J. de Barry

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.

Ectopic granule cell layer in mouse cerebellum after methyl-azoxy-methanol (MAM) treatment

SummaryPrevious results from our laboratory (Bejar et al. 1985) indicated that a single injection in mouse pups of the antimitotic/mutagenic agent methylazoxymethanol at postnatal day 5 typically produces hypogranular cerebella with no changes in foliation, in contrast to the severe alterations observed after the more usual injection on the day of birth. Here we report that injection of a higher dose (30 mg/kg) of methylazoxymethanol, always at postnatal day 5, leads to the additional presence of a ectopic cell layer in adult cerebellum. Immunostaining with several antibodies recognizing cell specific proteins ruled out the possibility that these ectopic cells were glial and electron microscopy indicated that they were morphologically mature granule cells. In the molecular layer of other cerebellar areas and apparently unrelated with granule cell ectopia, ectopic Golgi epithelial cells were observed. The reason for the presence of these ectopic cells of different type in the molecular layer was discussed in relation with analogous ectopias obtained by other means.

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.

A developmentally modified neurone-specific marker in rodent cerebellum

Out of several monoclonal antibodies secreted by hybridomas resulting from the fusion of a mouse myeloma cell line with spleen cells from mice immunized with cerebellar membranes from 12 day old rats, one, called 11.9, produced an unusual immunolabelling pattern when tested on sections of rat cerebellum. The cerebellar distribution of the antigenic sites recognized by this antibody using an immunoperoxidase technique at the optical and ultrastructural levels is described in detail in this report. The immunoreaction product was found in the adult rat to be associated with the microtubules and the zone immediately beneath the plasma membrane of parallel fibres. In young animals the density of immunostaining appears to be higher than in the adult, and the staining is detectable in addition in the perikaryal cytoplasm of granule cells. Biochemical studies using the Western immunoblot technique demonstrate that the antigens consist of two polypeptides of molecular weights 120 and 185 kD. The possible relation of the antigens to cytoskeletal structures is discussed and the labelling pattern is compared with that produced by other known monoclonal antibodies.

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.

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.

Developing rat cerebellum: Glutamine and glutamate influx correlated to the cellular distribution of glutamine synthetase.

Glutamate has been suggested to be the neurotransmitter of the granule cells in cerebellar cortex. Autoradiographic studies using very low concentrations (1μM range) of 2‐3‐[3H]l‐glutamate or 2‐3‐[3H]l‐glutamine have shown that both amino acids were preferentially taken up in the molecular layer of the cerebellar cortex of adult rats, but [3H]Glu accumulated essentially in glial cells, while Gln did not show a cellular preference (de Barry et al., Neuroscience 7,1289–1297,1982).