Vittorio Gallo

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 Eagles 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.

Expression of Excitatory Amino Acid Receptors by Cerebellar Cells of the Type‐2 Astrocyte Cell Lineage

Abstract: We have used postnatal rat cerebellar astrocyte‐enriched cultures to study the excitatory amino acid receptors present on these cells. In the cultures used, type‐2 astrocytes (recognized by the monoclonal antibodies A2B5 and LB1) selectively took up γ‐[3H]aminobutyric acid ([3H]GABA) and released it when incubated in the presence of micromolar concentrations of kainic and quisqualic acids. The releasing effect of kainic acid was concentration dependent in the range of 5–100 μM. Quisqualate was more effective than kainate in the lower concentration range but less effective at concentrations at which its releasing activity was maximal (∼50 μM). N‐Methyl‐d‐aspartic acid and dihydrokainate (100 μM) did not stimulate [3H]GABA release from cultured astrocytes. l‐Glutamic acid (20–100 μM) stimulated [3H]GABA release as effectively as kainate. The stimulatory effects of kainate and quisqualate on [3H]GABA release were completely Na+ dependent; that of kainate was also partially Ca2+ dependent. Kynurenic acid (50–200 μM) selectively antagonized the releasing effects of kainic acid and also that of l‐glutamate; quisqualate was unaffected. Quisqualic acid inhibited the releasing effects of kainic acid when both agonists were used at equimolar concentrations (50 μM). d‐[3H]aspartate was taken up by both type‐1 and type‐2 astrocytes, but only type‐2 astrocytes released it in the presence of kainic acid. Excitatory amino acid receptors with a pharmacology similar to that of the receptors present in type‐2 astrocytes were also expressed by the immature, bipotential progenitors of type‐2 astrocytes and oligodendrocytes.

A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings.

The effect of veratridine on neurotransmitter release was studied using rat brain synaptosomes superfused at 37°C. Veratridine (5–75 μ M) caused a concentration-dependent release of [3H]GABA from prelabeled synaptosomes in the presence of 2.7 mM Ca2+. In the whole range of veratridine concentrations, the release of [3H]GABA elicited by the drug was substantially increased rather than decreased in the absence of Ca2+ or with Ca2+ concentrations of 0.45 and 0.9 mM. The release of the amino acid was inhibited more by 5.4 mM than by 2.7 mM Ca2+. The effect on endogenous (chemically measured) GABA was similar to that on [3H]GABA. The inhibitory effect of Ca2+ on the veratridine-induced release of [3H]GABA was consistently seen in a variety of experimental conditions except one, namely when the experiment was run at room temperature (22–23°C) rather than at physiological temperature (37°C). In fact, at 22–23°C the release of GABA evoked by the alkaloid was somewhat potentiated by Ca2+. At 37°C, glutamate appeared to behave similarly to GABA, whereas the veratridine-induced release of [3H]noradrenaline and [3H]dopamaine was largely Ca2+-dependent. The mechanism of the release of transmitters elicited by veratridine is discussed. It is concluded that the evoked release of GABA and glutamate is due more to the veratridine-induced depolarization (Na+ influx) than to the accompanying influx of Ca2+, and it is suggested that the inhibitory effect of Ca2+ on the overall release of amino acids is due to the antagonism exerted by the divalent cation on the veratridine action at the Na+ channel. In contrast, in the case of catecholamines, the influx of Ca2+ would have a prominent role in triggering exocytotic release, whereas the depolarization itself would have slight or no importance.

Glutamate Receptor Subtypes in Cultured Cerebellar Neurons: Modulation of Glutamate and γ‐Aminobutyric Acid Release

