Nicotine Indirectly Inhibits [3H]Dopamine Uptake at Concentrations That Do Not Directly Promote [3H]Dopamine Release in Rat Striatum

The effects of both (-)- and (+)-nicotine isomers were examined on in vitro uptake and release of [3H]dopamine in rat striatum. Both isomers inhibited uptake of [3H]dopamine in chopped tissue at concentrations well below those necessary for promoting release of preloaded [3H]dopamine. (-)-Nicotine was more potent than (+)-nicotine both at inhibiting uptake and at promoting release. Unlike other dopamine uptake inhibitors, however, nicotine inhibited only 50% of the total uptake. In the presence of 1 nM nicotine, the residual [3H]dopamine uptake was less sensitive to inhibition by cocaine than uptake in the absence of nicotine. Nicotine did not compete against the binding of [3H]GBR 12935, a selective dopamine uptake inhibitor. The nicotinic receptor agonists carbachol and 1,1-dimethyl-4-phenylpiperazinium iodide also inhibited uptake, whereas the nicotinic antagonists chlorisondamine and mecamylamine blocked nicotine's effect. Thus, the effect of nicotine on dopamine uptake appears to be mediated by a receptor similar to the nicotinic acetylcholine receptor. These receptors do not seem to be on the terminals that are accumulating dopamine, however, since tetrodotoxin prevented the effect of nicotine on [3H]dopamine uptake and nicotine had no effect on uptake in a synaptosomal preparation.     

Nicotinic Modulation of [3H]Dopamine Release from Striatal Synaptosomes: Pharmacological Characterisation

Presynaptic nicotinic acetylcholine receptors on striatal nerve terminals modulate the release of dopamine. We have compared the effects of a number of nicotinic agonists and antagonists on a perfused synaptosome preparation preloaded with [3H]dopamine. (-)-Nicotine, acetylcholine, and the nicotinic agonists cytisine and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), at micromolar concentrations, stimulated the release of [3H]dopamine from striatal nerve terminals. Carbamylcholine was a much weaker agonist. The actions of (-)-nicotine, cytisine, and DMPP were inhibited by low concentrations of the nicotinic antagonists dihydro-beta-erythroidine, mecamylamine, pempidine, and neosurugatoxin; alpha-bungarotoxin was without effect, and extending the time of exposure to this toxin resulted in only very modest inhibition. This pharmacology points to a specific nicotinic receptor mechanism that is clearly distinct from that at the neuromuscular junction. Atropine failed to antagonise the effects of acetylcholine and carbamylcholine, suggesting that no muscarinic component is involved. The nicotinic receptor ligands (-)-[3H]nicotine and 125I-alpha-bungarotoxin bound to specific sites enriched in the synaptosome preparation. Drugs tested on the perfused synaptosomes were examined for their ability to interact with these two ligand binding sites in brain membranes. The differential sensitivity to the neurotoxins alpha-bungarotoxin and neosurugatoxin of the 125I-alpha-bungarotoxin and (-)-[3H]nicotine binding sites, respectively, leads to a tentative correlation of the (-)-[3H]nicotine site with the presynaptic nicotinic receptor on striatal nerve terminals.     

Solubilization of D2 Dopamine Receptor Coupled to Guanine Nucleotide Regulatory Protein from Bovine Striatum

D2 dopamine receptor from bovine striatum was solubilized in a form sensitive to guanine nucleotides, by means of a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). The presence of sodium ion markedly increased the solubilization yield. Treatment of the membranes with 10 mM CHAPS and 0.72 M NaCl solubilized 26% of the stereospecific [3H]spiperone binding sites in the original membrane preparations. The solubilized [3H]spiperone binding sites possessed characteristics of the D2 dopamine receptor: (a) localization of the site in the striatum but not in the cerebellum; (b) high affinity to nanomolar concentrations of [3H]spiperone; (c) displacement of [3H]spiperone binding by nanomolar concentrations of neuroleptics, but only by micromolar concentrations of dopamine and apomorphine; (d) equal activity of various dopamine agonists and antagonists in the soluble and membrane preparations. Guanine nucleotides decreased the affinity of the solubilized D2 dopamine receptor for dopamine agonists, but not for antagonists. The solubilized receptor complex was eluted in Sepharose CL-4B column chromatography as a large molecule, with a Stokes radius of approximately 90 A. These results indicate that the complex between the D2 dopamine receptor and GTP binding protein remains intact throughout the solubilization procedure.     

