Heterologous synapses upon purkinje cells in the cerebellum of the reeler mutant mouse: An experimental light and electron microscopic study

The projections of the spinal cord upon the cerebellum of normal and Reeler mutant mice were compared by light and electron microscopic methods after hemicordotomy. In both genotypes this afferent system projects to the cerebellar cortex and to the roof nuclei. In the Reeler, there is an additional projection among the Purkinje cells and interneurons of the central cerebellar mass. In both normal and Reeler cerebellar cortex this mossy fiber system terminates as large glomeruli. In Reeler the spinal projection also gives rise to a smaller terminal which is distributed both to the cortex and the central cerebellar mass. In both genotypes the dendrites of granule cells and the somata and dendrites of Golgi cells are synaptic targets of the glomeruli of the cortical projection. In Reeler both the glomeruli and smaller terminals also form heterologous synaptic contacts with dendritic spines of heterotopic intracortical and subcortical Purkinje cells. In both genotypes the synapses are exclusively type I. A second class of heterologous synapse, a type I junction between axons of Golgi cells and Purkinje cell spines, is also recognized in electron micrographs. The present study is the first unequivocal demonstration by experimental hodologic method of heterologous synaptic junctions in the mammalian central nervous system. The existence of such junctions in the cytoarchitectonically anomolous cerebellum of this mutant emphasizes the critical role played by the cellular environment in shaping neural circuits in the developing nervous system.     

Extraordinary synapses of the unipolar brush cell: An electron microscopic study in the rat cerebellum

A neglected type of neuron, termed the unipolar brush cell, was recently characterized in the granular layer of the mammalian cerebellar cortex with several procedures, including light and electron microscopic immunocytochemistry utilizing antibodies to calretinin and neurofilament proteins. Although certain features of the unipolar brush cells were highlighted in these studies, the internal fine structure was partially obfuscated by immunoreaction product. In this study, rat cerebella were prepared for electron microscopy after perfusion fixation and Araldite embedding, and folia of the vestibulo-cerebellum, where unipolar brush cells are known to be enriched, were studied by light microscopy in semithin (0.5–1 μ) sections and by electron microscopy in ultrathin sections. Unipolar brush cells were easily identified in semithin sections immunostained with antibodies to GABA and/or glycine, and cbunterstained with toluidine blue. The unipolar brush cells have a pale cytoplasm and are GABA and glycine negative, while Golgi cells are darker and appear positive for GABA and, for the most part, also for glycine. Sets of identification criteria to differentiate unipolar brush cells from granule and Golgi cells in standard electron micrographs are presented. The unipolar brush cells possess many distinctive features that make them easily distinguishable from other cerebellar neurons and form unusually conspicuous and elaborate synapses with mossy rosettes. The unipolar brush cell has a deeply indented nucleus containing little condensed chromatin. The Golgi apparatus is large and the cytoplasm is rich in neurofilaments, microtubules, mitochondria, and large dense core vesicles, but contains few cisterns of granular endoplasmic reticulum. In addition, unipolar brush cells contain an unusual inclusion, which invariably lacks a limiting membrane and is made up of peculiar ringlet subunits. The cell body usually emits a thin axon and is provided with a single, large dendritic trunk that terminates with a paintbrush-like bunch of branchlets. Numerous nonsynaptic appendages emanate from the cell body, the dendritic stem, and the branchlets. The appendages contain rare organelles and lack neurofilaments. The branchlets contain numerous mitochondria, neurofilaments, large dense core vesicles, and clusters of clear, small, and round synaptic vesicles. They form extensive asymmetric synaptic junctions with of a or two mossy fibers, which indicates minimal convergence of excitatory inputs. Under the postsynaptic densities, the branchlet cytoplasm displays a microfilamentous web. Besides their contact with mossy rosettes, the branchlets form symmetric and asymmetric synaptic junctions with presumed Golgi cell boutons that contain pleomorphic synaptic vesicles, indicating that the unipolar brush cells receive an inhibitory modulation. Some of these junctions are unusually extensive. The branchlets also form asymmetric synapses with granule cell dendrites, in which they represent the presynaptic elements, a feature never described before in the normal cerebellum. A minority of the unipolar brush cells receive mossy fiber contacts directly on the cell body, or on short dendritic branchlets emanating directly from the cell body. Such “enmarron” synapses were previously attributed to Golgi cells. Thus, the unipolar brush cells have complex synaptic features: Besides being specialized to form a powerful link with mossy rosettes, they may also have a paracrine function, and they participate with presynaptic dendrites in the cerebellar microcircuit. © 1994 Wiley-Liss, Inc.     

