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PubMed1990

Gender and gonadectomy influence specific proteins in the cerebellar cortex of the adult rat: a two-dimensional gel electrophoretic analysis.

Two-dimensional polyacrylamide gel electrophoresis was used to determine the influence of gender and gonadectomy on individual proteins of the 105,000 g supernatant (cytosol) of the cerebellar cortex of the adult rat. At age 60 days were gonadectomized or sham-gonadectomized. At age 140 days the rats were sacrificed and the 105,000 g supernatant prepared from the cerebellar cortex. Sham-operated female rats were sacrificed during the proestrus stage of the estrous cycle. Out of 200 proteins identified, 13 were influenced by gender and/or gonadectomy. Five of these proteins were influenced by both gender and gonadectomy. A total of 18 differences were observed. Apparent molecular weights and isoelectric points (pIs) for the 13 proteins ranged from 22,000 to 92,000 Da and pH 4.8-6.1, respectively. Sex differences occurred in the percent volumes (spot density) of 9 proteins between intact male and proestrous female rats. Sex differences in 4 proteins were not influenced by gonadectomy. These differences were presumed to be genetically programmed and/or to reflect the permanent organizational effects of gonadal steroids on the genome. Differences in the remaining 5 proteins depend upon the transient activational effects of the testicular sex steroids, i.e. these gender differences were eliminated by castration. The effects of gonadectomy on individual proteins were gender specific, i.e. proteins affected by ovariectomy were not influenced by castration and vice versa. This suggests intact gonads are required for the specific gender-dependent proteins identified in the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Early degeneration of the cerebellar cortex, particularly the granular cells.

An 8 month old infant, who died of severe gastroenteritis, presented a degeneration of the cerebellar cortex involving cells arising from the outer granular layer as well as Purkinje and Golgi II cells. Residual Purkinje cells showed vacuolar change of the cell body and dendritic abnormalities. Related lesions were atrophy of the inferior olives and degeneration of the mossy fibers.

Atrophy↗
PubMed1992

Normal serotonin but elevated 5-hydroxyindoleacetic acid concentration in cerebellar cortex of patients with dominantly-inherited olivopontocerebellar atrophy.

Beas-Zarate and coworkers (Eur. J. Pharmacol., 198 (1991) 7-14) recently reported markedly reduced concentration of presynaptic serotonin neurotransmitter markers in cerebellum of rodents which had suffered destruction of the inferior olivary-cerebellar (climbing fibre) projections by the neurotoxin 3-acetylpyridine; these experimental animal data suggested that serotonin might be one of the neurotransmitters released by climbing fibres. We measured the concentration of serotonin and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in autopsied cerebellar cortex of 14 patients with dominantly-inherited olivopontocerebellar atrophy (OPCA) who all had near-total degeneration of the inferior olivary climbing fibres. As compared with the controls, mean concentration of serotonin in cerebellar cortex of the OPCA patients was normal whereas 5-HIAA levels (+79%, P less than 0.02) and 'turnover' ratio 5-HIAA/serotonin (+148%, P less than 0.05), on average, were significantly elevated. These data do not support the notion that serotonin is a predominant neurotransmitter of the human climbing fibre. However, the markedly elevated serotonin turnover ratio suggests the possibility of increased serotonergic neuronal activity, which might alter, and perhaps improve, the functioning of the preserved cerebellar cortical neurones in OPCA.

Adult↗
PubMed1979

Influence of experimentally induced agranularity on the synaptogenesis of serotonin nerve terminals in rat cerebellar cortex.

The serotonin (5-HT) innervation of the posterior vermis was studied by high resolution radioautography in both normal and X-ray-induced agranular rat cerebella, following 3 h topical superfusion with 10(-4) M 3H-5-HT. In the normal cerebellar cortex, 5-HT axonal varicosities are scarce and only rarely exhibit the membrane differentiations characterizing synaptic contacts. In the agranular cerebellum, 5-HT terminals appear to have a much higher density than in normal controls, although their absolute number may not be significantly different when the important reduction in volume of this experimental cerebellum is taken into account. These terminals frequently show typical synaptic contracts. Most of them are established on the branchlet spines of Purkinje cell dendrites, but some are also observed on the shafts of Golgi cell dendrites. The 5-HT innervation of the cerebellar cortex thus undergoes important changes in the absence of granule cells. It is suggested that these modifications may be part of the general reorganization process of the cerebellar circuitry consequent on the early destruction of the external granular layer. This new example of synaptic remodelling could imply that the formation of cerebellar connectivity is modulated, to a certain extent, by the local cellular environment.

