Evidence for the Presence of Lectins with Mannose Specificity in the Rat Cerebellum

Two different methods were set up to detect the possible presence of lectin-like molecules with a specificity for mannose-rich glycans in the rat cerebellum. The first, affinity histochemistry, involved the isolation of a particular class of glycoproteins from the cerebella of 11-day-old rats followed by the formation of covalent complexes with horseradish peroxidase and then incubation with cerebellar slices. The second used in vitro interactions between [3H]leucine-labeled proteins, kept in solution, with insolubilized [14C]glucosamine-labeled glycoproteins. The results of both methods are compatible with the presence of lectin-like activities inhibited by high mannose concentrations, but not other sugars. However, the binding sites preferred by these molecules seem to be more than a single mannose residue.     

Decreased Benzodiazepine Receptor Density in Rat Cerebellum Following Neurotoxic Doses of Phenytoin

Abstract: The effect of acute and chronic administration of phenytoin on [³H]-flunitrazepam binding was examined in the rat cerebellum. There was no significant effect of phenytoin on [³H]flunitrazepam binding in the rat cerebellum 1 and 6 h after a single i.p. injection of 200 mg/kg of phenytoin. However, after 14 days and 28 days of chronic phenytoin administration, significant de-creases in [³H]flunitrazepam receptor density were observed, with no changes in apparent affinity constants in the rat cerebellum. This effect of phenytoin was dose-dependent, as lower doses of phenytoin (100 mg/kg/day) for 14 or 28 days produced no alterations in [³H]flunitrazepam binding in the rat cerebellum. Light-microscopic examination of the rat cerebellum treated with 200 mg/kg/day of phenytoin for 14 days showed degeneration of the Purkinje cells, with edematous Bergmann astrocytes. These data provide evidence for the neuronal localization of benzodiazepine receptors on cerebellar Purkinje cells.     

Differential Staining of Two Subpopulations of Purkinje Neurons in Rat Cerebellum with Acid Dyes

We present a new method that stains differently two subpopulations of Purkinje cells in the adult rat. Deparaffinized sections of cerebella, fixed by perfusion with buffered glutaraldehyde or Bouin's fluid were stained with 0.5% light green in 50% ethanolf 10-30 min). The excess dye was removed with saturated aqueous picric acid (10-30 min). At this point some Purkinje cells appeared as lightly stained neurons, while others were strongly stained. Slides were immersed in 0.5% aqueous acid fuchsin for approximately 1 min until the lightly stained neurons acquired a red color. Following immersion in 1% phosphotungstic acid, slides were rapidly dehydrated in ethanol, passed to xylene and mounted in Canada balsam. Two subpopulations of Purkinje cells differing in their protein content in somata and proximal dendrites stained differentially by this method. They occurred in all coronal and sagittal sections and in patches or stripes. Their relative proportion varied from lobule to lobule. A second staining method used potassium permanganate as the sole staining reagent. The staining reagent can be used on sections previously stained with the acid dyes. Purkinje cells appeared as subsets of brownish to deep brown stained neurons, the latter ones corresponding to green stained cells in the dichromic method. The results obtained indicated that the subpopulations reflect real differences among individual neurons and are not artifacts. The technique holds promise for identifying and localizing subsets of Purkinje cells differing in their protein content under normal and experimental conditions and for their further characterization by combined staining and histochemical procedures.     

Solubilisation of the Benzodiazepine Receptor from Rat Cerebellum by the Detergent n-Octylpentaoxyethylene

The detergent n-octylpentaoxyethylene is one in the series of tenside detergents developed for membrane solubilisation. We have used this detergent to solubilise benzodiazepine receptors from rat cerebellum. The soluble receptor has an affinity (KD) for [3H]flunitrazepam of 1.8 nM +/- 0.2, which is not significantly different from that observed in synaptic membranes. Under optimal conditions (0.6% wt/vol), the number of soluble flunitrazepam binding sites (Bmax) of 0.35 pmol/mg protein suggests an apparent solubilisation of 40% of sites from the membrane. However, the absence of the characteristic facilitation of [3H]flunitrazepam binding by gamma-aminobutyric acid (GABA), cartazolate, and pentobarbital in this soluble receptor preparation suggests that such a preparation is unlikely to be a useful preparation for further studies on the molecular mechanisms underlying GABAA receptor function.     

