Giovanni Fontana

Transmitter Release Associated with Long-term Synaptic Depression in Rat Corticostriatal Slices

Using a corticostriatal slice preparation, we have recently shown that tetanic stimulation of the corticostriatal pathway produces long‐term depression (LTD) of striatal excitatory synaptic transmission. In the present study we have analysed the relationship between LTD and the striatal release of different endogenous transmitters. Samples of perfusate were collected via a small cannula placed just above the surface of the striatal slice close to the recording electrode, and were analysed by HPLC. The high‐frequency stimulation (100 Hz, three trains, 3 s duration, 20 s intervals) used to induce LTD caused a significant but transient increase in the release of both excitatory (aspartate and glutamate) and inhibitory (glycine and GABA) amino acid transmitters. Tetanic stimulation also produced a significant, but transient increase in the release of endogenous dopamine. We conclude that the tetanic stimulation of the corticostriatal pathway is able to induce a large but transient release of excitatory amino acids and of dopamine, whose participation in the induction of striatal LTD has been demonstrated previously. Moreover, the maintenance of this form of synaptic plasticity does not seem to require a sustained change in transmitter release.

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons.

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N‐methyl‐D‐aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1–300 μM) increased in a concentration‐dependent manner (EC50 = 37.9 ± 12 μM) the release of [3H]‐GABA. The effect of 100 μM NMDA was prevented by 30 μM of the GABA transport inhibitor N‐(4,4‐diphenyl‐3‐butenyl)guvacine (SKF 100330A). Glycine (10 μM) or gp120 (0.01 μM) affected neither the basal nor the NMDA‐evoked [3H]‐GABA release. The NMDA (100 μM)‐evoked release was prevented by 5,7‐dichloro‐kynurenic acid (5,7‐DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration‐dependent manner (IC50 ≃ 0.3 μM). Glycine (3–10 μM) or gp120 (0.003–0.01 μM) produced reversal of the 5,7‐DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors. J. Neurosci. Res. 49:732–738, 1997.

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ‐aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [3H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC5017.2 μM) and Asp (EC50 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (‐)‐baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na+ dependent. Glu enhanced the release of [3H]GABA (EC50 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N‐methyl‐d‐aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D‐threo‐hydroxyaspartic acid (THA) and it was Na+ sensitive. Similarly to Glu, D‐Asp increased [3H]GABA release (EC50 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor antagonist 6‐cyano‐7‐nitroquinoxaiine‐2, 3‐dione, whereas the 6‐cyano‐7‐nitroquinoxaline‐ 2, 3‐dione‐insensitive portion of the effect was prevented by THA. GABA increased the [3H]D‐Asp outflow (EC50 13.7 μM) from hippocampal synaptosomes in a muscimol‐, (‐)‐ baclofen‐, bicuculline‐, and picrotoxin‐insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na+ dependent. Glu increased the release of [3H]‐ GABA from hippocampal synaptosomes (EC50 7.1 μM) in an N‐methyl‐d‐aspartic acid‐, kainic acid‐, or quisqualic acid‐insensitive way. The effect of Glu was prevented by THA and was Na+ dependent. As in the cortex, the effect of Glu was mimicked by D‐Asp in a THA‐sensitive manner. It is proposed that high‐affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA‐ BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.

Glycine stimulates [3H]noradrenaline release by activating a strychnine-sensitive receptor present in rat hippocampus

