Effects of neuroleptics on release of 3H-dopamine from slices of rat corpus striatum

Summary The characteristics of ³H-DA release from striatal slices by electrical stimulation were analyzed and the effects of a number of neuroleptics thereon were examined under different experimental conditions. The butyrophenones, haloperidol and spiroperidol, already at low concentrations (0.1–1 M) increased basal tritium efflux in a dose-dependent manner. The phenothiazines, chlorpromazine and fluphenazine, were much less effective in this respect.The butyrophenones strongly inhibited the electrically stimulated overflow of both ³H-DA and ¹⁴C-GABA, while the phenothiazines again had little effect. The action of 1 M haloperidol on ³H-DA release could be blocked by 10 M cocaine, but not with 1 M apomorphine. Apomorphine itself had no significant effect on ³H-DA release.Our data do not support the suggestion that presynaptic DA receptors on dopaminergic nerve terminals may modulate the release of newly taken-up ³H-DA. Some neuroleptics, particularly the butyrophenones may have presynaptic effects not related to interaction with DA receptors. It is suggested that different mechanisms may be involved in the local presynaptic receptor-mediated feedback regulation of transmitter release in noradrenergic and dopaminergic systems in the CNS.     

Activation of dopamine D4 receptors modulates [3H]GABA release in slices of the rat thalamic reticular nucleus

The thalamic reticular nucleus (nRt) is innervated by dopaminergic projections from the sustantia nigra compacta (SNc) and is rich in dopamine D4 receptors, however, the functional effects of dopamine on this structure are unknown. We examined whether the D1 receptor agonist SKF 38393, or the D2 class receptor agonist quinpirole, modify depolarization evoked Ca(2+)-dependent [3H]GABA release. SKF 38393 was without effects, whereas quinpirole inhibited [3H]GABA release with an IC50 of 81 +/- 33 nM. Dose-dependence determinations of agonists (quinpirole and PD 168, 077) and antagonists (L-745,870, U-101958, clozapine and raclopride) with different affinities for different D2 class subtype receptors showed that a D4 receptor mediates quinpirole inhibition. We used methylphenidate, an agent that acts by increasing interstitial dopamine, to determine whether endogenous dopamine modulates [3H]GABA release. Methylphenidate inhibited [3H]GABA release showing that the nRt contains sufficient endogenous dopamine to activate D4 receptors. This inhibition was completely reversed by selectively blocking D4 receptors with L-745,870 or U-101958 indicating that the catecholamine receptors that modulate GABA release are D4 receptors. Given the importance of the nRt in the control of attention, sensory processing and the generation of rhythmic activity during slow wave sleep, it is possible that abnormal nRt function may generate some of the manifestations of the disorders of dopaminergic transmission.     

Dietary cadmium exposure attenuates D-amphetamine-evoked [3H]dopamine release from striatal slices and methamphetamine-induced hyperactivity

Prolonged exposure to environmentally relevant amounts of CdCl2 results in cadmium accumulation in dopamine-rich brain regions, such as striatum. Exposure to these low levels of cadmium also diminishes cocaine-induced hyperactivity and conditioned reinforcement. The goal of the present study was to assess the effect of cadmium on amphetamine pharmacology. Direct application of cadmium (0.1-100 microM), within the concentrations reported in brain after chronic exposure, to preloaded rat striatal slices did not alter D-amphetamine-evoked [3H]dopamine release. To determine the effect of dietary cadmium exposure on amphetamines, rats received ad libitum access to diet containing CdCl2 (10 or 100 ppm) or to control diet for 30 days and then D-amphetamine-evoked [3H]dopamine release and methamphetamine-induced hyperactivity were measured. Dietary CdCl2 exposure produced a marked increase in cadmium blood and brain levels, approximate to environmental metal exposure. Dietary cadmium exposure was associated with decreased potency of D-amphetamine to evoke [3H]dopamine release. Cadmium-exposed rats were also less sensitive to the locomotor-activating effect of acute methamphetamine (0.3 or 1.0 mg/kg) injection. The present findings demonstrate that the presence of cadmium in brain is not sufficient for the inhibition of D-amphetamine-evoked dopamine release. This suggests that cadmium does not directly interfere with the mechanism of action for amphetamine pharmacology; rather, it suggests that long-term cadmium exposure induces a change in the number and/or function of striatal neurons.     