Using cerebellar, neuron‐enriched primary cultures, we have studied the glutamate receptor subtypes coupled to neurotransmitter amino acid release. Acute exposure of the cultures to micromolar concentrations of kainate and quisqualate stimulated D‐[3H]aspartate release, whereas N‐methyl‐D‐aspartate, as well as dihydrokainic acid, were ineffective. The effect of kainic acid was concentration dependent in the concentration range of 20–100 μM. Quisqualic acid was effective at lower concentrations, with maximal releasing activity at about 50 μM. Kainate and dihydrokainate (20–100 μM) inhibited the initial rate of D‐[3H]aspartate uptake into cultured granule cells, whereas quisqualate and N‐methyl‐DL‐aspartate were ineffective. D‐[3H]Aspartate uptake into confluent cerebellar astrocyte cultures was not affected by kainic acid. The stimulatory effect of kainic acid on D‐[3H]aspartate release was Na+ independent, and partly Ca2+ dependent; the effect of quisqualate was Na+ and Ca2+ independent. Kynurenic acid (50–200 μM) and, to a lesser extent, 2,3‐cis‐piperidine dicarboxylic acid (100–200 μM) antagonized the stimulatory effect of kainate but not that of quisqualate. Kainic and quisqualic acid (20–100 μM) also stimulated γ‐[3H]aminobutyric acid release from cerebellar cultures, and kynurenic acid antagonized the effect of kainate but not that of quisqualate. In conclusion, kainic acid and quisqualic acid appear to activate two different excitatory amino acid receptor subtypes, both coupled to neurotransmitter amino acid release. Moreover, kainate inhibits D‐[3H]aspartate neuronal uptake by interfering with the acidic amino acid high‐affinity transport system.

The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors

We have used an antibody raised against the bovine nasal cartilage proteoglycan chondroitin sulfate (CS) digested with chondroitinase ABC (anti-CS serum) to stain cerebellar glial cells maintained in culture. In cultures grown in the presence of serum, the antibody stained a subclass of GFAP+ astrocytes which we have previously shown to selectively bind the monoclonal antibodies A2B5 and LB1. Also the direct bipotential precursors of these cells, capable of differentiating into GFAP+ astrocytes or into Gal-C+, O1+ oligodendrocytes depending on the culture conditions, were stained, but stopped to produce CS when they differentiated into oligodendrocytes.

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

Abstract: The aim of the present paper was to determine whether the release of glutamate from putative “glutamergic” terminals in the cerebellum is influenced by γ‐aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+‐dependent way; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released in a Ca2+‐dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors.

Release of endogenous and newly synthesized glutamate and of other amino acids induced by non-N-methyl-D-aspartate receptor activation in cerebellar granule cell cultures.

Abstract: Amino acid release studies were performed by an HPLC procedure using differentiated rat cerebellar granule cell cultures. Kainic acid (KA; 50 μM) caused an increase (about threefold) in the release of endogenous glutamate and a lesser, but statistically significant, increase in the release of glutamine, glycine, threonine, taurine, and alanine. Quisqualic acid (QA) and, to a lesser degree, α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) (both 50 μM) enhanced the release of the following amino acids in the order glutamate > aspartate ≥ taurine, whereas the release of other amino acids was either unaffected or affected in a statistically nonsignificant way. The release of glutamate induced by KA was partially (43%) Ca2+ dependent. The other release‐inducing effects of KA and QA were not Ca2+ dependent. In all cases, the evoked release could be prevented by the non‐N‐methyl‐D‐aspartate (non‐NMDA) receptor antagonist 6‐cyano‐2,3‐hydroxy‐7‐nitroquinoxaline, and thus appeared to be receptor mediated. NMDA (5 and 50 μM) had no releaseinducing activity. The KA‐, QA‐, and AMPA‐evoked release of newly synthesized [3H]glutamate and [3H]aspartate (formed in the cells exposed to [3H]glutamine) was very similar to the evoked release of endogenous glutamate and aspartate. On the other hand, the release of preloaded D‐[3H]aspartate (purified by HPLC in the various fractions analyzed, before radioactivity determination) induced by 50 μM KA was twice as high as that of endogenous glutamate. In the case of high [K+] depolarization, in contrast, the release of preloaded D‐[3H]aspartate was ∼30% lower than that of endogenous glutamate. The reasons for the above differences in the susceptibility of the various glutamate pools to being released may be the following: Non‐NMDA receptor agonists cause essentially a carrier‐mediated efflux of glutamate from a cytoplasmic pool that is preferentially labeled by exogenous D‐[3H]aspartate, whereas depolarization with high [K+] causes mainly an exocytotic‐like release from a vesicular pool to which exogenous D‐[3H]aspartate has a more limited access.