Rat hippocampus slices were prelabeled with [3H]noradrenaline ([3H]NA) and depolarized by superfusion with KCl. The release evoked by 12 mM K+ was totally calcium-dependent and more than 90% tetrodotoxin (TTX)-sensitive. Glycine (0.1-1 mM) increased the K(+)-evoked [3H]NA overflow in a concentration-dependent manner. The effect of 1 mM glycine reached 300%. Strychnine (0.3 microM) shifted to the right the concentration-response curve for glycine. The effect of glycine (0.1 or 1 mM) was totally abolished by 3 microM strychnine but was unaffected by the GABAA receptor antagonist, bicuculline (10 microM), or by 100 microM of 1-hydroxy-3-aminopyrrolidone-2 (HA-966), a proposed antagonist of glycine at the strychnine-insensitive site located on the N-methyl-D-aspartate (NMDA) receptor. The effect of glycine was mimicked by L-serine, although less potently; the release of [3H]NA was enhanced by 200% in presence of 3 mM L-serine. At this concentration D-serine was ineffective. Strychnine shifted to the right the concentration-response curve for L-serine. Glycine (1 mM) had only a minor effect (less than 20% potentiation) on the release of [3H]NA evoked by 12 mM KCl in hippocampal synaptosomes. While the effect of glycine in slices was increased by decreasing the depolarizing concentration of K+ (about 500% potentiation at 9 mM K+), the response of synaptosomes remained minimal, even in presence of 9 mM KCl. Hippocampal synaptosomes prelabeled with [3H]glycine released the radiolabeled amino acid when exposed to superfusion with 12 mM KCl. The release of [3H]glycine was more than 75% calcium-dependent. The results suggest that the release of NA in rat hippocampus may be enhanced by glycine through the activation of a strychnine-sensitive receptor. This receptor does not seem to be located on noradrenergic terminals.

GABAB autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions

Rat cerebral cortex synaptosomes prelabeled with [3H]gamma-aminobutyric acid [( 3H]GABA) were exposed in superfusion to various concentrations of KCl (9-50 mM). The evoked release of [3H]GABA reached a plateau at about 35 mM KCl. The K+-induced release was Ca2+-dependent, particularly at the lowest K+ concentrations. The GABAB agonist (-)-baclofen concentration dependently inhibited the release of [3H]GABA evoked by K+; this effect decreased with increasing K+ concentration and disappeared at 35 mM KCl. The GABAA agonist muscimol (1-100 microM) was totally ineffective to inhibit the release of [3H]GABA. Veratrine (1-30 microM) induced the release of [3H]GABA and the effect was tetrodotoxin-sensitive. (-)-Baclofen, but not muscimol, decreased the veratrine-induced [3H]GABA release; the GABAB agonist was particularly effective in presence of low concentrations of veratrine (1-3 microM) but the effect disappeared when 30 microM of the alkaloid was used. The inhibitory effect of (-)-baclofen on the release of [3H]GABA evoked by 15 mM KCl was dependent on the concentration of Ca2+: the effect increased as the concentration of Ca2+ was raised, reaching a plateau at 0.6 mM Ca2+. Exogenous GABA, in presence of the GABA uptake blocker SK & F 89976A, inhibited the release of [3H]GABA evoked by K+; this effect was antagonized by phaclofen. The data support the idea that terminal GABA autoreceptors in the rat cerebral cortex are of the GABAB type.

Presynaptic Mechanisms Underlying the γ-Aminobutyric Acid-Evoked Receptor-Independent Release of [3H]Norepinephrine in Rat Hippocampus

Abstract: The effects of γ‐aminobutyric acid (GABA) on the spontaneous efflux of [3H]norepinephrine ([3H]NE) were studied in synaptosomes prepared from rat hippocampus and prelabelled with [3H]NE. It had been observed previously that, when synaptosomes were exposed in superfusion to GABA, the basal release of the tritiated catecholamine was enhanced, apparently with no involvement of the known GABA receptors. The mechanisms underlying this effect have now been investigated. The potency of GABA as a releaser of [3H]NE was decreased by lowering the Na+ content of the superfusion medium, and its effect disappeared at 23 mM Na+. The GABA‐induced [3H]NE release was counteracted by the GABA uptake inhibitor N‐(4,4‐diphenyl‐3‐butenyl)nipecotic acid (SKF 89976A), but it was unaffected by the NE uptake blockers desmethylimipramine and nisoxetine. The GABA‐induced release of [3H]NE was Ca2+‐dependent and tetrodotoxin‐sensitive. The data support the hypothesis that GABA provoked [3H]NE release by a novel mechanism which involves penetration into the noradrenergic nerve terminals through a GABA carrier located on the NE terminals themselves. This uptake process might be electrogenic and provoke depolarization of the nerve terminals, causing an exocytotic release of [3H]NE.

Role of External and Internal Calcium on Heterocarrier‐Mediated Transmitter Release

Abstract: Release‐regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA‐evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2‐Bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA‐AM, prevented the effect of GABA on [3H]dopamine release. Autoreceptor activation inhibited the GABA‐evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]‐independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier‐mediated vesicular release linked to entry of extracellular Ca2+.