GABA potentiates potassium-stimulated 3H-dopamine release from slices of rat substantia nigra and corpus striatum

GABA (10-5--10-3 M) had no effect on the spontaneous outflow of previously accumulated 3H-DA or 3H-5HT from rat nigral or striatal slices. However, GABA markedly potentiated the potassium-stimulated release of 3H-DA in both brain regions, while the depolarization-induced output of 3H-5HT was only slightly increased. This action of GABA was blocked by pictotoxin but not by bicuculline. Amphetamine likewise evoked a dose-related efflux of 3H-DA and 3H-5HT from nigra and striatum, but these releases were unchanged by GABA. The data suggest that GABA acts presynaptically in these areas to regulate dopaminergic cell function.     

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.     

Altered characteristics of [3H]dopamine release from superfused slices of corpus striatum obtained from rats receiving ethanol in vivo

Ethanol, 50 mM, in vitro inhibited the release of [3H]dopamine ([3H]DA) induced by depolarisation with 40 mM K+ from slices of corpus striatum of the rat. In contrast, the release of [3H]DA induced by the Ca2+ ionophore (A23187) was enhanced by the presence of ethanol in vitro. When similar preparations were obtained from brains of rats which had received ethanol in vivo chronically by inhalation for 5-7 days the characteristics of release of [3H]DA were altered. Thus, the inhibitory effect of ethanol in vitro on release induced by K+-depolarisation was lost, as was the enhancing effect of ethanol on the release induced by A23187. When release of [3H]DA was studied in the absence of added ethanol the fraction of stored 3H released either by K+-depolarisation or by A23187 was increased in the preparations from animals which had received ethanol in vivo. Similar changes in release induced by A23187, though of lesser magnitude, could be seen in rats which had received ethanol acutely (3 g kg-1 i.p.; 30 min). An even greater fraction of [3H]DA was released by A23187 in preparations from rats which had been made physically dependent on ethanol. These changes in the release characteristics of [3H]DA were still apparent in animals undergoing a physical syndrome of withdrawal from ethanol. The results are discussed in relation to the cellular basis for the development of tolerance to and dependence on ethanol.     

Cholecystokinin-8 increases K-evoked [H]γ-aminobutyric acid release in slices from various brain areas

[³H]γ-Aminobutyric acid (GABA) release was studied in rat brain slices in the absebce or presence of cholecystokinin-8 (CCK-8). [³H]GABA release under the conditions used was Ca²⁺-dependent and insensitive tot he presence of the glial uptake blocker β-alanine. While the basal release of [³H]GABA was not affected by CCK-8, the K⁺-stimulated release of [³H]GABA wasm significantly enhanced by 300 nM of CCK-8 in the caudate putamen, the substantia nigra, the hippocampal formation and the parietofrontal cortex. In the cerebral cortex the CCK-8 enhancement of [³H]GABA release was concentration-dependent and abolished by the CCK_B receptor antagonists PD135,158 (1.0 nM) and L-365,260 (100 nM). A significant counteraction of the CCK-8 action was also found with the CCK_A receptor antagonist L-364,718 (100 nM) but only in concentrations at which both CCK_A and CCK_B receptors are blocked. No CCK-8 effects on [³H]GABA release were observed when tetrodotoxin was superfused 5 min before the K⁺-induced [³H]GABA release. It is suggested that the enhancing actions of CCK-8 on K⁺-stimulated [³H]GABA release is mainly related to an activation of CCK_B receptors.     