Animals↗
PubMed2004

Flavoprotein autofluorescence imaging of neuronal activation in the cerebellar cortex in vivo.

Autofluorescence has been used as an indirect measure of neuronal activity in isolated cell cultures and brain slices, but only to a limited extent in vivo. Intrinsic fluorescence signals reflect the coupling between neuronal activity and mitochondrial metabolism, and are caused by the oxidation/reduction of flavoproteins or nicotinamide adenine dinucleotide (NADH). The present study evaluated the existence and properties of these autofluorescence signals in the cerebellar cortex of the ketamine/xylazine anesthetized mouse in vivo. Surface stimulation of the unstained cerebellar cortex evoked a narrow, transverse beam of optical activity consisting of a large amplitude, short latency increase in fluorescence followed by a longer duration decrease. The optimal wavelengths for this autofluorescence signal were 420-490 nm for excitation and 515-570 nm for emission, consistent with a flavoprotein origin. The amplitude of the optical signal was linearly related to stimulation amplitude and frequency, and its duration was linearly related to the duration of stimulation. Blocking synaptic transmission demonstrated that a majority of the autofluorescence signal is attributed to activating the postsynaptic targets of the parallel fibers. Hypothesized to be the result of oxidation and subsequent reduction of flavoproteins, blocking mitochondrial respiration with sodium cyanide or inactivation of flavoproteins with diphenyleneiodonium substantially reduced the optical signal. This reduction in the autofluorescence signal was accomplished without altering the presynaptic and postsynaptic components of the electrophysiological response. Results from reflectance imaging and blocking nitric oxide synthase demonstrated that the epifluorescence signal is not the result of changes in hemoglobin oxygenation or blood flow. This flavoprotein autofluorescence signal thus provides a powerful tool to monitor neuronal activity in vivo and its relationship to mitochondrial metabolism.

Action Potentials↗
PubMed2001

Glucose-6-phosphate dehydrogenase supports the functioning of the synapses in rat cerebellar cortex.

This study investigates heterogeneous glucose-6-phosphate dehydrogenase (G6PD) expression in the rat cerebellar cortex. G6PD activity and its electrophoretic pattern, evaluated on the cerebellar homogenate, were found to be similar to those of other brain areas. However, histochemical and immunohistochemical analyses revealed that the highest expression of G6PD activity and protein was in Purkinje's cells, followed by the molecular and granular layers. Electron microscopy analysis showed that, in Purkinje's cells, the G6PD reaction products were concentrated in the neurites while in the basket cells in the cell body. The granules showed a weaker activity everywhere. The quantitative distribution of G6PD is discussed in the light of the neurochemical function of these cells.

Animals↗
PubMed1988

Topographical distribution of muscarinic cholinergic receptors in the cerebellar cortex of the mouse, rat, guinea pig, and rabbit: a species comparison.

