Disturbed Purkinje cell migration due to reduced expression of Reelin by X-irradiation in developing rat cerebellum.

The major histogenetic events of the rat cerebellum take place in the early postnatal days. During this period, precursors of microneurons, such as granule cells, form the external granular layer (EGL), extend over the surface of the primordial cerebellum, and actively proliferate. Postmitotic granule cells leave the EOL and migrate to the internal granular layer (IGL). On the other hand, guided by radial glial fibers, immature Purkinje cells migrate from the ventricular zone of the fourth ventricle and settle in the Purkinje cell plate with thickness of several cells. Various cell adhesion molecules are involved in the interaction between the migratory immature Purkinje cells and processes of the radial glia as the basis for contact guidance. The second process is the formation of immature Purkinje cells to the monolayer. This process takes place at the first week after birth of the rat and cell adhesion molecules such as neural cell adhesion molecule (NCAM), fibronectin, tenascin and Reelin are also suggested to play an important role for the cell patterning. When rat fetuses are exposed to X-radiation in the last gestation period, abnormal foliation of the cerebellum develops with ectopic Purkinje cells. The molecular mechanism that contributes to abnormal migration of Purkinje cells and foliar malformation induced by X-irradiation in the cerebellum are not yet clear. This study was undertaken to elucidate the mechanisms of ectopic Purkinje cell formation by examining the expression of cell adhesion molecules.     

Granule cell raphes in the developing mouse cerebellum

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.     

Modulation of synaptic plasticity and memory by Reelin involves differential splicing of the lipoprotein receptor Apoer2

Apolipoprotein E receptor 2 (Apoer2), a member of the LDL receptor gene family, and its ligand Reelin control neuronal migration during brain development. Apoer2 is also essential for induction of long-term potentiation (LTP) in the adult brain. Here we show that Apoer2 is present in the postsynaptic densities of excitatory synapses where it forms a functional complex with NMDA receptors. Reelin signaling through Apoer2 markedly enhances LTP through a mechanism that requires the presence of amino acids encoded by an exon in the intracellular domain of Apoer2. This exon is alternatively spliced in an activity-dependent manner and is required for Reelin-induced tyrosine phosphorylation of NMDA receptor subunits. Mice constitutively lacking the exon perform poorly in learning and memory tasks. Thus, alternative splicing of Apoer2, a novel component of the NMDA receptor complex, controls the modulation of NMDA receptor activity, synaptic neurotransmission, and memory by Reelin.     

The ApoE receptors Vldlr and Apoer2 in central nervous system function and disease: Thematic Review Series: ApoE and Lipid Homeostasis in Alzheimer's Disease

The LDL receptor (LDLR) family has long been studied for its role in cholesterol transport and metabolism; however, the identification of ApoE4, an LDLR ligand, as a genetic risk factor for late-onset Alzheimer's disease has focused attention on the role this receptor family plays in the CNS. Surprisingly, it was discovered that two LDLR family members, ApoE receptor 2 (Apoer2) and VLDL receptor (Vldlr), play key roles in brain development and adult synaptic plasticity, primarily by mediating Reelin signaling. This review focuses on Apoer2 and Vldlr signaling in the CNS and its role in human disease.     

Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis

Angiostrongylosis is a neurological disorder caused by invasion of the central nervous system by developing larvae of Angiostrongylus cantonensis. Purkinje cells in infected mouse cerebellums are small and irregular with degenerative atrophy or partial loss. Ultrastructural changes in degenerative cells included enlarged vacuolar structures and swollen mitochondria within the cytoplasm. The matrix metalloproteinase-9 mRNA which is low in normal cerebellums was expressed in A. cantonensis-infected mice cerebellum prior to Purkinje cell degeneration. Matrix metalloproteinase-9 protein level and enzyme activity increased when the Purkinje cells appeared degenerated. Using immunohistochemistry, matrix metalloproteinase-9 was localised within degenerative Purkinje cells. In addition, when the specific matrix metalloproteinase inhibitor, GM6001, was added, matrix metalloproteinase-9 enzyme activity was reduced by 41.6%. The numbers of degenerative Purkinje cells increased significantly upon establishment of infection but subsided upon inhibition. These results suggested that the expression of matrix metalloproteinase-9 may be associated with degeneration of Purkinje cells in mouse cerebellum infected by A. cantonensis.     

Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum

Purkinje cells (PCs) are the projection neurons of the cerebellar cortex. They receive two major types of synaptic input - that from the inferior olive via climbing fibres and that from the granule neurons via parallel fibres. The precursors of granule neurons proliferate at the surface of the developing cerebellumin the external granule layer (EGL), which persists until postnatal day 14 in the mouse [1]. PCs are thought to provide trophic support for granule neurons [2][3] and to stimulate the proliferation of cells in the EGL [4], but the signalling molecules that mediate these cell-cell interactions have not been identified. I show here that PCs in the developing mouse cerebellum express the gene encoding the morphogen Sonic hedgehog (Shh) and that dividing cells in the EGL express Patched (Ptc) and Gli1, two target genes of which expression is upregulated in response to Hedgehog signalling (see [5] and references therein). Treatment of developing mice with hybridoma cells that secrete neutralizing anti-Shh antibodies [6] disrupted cerebellar development and reduced bromodeoxyuridine (BrdU) incorporation in the EGL of neonatal mice, whereas treatment of dissociated granule neuron cultures with recombinant Shh stimulated BrdU incorporation. These results suggest that PC-derived Shh normally promotes the proliferation of granule neuron precursors in the EGL.     