Extracellular matrix of cultured glial cells: Selective expression of chondroitin 4-sulfate by type-2 astrocytes and their progenitors☆

We have studied the extracellular matrix composition of cultured glial cells by immunocytochemistry with different monoclonal and polyclonal antibodies. Double immunofluorescence experiments and metabolic labeling with [3H]glucosamine performed in different types of cerebellar and cortical cultures showed that bipotential progenitors for type-2 astrocytes and for oligodendrocytes (recognized by the monoclonal antibody LB1 at early stages of their development) synthesize chondroitin sulfate (CS) and deposit this proteoglycan in their extracellular matrix. The distribution of the various [3H]glucosamine-labeled glycosaminoglycans between the intracellular and the extracellular space was different. CS was present both within the cells and in the culture medium, although in different amounts. Bi-potential progenitors became also O4-positive during their development in vitro. At the stage of O4-positivity they were still stained with antibodies against CS. However, when the progenitor cells were maintained in serum-free medium and differentiated into Gal-C-positive oligodendrocytes, they became CS-negative. In the presence of fetal calf serum in the culture medium, the bipotential progenitors differentiated into GFAP-positive type-2 astrocytes. These cells still expressed CS: their Golgi area and their surface were stained with anti-CS antibodies. Staining with monoclonal antibodies specific for different types of CS (4-sulfate, 6-sulfate, and unsulfated) revealed that both bipotential progenitors and type-2 astrocytes synthesized only chondroitin 4-sulfate. Type-1 astrocytes were negative for both the polyclonal and the monoclonal anti-CS antibodies. Finally, type-2 astrocytes and their progenitors were weakly stained with anti-laminin antibodies and unstained with anti-fibronectin. Type-1 astrocytes were positive for both anti-laminin and anti-fibronectin antibodies and appeared to secrete fibronectin in the extracellular space.

Modulation of Non-N-Methyl-D-Aspartate Receptors in Cultured Cerebellar Granule Cells

Abstract: Kainic acid (KA), quisqualic acid (QUIS), and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) stimulated D‐[3H]aspartate release from cultured cerebellar granule cells in a concentration‐dependent way. The EC50 values were 50 μM for KA (Gallo et al., 1987) and 20 μM for both QUIS and AMPA, but the efficacy of QUIS appeared to be greater than that of AMPA. The release of D‐[3H]aspartate induced by KA, QUIS, and AMPA was blocked, in a dose‐dependent way, by the new glutamate receptor antagonist 6‐cyano‐2,3‐dihydroxy‐7‐nitroquinoxaline (CNQX); IC50 values were 0.7 μM in the case of AMPA (50 μM) and 1 μM in the case of KA (50 μM). AMPA (50–300 μM) inhibited the effect of 50 μM KA on D‐[3H]aspartate release. At 300 μM AMPA, the effect of KA plus AMPA was not antagonized by the KA receptor antagonist kynurenic acid (KYN). In contrast, when KA was used at an ineffective concentration (10 μM), the addition of AMPA at concentrations below the EC50 value (10–20 μM) resulted in a synergistic effect on D‐[3H]aspartate release. In this case, the evoked release of D‐[3H]aspartate was sensitive to KYN. KA stimulated the formation of cyclic GMP, whereas QUIS, AMPA, and glutamate were ineffective. The accumulation of cyclic GMP elicited by KA (100 μM) was prevented not only by the antagonists CNQX (IC50= 1.5 μM) and KYN (IC50= 200 μM), but also by the agonists AMPA (IC50= 50 μM), QUIS (IC50= 3.5 μM), and glutamate (IC50= 100 μM). We conclude that AMPA, like QUIS, may act as a partial agonist at KA receptors. Moreover, CNQX effectively antagonizes non‐N‐methyl‐D‐aspartate receptor‐mediated responses in cultured cerebellar granule cells.

Neuronal fodrin proteolysis occurs independently of excitatory amino acid-induced neurotoxicity

In cultured cerebellar granule cells, the total amount of fodrin alpha subunit increased 3-fold between 0 and 10 days in vitro and fodrin mRNA increased 5-fold. The exposure of cerebellar neurons to NMDA induced the accumulation of a 150 kd proteolytic fragment of fodrin. The NMDA-induced breakdown of fodrin was time-, concentration-, and Ca2(+)-dependent and was inhibited by APV, Mg2+, or the calpain I inhibitor N-acetyl-Leu-Leu-norleucinal. Kainate caused fodrin proteolysis through indirect activation of NMDA receptors. Quisqualate was ineffective. The NMDA-induced degradation of fodrin occurred under conditions that did not cause degeneration of cultured cerebellar neurons. These results show that Ca2+/calpain I-dependent proteolysis of fodrin is selectively associated with NMDA receptor activation; however, fodrin proteolysis per se does not play a causal role in NMDA-induced toxicity in cerebellar granule cells.