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton. © 1996 Wiley-Liss, Inc.     

Glutamate receptor subunits at mossy fiber-unipolar brush cell synapses: Light and electron microscopic immunocytochemical study in cerebellar cortex of rat and cat

The present study provides a survey of the immunolocalization of ionotropic glutamate receptor subunits throughout the rat and cat cerebellar cortex, with emphasis on the unipolar brush cell (UBC), a hitherto neglected cerebellar cell that is densely concentrated in the granular layer of the vestibulocerebellum and that forms giant synapses with mossy fibers. An array of nine previously characterized antibodies has been used, each of which stained a characteristic profile of cerebellar cells. The UBCs of both rat and cat were strongly immunostained by an antibody against the α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA) receptor subunits, GluR2 and GluR3; were moderately immunostained by a monoclonal antibody to kainate receptor subunits, GluR5/6/7; were weakly immunostained by antibodies to NR1 subunits; and were not stained by antibodies to GluR1, GluR4, GluR6/7, KA-2, and NR2A/B. Postsynaptic densities of the giant mossy fiber-UBC synapses were GluR2/3, GluR5/6/7, and NR1 immunoreactive. The other cerebellar neurons were all immunolabeled to some extent with the GluR2/3 and NR1 antibodies. In addition, Purkinje cells were immunopositive for GluRl and GluR5/6/7; granule cells were immunopositive for GluR5/6/7, GluR6/7, KA-2, and NR2A/B. The Golgi-Bergmann glia was densely stained by GluRl and GluR4 antibodies, whereas astrocytes of the granular layer were stained by the GluR4 antiserum. Data provided herein may guide further electrophysiological and pharmacological studies of cerebellar cells in general and the UBCs in particular. © 1995 Wiley-Liss, Inc.     

Protein kinase C: Its role in activity-dependent purkinje cell dendritic development and plasticity

The cerebellum is a central organ in the control of motor learning and performance. In this respect, the cellular plasticity model systems of multiple climbing fiber elimination and long-term depression have been intensively studied. The signalling pathways involved in these plastic changes are now well understood on a molecular level and protein kinase C (PKC) activity appears to be crucially involved in both processes. Furthermore, as shown in recent studies, Purkinje cell dendritic development also critically depends on the activity of PKC. Thereby, the Ca(2+)-dependent PKC subtypes, activated by synaptic inputs through metabotropic glutamate receptors, trigger functional changes as well as long-term anatomical maturation of the Purkinje cell dendritic tree during cerebellar development at different time levels. This review summarizes these findings and forwards the hypothesis of a link between the functional mechanisms underlying LTD and the differentiation of Purkinje cell dendrites.     

Immature chemodifferentiation of purkinje cell synapses revealed by 5′-nucleotidase ecto-enzyme activity in the cerebellum of the reeler mouse

During postnatal development of the rodent cerebellum, a transient enzyme activity of ecto-5′-nucleotidase has been shown in the asymmetrical synapses of Purkinje cells. The alterations of the afferent circuitry and microenvironment of the ectopic Purkinje cells present in the cerebellum of the reeler mutant mouse could enlighten parameters that influence the synaptic 5′-nucleotidase activity of these cells. Ecto-enzyme cytochemistry reveals intense 5′-nucleotidase activity in 43% of synapses of the Purkinje cells throughout the cortex and the core of the reeler cerebellar vermis, although the molecular layer displays large areas with less than 1% of labelled synapses. However, enzymatic labelling is found in considerably more Purkinje cells synapses (73%) throughout the granular layer and the subcortical mass. Climbing fiber synapses of monoinnervated Purkinje cells are labelled by 5′-nucleotidase activity in the molecular layer, as well as asymmetrical synapses made on the subjacent ectopic Purkinje cells by the multiple climbing fibers and by the heterologous afferences. The non-innervated dendritic spines of these cells are also labelled, suggesting that 5′-nucleotidase activity at postsynaptic sites of reeler Purkinje cells does not depend on the presynaptic innervation. Rather, 5′-nucleotidase enzyme activity is enhanced at theses sites when the Purkinje cells have not achieved chemodifferentiation but have conserved immature wiring, i.e., low parallel fiber and multiple climbing fiber inputs. Synapse 29:279–292, 1998. © 1998 Wiley-Liss, Inc.     

