Alain Rabié

Calcium-binding protein in the developing rat cerebellum

SummarySpecific antibodies raised against a human 28 000 dalton cerebellar calcium-binding protein (CaBP) were used in an immunocytochemical study during development of the rat cerebellum. Both light and electron microscopy showed (1) that labelling was entirely restricted to the Purkinje cells, (2) that it appeared very early in Purkinje cell development, (3) that the entire cell was labelled from the tip of the smallest dendrites to the axonal terminals, and (4) that with increasing age, the immunoreaction appeared to be progressively restricted to the cell and organelle membranes.

Immunocytochemical detection of calcium-binding protein in the cochlear and vestibular hair cells of the rat.

SummarySpecific antibodies raised against human cerebellar calciumbinding protein (CaBP) intensely labelled the cochlear hair cells of the rat. The vestibular hair cells also stained weakly. In both inner and outer cochlear hair cells, the cuticular plate was the most stained area. These results suggest that CaBP may prevent excessive concentrations of intracellular calcium and thus modulate some Ca2+-mediated biochemical processes, especially at the level of the cuticular plate and stereocilia; CaBP could be involved in the mechanochemical coupling of hearing or vestibular function.

The V1a and V1b, But Not V2, Vasopressin Receptor Genes Are Expressed in the Supraoptic Nucleus of the Rat Hypothalamus, and the Transcripts Are Essentially Colocalized in the Vasopressinergic Magnocellular Neurons

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Sequential effects of thyroxine on the developing cerebellum of rats made hypothyroid by propylthiouracil

Young rats made hypothyroid by propylthiouracil (PTU) received a daily physiological dose of thyroxine (T4) from day 0, 4, 6, 8, 10, 11, 12 or 13 and their cerebella were studied on day 14. With the very low doses of T4 used and when the treatment was started at birth, cerebellar development was nearly normal in terms of the parameters studied (cell formation, migration, maturation and death). The effect of T4 on cell formation appeared after two days. With the same latency, T4 induced migration of the newly-formed granule cells. The effects on those processes requiring cell movements over long distances, e.g. the number of cells in the internal granular layer or the thickness of the molecular layer, were longer to appear. The most rapidly affected parameter was the pyknotic index in the internal granular layer. This index was half. The increased cell death in the cerebellum of hypothyroid rats is probably related to the decreased synaptogenetic competence of Purkinje cells. The rapidity of the effect of T4 on the pyknotic index may be related to an important effect of this hormone on the formation of synapses and, more generally, on the mechanisms of neuronal maturation.

Immunocytochemical detection of 28000-MW calcium-binding protein in horizontal cells of the rat retina

SummaryHorizontal cells of rat retina were labeled intensely by a specific antibody to cerebellar calcium-binding protein. The amacrine cells stained very weakly. The presence of calcium-binding protein in horizontal cells could be of interest for the understanding of the feedback action of these cells on photoreceptors.

The Vasopressin Receptors Colocalize with Vasopressin in the Magnocellular Neurons of the Rat Supraoptic Nucleus and Are Modulated by Water Balance

Activity of the magnocellular neurons that synthesize vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus is modulated by local release of the neuropeptide within the nuclei. V1a and V1b vasopressin receptor genes are expressed in these cells. The present study reports the localization of V1a and V1b receptors using multiple labeling immunocytochemistry. Both receptors are mainly located in vasopressinergic magnocellular neurons and colocalized with vasopressin in cytoplasmic vesicles dispersed throughout the cell. Possible functional modifications of the mRNA and protein levels of the V1a receptor, the major isoform, were also investigated by semiquantitative in situ hybridization and immunocytochemistry in rats submitted to reduced or increased water intake. V1a mRNA and receptor levels varied with water balance. V1a mRNA level dropped in rats submitted to high water intake. Conversely, dehydration up-regulated the V1a receptor content. These observations suggest that the pathwa...

