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V Gallo

Publications and source records attributed to V Gallo.

At least 91 records · Page 5Linked to original sources
PubMed1986

Developmental features of rat cerebellar neural cells cultured in a chemically defined medium.

We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue 3H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of 3H-D-aspartate previously observed in this type of culture. In contrast, 3H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: 3H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. 3H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells exhibited a stellate morphology, were stained by the monoclonal antibody A2B5, and have been characterized elsewhere [Levi et al, 1986] as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity 3H-GABA uptake.

Animals↗
PubMed1986

Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and "neuron-like" gamma-aminobutyric acid transport.

When postnatal rat cerebellar cells were cultured in a chemically defined, serum-free medium, the only type of astrocyte (defined by the expression of the glial fibrillary acidic protein, GFAP) present was unable to accumulate gamma-[3H]aminobutyric acid (GABA), did not express surface antigens recognized by two monoclonal antibodies, A2B5 and LB1, and showed minimal proliferation. In these cultures, nonneuronal A2B5+, LB1+ stellate cells exhibiting "neuron-like" [3H]GABA uptake formed cell colonies of increasing size and were GFAP-. After about one week of culturing, the A2B5+, LB1+, GABA-uptake positive cell groups became galactocerebroside (GalCer) positive. Immunocytolysis of the A2B5+ cells at 3 and 4 days in vitro prevented the appearance of the A2B5+, LB1+, GABA-uptake positive cell colonies, and also of the GalCer+ cell groups. If 10% (vol/vol) fetal calf serum was added to 6-day cultures, the A2B5+, LB1+, GABA-uptake positive cell groups expressed GFAP and not GalCer. If the serum was added to the cultures 2 days after lysing the A2B5+ cells, only A2B5-, LB1-, GABA-uptake negative astrocytes proliferated. It is concluded that the putative fibrous astrocytes previously described in serum-containing cultures (which had a stellate shape and were A2B5+, LB1+, GABA-uptake positive) derive from bipotential precursors that differentiate into oligodendrocytes (GalCer+) in serum-free medium or into astrocytes (GFAP+) in the presence of serum, while the epithelioid A2B5-, LB1-, GABA-uptake negative astrocytes originate from a different precursor not yet identified.

Animals↗
PubMed1985

Survival, morphology and adhesion properties of cerebellar interneurones cultured in chemically defined and serum-supplemented medium.

Cultures obtained from early postnatal rat cerebellum, grown in either chemically defined or in serum-supplemented medium containing 25 mM K+, contained predominantly (greater than 90%) small interneurones, mostly granule cells, with good and comparable viability (assessed by the retention of preloaded 51Cr). Neuronal survival was prolonged in the chemically defined medium, nerve cells living up to two weeks longer than in serum-supplemented medium, although the proportion of non-neuronal cells was not greatly increased. In the serum-supplemented medium neurones became organised into clumps connected by thick, fasciculated bundles of neurites by about one week in vitro. In comparison, in the chemically defined medium aggregation of neurones and fasciculation of neurites was markedly reduced even after 4 weeks in culture. The possible relationship between the organisation of neurones and the nature of the substratum, chemical factors in the medium as well as the surface properties of the cells is discussed.

Animals↗
PubMed1985

Cell surface proteins of cerebellar interneurones and astrocytes cultured in chemically defined and serum-supplemented media.

Lactoperoxidase catalysed 125I-iodination of cerebellar interneurone enriched cultures grown in serum-supplemented or in serum-free, chemically defined medium was studied. It was observed that the differences in the adhesion properties of nerve cells under these conditions are accompanied by differences both in the degree of 125I-iodination of the proteins on the plasma membrane of nerve cells and in the profile of the labelled polypeptides resolved by SDS-polyacrylamide gel electrophoresis. The relative labelling of the major 125I-iodinated polypeptides changed with time in both types of cultures, suggesting that alterations in the overall organisation of the neuronal plasma membrane occur during the development of the cells under both culture conditions. The developmental changes affecting the D2 protein (which is nerve cell specific in these cultures) were significantly retarded in the neuronal cultures grown in the serum-free medium compared with those grown in the serum-containing medium: the increase in D2 content was reduced by 7 DIV and the maturational change in the molecular form of D2 was retarded significantly during the whole cultivation period. The degree of surface 125I-iodination of cerebellar astrocytes in culture was only a fraction (7-20% depending on cultivation time and conditions) of that of the neuronal cultures and the labelled polypeptide profiles obtained from the two types of cultures were markedly different. In comparison with cultures in the serum-supplemented medium, astrocytes under the serum-free conditions showed only minor and transient differences in the profile of surface 125I-iodinated proteins, although the morphology of the cells was markedly different.