Neural circuits revealed by axon tracing and mapping cadherin expression in the embryonic chicken cerebellum

Fiber connections of the cerebellar cortex are organized into distinct parasagittal domains. Each domain expresses a unique subset of various genes. Brain structures that are directly connected to the cerebellar cortex, such as the deep cerebellar nuclei and the inferior olivary nucleus, show a similarly differential pattern of connectivity and gene expression. For example, several members of the cadherin family of adhesion molecules are expressed differentially in the subdivisions of the cerebellar system in chicken and mouse. Little is known, however, about how the molecular maps in the different parts of the cerebellum relate to each other in terms of connectivity. Here, we mapped the expression of three cadherins (cadherin-8, protocadherin-7, and protocadherin-10) in the cerebellar system of the chicken embryo. By simultaneously tracing axonal connections with biotinylated dextran amine, we demonstrate that cortical domains and deep nuclear portions as well as their fiber connections have a matching expression profile for protocadherin-10 in the posterior part of the cerebellum. Based on the tracing results for protocadherin-10 and the comparative expression mapping of all three cadherins, the cortical projection domains of the three deep cerebellar nuclei were determined in the posterior part of the cerebellum. Results were extrapolated to the rest of the cerebellar cortex. Our results provide direct experimental support for the notion that cadherins are markers for neural circuits in the brain. Moreover, we show that the expression pattern of all three cadherins confers unique identities to the Purkinje cell domains.     

Formation and maturation of parallel fiber‐purkinje cell synapses in the staggerer cerebellum ex vivo

In vivo, homozygous staggerer (Rora^sg/sg) Purkinje cells (PCs) remain in an early stage of development with rudimentary spineless dendrites, associated with a lack of parallel fiber (PF) input and the persistence of multiple climbing fibers (CFs). In this immunocytochemical study we used cerebellar organotypic cultures to monitor the development of Rora^sg/sg PF‐PC synapses in the absence of CF innervation. Ex vivo the vesicular glutamate transporters VGluT1 and VGluT2 reactivity was preferentially localized around the Rora^sg/sg PC soma and proximal dendrites, which are typically CF domains. The shift from VGluT2 to VGluT1 in PF terminals during development was delayed in Rora^sg/sg slices. The postsynaptic receptors mGluR1 and GluRδ2 were differently distributed on Rora^sg/sg PCs. mGluR1 reactivity was evenly distributed in PC soma and dendrites, whereas GluRδ2 reactivity, normally restricted at PF synapses, was dense in Rora^sg/sg PC somata. The presynaptic distribution of VGluT1 and VGluT2 on Rora^sg/sg PCs matched the postsynaptic distribution of the glutamate receptor GluRδ2, but not mGluR1. J. Comp. Neurol. 512:467–477, 2009. © 2008 Wiley‐Liss, Inc.     

Localization of transferrin binding protein in relation to iron, ferritin, and transferrin receptors in the chicken cerebellum

We have demonstrated that transferrin binding protein (TfBP), ferritin, and iron, are specifically localized in Bergmann glia, while the transferrin receptor is confined to Purkinje cells in the chicken cerebellum. The results of this study suggest that Bergmann glia have previously undescribed functions related to iron regulation such as sequestration of iron and the maintenance of iron homeostasis in the cerebellum.     

Neuroprotective effect of adenine on purkinje cell survival in rat cerebellar primary cultures

Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism.     