Modeling of Myelination and Demyelination Processes in Cell Culture of the Rat Cerebellum

We studied the structural features of the myelination process under conditions of culturing of dissociated cerebellar tissue obtained from newborn rats, and we made a comparative analysis of the modifications of the structure of myelin sheaths under the influence of a demyelinating factor – blood serum obtained from patients with multiple sclerosis (MS) in different stages of the disease was added to the medium. Under the conditions of our experiments, the optimum term for the formation of full-value myelinated sheaths in vitro was 26 days of culturing; thus, we used this time interval for studying the demyelinating influence. Addition of the blood serum of MS patients in the remission or acute stages very quickly (in a few hours) evoked significant morphological damage to the myelin sheaths of the axons. In general, the demyelinating effects of the blood serum of acute-stage MS patients were more significant.     

Thyroid Hormone Mediates Syndecan Expression in Rat Neonatal Cerebellum

Thyroid hormone (T₃) plays an essential role in the central nervous system development. Astrocytes mediate many of the T₃ effects in the growth and differentiation of cerebellum. In culture, T₃ induces cerebellar astrocytes to secrete growth factors, mainly FGF₂, and alters the expression and organization of the extracellular matrix (ECM) proteins, laminin, and fibronectin. In addition, T₃-treated astrocytes promote neuronal differentiation. In this study, we have investigated whether other ECM molecules, such as syndecans, are involved in T₃ action. Thus, we analyzed the expression of syndecans (1–4) by RT-PCR in astrocyte cultures from cerebellum, cortex, and hippocampus of newborn rats. Our results showed that syndecans (1–4) are expressed in astrocytes of cerebellum and cortex, whereas in hippocampus only syndecans 2 and 4 were detected. Semi-quantitative RT-PCR analysis revealed the reduced expression of syndecans 1, 2, and 4, and increased expression of syndecan 3 in hypothyroid cerebellum, when compared to the euthyroid tissue. Furthermore, we observed a reduced expression of syndecans 2 and 3 in T₃-treated cerebellar astrocytes, when compared to control cultures. This balance of proteoglycans may be involved in T₃ action mediated by FGF₂ signaling, possibly affecting the formation of the trimeric signaling receptor complex composed by syndecan/FGF/FGF-receptor (FGFR), which is essential for FGFR dimerization, activation, and subsequent cell signaling.     

Mtss1抗体制备及小脑中不同亚型磷酸化水平的检测

肿瘤转移抑制蛋白(Metastasis suppressor1,Mtss1),又名肿瘤转移消失蛋白(Missing in metastasis,MIM)在小脑神经元发育中受到调控,依次表达两种亚型:包含Src磷酸化位点的广泛表达亚型;含Src磷酸化位点片段经RNA剪接去除的神经元特异亚型.为检测这两种Mtss1亚型的酪氨酸磷酸化水平是否因Src磷酸化位点的去除存在明显区别,制备了灵敏度较高并可特异性沉淀外源和内源表达的Mtss1的兔多克隆抗体,对发育时期与成年大鼠小脑内源Mtss1酪氨酸磷酸化水平进行检测,发现成年后的Mtss1与出生后发育时期的Mtss1均发生明显的酪氨酸磷酸化,表明剪接去除包含有Src磷酸化位点中的神经元特异亚型中,还有其他的酪氨酸残基被磷酸化,提示其他酪氨酸磷酸化激酶信号通路对Mtss1神经元亚型的调控作用.     

Synaptic Membrane Antigens in Developing Rat Brain Cerebral Cortex and Cerebellum

Abstract: The contents of five synaptic membrane antigens (56K, 58K, 62K, 63K, and 64K) were determined in rat cerebral cortex and cerebellum at eight developmental time points: E9, E14, P < 1, P5, P14, P28, P60, and P180 (E, embryonic; P, postnatal). In cerebral cortex, the five antigens showed five different developmental patterns with respect both to specific content (i.e., quantity per unit of membrane) and total content (i.e., quantity per cortex). The 56K, 58K, and 62K polypeptides were first detected at E14, increased slightly to P5, then increased rapidly from P5 to P28 by 14-, 11-, and 18-fold, respectively. From P28 to PI80, the patterns of these antigens showed very large differences. The 63K and 64K antigens were first detected at P14 and P28, respectively. The specific content of 63K antigen continued to increase steadily throughout adult life; in contrast, the specific content of the 64K antigen did not change appreciably. In cerebellum only three antigens (56K, 58K, and 62K) were detected. These three antigens showed different developmental patterns. The 56K polypeptide was first detected at E14; its specific content increased very rapidly to a maximum at P < 1; it then decreased, first slowly, and then more rapidly, disappearing at P60. The 58K polypeptide also was detectable at E14 and increased very rapidly to a maximum at P < 1. It then decreased markedly to P5, followed by an increase, returning almost to its maximum level at P14. It then slowly decreased disappearing at P180. The 62K antigen was first detected at P14 and then it slowly decreased with disappearance at P60. The patterns with respect to total contents per cerebellum were similar for the three antigens, with a maximum at P28. We conclude that the highest increase in the contents of these antigens roughly corresponds to the period of maximal synaptogenesis (P9 to P28) in both regions. Differences among developmental patterns probably reflect changing molecular machinery required for development and functional differentiation of synapses in different brain regions. The fine structure of these patterns suggests that the quantitative measurement of synaptic membrane antigens will be useful for delineating complex processes occurring during synaptogenesis.     