Animals↗
PubMed1985

Does thyroid hormone influence the maturation of cerebellar granule neurones?

The following hypotheses were tested: is the degeneration of differentiating granule cells in the internal granular layer of the thyroid deficient cerebellum due to a direct requirement of these cells for thyroid hormone, or is it mediated through the failure of some of these cells to make synaptic contact with the hypoplastic Purkinje cells? The effect of thyroid hormone (T3) was studied in rat cerebellar cultures which contain predominantly granule cells. The cultures were grown in a chemically defined medium (S-) in the presence or absence of T3, and were also compared with serum (and thus thyroid hormone) containing cultures (S+). It would appear that T3 is not essential for the relatively long-term survival of the granule cells. Furthermore, cell growth in terms of protein accretion, and the morphological appearance of the cultures were also similar in S- in the presence and absence of T3. Maturation of granule cells was followed by estimating indices, which in the cerebellum in vivo are influenced by the hormone. However, developmental changes affecting the D2 protein, which is implicated in adhesion among nerve cells, and muscarinic receptor binding were not influenced by T3 in vitro. The voltage (veratridine)-sensitive uptake of 22Na was also unaffected, although T3 increased the rate of the relatively small veratridine insensitive component of the 22Na-influx. However, in comparison with cells grown in S+, the rate of both the veratridine sensitive and insensitive component of 22Na-influx was similar under serum-free conditions, whereas the maturation of the D2 protein and muscarinic receptor binding was retarded. The failure of thyroid hormone to influence the differentiation of granule cells, is not due to the in vitro conditions, since T3 is known to have a marked effect on the maturation of certain other classes of neural cells in culture. The results are consistent with the view that the effect of thyroid hormone on neural maturation is cell-type specific, and that granule cells are not targets of thyroid hormone action. They support our second hypothesis, that is, that the degeneration of granule cells in thyroid deficiency is a consequence of the reduction in available postsynaptic sites for the granule cell axons due to the retarded differentiation of Purkinje cells.

Animals↗
PubMed1985

gamma-Aminobutyric acid- and benzodiazepine-induced modulation of [35S]-t-butylbicyclophosphorothionate binding to cerebellar granule cells.

t-Butylbicyclophosphorothionate (TBPS) is a bicyclophosphate derivative with potent picrotoxin-like convulsant activity that binds with high affinity and specificity to a Cl- channel-modulatory site of the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex. Using intact cerebellar granule cells maintained in primary culture, we have studied the modifications induced by GABA and diazepam on the ion channel-modulatory binding site labeled by [35S]TBPS. At 25 degrees C, and in a modified Locke solution, the [35S]TBPS specific binding, determined by displacing the radioligand with an excess (10(-4) M) of picrotoxin, was approximately 70% of the total radioactivity bound to the cells. [35S]TBPS specific binding was saturable with a Kd of approximately 100 nM, a Bmax of approximately 440 fmol/mg of protein, and a Hill coefficient of 1.18. Neither cerebellar astrocytes maintained in culture for 2 weeks nor a neuroblastoma cell line (NB-2A) exhibited any specific [35S]TBPS binding. Muscimol (0.3 to 5 microM) enhanced and bicuculline (0.1 to 5 microM) inhibited [35S]TBPS specific binding to intact cerebellar granule cells. The effect of muscimol and bicuculline on [35S]TBPS binding was noncompetitive. Muscimol (0.1 to 5 microM) reversed bicuculline inhibition in a dose-dependent fashion but failed to reverse picrotoxin-induced inhibition. [35S]TBPS binding was also modulated by benzodiazepine receptor ligands. The binding was increased by diazepam and decreased by 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methylester. Muscimol (0.05 microM) failed to reverse bicuculline inhibition in the absence of diazepam, but it became effective in the presence of 0.1 to 1 microM diazepam.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Autoradiographic localization and depolarization-induced release of acidic amino acids in differentiating cerebellar granule cell cultures.

Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum, for 2,5,8 or 12 days in vitro (DIV), in conditions giving a purity greater than 90%. The results obtained can be summarized as follows: (1) Light microscopic autoradiography showed that cultured granule cells and their processes can accumulate the glutamate analog [3H]D-aspartate once they have reached an advanced degree of morphological differentiation (8 and 12 DIV), but, even then, only a limited number of cells was heavily labeled. In contrast, astrocytes were heavily labeled at all stages. (2) Calcium-dependent, high [K+]-induced release, or tetrodotoxin-sensitive, veratridine-induced release of [3H]D-aspartate from granule cell-enriched cultures was detectable only in cultures of 8 or 12 DIV. (3) When subject to 3 consecutive depolarizations, cultured granule cells maintained their ability to release [3H]D-aspartate and endogenous glutamate almost unchanged. (4) Newly synthesized [3H]glutamate was autoradiographically localized in both neurons and astrocytes (the latter, however, were not preferentially labeled as with [3H]D-aspartate), but was specifically released from neuronal structures (perikarya and processes) by depolarizing stimuli.

