G. Gombos

Different effect of methylazoxymethanol on mouse cerebellar development depending on the age of injection

SummaryMethylazoxymethanol (MAM), a powerful antimitotic, has been extensively used to affect rodent CNS development. Here we show that MAM causes different effects on mouse cerebellum depending on the age of the injected pup. Sublethal doses were determined for each age. A single injection at birth permanently reduces the number of cells. In addition, the cytoarchitecture was greatly perturbed: Purkinje cells retained an immature aspect and were dispersed through the cerebellar cortex. A single dose of MAM injected into 5 day old mice also affected the number of cells but, at the level of light microscopy, the cytoarchitecture of the cerebellar cortex appeared not to be altered. Purkinje cells, however, showed some immaturity and degenerated around the 22nd postnatal day. This modulation of MAM effect appears to provide a good model for studying cerebellar ontogeny and neuronal plasticity.

l-glutamate andl-glutamine uptake in adult rat cerebellum: An autoradiographic study

The compartmentation of L-glutamate in the central nervous system has been extensively studied and L-glutamine is believed to be the precursor of the neuronal releasable pool of the L-glutamate. In order to localize the sites of uptake of both L-glutamate and L-glutamine, autoradiography was used in tissue slices of adult rat cerebellum, where granule cells are considered to be glutamatergic. Incubation of the tissue with low concentrations of [3H]L-glutamate or [3H]L-glutamine produces in both cases a heavy labelling of the molecular layer. [3H]L-glutamate uptake seems to be essentially glial (Golgi epithelial cells and Bergmann fibres) while [3H]L-glutamine is more diffusely distributed over the molecular layer. Although no conclusions can be drawn on the nature of L-glutamine uptake, these results are in agreement with the model which considers L-glutamate uptake by glial cells to be the inactivating process of glutamatergic synapses.

Distribution of the neural antigen BSP-2 in the cerebellum during development

The monoclonal antibody anti‐BSP‐2 recognizes three glycosylated peptide chains of 180,000, 140,000 and 120,000 daltons in extracts from adult mouse forebrain and cerebellum. In extracts of embryonic or neonatal brain, it recognizes a different form, migrating as a broad band of higher molecular weight on SDS polyacrylamide gels. This report describes the distribution of the antigen BSP‐2 in developing mouse cerebella using a sensitive immunoperoxidase technique at the electron microscope level. As early as 3 days after birth the antigen can be detected on the surface of all cerebellar neurons, including neuroblasts in the external granular layer, basket and stellate neurons and Purkinje cells. In addition, radial glial fibres (astrocytes) terminating on the pial surface contain BSP‐2. At later stages a pronounced surface labelling of parallel fibres is observed. Migrating granule cell perikaryal membranes possess the glycoprotein. The results are discussed in relation to the apparent identity of BSP‐2 and the cell adhesion molecule N‐CAM and its physiological properties.

Recent Methods for the Separation and Analysis of Central Nervous System Glycoproteins

The methodology for fractionation and analysis of brain glycoproteins has already been thoroughly reviewed in a previous volume of this series (Margolis and Margolis, 1972). In this chapter we intend to add some more recent techniques with particular emphasis on those developed for studies on membrane-bound glycoproteins.

Étude de la fraction protéique neurospécifique S 100 : II. — Hétérogénéité moléculaire de la fraction protéique S 100

Summary A method, involving only two steps, has been developed for the rapid preparation of the neurospecific S-100 protein fraction with high yields. The method involves a fractionated ammonium sulphate precipitation followed by chromatography on DEAE-cellulose. Subsequently the slow and fast migrating electrophoretic components that have demonstrated in vivo can be separated. The comparison of tryptic peptide maps indicates that these two components are not different conformational states of one single protein, but that there are at least two proteins of distinct primary structure in the S-100 protein fraction. A more detailed study has shown that the fraction is even more heterogeneous. This conclusion contradicts previous authors who have claimed that the « S-100 proteinwas homogeneous. Taking into account these results and our previous immunological studies, we suggest that the heterogeneity may be due to the existence of isoproteins of S-100, analogous to the lactate dehydrogenase isoenzymes. In electrophoretic systems which contain EDTA, above a minimal concentration which depends upon the buffer system, the isoproteins separate into two classes, the fast and slow components. The interspecific and regional differences of the two components in the central nervous system could be due to varying proportions of these isoproteins. The complexity of the S-100 fraction demonstrated by electrophoretic analysis in the absence of EDTA should, in our opinion, be related to the existence of these isoproteins and to the effect of calcium ions upon their conformation.

Uptake and metabolism of L-[3H]glutamate and L-[3H]glutamine in adult rat cerebellar slices.