Neurotensin effects on evoked release of dopamine in slices from striatum,nucleus accumbens and prefrontal cortex in rat

Summary The effects of neurotensin (NT) on the K⁺-evoked release of endogenous and tritiated dopamine in striatum and on ³H-dopamine in slices from nucleus accumbens and prefrontal cortex were investigated. In striatum, NT (1–1000 nM) elicited a dose-dependent increase in endogenous and ³H-dopamine release. The dose-response curves were comparable with the two methods. Concerning the comparison of NT modulation of ³H-dopamine release in the three cerebral structures, the peptide induced a more marked effect in striatum with a maximal effect of 150% increase. In accumbens, NT (1–1000 nM) potentiated the K⁺-evoked 3H-dopamine release, but in contrast with striatum, the plateau corresponded to a 50% increase. In prefrontal cortex, NT (1–1000 nM) induced small but significant effects, with a maximal increase of 50% at 100 nM. Acetyl-NT (8–13) displayed an action similar to the natural peptide while NT (1–8) did not exhibit any effect, suggesting that the action of NT involved a receptor. The presence of tetrodotoxin did not alter the facilitating effects of NT in the three structures, indicating that interneurons were not involved in the action of NT. The comparison of the effects of NT showed that in terms of efficacy, NT induced an increase in dopamine release more marked in striatum than in nucleus accumbens and prefrontal cortex. These results are consistent with differences in NT receptors localization in these three dopaminergic structures. Send offprint request to: A. Boireau     

Modification of GABA turnover in the striatum and hippocampus of the rat after zopiclone

Summary The effects of zopiclone, a non-benzodiazepine compound that interacts with benzodiazepine receptors, on GABA turnover rate and GABA content in the rat striatum and hippocampus have been studied. Intraperitoneal administration of zopiclone reduced the GABA turnover rates in both the striatum and hippocampus, as estimated from the rate of GABA accumulation after inhibition of GABA transaminase by aminooxyacetic acid (AOAA). The effect of zopiclone on AOAA-induced accumulation of GABA in the hippocampus and striatum was blocked by the intraperitoneal injection of the benzodiazepine receptor antagonist Ro 15-3505. Furthermore, zopiclone slightly but significantly decreased GABA content in the hippocampus, the decrease being blocked by coadministration of the benzodiazepine receptor antagonist Ro 15-1788. Our results confirm that the GABAergic system plays a role in the mechanism of action of zopiclone.This work was supported by MPI 60% grant Send offprint requests to F. Zambotti at the above address     

Characterization of nicotinic acetylcholine receptor-mediated [(3)H]-dopamine release from rat cortex and striatum

The objective of this study was to use a new high throughput method to compare nicotinic acetylcholine receptor (nAChR)-mediated [(3)H]-dopamine (DA) release from slices of rat striatum and cortex. (-)Nicotine, (-)-cytisine, 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and (+/-)-epibatidine evoked release of striatal [(3)H]-DA with pEC(50) values of 6.7, 8.25, 5.11, and 9.08, respectively. The same agonists evoked release of cortical [(3)H]-DA with pEC(50) values of 6.98, 8.06, 5.58, and 9.59. Relative to (-)-nicotine, (-)-cytisine was a partial agonist in both tissues. In contrast, the maximal response evoked by DMPP differed between the two tissues. The rank order of potency for antagonists to block DA release was the same (mecamylamine (Mec)>dihydro-beta-erythroidine (DHbetaE)>hexamethonium (Hex)>D-tubocurarine (D-TC)); however, the pIC(50) values varied between the two regions. Whereas Mec potently antagonized (-)-nicotine-evoked DA release similarly from striatum and cortex, with pIC(50) values of 6.07 and 6.53 respectively, the values obtained for DHbetaE, D-TC and Hex differed. Additionally, the present study was able to distinguish exocytotic vesicular-mediated from transporter-mediated DA release, by altering temperature of the incubation and exclusion of calcium. Assays carried out under these conditions indicate that approximately 60% of nicotine-evoked cortical DA release was likely mediated through the DA transporter. In contrast, under the same conditions only 15%-20% of striatal release appeared to be transporter-mediated. We conclude that the relative contributions of the mechanisms by which (-)-nicotine evokes DA release differ between striatum and cortex. In addition, the data suggest that the subtypes of nAChRs involved in regulating [(3)H]-DA release may be somewhat different in the two tissues.     