Animals↗
PubMed1983

Substrate specificity and developmental aspects of a presynaptic GABA receptor regulating glutamate release in the rat cerebellum.

In order to better characterize the presynaptic GABA receptors regulating glutamate release in the cerebellum [Levi and Gallo, 1981], a number of GABA agonists and GABA transport inhibitors were tested for their ability to potentiate the depolarization-induced release of the glutamate analog D-[3H]aspartate from superfused cerebellar synaptosomes. Of all the compounds tested, only those which are known to interact specifically with GABA receptors were effective when tested on synaptosomal preparations. The order of effectiveness found was the following: muscimol congruent to 3-APS greater than or equal to P4S greater than isoguvacine greater than THIP. GABA uptake inhibitors were unable to enhance D-[3H]aspartate evoked release from synaptosomes, but were effective when tested in cerebellar slices; in the latter case, the activation of the GABA receptors may be achieved indirectly, through an increase of the extracellular GABA concentrations. The substrate specificity of the presynaptic GABA receptors regulating cerebellar acidic amino acid release appears to be similar to that reported for GABA receptors in radioligand binding studies and for GABA autoreceptors. Studies on synaptosomes from immature cerebella suggested that the presence of the potentiating effect on the acidic amino acid release by GABA agonists is correlated with the development of the parallel fiber terminals, which are believed to be the main site from which glutamate is released in the adult cerebellum.

Animals↗
PubMed1983

Muscimol potentiation of acidic amino acid release from cerebellar synaptosomes is chloride dependent.

In previous studies we have shown that the depolarization-induced release of preaccumulated acidic amino acids and newly synthesized glutamate from cerebellar synaptosomal preparations is potentiated by gamma-aminobutyric acid (GABA) agonists through a GABAergic presynaptic mechanism. Here we report a systematic analysis of the ionic requirements of the potentiating effect of muscimol on the high K+-evoked release of D-[3H]aspartate. Our studies show that: Ca2+, Na+, and Mg2+ are not required for muscimol to exert its effect; a depolarizing concentration of K+ is a necessary, but not sufficient, condition to observe the presynaptic effect in question; and a minimal Cl- concentration (50-70 mM) is also required. A possible model based on these findings is proposed.

Animals↗
PubMed1982

Putative acidic amino acid transmitters in the cerebellum. I. Depolarization-induced release.

In the present investigation we studied the autoradiographic localization and the characteristics of the depolarization-induced release of acidic amino acids in in vitro rat cerebellar preparations. Light microscopy autoradiography of cerebellar slices preincubated in the presence of the non-metabolized glutamate analogue D-[3H]aspartate showed a large accumulation of radioactivity over glial cells, and very little labelling of the granule cells, whose putative neurotransmitter may be glutamate. In spite of its predominant localization in glia, D-[3H]aspartate (and [14C]glutamate) was released from cerebellar slices depolarized with high [K+] in a Ca2+-dependent way, and the release elicited by veratrine was prevented by TTX. These findings, together with the observation that freshly isolated or cultured glial cells did not show any Ca2+-dependent, depolarization-induced release of D-[3H]aspartate, suggest that the radioactive amino acid released from slices has a neuronal origin. The high [K+]-induced release of exogenous radioactive acidic amino acids from superfused cerebellar synaptosomal preparations exhibited, as best, a modest Ca2+-dependence, a result probably due to the existence of a substantial non-Ca2+-dependent release of the amino acid from glial fragments contaminating the preparation. However, both the K+-evoked release of endogenous glutamate, and that of [14C]glutamate previously synthesized from [14C]glutamine were largely Ca2+-dependent, suggesting that nerve endings are the main sites involved in the stimulus-coupled secretion. In the experiments in which synaptosomes had been prelabelled with [14C]glutamine, a study of the specific radioactivity of the glutamate released and of that present in synaptosomes at the beginning and at the end of superfusion period provided evidence in favour of a preferential release of the newly synthesized [14C]glutamate. In contrast to glutamate, endogenous aspartate was not released in a Ca2+-dependent manner, and the efflux of newly formed [14C]aspartate was only slightly potentiated by Ca2+, which suggests that glutamate and aspartate are not released from the same sites. Studies on preparations (slices and synaptosomes) from immature, 8-day-old cerebella showed that neither the K+-evoked release of D-[3H]aspartate, nor that of endogenous glutamate was Ca2+-dependent. In conclusion, the data presented are consistent with the proposition that glutamate has a neurotransmitter role in the cerebellum.U

Amino Acids↗
PubMed1982

Evidence in favor of a neurotransmitter role of glycine in the rat cerebral cortex.