Using very low concentrations (1 μmol range) ofl-2-3-[3H]glutamate, (3H-Glu) orl-2-3-[3H]glutamine (3H-Gln), we have previously shown by autoradiography that these amino acids were preferentially taken up in the molecular layer of the cerebellar cortex. Furthermore, the accumulation of3H-Glu was essentially glial in these conditions. We report here experiments in which uptake and metabolism of either (3H-Glu) or (3H-Gln) were studied in adult rat cerebellar slices. Both amino acids were rapidly converted into other metabolic compounds: after seven minutes of incubation in the presence of exogenous3H-Glu, 70% of the tissue accumulated radioactivity was found to be in compounds other than glutamate. The main metabolites were Gln (42%), α-ketoglutarate (25%) and GABA (1,4%). In the presence of exogenous3H-Gln the rate of metabolism was slightly slower (50% after seven minutes of incubation) and the metabolites were also Glu (29%), α-ketoglutarate (15%) and GABA (5%). Using depolarizing conditions (56 mM KCl) with either exogenous3H-Glu or3H-Gln, the radioactivity was preferentially accumulated in glutamate compared to control. From these results we conclude: i) there are two cellular compartments for the neurotransmission-glutamate-glutamine cycle; one is glial, the other neuronal; ii) these two cellular compartments contain both Gln and Glu; iii) transmitter glutamate is always in equilibrium with the so-called “metabolic” pool of glutamate; iv) the regulation of the glutamate-glutamine cycle occurs at least at two different levels: the uptake of glutamate and the enzymatic activity of the neuronal glutaminase.

Developmentally regulated changes of glutamate binding sites in mouse deep cerebellar nuclei

The expression of L-[3H]glutamate binding sites of different ionic and pharmacological sensitivities was studied in mouse deep cerebellar nuclei during early postnatal development by means of in vitro autoradiography. Ca2+/Cl(-)-dependent, quisqualate/AMPA/ibotenate-sensitive, and APB-insensitive binding sites are present at high density in the deep cerebellar nuclei of young animals, but greatly decrease between the 10th and 25th postnatal day and remain low in the adult. The density of Ca2+/Cl(-)-independent binding sites remains low and constant during the whole of postnatal development. The possible involvement of the Ca2+/Cl(-)-dependent binding sites in brain development is discussed.

Alteration of benzodiazepine receptors in mouse cerebellum following methylazoxymethanol treatment during development

The specific binding of [3H]flunitrazepam was studied to biochemically specify the morphological alterations induced in mouse cerebellum by a single injection of an antimitotic agent, methylazoxymethanol (MAM) performed at the beginning of the postnatal life. The MAM injection causes a general reduction of the benzodiazepine receptors in the adult mice which is particularly severe in mice having been injected the 1st day of postnatal life (so-called MAM0 mice) as compared to animals injected the 5th day (MAM5 mice): in MAM0 mice the benzodiazepine receptor is reduced to half of the control value. The affinity of the benzodiazepine towards its receptor was not affected and the topographic and biochemical action of MAM in the central nervous system was ascertained. Correlations could be made between the biochemical modifications and the morphological alterations otherwise described.

Étude de la fraction protéique neurospecifique S-100 : I. Présence in vivo des composants rapide et lent de la fraction protéique S-100

Summary The possibility that fast and slow migrating components of S-100 protein fraction, a neurospecific protein, were artifactual, was considered. Several putative causes of artifacts, particularly in relation to conditions of extraction and electrophoresis of brain soluble proteins were examined and ruled out. The two components are neither aggregates nor subunits since they have the same sedimentation coefficients as whole S-100 protein fraction. They are not due to post mortem changes, since there were no differences whether the brain was homogenized immediately or incubated first for 4 h at 37°. Nor are they due to the oxidation of SH-groups, since 2-mercaptoethanol in the extraction buffer or in the electrophoretic system had no effect on the two band pattern. In addition, prerunning of the gels had no effect. Above all, the effect of low (physiological) and high Ca++ ion concentration on the S-100 protein fraction was examined. The two components were not related to the electrophoretic bands induced by Ca++ ions which have been described by certain authors. All the data are in favour of the presence in vivo of fast and slow migrating components of S-100 protein fraction.

An improved method for the preparation of rat cerebellar glomeruli

Cerebellar glomeruli consist of large portions of the mossy fiber giant terminal, granule cell dendrites and Golgi neuron terminals. By modifying previously reported procedures we have developed a new method for bulk preparation of this polysynaptic complex from rat cerebellum. We obtained well preserved isolated glomeruli of satisfactory purity and homogeneity as indicated by electron microscopy and by determination of appropriate biochemical markers. The method is fast and simple, and it provides a glomerular fraction suitable for investigation of neurotransmitter receptors.