Isabelle Chaudieu

Interleukin-2 Modulates Evoked Release of [3H]Dopamine in Rat Cultured Mesencephalic Cells

Abstract: Mesencephalic cell cultures were used as a model to investigate the effects of interleukin‐2 (IL‐2) on evoked release of [3H]dopamine ([3H]DA) and γ‐[3H]‐aminobutyric acid ([3H]GABA). At low concentrations (10−13‐10−12M), IL‐2 potentiated [3H]DA release evoked by the excitatory amino acids N‐methyl‐D‐aspartate (NMDA) and kainate, whereas higher IL‐2 concentrations (10−9‐10−8M) had no effect. IL‐2 (10−14‐10−8M) modulated K+‐evoked [3H]DA release in a biphasic manner, with low concentrations (10−12‐10−11M) of IL‐2 potentiating and higher concentrations (10−9‐10−8M) inhibiting K+‐induced [3H]DA release. IL‐2 (10−14‐10−8M) by itself failed to alter spontaneous [3H]DA release. The inhibition by IL‐2 of K+‐evoked [3H]DA release was reversible and not due to neurotoxicity, as preexposure to IL‐2 (10−8M) had no significant effect on the subsequent ability of dopaminergic cells to take up and to release [3H]DA. Under our experimental conditions, IL‐2 (10−8 M) did not alter Ca2+‐independent [3H]GABA release evoked by either K+ or NMDA. The results of this study indicate that IL‐2 is able to potentiate [3H]DA release evoked by a number of different stimuli, including K+ depolarization and activation of both NMDA and non‐NMDA receptor subtypes in mesencephalic cell cultures. IL‐2 is active at very low concentrations, a finding that indicates a potent effect of IL‐2 on dopaminergic neurons and implicates a physiological role for this cytokine in the modulation of DA release.

Alterations in spinal cord excitatory amino acid receptors in amyotrophic lateral sclerosis patients

Excitatory amino acids (EAA) have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). We have analyzed the distribution of the N-methyl-D-aspartate (NMDA) 1-(1-(2-thienyl)-cyclohexyl) piperidine (TCP), kainate and alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) quisqualate subtypes of EAA receptors using quantitative receptor autoradiography in the cervical and thoracic spinal cords of patients who have died with ALS, and of controls. We observed that in control spinal cords [3H]TCP/NMDA binding sites were located both in the ventral and dorsal horns with the highest densities being situated in lamina II. [3H]AMPA and [3H]kainate binding sites were present almost exclusively in the substantia gelatinosa of the dorsal horn. In ALS, the distribution of these 3 types of receptors was unchanged, but [3H]TCP/NMDA binding was decreased both in the dorsal and ventral horns. [3H]kainate binding was possibly decreased in substantia gelatinosa, of ALS cords. However, the limited sample size available for [3H]kainate binding did not permit statistical analysis. [3H]AMPA binding sites were unaltered in ALS. These results indicate that there is a preferential reduction in NMDA receptors in ALS. We suggest that should an excitotoxic mechanism be involved in the pathogenesis of ALS, then NMDA receptors may be the target of this effect.

GABAA receptor-mediated inhibition of N-methyl-D-aspartate-evoked [3H]dopamine release from mesencephalic cell cultures

Direct activations of both GABAA and GABAB receptors are known to hyperpolarize dopaminergic neurons. However systemic or intra-ventral tegmental administration of a GABAA receptor agonist produces paradoxical depolarization of mesencephalic dopaminergic neurons and increases dopamine release. Thus indirect excitation appears to preclude observation of inhibitory GABAA effects on dopamine release in intact tissue. The present study used cultures of isolated cells from rat ventral mesencephalon to characterize effects of GABAA and GABAB receptor activation on evoked dopamine release. The GABAA receptor agonist, muscimol, produced a potent and complete inhibition of N-methyl-D-aspartate (NMDA)-evoked [3H]dopamine release. This effect was blocked by the GABAA receptor antagonist, picrotoxin, and enhanced by flunitrazepam. Omission of Mg2+ greatly reduced the inhibitory effect of muscimol on NMDA-evoked [3H]dopamine release. Muscimol had little or no effect on [3H]dopamine release evoked by the non-NMDA receptor agonists, quisqualate and kainate. The GABAB receptor agonist, baclofen, slightly inhibited NMDA-evoked [3H]dopamine release and had no effect on release evoked by quisqualate or kainate. Endogenous GABA released by the mesencephalic cells also appeared to inhibit NMDA-evoked [3H]dopamine release mainly via a GABAA receptor-mediated mechanism. This is suggested by the observations that NMDA-evoked [3H]dopamine release was potentiated by picrotoxin but not by the GABAB receptor antagonist, phaclofen, and that blockade of extracellular GABA removal, with amino-oxyacetic acid and beta-alanine, inhibited NMDA-evoked [3H]dopamine release in a picrotoxin-sensitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of L- and N-type Ca2+ channel antagonists on excitatory amino acid-evoked dopamine release.