1-Hydroxy-3-aminopyrrolid-2-one (HA-966) and kynurenate antagonize N-methyl-D-aspartate induced enhancement of [3H]dopamine release from rat striatal slices

Excitatory amino acid receptors, including those of conventional N-methyl-D-aspartate (NMDA) type, are believed to be located on terminals of the nigrostriatal dopaminergic projection in the rat. Activation of these receptors enhances depolarization-induced dopamine release. Rat striatal slices were preloaded with [3H]dopamine (DA) and its subsequent release and possible modulation during excitatory amino acid receptor activation were investigated. Superfusion of slices with K+ (20 mM) produced a robust increase in [3H]DA release, which was markedly enhanced by the inclusion of N-methyl-D-aspartate (NMDA) or NMDLA in the buffer. No enhancement was observed following addition of glycine, suggesting that either the glycine binding site on the NMDA receptor complex was lacking, or that it was tonically fully activated. The latter appears to be the case since 1-hydroxy-3-aminopyrrolid-2-one (HA-966) (a purported antagonist at strychnine-insensitive glycine receptors) was able to antagonize the NMDA-induced enhancement of [3H]dopamine release. This action of HA-966 could subsequently be reversed by inclusion of glycine in the medium. While the action of NMDA was readily prevented by the inclusion of the competitive antagonist 2-amino-7-phosphonoheptanoate (AP7), this antagonism could not be reversed by either glycine or D-serine. Kynurenate behaved in an apparently identical manner to HA-966. Strikingly, the enhancement of dopamine release by kainate was unaffected by HA-966. These data indicate that NMDA receptor-mediated enhancement of [3H]DA release from striatal dopaminergic terminals can be modulated through strychnine-insensitive sites.     

Effect of soman on N-methyl-d-aspartate-stimulated [3H]norepinephrine release from rat cortical slices

Effects of soman on N-methyl-d-aspartate (NMDA) evoked [³H]norepinephrine (NE) release were examined in rat brain cortical slices. NMDA increased [³H]NE release in a concentration-dependent manner. Soman could inhibit the increase evoked by NMDA, but carbachol, an agonist of cholinergic receptor, could potentiate the increase evoked by NMDA. Atropine (a selective muscarinic antagonist) attenuated the release of [³H]NE induced by NMDA in the presence of carbachol or acetylcholine (ACh), but had no effect on the release of [³H]NE induced by NMDA alone. Both d-tubocurarine (an antagonist of nicotinic receptor) and atropine had no effect on the release of [³H]NE induced by NMDA in the presence of soman. These results suggested that soman has a direct action at non-cholinergic sites, probably at NMDA receptors.     

Effects of the enantiomers of MDA, MDMA and related analogues on [3H]serotonin and [3H]dopamine release from superfused rat brain slices

The primary amines 3,4-methylenedioxyamphetamine (MDA), and 1-(1,3-benzodioxol-5-yl)-2-butanamine (BDB) were measured for efficacy in release of [3H]serotonin (5-HT) from rat hippocampal slices, and release of [3H]dopamine (DA) from rat caudate nucleus slices. The N-methyl derivatives of MDA and BDB, MDMA and MBDB, respectively, and the optical antipodes of these four agents were compared in this paradigm. All of the test compounds demonstrated a similar efficacy of [3H]5-HT release in the micromolar concentration range. No significant stereoselectivity was seen in measurements of 5-HT release. However, striking differences were found between the test compounds when [3H]DA release was studied. N-methylation of racemic MDA resulted in a decreased ability to release DA, while side chain extension from alpha-methyl to alpha-ethyl completely abolished this activity. Stereoselectivity for the S-(+)-isomers of MDA and MDMA was also demonstrated in the DA release studies. Correlation of these biochemical findings with human subjective reports indicates that serotonin release may play a more important role in the mechanism of action than does dopamine release.     