Alterations in the Development of Rat Cerebellum and Impaired Behavior of Juvenile Rats after Neonatal 6-OHDA Treatment

The effects of neonatal systemic administration of the neurotoxin 6-hydroxydopamine (6-OHDA) on cerebellum development and behavior were studied in juvenile rats. The methods employed were immunohistochemistry, in situ hybridization, ligand binding, and behavioral testing. The results revealed, for the first time, that 6-OHDA treatment alters Bergmann glial cells and reduced the expression GABA_A receptor subtypes α1 and α6 especially in granule cells. The Bergmann glial cells were abnormally located and structurally different (e.g., no intimate associations with Purkinje cells). Significant microglial activation was also observed. The animals showed impairment in behavior, especially in their orientation to a novel environment. Recent data on neuron–glia interactions support the conclusion that the observed structural changes in Bergmann glia and granular neurons disrupted the normal functioning of the Purkinje cells which then in turn resulted in the impaired sensory-motor coordination at least in juvenile rats. This paper is a summary of previously published work and some recent data in this field obtained at our laboratory. Special issue dedicated to Dr. Simo S. Oja     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

Palmdelphin在出生后发育的大鼠小脑中的表达模式研究

Palmdelphin是参与质膜的动态变化与细胞形态的调控的paralemmin家族新成员,与神经发育的相关性尚不明确.前期工作提示,它与调控小脑发育的一种肌动蛋白结合蛋白Mtss1(metastasis suppressor1)具有一定相关性.为了探索该基因与小脑出生后发育的相关性,利用原位杂交技术研究Palmdelphin在小脑中的时空表达,结果表明,Palmdelphin在出生后第7d大鼠小脑中有明显的表达,且分布主要集中在浦肯野神经元.半定量RT-PCR的结果进一步表明Palmdelphin的转录水平在小脑发育过程中受到调控,在出生后7d有表达高峰.这些结果显示Palmdelphin与小脑出生后神经元发育存在一定相关性.     

Impaired Development of Rat Cerebellum Induced by Neonatal Injection of the Glycoprotein Synthesis Inhibitor, Tunicamycin

The effects of the protein glycosylation inhibitor tunicamycin on the postnatal development of the rat cerebellum were examined in vivo. Tunicamycin (0.2 micrograms) was injected intracranially into 1-day-old rats. Inhibition of glycosylation of the macromolecules in the cerebellum by tunicamycin treatment was suggested by a reduced incorporation of [3H]glucosamine into the trichloroacetic acid (TCA)-insoluble fraction. The tunicamycin treatment did not affect gain in body weight significantly. However the cerebellar weight was significantly reduced by 30-40% compared with that of the controls. Development of GABAergic and cholinergic innervations in the hypoplastic cerebellum was examined by measuring the activities of glutamate decarboxylase (GAD) and choline acetyltransferase (ChAT). The specific activity and the total activity of GAD were significantly reduced in the tunicamycin-treated cerebellum. In contrast the specific activity of ChAT was significantly increased, whereas the total activity of ChAT per cerebellum was identical with that of the controls. These results suggest that the intracranial injection of tunicamycin affects the postnatal development of rat cerebellum, such as GABAergic and cholinergic innervations.     

Degranulation and appearance of vesicular inclusions in canine C cells after administration of antithyroid drug

Summary When stimulated by the feeding of ethylenethiourea for a period of 6 months, dog thyroid glands increased to approximately 30 times their normal size. Not only follicular cells but also C cells had hyperplastic and hypertrophic features. Compared with normal controls there was in the C cells a marked decrease in secretory granules immunoreactive for calcitonin. Furthermore, vesicular inclusions of various sizes and dilated nuclear envelopes, which showed a positive immunoreaction for calcitonin, were observed in the C cells. These findings indicate that the antithyroid drug interferes with synthesis of calcitonin by C cells, probably by inhibiting the conversion of a larger precursor to calcitonin.     