Biphasic dispersion of clones containing Purkinje cells and glia in the developing chick cerebellum.

The cerebellum is a highly conserved structure which exhibits patterns of gene expression and axonal connections that are organized into parasagittal domains. These aspects of the mature cerebellum are presaged during embryonic development by the expression patterns of vertebrate homologs of Drosophila segmentation genes. We wished to determine whether the parasagittal domains of gene expression are compartments of lineage restriction. To this end, a clonal analysis of the chick cerebellum was conducted with a complex retroviral library. From embryonic day (E) 8 to E12, clones derived from the more medial portion of the cerebellar ventricular zone (VZ) were observed to spread preferentially in the mediolateral direction, crossing the boundaries of the parasagittal domains of gene expression. In late embryonic and posthatch periods, VZ clones were found to comprise Purkinje cells, glial cells, or both types of cells. At these later times, clonally related glial cells formed tight parasagittal clusters, while clonally related Purkinje cells were scattered extensively in the anteroposterior direction. We propose that a subset of the cerebellar VZ clones, those with medial origins, undergoes a biphasic dispersion: an early phase of mediolateral dispersion and a late phase of anteroposterior dispersion. This novel pattern of clonal dispersion suggests that the cerebellar VZ is not partitioned into parasagittal domains of lineage restriction. It leaves open the possibility that the later dispersion along the anteroposterior axis results from the parasagittal patterns of gene expression in the developing cerebellar cortex.     

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.     

Expression of BDNF and TrkB receptor subtypes in the postnatal developing Purkinje cells of monkey cerebellum

In the previous study, we have shown the complementary expression of TrkB subtypes (TK+ and T1) in the adult monkey cerebellar cortex. In this study, to clarify when that expression pattern appeared, we examined the expressions of TrkB subtypes and its ligand brain-derived neurotrophic factor (BDNF) by immunohistochemistry and Western blot analysis. At the newborn stage, both TK+ and T1 were expressed uniformly in the cerebellar cortex. At postnatal month 3.5, the uneven expression of TrkB subtypes was observed, while the BDNF immunoreactivity was strongly detected in all regions of the cerebellar cortex. The expression patterns of TrkB subtypes and BDNF at both postnatal month 6 and year 7 were the same as those at postnatal month 3.5. Western blot analysis demonstrated that TK+ and T1 were expressed at high levels in the synaptic membrane from newborn to adult stages. Furthermore, the dimerization of TrkB subtypes changed at postnatal month 3, which was similar to the adult pattern: at the newborn stage, the TK+ and TK- homodimers; after postnatal month 3.5, the TK+ and TK- homodimers, and the TK+/TK- heterodimer. These findings suggest that the localization of TrkB subtypes in each Purkinje would be changed at postnatal month 3.5, resulting in the uneven expression of TrkB subtypes and the change of TrkB dimerization.     

Vertex analysis of Purkinje cell dendritic trees in the cerebellum of the rat

All networks are made up of vertices (points interconnected by segments), which include terminals interconnected by terminal segments, nodes interconnected by link segments and the root point connected to the tree by the root segment. All nodes may be classified into unique types according to the number of terminal and link segments they drain. For example, there are three distinct dichotomous nodes, a 'primary' node draining two terminal segments, a 'secondary' node draining one terminal segment and a link segment, and a 'tertiary' node draining two link segments. The numbers of primary and tertiary nodes approximate to equality in large networks and thus the ratio of primary to secondary nodes defines topology. All higher order nodes ( trichotomous and beyond) may be resolved into dichotomous forms and incorporated into the analysis. Different forms of growth may thus be analysed by comparing the frequency distributions of nodes with those generated by computer simulated growth models. Moreover, all vertices can be ordered so that metrical parameters are easily incorporated and the hierarchical arrangements of vertices of different order discerned. The dendritic trees of 48 Purkinje cells, taken from folia along the primary fissure, were analysed using vertex analysis. The mean number of segments in Purkinje cell trees was 881 +/- 23 (s.e.) and mean total dendritic length 7959 +/- 233 (s.e.) micrometers. Segment lengths were longest over proximal segments but over most of the tree segment lengths were constant at 10 +/- 0.2 (s.e.) micrometers. Vertex, segment and terminal frequency distributions of equivalent orders were all normal with a slight positive skew. Peak frequencies were recorded at the 12th equivalent order. The mean primary/secondary nodal vertex ratio was 0.93 and the proportion of trichotomous branch points in the tree was 5%. Comparison of the frequency distribution of all vertices with computer generated models showed that growth of the Purkinje cell was most closely simulated by a random terminal growth model, incorporating 5% trichotomy , in which the branching of high order terminals was more likely than low order terminals. It was concluded that growth of the Purkinje cell tree could proceed by random terminal branching with growth occurring preferentially over a front composed of terminals that are ascending through a corridor in the molecular layer whose margins are defined by neighbouring trees.     