Gamma-aminobutyric acid enhancement of potassium-stimulated release of [3H]norepinephrine by multiple mechanisms in rat cortical slices

Gamma-Aminobutyric acid (GABA) and GABAA agonists enhance stimulated release of [3H]norepinephrine [( 3H]NA) in several regions of the rat brain. In this study, the mechanisms by which GABA and GABAergic agonists augment potassium-stimulated release of [3H]NA from rat frontal cortical slices were examined. GABA enhanced potassium-stimulated [3H]NA release, but did not alter release of [3H]NA evoked by the calcium ionophore A23187, 10(-5) M, either in the presence or the absence of extracellular calcium. The effect of GABA on potassium-stimulated [3H]NA release was apparently reduced by the GABAA antagonist bicuculline methiodide, 10(-4) M, and by the selective inhibitor of GABA uptake SKF 89976A, 10(-5) M, but was abolished only when bicuculline methiodide and SKF 89976A were present in combination. The GABAA agonist muscimol enhanced potassium-stimulated release of [3H]NA in a manner similar to GABA. In addition, nipecotic acid, a substrate for GABA uptake, enhanced potassium-stimulated [3H]NA release. Thus, GABA appears to enhance potassium-stimulated [3H]NA release by acting upon both GABA uptake and GABAA receptors. The GABAA receptors involved in this effect may be a subtype of GABAA receptors since they are not modulated by benzodiazepines. These results support the involvement of the GABA uptake carrier and the GABAA receptor in mediating the enhancement by GABA of potassium-stimulated [3H]NA release in the cortex of the rat.     

Beware the builders: Construction noise changes [C]GABA release and uptake from amygdaloid and hippocampal slices in the rat

The effects of exposure to chronic noise and vibration, produced by construction work, on the release and uptake of [³H]5-hydroxytryptamine ([³H]5-HT) and [¹⁴C]γ-aminobutyric acid ([¹⁴C]GABA) from rat amygdaloid and hippocampal slices were investigated. Noise-exposure resulted in increased release, with no significant change in uptake, of [¹⁴C]GABA from amygdaloid slices. In hippocampal slices, [¹⁴C]GABA release was also increased, but the changes in release were dependent upon the marked decrease in uptake of [¹⁴C]GABA into these slices. There was an increase in peak K⁺-evoked release of [³H]5-HT from hippocampal slices, but no other changes in [³H]5-HT release or uptake in either region were observed following noise-exposure. These findings may have important practical implications for the research carried out in laboratories exposed to construction noise and vibrations.     

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

Summary Dendrosomes prepared from substantia nigra are able to take up and release [³H]dopamine in a Ca²⁺-dependent manner. The V_max 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.Part of this work was presented at a satellite meeting of the 11th International Congress of Pharmacology: Dopamine '90 held in Como, Italy (July 1990) Send offprint requests to M. Raiteri at the above address     

Mechanism of aminopyridine-induced release of [3H]dopamine from rat brain synaptosomes.

1. Aminopyridines (APs) induced the release of [3H]dopamine (3H-DA) from rat synaptosomal preparations. 2. 4-AP and 3,4-DAP were of equal efficacy in inducing release of 3H-DA; 3-AP, 2-AP and 2,6-AP were less active; pyridine and pyridine-4-carboxylamide were inactive. 3. Cd2+ was more effective in inhibiting 4-AP-induced release of 3H-DA (IC50 approximately 4 microM) than Co2+ and Ni2+ (IC50s approximately 500 microM). 4. While 4-AP increased the 45Ca2+ content of whole synaptosomal preparations, no effect of 4-AP on 45Ca2+ content was observed in lysed synaptosomal preparations. 5. 4-AP-induced 45Ca2+ uptake was inhibited by Cd2+, Ni2+ and Co2+ in concentration ranges similar to those inhibiting 3H-DA release.