Dopamine receptors in the central nervous system.

Dopamine receptors in the central nervous system can be studied by measuring the specific binding of [3H]dopamine, [3H]haloperidol, d-[3H]LSD, [3H]dihydroergocryptine or [3H]apomorphine. The receptors are stereoselectively blocked by +)-butaclamol, a neuroleptic. All neuroleptics inhibit the specific binding of [3H]haloperidol in relation to their clinical potencies. The radioligand that desorbs most slowly from the receptor is [3H]apomorphine, thus making it a reliable ligand for dopamine receptors. Dopamine agonists that compete for [3H]apomorphine binding do so at concentrations that correlate with their potency in stimulating striatal adenylate cyclase. Structure-activity analysis, using [3H]apomorphine, confirms that the active dopamine-mimetic conformation is the beta rotamer of dopamine. Prolonged exposure in vitro of caudate homogenate to high concentrations of dopamine leads to increased binding of [3H]apomorphine or [3H]haloperidol, suggesting receptor "sensitization." Chronic haloperidol treatment of rats leads to an increased number of dopamine/neuroleptic receptors in the striatum, but a decrease in the pituitary.     

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.     

Evidence for D2 Receptor Regulation of Dopamine Release in the Goldfish Retina

The possible existence of a dopamine D₂ receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [³H]dopamine and superfused with D₂ dopamine receptor agonists or antagonists to determine if there was an effect on [³H]dopamine release. The D₂ receptor antagonist sulpiride increased both baseline [³H]- dopamine release and [³H]dopamine release induced by an increase in extracellular potassium concentration. The D₂ receptor agonists LY-171555 and RU-24213 did not reduce baseline [³H]dopamine release but completely inhibited [³H]dopamine release induced by an increase in [K^±]_o. This action of the D₂ agonists was blocked by sulpiride. These studies demonstrate the existence of D₂ receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.     

Direct evidence that release-stimulating alpha7* nicotinic cholinergic receptors are localized on human and rat brain glutamatergic axon terminals

The existence on glutamatergic nerve endings of nicotinic acetylcholine receptors (nAChRs) mediating enhancement of glutamate release has often been suggested but not demonstrated directly. Here, we study the effects of nAChR agonists on [3 H]-d-aspartate ([3 H]-d-ASP) release from synaptosomes superfused in conditions known to prevent indirect effects. Nicotinic receptor agonists, while unable to modify the basal [3 H]-d-ASP release from human neocortex or rat striatal synaptosomes, enhanced the Ca2+ -dependent exocytotic release evoked by K+ (12 mm) depolarization. Their rank order of potency were anatoxin-a > epibatidine > nicotine > ACh (+ atropine). The anatoxin-a effect, both in human and rat synaptosomes, was antagonized by mecamylamine, alpha-bungarotoxin or methyllycaconitine. The basal release of [3 H]ACh from human cortical synaptosomes was increased by (-)-nicotine (EC50 = 1.16 +/- 0.33 microm) or by ACh plus atropine (EC50 = 2.0 +/- 0.04 microm). The effect of ACh plus atropine was insensitive to alpha-bungarotoxin, methyllycaconitine or alpha-conotoxin MII, whereas it was totally antagonized by mecamylamine or dihydro-beta-erythroidine. To conclude, glutamatergic axon terminals in human neocortex and in rat striatum possess alpha7* nicotinic heteroreceptors mediating enhancement of glutamate release. Release-enhancing cholinergic autoreceptors in human neocortex are nAChRs with a pharmacological profile compatible with the alpha4beta2 subunit combination.     

Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.     

Three distinct Ca(2+) influx pathways couple acetylcholine receptor activation to catecholamine secretion from PC12 cells

Amperometry and microfluorimetry were employed to investigate the Ca(2+)-dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine-evoked exocytosis was entirely dependent on extracellular Ca(2+) but was only partly blocked by Cd(2+), a nonselective blocker of voltage-gated Ca(2+) channels. Secretion and rises of [Ca(2+)](i) observed in response to nicotine could be almost completely blocked by methyllycaconitine and alpha-bungarotoxin, indicating that such release was mediated by receptors composed of alpha7 nicotinic acetylcholine receptor subunits. Secretion and [Ca(2+)](i) rises could also be fully blocked by co-application of Cd(2+) and Zn(2+). Release evoked by muscarine was also fully dependent on extracellular Ca(2+). Muscarinic receptor activation stimulated release of Ca(2+) from a caffeine-sensitive intracellular store, and release from this store induced capacitative Ca(2+) entry that could be blocked by La(3+) and Zn(2+). This Ca(2+) entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca(2+)](i) and exocytosis via Ca(2+) influx through voltage-gated Ca(2+) channels, alpha7 subunit-containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca(2+) entry.     

Dopamine Efflux from Striatum After Chronic Nicotine: Evidence for Autoreceptor Desensitization

We examined the effect of chronic nicotine treatment on dopaminergic activity by measuring the effects of D1 and D2 dopamine (DA) receptor agonists and antagonists on tritium release from mouse striatum preloaded with [3H]DA. The radioactivity released during superfusion was separated on alumina columns and the distribution and efflux of [3H]DA and its main 3H-labeled metabolites were quantified. After preloading by incubation with [3H]DA, the electrical stimulation-evoked tritium overflow was higher in striatum prepared from nicotine-treated mice, whereas in vitro addition of nicotine caused a similar increase in tritium release from striatum of untreated and chronic nicotine-treated mice. The overflow of [3H]DA and its 3H-metabolites exhibited similar distribution patterns in [3H]DA-preloaded striatum dissected from untreated and chronic nicotine-pretreated mice, indicating that repeated injections with nicotine did not alter the metabolism of [3H]DA taken up by the tissue. (-)-Quinpirole, a selective agonist for D2 DA receptors, and apomorphine, a nonselective D1/D2 agonist, inhibited the electrical stimulation-induced tritium efflux from striatum of untreated mice, whereas (+/-)-sulpiride, a D2 DA receptor antagonist, enhanced the evoked release of tritium. These changes in tritium efflux effected by (-)-quinpirole and (+/-)-sulpiride reflected changes in [3H]DA release and not in DA metabolism, as shown by separation of the released radioactivity on alumina columns. The D1 receptor agonist (+/-)-SKF-38393 did not affect the tritium overflow, whereas the D1 receptor antagonist (+)-SCH-23390 exerted a stimulatory action but only at a high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the 5-Hydroxytryptamine Receptor Modulating the Release of 5-[3H]Hydroxytryptamine in Slices of the Human Neocortex

In the rat brain, the presynaptic 5-hydroxytryptamine (5-HT) autoreceptors located on 5-HT terminals correspond to the 5-HT1B subtype. The presence of a 5-HT receptor probably located on 5-HT nerve endings and modulating transmitter release in the human neocortex has been reported, but its detailed pharmacological characterization is not yet available. On the other hand, receptor binding and autoradiographic results indicate that the 5-HT1B receptor subtype is not present in the human brain. We, therefore, studied the modulation of the electrically evoked release of [3H]5-HT by various 5-HT receptor agonists and antagonists in preloaded slices of human neocortex obtained from 18 patients undergoing neurosurgery. The nonselective 5-HT1A/1B/1D receptor agonist 5-carboxamidotryptamine produced a potent inhibition (70% at 0.03 microM) of the electrically evoked release of [3H]5-HT which was blocked by 5-HT receptor antagonists with the following relative order of potency: methiothepin greater than metergoline = methysergide greater than propranolol. The selective 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin at 0.1 microM did not modify the electrically evoked release of [3H]5-HT. The 5-HT1A/1B receptor agonist RU 24969 was 10 times more potent at inhibiting [3H]5-HT overflow in the rat frontal cortex than in the human neocortex. The potent 5-HT1B receptor antagonist cyanopinodolol did not modify the 5-carboxamidotryptamine-induced inhibition of the electrically evoked release of [3H]5-HT in slices of the human neocortex, but produced by itself a small inhibition of [3H]5-HT overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]Fluphenazine Binding to Brain Membranes: Simultaneous Measurement of D-1 and D-2 Receptor Sites

