not competition.

Many mossy fiber afferent projections to the rat cerebellar cortex terminate in parasagittal bands. In particular, the anterior lobe vermis of the cerebellum contains alternating bands of mossy fibers from the spinal cord and external cuneate nuclei. The cerebellar cortical efferents, the Purkinje cells, are also organized in parasagittal bands. These can be revealed by immunochemical staining for the antigen zebrin II, which is selectively expressed by bands of Purkinje cells. In some cases, the boundaries between mossy fiber terminal fields align with identified transitions between zebrin+/- sets of Purkinje cells, whereas others are located within apparently homogeneous Purkinje cell compartments. Two theories can explain the terminal-field topography: In one view, mossy fiber terminals segregate during development, because growth cones from different sources compete for common territory. Alternatively, mossy fiber growth cones directly recognize chemically distinct target territories, and activity-dependent mechanisms play only minor roles. To explore these issues, two sets of experiments were performed. First, the terminal-field map of the neonatal spinocerebellar projection was compared to the Purkinje cell compartmentation as revealed by anticalbindin immunocytochemistry. Second, subsets of spinocerebellar mossy fiber afferents were ablated early in postnatal development, and the consequences for the neighboring cuneocerebellar terminal fields were mapped in the adult with reference to the zebrin II+/- compartments. These experiments revealed no evidence that competitive interactions constrain the mossy fiber terminal-field distribution but, rather, suggest that the organization of the mossy fiber projections follows the compartmentation of the Purkinje cells.

Analysis of Variance↗
PubMed1995

Molecular compartmentation expressed in cerebellar cultures in the absence of neuronal activity and neuron-glia interactions.

The purpose of the study was to determine if zebrin compartmentation developed in permanently isolated cerebellar cultures, in the presence of agents that block neuronal activity and in the absence of myelination and astrocytic ensheathment of Purkinje cells. Parasagittally oriented organotypic cultures derived from newborn mice and carefully undercut at explantation to exclude extracerebellar afferents were subjected to three conditions: 1) Some were maintained in standard nutrient medium; 2) some were chronically exposed to tetrodotoxin and elevated levels of magnesium to block neuronal activity; and 3) some were exposed to cytosine arabinoside for the first 5 days in vitro (DIV) to destroy granule cells and oligodendrocytes and functionally compromise astrocytes, so that the astrocytic survivors did not ensheath Purkinje cells. Cultures fixed as whole-mount preparations were reacted with antibody to zebrin II. Cultures that were cryostat sectioned were dually reacted with antibody to zebrin II and calbindin. Groups of zebrin+ and zebrin- Purkinje cells were evident after 14 DIV in all of the experimental conditions, indicating that zebrin compartmentation developed 1) in isolated cerebellar explants, 2) in the absence of neuronal activity, and 3) in the absence of neuron-glia interactions such as myelination and glial ensheathment of Purkinje cell somata and dendrites. These results are consistent with the concept that expression of the zebrin+ and zebrin- phenotypes is an intrinsic property of Purkinje cells. The fact that zebrin expression seems to depend on an intrinsic program of differentiation in Purkinje cells suggests some role for zebrin compartmentation in cerebellar function.

Animals↗