Development of cadherin-Defined parasagittal subdivisions in the embryonic chicken cerebellum

The expression of three cadherins (cadherin-6B, cadherin-7, and R-cadherin) was analyzed by immunohistochemistry at early to intermediate stages of chicken cerebellar development (4.5–12 days of incubation [E4.5–E12]). Expression was first detected at approximately E5. On the cerebellar surface, expression of cadherin-6B and cadherin-7 is initially observed in transverse domains that subsequently elongate into parasagittal stripes. The sequence of emergence, the borders, and the orientation of these expression domains suggest a parcellation of the cerebellum into distinct medial, lateral, and caudal embryonic subdivisions. These subdivisions relate to histologic features, to the expression of gene regulatory proteins, and, possibly, to patterns of clonal restriction. Individual cadherin-expressing cell clusters can be observed to split into cortical and nuclear subdivisions, which are connected by nerve fibers expressing the same cadherin, thus establishing the parasagittal corticonuclear connectivity pattern found at later developmental stages. Our results suggest that cadherins may play a role in the transition from the early embryonic to the later functional organization of the cerebellum by providing a scaffold of potential adhesive cues. J. Comp. Neurol. 401:367–381, 1998. © 1998 Wiley-Liss, Inc.     

The postnatal development of glial fibrillary acidic protein and neurofilament triplet proteins in rat brain stem

Cytoskeletal preparations containing both the glial fibrillary acidic protein and the neurofilament triplet proteins were prepared from brain stems of rats at different ages and the individual peptides separated in polyacrylamide gels. Stained peptide bands were quantitated as the area under peaks generated by densitometric scanning. Peak areas were converted to grams of protein based on total gel dye binding and total protein applied to the gels. Between 5 and 30 days, the concentration of the peptide (g of peptide/mg of tissue protein) of apparent molecular weight 51,000 (corresponding to the glial fibrillary acidic protein), increased 3 fold. The corresponding increase in total concentration of the three peptides corresponding to the neurofilament proteins was 4.5 fold. However, the increase in concentration of the individual neurofilament peptides was each different. Very little of the apparent molecular weight 210,000 neurofilament peptide was present at 5 days and its concentration increased 11 fold by 30 days compared to about 3.5 fold for the other two neurofilament peptides. These results are in general agreement with studies using immunological techniques and the methods have the advantages of using readily available techniques and allowing the simultaneous comparison of both neuronal and glial specific filaments during development.     

Fiber formation and myelinization of cultivated dissociated neurons from chicken dorsal root ganglia: an electron microscopic and scanning electron microscopic study

Dissociated neurons from chicken embryo dorsal root ganglia were cultivated in Rose chambers for up to 5 weeks. Newly formed fibers appeared as single fibers or grouped in bundles. During the first week of cultivation microtubules and microfilaments were frequently observed. The diameters of the fibers increased progressively and a number of varicosities appeared. In some distal portions of fibers large vesicles could be observed. During the second week in culture Schwann cells were easily recognized. They appeared as both dark and light cells. During this period myelinization of some fibers was seen to commence. High glucose concentrations were not observed to influence the process of myelinization. Essentially the important factors were the number of cells present in the culture and also the presence of NGF in the medium. Schwann cells myelinated nerve fibers exclusively. Two types of abnormalities in myelin formation were recognized: one Schwann cell myelinating two fibers and one fiber being myelinated twice, by two Schwann cells. Concomitantly with myelinization, myelin degeneration was observed. Histiotypic fascicles, typical constituents of the outgrowth zone of cultivated intact dorsal root ganglia, bundles of fibers, surrounded by connective tissue, are not formed. The surface ultrastructure of nerve fibers, as studied by scanning EM, was seen to be covered by numerous spherical elements as well as by small fibers and irregular elements. The growth cones of fibers were void of any glial contact. Myelinization occupied individual isolated segments along with nerve fibers and evidenced the absence of nodes of Ranvier. Relationships between single elements in the dissociated culture system are discussed, with respect to the possibilities for analysis of some of the elementary mechanisms of cellular and molecular interaction responsible for the development of the peripheral nervous system.     