Expression of Tropomyosin Genes During the Development of the Rat Cerebellum

The expression of the tropomyosin genes in the rat nervous system was examined during the postnatal development of the cerebellum, using human-specific alpha-, beta-, gamma-, and delta-tropomyosin cDNA probes and rat-specific alpha-, beta-, and delta-tropomyosin oligonucleotide probes. The beta- and gamma-genes do not seem to be expressed in the rat brain. The delta-tropomyosin gene produces two mRNAs: a major one of 2.4 kb, which is highly concentrated during the first postnatal week and then decreases fourfold in level until the age of 35 days, and a minor one of 2 kb, with the same developmental profile as the 2.4-kb mRNA. A 3-kb mRNA is expressed by the alpha-tropomyosin gene and is characteristic of the mature rat. The expression of the tropomyosin genes during the development of the rat cerebellum does not seem to be regulated through alternative splicing but rather implies the differential expression of two different isogenes. The multiple isoforms of tropomyosin produced during neuronal differentiation may be intimately involved in the regulation of the organization and function of actin microfilaments.     

Endothelins Induce Fos Expression in Neurons and Glia in Organotypic Cultures of Rat Cerebellum

Abstract: Endothelins (ETs) and their receptors are present in high levels in the brain and have been proposed to act as neuromodulators or neurotransmitters. However, neither their role nor their precise mechanism of action in the brain is understood. In this study, c- fos expression was used as a marker of neuronal activation in organotypic cultures of rat cerebellum. ETs induced Fos protein expression in both granule cells and glia but not in Purkinje cells. Granule cells and glia were both very sensitive to ETs, but different receptor subtypes appeared to be involved, because granule cells did not respond to ET-3. However, they did respond to the ET_B-selective agonist BQ3020, suggesting the possible existence of a novel neuronal ET_B-like receptor. The induction of Fos in granule cells was independent of extracellular calcium ion concentration, but the ryanodine receptor antagonist dantrolene significantly inhibited the response to ETs, suggesting that the mobilisation of calcium ions from intracellular stores is important. These data support previous evidence that ETs act directly on neurones and show that the intracellular pathways after ET receptor activation are complex. It appears likely that ETs play an important neuromodulatory role in the cerebellum.     

Agonist-Stimulated Inositol Polyphosphate Formation in Cerebellum

The accumulation of inositol polyphosphates in the cerebellum in response to agonists has not been demonstrated. Guinea pig cerebellar slices prelabeled with [3H]inositol showed the following increases in response to 1 mM serotonin: At 15 s, there was a peak in 3H label in the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], decreasing to a lower level in about 1 min. The level of 3H label in the putative second-messenger inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] increased rapidly up to 60 s and increased slowly thereafter. The accumulation of 3H label in various inositol phosphate isomers at 10 min, when steady state was obtained, showed the following increases due to serotonin: inositol 1,3,4-trisphosphate [Ins(1,3,4)P3], eight-fold; Ins(1,3,4,5)P4, 6.4-fold; Ins(1,4,5)P3, 75%; inositol 1,4-bisphosphate [Ins(1,4)P2], 0%; inositol 3,4-bisphosphate, 100%; inositol 1-phosphate/inositol 3-phosphate, 30%; and inositol 4-phosphate, 40%. [3H]Inositol 1,3-bisphosphate was not detected in controls, but it accounted for 7.2% of the total inositol bisphosphates formed in the serotonin-stimulated samples. The fact that serotonin did not increase the formation of Ins(1,4)P2 could be due to the fact that Ins(1,4)P2 is rapidly degraded or that Ins(1,4,5)P3 is metabolized primarily by Ins(1,4,5)P3-3'kinase to form Ins(1,3,4,5)P4. In the presence of pargyline (10 microM), [3H]Ins(1,3,4,5)P4 and [3H]Ins(1,3,4)P3 levels were increased, even at 1 microM serotonin. Ketanserin (7 microM) completely inhibited the serotonin effect, indicating stimulation of serotonin2 receptors. Quisqualic acid (100 microM) also increased the levels of [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3, but the profile of these increases was different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cerebellum Lipids in Rats After Chronic Ethanol Treatment

Eighteen male Wistar rats weighing approximately 200 g were divided into three groups of six animals each. The experimental animals were maintained on nutritionally complete diets in which ethanol comprised 45% of the available energy. Control animals were pair-fed an equivalent diet in which sucrose was substituted isocalorically for ethanol. An additional control group received unlimited access to standard pelleted laboratory food and water. The investigations were carried out over 24 weeks. The effects on phospholipid, monogalactosyl glycolipid, and ganglioside composition after 24 weeks of feeding 43% alcohol were studied. There is abundant evidence that the changes in the cerebellum membrane phospholipids (phosphatidylethanolamine and phosphatidylcholine), gangliosides (GT1b), and myelin lipids (phosphatidylserine, sphingophospholipid, phosphatidylinositol, cerebrosides with hydroxy fatty acids, sulfoglycolipids, and monosialoganglioside GM1) occur as a result of chronic ethanol treatment.