Cholinergic modulation of [3H]dopamine release from dendrosomes of rat substantia nigra

SummaryDendrosomes prepared from substantia nigra are able to take up and release [3H]dopamine in a Ca2+-dependent manner. The Vmax values of [3H]dopamine uptake in substantia nigra dendrosomes was about 5 times lower than that in caudate putamen synaptosomes. The pattern of the K+-dependency of the [3H]dopamine release in substantia nigra dendrosomes was significantly different from that found in caudate putamen synaptosomes. The release of [3H]dopamine evoked by 15 mmol/l KCl from superfused dendrosomes was increased in a concentration-dependent manner by acetylcholine. The maximal potentiation produced by acetylcholine was about 40%. The potentiation of [3H]dopamine release by 10 µmol/l acetylcholine was insensitive to mecamylamine but antagonized by atropine and by pirenzepine. The effects of acetylcholine on the release of [3H]acetylcholine from substantia nigra nerve endings was also studied. Exogenous acetylcholine added to the superfusion medium decreased in a concentration-dependent manner the release of acetylcholine. This effect was not antagonized by mecamylamine or pirenzepine but fully antagonized by atropine. The data suggest the existence, in the substantia nigra of the rat, of two distinct muscarinic receptor subtypes regulating respectively dopamine release from dopamine dendrites and acetylcholine release from cholinergic nerve terminals.

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l‐Glutamate, NMDA, dl‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin‐like immunoreactivity (SRIF‐LI) from primary cultures of rat hippocampal neurons. In Mg2+‐containing medium, the maximal effects (reached at ∼100 µM) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µM (AMPA), 39 µM (glutamate), 41 µM (KA), and 70 µM (NMDA). The metabotropic receptor agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylate did not affect SRIF‐LI release. The release evoked by glutamate (100 µM) was abolished by 10 µM dizocilpine (MK‐801) plus 30 µM 1‐aminophenyl‐4‐methyl‐7,8‐methylenedioxy‐5H‐2,3‐benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK‐801 but insensitive to GYKI 52466. The AMPA‐ and KA‐evoked releases were blocked by 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) or by GYKI 52466 but were insensitive to MK‐801. The release of SRIF‐LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA‐evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF‐LI release, an effect abolished by MK‐801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.

Native human neocortex release-regulating dopamine D2 type autoreceptors are dopamine D2 subtype.

Dopamine (DA) autoreceptors expressed at DA nerve terminals regulate DA release. Considerable evidence has indicated that, in rodents, these autoreceptors belong to the D2 type of the DA receptor family, which, in turn, comprises the D2, D3 and D4 subtypes. We investigated here, for the first time, the subclassification of native human DA autoreceptors by studying the release of [3H]DA evoked by electrical stimulation in fresh human neocortical slices. The results have been compared with those obtained in three animal systems: rat neocortical and striatal slices and rat mesencephalic neuronal cultures. In human neocortical slices, the D2/D3 receptor agonist quinpirole (1 n m–10 μm) inhibited tritium release with a calculated EC50 of 17 n m and a maximal inhibition of ≈ 75% reached at 1 μm. In the presence of the D2/D3 receptor antagonist (–)‐sulpiride (0.1 and 1 μm), the concentration–response curve of quinpirole was shifted to the right, and the apparent pA2 mean value was 8.5 (8.14–8.77); on the other hand, the inhibitory effects of quinpirole were not affected by the D3 receptor‐selective antagonist [7‐N,N‐dipropylamino‐5,6,7,8‐tetrahydro‐naphtho(2,3b) dihydro,2,3‐furane] (S 14297) and the D4 receptor‐selective antagonist 3‐(4‐[4‐chlorophenyl]piperazin‐1‐yl)‐methyl‐1H‐pyrrolo [2,3‐b]pyridine (L‐745,870) (0.01–1 μm in each case). Superimposable results have been obtained when the release was elicited from rat striatal slices or dopamine mesencephalic neurons in culture, whereas quantitative differences emerged in the case of rat cortical slices. It is concluded that in human brain, as well as in rat brain, the release of DA in the terminal region of midbrain dopaminergic neurons is regulated through autoreceptors of the D2 subtype.