Aging of the human cerebellum: a stereological study

Cerebella from 19 normal Caucasian males, ages 19-84 years, were studied using stereological methods. Cerebellum was divided into four different regions: the anterior and posterior lobe, the vermis, and the flocculonodular lobe. Total volume of the cerebellar cortex and white matter, cerebellar surface area, total Purkinje and granule cell number, and the distribution of the volumes of the Purkinje cells and their nuclei were estimated in all four regions. The global white matter was reduced by 26% with age; the mean volume of the Purkinje cell body was decreased by 33% with no decrease in the volume of the Purkinje cell nuclei. A tendency towards a 16% total cerebellar volume loss was seen without a concomitant neuron loss. No global Purkinje or granule cell loss was detected with age, total Purkinje cell number being 28 x 10(6) (coefficient of variation, CV = 0.16) and total granule cell number 109 x 10(9) (CV = 0.17). However, a significant change was observed with age in the anterior lobe, where a selective 40% loss of both Purkinje and granule cells was found. Furthermore, a 30% loss of volume, mostly due to a cortical volume loss, was recorded in the anterior lobe, which is predominantly involved in motor control.     

The dendritic tress of purkinje cells: A computer assisted analysis of HRP labeled neurons in organotypic cultures of kitten cerebellum

Purkinje cell dendritic arborizations were studied in HRP intracellularly stained mature neurons grown during 30–50 days in organotypic cerebellar cultures from newborn kittens. The effects of afferent fiber depletions on the final dendritic topological parameters were investigated by computer assisted methods. Three differently deafferented models lacking both parallel and climbing fibers (PF and CF), only PF or only CF were thus studied. Qualitative data were found to be common to these 3 in vitro models and similar to those following other in vivo deafferenting procedures. Quantitative data obtained from 16 measurable parameters showed that all dendritic trees were markedly reduced in size as indicated by a decrease in total dendritic length and total number of segments although in all models individual segment lengths remained largely unaltered. A discriminant analysis permitted the recognition of 3 populations (100% well classified cells) based on 3 selected variables: dendritic field area, mean path length and total number of segments (with the lowest values for the model lacking only PF). These results are shown to lend further support to the role played in the final dendritis arrangement by both intrinsic (non-discriminant) and extrinsic (discriminant) factors and more precisely by the two distinct specific afferent systems (PF and CF).     

Cerebellar alterations induced by chronic hypoxia: an immunohistochemical study using a chick embryonic model

A model of fetal aerogenic hypoxia was developed in which fertilized chicken eggs were half-painted with melted wax and incubated under normal conditions. The cerebellum of the hypoxic chick embryos at a later stage of development (E18-20) was analyzed immunochemically. Hypoxic insult resulted in considerable neurocytological deficits of the Purkinje cells and altered glial fibrillary acid protein (GFAP) immunoreactivity in the fetal cerebellum. Purkinje cells in the hypoxic embryos were marked by small cell size, poorly developed dendrites, low cell density, deletion and ectopia. On the other hand, enhanced GFAP immunoreactivity was found in astrocytes and Bergmann glia of the hypoxic embryos. Our results indicate that chronic hypoxia in the chick fetus can cause severe disorders of neuronal development as well as glial activation. We suggest that our hypoxic model of chick embryos could be an accessible animal model for further elucidating fetal hypoxia.     

Immunocytochemical localization of parvalbumin- and neurofilament triplet protein immunoreactivity in the cat retina: colocalization in a subpopulation of AII amacrine cells

Using antibodies against parvalbumin and neurofilament triplet protein, colocalization of these two neuronal markers was revealed in all of type A horizontal cells and alpha ganglion cells and in a small number of AII amacrine cells of the cat retina. Besides the double-labeled neurons, parvalbumin alone was present in type B horizontal cells, in small numbers of starburst- and A13-like amacrine cells and in the somata of unidentified ganglion cells. The processes of the double- or single-labeled amacrine cells did not have a continuous retinal cover. Although the parvalbumin- and neurofilament-immunolabeled amacrine cells belonged to groups of neurons with well-defined cell morphologies, their neurochemical features differed from other AII, starburst and A13 amacrine cells. The presence of these cells may be due to an accidental expression of an unusual combination of neurochemical features during retinal development. It is also possible that these cells support the functioning of ganglion cells with rarely occurring complex receptive fields.     