Purkinje cell size is reduced in cerebellum of patients with autism

1. The authors' goal was to compare the size and density of Purkinje cells in the cerebellum of subjects with and without autism. Blocks of cerebellum were dissected at autopsy from the brains of age, sex- and postmortem-intervaled (PMI) groups of autistic and normal control individuals (N = 5 per group). Frozen, unfixed blocks were sectioned and stained with 1% cresyl violet.2. The linear, molecular, granular densities and cross-sectional area of Purkinje cells were measured using computer-assisted image analysis. The average cross-sectional areas of Purkinje cells of the patients with autism were smaller by 24% when compared to the normal subjects. Two of the five autistic subjects had mean Purkinje cell sizes that corresponded to greater than 50% reduction in size. There was a substantial effect size difference in Purkinje cell size (² = 0.29) between control and autistic brains (F(1, 8) = 3.32, P = 0.106). No differences in Purkinje cell densities were observed between the two groups.3. These data indicate the possibility of Purkinje cell atrophy in autism with significant neurohistological heterogeneity among individuals diagnosed with this disorder.     

Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum.

Large-scale mouse mutagenesis experiments now under way require appropriate screening methods. An important class of potential mutants comprises those with defects in the development of normal cerebellar patterning. Cerebellar defects are likely to be identified often because they typically result in ataxia. Immunohistochemistry (IHC) is commonly used to reveal cerebellar organization. In particular, the antigen zebrin II (=aldolase C), expressed by stripes of Purkinje cells, has been valuable in revealing cerebellar pattern abnormalities. The development of whole-mount procedures in Drosophila, chick, and Xenopus embryos allows complex patterns to be studied in situ while preserving the integrity of the structure. By combining procedures originally designed for embryonic and early postnatal tissue analyses, we have developed a whole-mount IHC protocol using anti-zebrin II, which reveals the complex topography of Purkinje cells in the adult mouse cerebellum. Furthermore, the procedure is effective with a number of other antigens and works well on both perfusion-fixed and immersion-fixed tissue. By use of this approach, normal adult murine cerebellar topography and patterning defects caused by mutation can be studied without the need for three-dimensional reconstruction.     

Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.     

Autoradiographic localization of angiotensin II receptors in developing rat cerebellum and brainstem

The role of Angiotensin II (Ang II) as a growth promoting or modulating factor has recently become a field of intensive research. A central issue in developmental neurobiology is the understanding of mechanisms governing the formation of spatially ordered connections. In this study, we show the localization of Ang II receptor subtypes by autoradiography in 2-week-old rat hindbrains confronting these data with membrane binding assays. Competition studies done on membrane preparations evidence no major changes on the relative affinities for both receptor subtypes between 2-week-old and adult rat tissues. By autoradiography, we found that all the areas (1-10) of the 2-week-old cerebellum showed both receptor subtypes present in complementary adjacent layers. Areas expressing a high level of AT2 receptors follow: inferior colicullus (IC), dorso tegmental nucleus, central (DTgC), subcoeruleus, alpha, sensory root of the trigeminal nerve, principal sensory root trigeminal nucleus (Pr5, Pr5VL) supragenual nucleus, genu facial nerve, facial nucleus, cerebellar peduncles, vestibular and lateral nuclei. Spinal trigeminal, (oral) and Raphe nuclei express AT1 receptor subtype. The high level of Ang II AT2 receptors present in the cerebellar peduncles might have a meaning on the establishment of the olivo-cerebellar connection. The high expression of Ang II AT2 receptors on 2-week-old rat hindbrains, a critical age on development, as well as its disappearance in the adult, strongly suggests a probable role of these receptors in cell migration and neuronal synaptogenesis.     

The Reelin signaling pathway in mouse cortical development

Most of the cerebral cortex derives from the cortical plate which, in all mammals, is radially organized and develops from inside to outside. Several genes involved in the organization and inside-outside development of the embryonic cortical plate in the mouse form the so-called Reelin signaling pathway. Biochemical and genetic arguments show that the extracellular matrix protein Reelin binds to two lipoprotein receptors (VLDLR and ApoER2), which relay the Reelin signal inside target neurons by docking the tyrosine kinase adapter disabled-1 (Dab1). In addition, biochemical evidence suggests that the integrins alpha 3/beta 1 and protocadherins of the CNR family may also modulate the Reelin signal. The mechanisms by which the presence of Reelin stops migration and instructs the radial organization of cortical plate cells remains unknown.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.