In the present study we analyze whether glycine satisfies some electrophysiological and biochemical criteria to consider it as a putative transmitter in the rat cerebral cortex. Intracellular recordings from rat sensory-motor cortex showed that in 15-20% of the tested neurons glycine hyperpolarized the cell membrane, decreased the firing rate and flattened the evoked EPSP-IPSP sequence by increasing the membrane conductance. The iontophoretic application of strychnine antagonized the block of 'spontaneous' firing and the membrane hyperpolarization induced by glycine. Moreover, in a group of neurons, strychnine decreased the amplitude and duration of the IPSP and brought back the membrane potential to resting values. Previously accumulated [3H]glycine and endogenous glycine were released from cortical synaptosomal preparations by depolarizing stimuli in a Ca2+-dependent way. The release pattern of glycine was qualitatively similar in cortical and in spinal synaptosomes. [14C]Glycine was rapidly synthetized from [14C]serine in cortical synaptosomal preparations, and the newly formed [14C]glycine was released by depolarizing stimuli in a Ca2+-dependent way. It is concluded that glycine, which is generally considered as an inhibitory neurotransmitter in the spinal cord, medulla and pons, may also have a transmitter role in a discrete number of cortical neurons of some mammalian species.U

Animals↗
PubMed1982

Selective release of glutamate from cerebellar granule cells differentiating in culture.

The aim of the present study was to assess whether endogenous and newly synthesized glutamate can be released from differentiating cultured cerebellar granule cells in a way compatible with a neurotransmitter role. Granule cells from 8-day-old rat cerebella were grown in basal Eagle's medium with 10% fetal calf serum for 2-12 days in vitro (DIV), then washed with Krebs-Ringer medium, and labeled for 45 min with tracer amounts of radioactive glutamine. Subsequently, the release of endogenous glutamate and of newly formed radioactive glutamate was measured in basal conditions and upon depolarization with elevated K(+) concentration or veratridine. At 2 DIV, the release of endogenous and newly synthesized glutamate evoked by high K(+) concentration was small and Ca(2+) independent, but it progressively and steadily increased (up to 8- to 10-fold) and became Ca(2+) dependent (up to 80-85%) at later stages (4, 8, and 12 DIV). Veratridine was almost ineffective with cells at 2 DIV but greatly increased glutamate release (endogenous and neosynthesized) at 8 DIV, and its action was totally antagonized by tetrodotoxin. The level and synthesis of glutamate remained fairly constant in cells from 2 to 12 DIV. gamma-Aminobutyric acid synthesis from radioactive glutamine was about 3% of that of glutamate, and gamma-aminobutyric acid release (endogenous and neosynthesized) was not measurable. Aspartate synthesis was about 10% of that of glutamate, and the high K(+) concentration-evoked release of this amino acid was modest and scarcely affected by Ca(2+). Neither high K(+) concentration nor veratridine was able to induce glutamate release from confluent cerebellar astrocyte cultures at 14 DIV, although the level and synthesis of the amino acid were comparable to those in granule cells. In conclusion, the data show that a stimulus-coupled release of endogenous and neosynthesized glutamate is progressively expressed by cerebellar granule cells differentiating in culture, and this strongly supports the concept that glutamate is the neurotransmitter of these cells.

Animals↗
PubMed1982

Treatment of acute alcohol withdrawal syndrome with carbamazepine: a double-blind comparison with tiapride.

A double-blind, randomized trial is described which was designed to compare the clinical effect of 600 mg daily of carbamazepine (Tegretol) and of tiapride (Sereprile) in hospitalized patients with pre-delirium tremens. Physicians' assessment of patients' progress was made following 2, 4 and 7 days of treatment. Of the sixty patients admitted to the study, five dropped out for various reasons, leaving fifty-five patients who completed the study. Both drugs were effective in the treatment of alcohol withdrawal symptoms; no significant difference was found between the two treatments with respect to total symptoms, score and visual analogue scale assessment. Carbamazepine gave faster relief of symptoms and demonstrated a preferential action on symptoms like fear and hallucinations. No case of delirium tremens was observed in those patients who completed the trial.

Adult↗