Light microscopic autoradiography of [3H]quinuclidinyl benzilate (QNB) binding sites was used to study the distribution of muscarinic acetylcholine receptors in the mouse, rat, guinea pig, and rabbit cerebellar cortex. In the mouse, the laminar distribution of grain density was similar throughout the cortex, with slightly higher levels over lobules IX and X. The highest [3H]QNB labeling was present over the granule cell layer, and low levels were observed over the molecular layer. In the rat, the general distribution was similar to that of the mouse in that the granule cell layer was most densely labeled and the highest concentration of [3H]QNB binding sites was present in lobules IX and X of the archicerebellum. In these lobules, however, the laminar distribution of grain density was reversed so that the molecular layer was more densely labeled than the granule cell layer. In addition, several discrete columns of elevated grain density traversed the granule cell layer in caudal regions of lobule IX. The distribution of [3H]QNB binding sites in the guinea pig cerebellum was similar to that of the rat in that the molecular layer of lobules IX and X was again more intensely labeled than other cerebellar regions. In the remaining lobules, grain density was equal over the granule cell and molecular layers. In the rabbit cerebellar cortex, slightly higher grain density was observed in the granule cell layer than in the molecular layer. In lobules IX and X and in the hemisphere of X, the Purkinje cell layer was most densely labeled; parasagittal columns of very high grain density were present over the molecular layer of several cortical regions, including lobules, I, II, III, IV, V, IX, X, and the hemispheres of IX and X. Since muscarinic receptors have previously been found on blood vessels, there is a possibility that some proportion of receptor labeling may be localized to these structures. Microvessels and capillaries in each of the species examined were more numerous in the granule cell layer than in the molecular layer and white matter. The distribution of blood vessels in many cerebellar lobules of mice, rats, and guinea pigs corresponded quite closely to the general distribution of [3H]QNB binding sites. Unique patterns of labeling in lobules IX and X were not accompanied by corresponding patterns of blood vessel distribution, however. In the mouse, there was a slight increase in muscarinic receptor density observed in the archicerebellum, with no corresponding increase in the density of blood vessels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1980

Histofluorescence studies of the effect of AOAA and etomidate on catecholamine terminals. Possible GABA-NA interaction in the cerebellar cortex.

The effect of two GABA-ergic drugs: aminooxyacetic acid (AOAA) (37,5 mg/kg) and etomidate (2 X 10 mg/kg) on noradrenergic (NA) and dopaminergic (DA) neurons in the rat brain was studied using the Falck-Hillarp histofluorescence method for monoamines. Both drugs enhanced the catecholaminergic fluorescence in NA terminals in the cerebellar cortex, which suggests an increase in NA content there. This effect was blocked by pretreatment with picrotoxin, a Gaba receptor blocking compound. The increase in fluorescence was also observed in DA terminals in nucleus caudatus-putamen after AOAA (but not after etomidate) and this effect was blocked by picrotoxin in 3 out of 5 rats. The obtained results indicate that apart from previously described GABA-DA interaction in the striatum, there exists also GABA-NA interaction seen in the cerebellar cortex.

Acetates↗
PubMed2005

Localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex with imaging TOF-SIMS equipped with a bismuth cluster ion source.

Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of co-localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex. Rat cerebellum was fixed, freeze-protected by sucrose, frozen and sectioned by cryoultramicrotomy and dried at room temperature. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi(1-7)+-source. The cholesterol signal (m/z 369 and 385) was localized in Purkinje cells and in nuclei of granular layer cells. The phosphocholine headgroup of phosphatidylcholine and sphingomyelin was localized by imaging a specific fragment (m/z 86). This signal was localized in the molecular layer of cerebellar cortex, in Purkinje cells and in parts of the granular layer probably representing the synapse-rich glomeruli. The galactosylceramide was localized by imaging the quasi-molecular ions at m/z 835 and 851, showed a clear colocalization with cholesterol, but also a specific localization in dots (diameter <or=700 nm) in the molecular layer in the vicinity of Purkinje cells, at Purkinje cells and at cells in the granular layer. The results show a heterogeneous distribution of lipids between different cell types not previously described. In order to avoid redistribution artefacts, controls were made by a technique, based on high pressure freezing ,freeze fracturing and freeze drying of samples which were then analyzed by bombardment with a Bi3+ liquid metal ion gun. The galactosylceramide and cholesterol were found distributed as spots in the granular layer. The spots were of homogeneous size with a diameter of <700 nm. Although the galactosylceramide and cholesterol were localized to the same areas, there were clear differences in their distribution at higher resolution.

Animals↗
PubMed1994

GABAA/benzodiazepine receptor alpha 6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: expression in developing and mutant mice.