In the present study we tested the effect of dihydropyridine (DHP) Ca2+ channel antagonists and of omega-conotoxin GVIA on [3H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca2+ channels in these EAA-mediated processes. Micromolar concentrations (10-30 microM) of DHP L-type Ca2+ channel antagonists inhibited [3H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [3H]DA release evoked by the L-type Ca2+ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1-1 microM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, (+)-PN 200-110, was more potent than (-)-PN 200-110 in inhibiting [3H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca2+ channels is able to evoke [3H]DA release. However activation of L-type channels is not involved in EAA-induced [3H]DA release and therefore inhibition of EAA-induced [3H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca2+ channels. omega-Conotoxin GVIA (3 microM) had no effect on [3H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient postnatal increases in excitatory amino acid binding sites in rat ventral mesencephalon

The developmental profile of three sub-types of excitatory amino acid (EAA) binding sites was determined in the ventral mesencephalon and the striatum of rats from prenatal day 19 to adult (3 months) using membrane binding assays. In the ventral mesencephalon, there was a transient increase of EAA receptor binding sites beyond adult levels, which peaked at postnatal day 7 (P7) for [3H]glutamate binding to NMDA receptors and at P14 for [3H]AMPA and [3H]kainate binding. In the striatum, [3H]glutamate/NMDA and [3H]kainate binding reached adult levels during the early postnatal period, stabilizing at this level with no transient overexpression beyond adult levels. [3H]AMPA binding also showed an increase above adult levels at P14 in the striatum. These results raise the possibility that the transient overexpression of EAA receptors in the ventral mesencephalon may affect the developmental fate of dopaminergic and other neurons in this region.

Phorbol ester enhances excitatory amino acid-induced dopamine release from mesencephalic cell cultures.

The hypothesis that protein kinase C activation can modulate excitatory amino acid-induced dopamine release was tested by investigating effects of phorbol esters, direct activators of protein kinase C, on dopamine release stimulated by N-methyl-D-aspartate (NMDA) and non-NMDA sub-types of excitatory amino acid agonists in fetal rat mesencephalic cell cultures. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), enhanced dopamine release evoked by NMDA, kainate, quisqualate and by K+ depolarization. Release in the presence of NMDA and TPA was completely abolished by the NMDA antagonist, MK-801. TPA enhancement of NMDA-stimulated dopamine release was likely due to protein kinase C activation by the phorbol ester since (1) the NMDA response was enhanced by nanomolar concentrations of TPA, (2) two phorbol esters capable of activating protein kinase C enhanced the NMDA response while an inactive phorbol ester did not, (3) staurosporine, a potent protein kinase C inhibitor, blocked TPA enhancement of the NMDA response. TPA enhancement of NMDA-stimulated dopamine release was not blocked by H8, an inhibitor with high affinity for cyclic nucleotide dependent kinases, while forskolin, a direct activator of adenylate cyclase, had no effect on NMDA-stimulated release, indicating a lack of involvement of cAMP-dependent kinase in the TPA effect. TPA enhanced NMDA-stimulated release both in the presence and absence of Mg2+, indicating that TPA enhancement was not due to reversal of a Mg2+ blockade of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory Amino Acid Receptors in Human Spinal Cord Evaluation in Amyotrophic Lateral Sclerosis Patients

Amyotrophic lateral sclerosis (ALS) is a progressive id neuromuscular disease that attacks nerve cells and pathways in the brain and spinal cord. Its etiological basis is st i l l unknown. Recent data’-3 suggest that ALS may result h m motoneuron damage induced by endogenous or exogenous excitotoxins, and particularly by excitatory amino acids.‘ A similar neuroexcitotoxic mechanism has been suggested as contributing to neurodegenerative processes in disorders such as Huntington’s and Alzheimer‘s