A monoclonal antibody to mammalian neurofilament protein stains somata and dendrites in gymnotid fish

Monoclonal antibody N210 (mabN210) recognizes the 210 kdalton neurofilament protein in mammals and gives characteristic immunocytochemical staining of neurofilament-rich processes. For example, in the cerebellum it recognizes myelinated axons and the calyx formed by basket cell axon collaterals. The distribution of mabN210 immunoreactivity was studied in the gymnotid brain (Apteronotus albifrons). In contrast to the mammalian distribution, mabN210 immunoreactivity was not found in most axons of the gymnotid brain. Instead, deposits of reaction product were present in the somata and dendrites of most neurons and were especially dense in those neurons with extensive dendritic trees, the Purkinje cells, pyramidal cells of the electrosensory lateral line lobe, the crest cells of the nucleus medialis and the pyramidal cells of the tectum. Electrosensory lateral line lobe pyramidal cells are known to contain few, if any, neurofilaments in their dendrites. Western blots of whole gymnotid brain proteins demonstrated that mabN210 recognizes two polypeptides apparent molecular weights 60 and 19 kdaltons. These proteins are thus antigenically similar to neurofilament protein and their expression in the gymnotid brain may be related to the peculiar dendritic branching pattern of Purkinje cells and similar cell types.     

Quantification of cerebellar hemispheric purkinje cell linear density: 32 ET cases versus 16 controls

Although essential tremor (ET) is among the most prevalent neurological diseases, its precise pathogenesis is not understood. Purkinje cell loss has been observed in some studies and is the focus of interest and debate. Expressing these data as Purkinje cells/layer length allows one to adjust for the inherent curved nature of the cerebellar folia. Capitalizing on the Essential Tremor Centralized Brain Repository, we quantified Purkinje cell linear density in cases versus controls. Free‐floating 100‐μm parasagittal cerebellar hemispheric sections were subjected to rabbit polyclonal anti‐Calbindin D28k antibody, and 10 random fields/brain were selected for quantification of Purkinje cells/mm^?1 Purkinje cell layer. Purkinje cell linear density was lower in 32 ET cases than in16 controls (1.14 ± 0.32 vs. 1.35 ± 0.31/mm^?1, P = 0.03). Purkinje cell linear density was inversely associated with torpedo count (r = ?0.38, P = 0.028). The current sample of ET cases demonstrates a reduction in Purkinje cell number relative to that of controls. Greater Purkinje cell axonal remodeling (torpedoes) was found in individuals who had the most Purkinje cell drop out. The role of Purkinje cell loss in the pathogenesis of this disorder merits additional study. © 2013 International Parkinson and Movement Disorder Society     

Effects of brain endogenous insulin on neurofilament and MAPK in fetal rat neuron cell cultures

We demonstrated the ‘de novo' synthesis of insulin within the fetal nervous system in vivo and in vitro. We undertook this study to show a role for brain endogenous insulin within the fetal brain. We used neuron cell cultures (NCC) from 19 days gestational age fetal rat brains incubated in an insulin free/serum free defined medium. The neurons showed the presence of preproinsulin I and II mRNA using polymerase chain reaction and insulin immunoreaction employing peroxidase anti-peroxidase and radioimmunoassay techniques. Using an anti-pan neurofilament antibody (that recognizes non-phosphorylated neurofilaments) neurofilament immunoreaction (NFI) was observed within the neuron body, dendrites and axon. Either insulin antibody or isoproterenol treatment induced the neurites to retract and most of the neurons become round, with NFI confined to the neuron body. The antibody treatments induced the neurons to become hypertrophic and vacuolated. With PD98059 treatment NFI was only observed within the neuron body. The addition of insulin reversed the effects of isoproterenol and PD98059, but not those of the insulin antibody. Treatment with wortmannin had no effect. Western blot analysis showed that the basal level of mitogen activated protein kinase (MAPK) phosphorylation was inhibited by the treatment of the NCC with isoproterenol or trypsin, but was significantly increased by treatment with exogenous insulin, demonstrating that brain endogenous insulin phosphorylated MAPK (p<0.05). Thus, brain endogenous insulin promotes neurite outgrowth, probably via MAPK and by stimulating neurofilament distribution via this mechanism participates in neuron differentiation.