The gamma aminobutyric acidA/benzodiazepine (GABAA/BZ) receptor is a multisubunit (alpha, beta, gamma, delta, and rho) ligand-gated chloride channel; there are several variants of the alpha, beta, and gamma subunits, each of which has been localized throughout the central nervous system. A large number of GABAA/BZ subunit variants are expressed within the cerebellar cortex. In previous studies from other laboratories, alpha 6 subunit mRNA has been reported to be present exclusively in cerebellar granule cells. The developmental expression of alpha 6 mRNA in cerebellar and cochlear granule cells is of interest because it has been suggested that each of these cell types is derived from a common precursor pool. The polymerase chain reaction was used to generate a cDNA fragment encoding a portion of the M3-M4 intracellular loop of the alpha 6 subunit of the GABAA/BZ receptor. A [35S] riboprobe, transcribed from this cDNA fragment, was used to examine the expression of the alpha 6 subunit mRNA by in situ hybridization in developing normal mice and in adult mutant mice with known deficits in synaptic circuitry. A strong hybridization signal was observed over the granule cell layers of both the cerebellum and cochlear nuclei in adult mice. The signal over the cochlear nuclei appeared after birth toward the end of postnatal week 1, coinciding with the appearance of labeling in the cerebellar cortex. The intensity of the hybridization signal in both regions increased rapidly until postnatal day 14, after which it increased more gradually, reaching adult levels during postnatal week 3.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

[Quantitative changes in piriform neurons of human cerebellar cortex from birth to 20 years].

Age changes of size parameters of piriform (Purkinje) neurons in visual cerebellar cortex (pyramid and lateral areas of right and left digastric lobes) were studied at monthly intervals (during the first postnatal year) and at annual intervals (since birth up to 20 yrs) using computer optic image analyzer. Postnatal development of piriform neurons from human cerebellar visual cortex was found to be periodic (cyclic); each morphological cycle is characterised by certain duration and a series of quantitative and qualitative changes.

Adolescent↗
PubMed1998

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione-S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.

Animals↗
PubMed1999

Neurotrophic activity of pituitary adenylate cyclase-activating polypeptide on rat cerebellar cortex during development.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors are present in the external granule cell layer of the rat cerebellum during postnatal development. In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth on immature cerebellar granule cells in primary culture. In the present study, we have investigated the effect of PACAP on the development of the cerebellar cortex of 8-day-old rats. Incubation of cultured granule cells for 12 or 18 h with PACAP provoked a significant increase in the rate of incorporation of [(3)H]thymidine in cultured granule cells, suggesting that PACAP could stimulate the proliferation of granule cells. After 96 h of treatment, in vivo administration of PACAP provoked a transient increase in the number of granule cells in the molecular layer and in the internal granule cell layer. In contrast, PACAP did not affect the number of Purkinje cells. The augmentation of the number of granule cells evoked by PACAP was significantly inhibited by the PACAP receptor antagonist PACAP(6-38). Administration of PACAP also caused a significant increase in the volume of the cerebellar cortex. The present study provides evidence that PACAP can act in vivo as a trophic factor during rat brain development. Our data indicate that PACAP increases proliferation and/or inhibits programmed cell death of granule cells, as well as stimulating neuronal migration from the external granule cell layer toward the internal granule cell layer.

Aging↗
PubMed1993

Differential localization of class III, beta-tubulin isotype and calbindin-D28k defines distinct neuronal types in the developing human cerebellar cortex.

This immunohistochemical study compares the localization of the neuronal class III beta-tubulin isotype (beta III) to that of calbindin-D28k in 40 human fetal and postnatal cerebella ranging from 12 weeks gestation to adulthood. In the external granule layer of the developing cerebellar cortex, beta III staining was present in the premigratory (postmitotic) zone of horizontal neurons but was absent in "epithelioid" cells of the subpial proliferative mitotic zone. In the molecular layer, intense beta III staining was associated with parallel fibers, stellate/basket neurons and migrating fusiform granule neurons. beta III staining was also present in internal granule neurons. In contrast, beta III was not detectable in fetal and neonatal Purkinje neurons and Golgi II neurons, but was evident in these neurons from juvenile and adult cerebella. Calbindin-D28k staining was present in Purkinje neurons also delineating their somatic spines ("pseudopodia"), lateralizing and apical dendrites (including dendritic spines), subpopulations of small to intermediate-sized Golgi II neurons in the internal granule layer ("synarmotic cells" of Landau), large to medium-sized subcortical Golgi II neurons and neurons of cerebellar roof nuclei, at various gestational stages and postnatally. It was absent in the external granule layer, parallel fibers, stellate/basket and internal granule neurons. Variable degrees of beta III and calbindin-D28k staining were detected in subpopulations of immature neuroepithelial cells of the ventricular matrix at the roof of the fourth ventricle. Glial (including Bergmann glia) and mesenchymal cells were not stained for either antigenic determinants. The differential expression of calbindin-D28k and beta III defines distinct populations of neurons in the developing human cerebellar cortex and supports the ontogenetic concept of Ramon y Cajal.