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells

To characterize the volume‐sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [3H]‐taurine efflux from acutely isolated SON. Taurine release induced by hypotonic stimulus (250 mosmol l−1) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, confirming the lack of implication of the transporter. The osmodependent release of taurine was blocked by a variety of Cl− channel inhibitors with the order of potency: NPPB>niflumic acid>DPC>DIDS>ATP. On the other hand, release of taurine was only weakly affected by other compounds (dideoxyforskolin, 4‐bromophenacyl bromide, mibefradil) known to block volume‐activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. Although the molecular identity of volume‐sensitive anion channels is not firmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC3, phospholemman and VDAC1, and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC2. In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological profile, suggesting the expression of a particular type or subtype of volume‐sensitive anion channel, which is likely to be formed by yet unidentified proteins.

Effects of thyroid dysfunction on the development of the rat cerebellum, with special reference to cell death within the internal granular layer

The increased cell death within the internal granular layer of the cerebellar cortex, previously demonstrated by other investigators in 12-day-old rats treated with propylthiouracil, was found again in 10-, 14- and 21-day-old similarly treated young rats. In thyroid-deficient as well as in normal animals, cell death was maximal at 10 days. In hypothyroid rats, the greatest difference with the normal animals was at 14 days, when there was an increase by a factor of 20 of the number of dying cells. On day 13, the ratio of free to total N-acetyl-beta-D-glucosaminidase activities was also increased by 34%. Cell death predominantly occurred in the lower part of the internal granular layer at 10 days, in the middle part at 14 days and the upper part at 21 days. The increase in thickness of the molecular layer, which reflects the development of the Purkinje cell dendritic arborizations, was also more retarded than the acquisition of a normal ratio of granule cells to Purkinje cells. Administration of a daily dose of 0.10 mug thyroxine to thyroid-deficient animals was sufficient (and a lower dose insufficient) to return to normal the number of dying cells as well as the development of the molecular layer and the evolution of the ratio of granule cells to Purkinje cells. A daily dose of T4 as low as 0.20 or 0.25 mug already induced a marked hyperthyroid state resulting in a decrease in granule cells formation without increased cell death. Indeed, an increased cell death seemed to occur only when the normal synchronism between the development of the Purkinje cell arborizations and the laying down of granule cells was suppressed, as is the case in thyroid deficiency but not in neonatal hyperthyroidism.

Corrective effects of thyroxine on cochlear abnormalities induced by congenital hypothyroidism in the rat. II. Electrophysiological study

In order to study the corrective effects of thyroxine (T4) on functional abnormalities induced by congenital hypothyroidism, small doses of T4 were injected to propylthiouracil-treated (PTU-treated) rat pups for 2 consecutive days on selected periods of development (days 3 and 4, 6 and 7, 9 and 10, 12 and 13, 18 and 19). Some animals also received thyroid replacement therapy from days 12 to 17. The animals were tested electrophysiologically on day 30, by recording the compound action potential and the cochlear microphonic from the round window after click and tone burst stimulation. PTU-treated animals given T4 for 2 consecutive days demonstrated both AP and CM threshold shifts. On the contrary, PTU-treated animals given T4 from days 12 to 17 demonstrated a normal CM output of the cochlea, but still showed elevated AP thresholds. These results are discussed with previous data concerning the corrective effects of T4 on cochlear structures in PTU-treated rats previously described.

Effect of Thyroid Deficiency on the Growth of the Hippocampus in the Rat

The growth of the hippocampus was studied in normal and hypothyroid rats using both biochemical and morphological techniques, and the results were compared with observations on the whole forebrain or on the cerebral cortex. The longitudinal growth, area and the volume of the hippocampus was severely reduced in thyroid deficiency. In the cerebral cortex the longitudinal growth and some of the parameters of transverse development were significantly decreased at the rostral but not at the caudal level. On rehabilitation from day 35 to 160 the longitudinal growth remained decreased in both the brain parts while the transverse growth was restored to normal in the cerebral cortex but not in the hippocampus. In the normal hippocampus about 60% of the cells were formed during the first 3 postnatal weeks. This developmental increase was significantly depressed in hypothyroid rats; the final deficit in cell number was about 13%. The rate of cell acquisition was calculated from the slopes of the logistic curves fitted to the data of DNA content. At the age of maximal cell acquisition (at day 2-3) the daily deposition was 0.44x106 cells in controls and 0.34x106 cells in the hypothyroid rats. In controls the concentration of DNA decreased in the hippocampus during maturation. Thyroid deficiency did not influence this developmental trend. In contrast, a severe reduction was observed in the cellular composition of RNA and protein.