Adolescent↗
PubMed1997

Cerebellar cortex delayed maturation in sudden infant death syndrome.

The cerebellum shows afferent and efferent connections with intrinsic bulbar nuclei and plays an important role in respiration and cardiovascular control. Pathological and neurochemical abnormalities of bulbar nuclei including the arcuate nucleus have been postulated in sudden infant death syndrome (SIDS). Most of these abnormalities have been related to impairment in brain development. The cerebellar cortex has a well-documented evolution from fetal life until infancy; thus, it may be a very good model to assess brain maturation in SIDS. The present study was conducted to investigate changes in the cerebellar cortex in 19 SIDS cases compared with 12 age-related controls using morphological, quantitative, and statistical approaches. Five-microns paraffin sections from the midsagittal cerebellar vermis were stained with hematoxylin and eosin (H&E). Immunohistochemical staining was carried out using a polyclonal antiserum to glial fibrillary acidic protein (GFAP). Each case consisted of a 25-microns parallel paraffin section stained with H&E, where the cerebellar external granular layer (EGL) cell density was obtained in one field magnification (x1,000) using an optical dissector procedure on the basis of a stereological method. A statistically significant high EGL cell density, mostly related to the presence of immature bipolar, elongated neuronal cells of the premigratory zone with hyperchromatic, oval or poor differentiated nuclei, was observed in SIDS. In these cases, EGL expressed immunoreactivity for GFAP mainly in the subpial and the postmitotic zone. These findings demonstrate a delayed or slower decline in the number of EGL neurons in SIDS, suggesting either a prolongation of the growth phase related to postnatal cerebellar foliation or a delay in inward migration. These results suggest that in SIDS there is delayed maturation of the cerebellar cortex/EGL, which may support the hypothesized cardiopulmonary control dysfunction, leading to death in a vulnerable period of postnatal development.

Adult↗
PubMed2001

Quantitative immunocytochemistry of glia in the cerebellar cortex of old ethanol-fed rats.

It is clear from results of studies in this laboratory that chronic ethanol consumption causes regression of the extensive Purkinje neuron (PN) dendritic arbor. There are, however, a paucity of studies on the effects of chronic ethanol consumption on glia cells that reside in the molecular layer of the cerebellar cortex with PN dendrites. The purpose of the present study was to investigate the possibility that chronic ethanol consumption in old F344 rats results in gliosis within the molecular layer of the cerebellar cortex. Ten 12-month-old, male, F344 rats received a liquid diet containing 35% ethanol for 40 weeks. Pair-fed controls (n=10) received a liquid diet in which maltose dextrins were substituted for ethanol. Chow-fed rats (n=10) served as controls for age. At the end of the treatment period, rats were euthanized and perfused through the aorta, and cerebella were prepared for immunocytochemistry. Free floating sections were stained with (1) glial fibrillary acidic protein antibody for labeling of Bergmann glial cells and fibers, (2) OX-42 antibody for labeling of microglia, and (3) 0.5% cresyl violet for estimates of molecular layer volume. Results indicate that the densities of Bergmann glial cell processes and microglia within the cerebellar molecular layer are not altered by ethanol consumption.

Animals↗
PubMed1989

Light and electron microscope immunolocalization of cytosolic malic enzyme-like activity in the rat's cerebellar cortex.

The immunocytochemical localization of malic enzyme-like activity in the cerebellar cortex of the rat at light and electron microscope levels using the PAP method was investigated. The antiserum against the malic enzyme was obtained from rabbit after having injected the chicken liver malic enzyme. The microscope observations have demonstrated the presence of a positive immunocytochemical reaction against malic enzyme in the cytosol of Bergmann glial cells and in the astrocytes of granular layer and also in several dendrites, but not in all, in the postsynaptic site. A possible role of cytosolic malic isoenzyme (cME) in the several synapses is suggested.

Animals↗