[3H]Fluphenazine was used to label both D-1 and D-2 dopamine receptors in mouse striatal membranes. The D-1 and D-2 specific binding of [3H]fluphenazine was discriminated by the dopamine antagonists SCH-23390 (D-1 selective) and spiperone (D-2 selective). Saturation analyses of these two sites yielded a D-1 receptor density in mouse striatum of 1,400 fmol/mg of protein and a D-2 receptor density of 700 fmol/mg of protein. The affinity of [3H]fluphenazine for the D-2 site was slightly greater than for the D-1 site; the equilibrium dissociation constant (KD) was 0.7 versus 3.2 nM, respectively. Assay conditions are described that reduce nonspecific binding of [3H]fluphenazine to acceptable levels (35% of total binding at 1 nM [3H]fluphenazine). By comparison of displacement curves from a series of dopaminergic and nondopaminergic ligands, the pharmacological specificity of [3H]fluphenazine binding in mouse striatum was demonstrated to be dopaminergic. Only small amounts of dopamine-specific (apomorphine-sensitive) [3H]fluphenazine binding were found in other brain regions. However, chlorpromazine displaced considerable [3H]fluphenazine from all brain regions, including cerebellum, suggesting the presence of a [3H]fluphenazine binding site with a phenothiazine specificity.     

Nicotine-Stimulated Release of [3H]Norepinephrine from Fetal Rat Locus Coeruleus Cells in Culture

Abstract: Acute nicotine administration stimulated [³H]norepinephrine ([³H]NE) release from cultured fetal locus coeruleus (LC) cells. The effect was concentration dependent, with an EC₅₀ of 0.9 µ M , and was abolished by removal of calcium from, or addition of tetrodotoxin (500 n M ) to, the assay buffer. Other nicotinic receptor agonists stimulated [³H]NE release, with the rank order of potency being (±)-epibatidine > (−)-nicotine > 1,1-dimethyl-4-phenylpiperazinium (DMPP). Whereas (−)-nicotine and (±)-epibatidine exhibited equal maximal responses, DMPP was a partial agonist and (−)-cytisine had no agonist activity. Nicotine-stimulated release of [³H]NE was blocked by nicotinic receptor antagonists, with an order of potency of mecamylamine > lobeline > cytisine > methyllycaconitine > dihydro-β-erythroidine. The pharmacological profile of this nicotinic receptor is largely consistent with that described previously for an α4β2 subunit combination, although discrepancies in the efficacies of agonists were observed. No additivity in NMDA- and nicotine-stimulated [³H]NE release was observed, suggesting a common signal transduction mechanism. However, the pharmacological characteristics of MK-801 blockade of nicotine-induced responses were not consistent with those of an NMDA receptor. We therefore conclude that nicotine directly releases [³H]NE from LC cells and does not act indirectly via activation of glutamate release.     

Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon

In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10(-5)-10(-3) M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis-2,3-piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by veratridine, an activator of voltage-sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate-stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N-methyl-D-glucamine HCl. Glutamate-stimulated [3H]-dopamine release was well maintained (60-82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.     

Stimulation of D-2 Dopamine Receptors Decreases the Evoked In Vitro Release of [3H] Acetylcholine from Rat Neostriatum: Role of K+ and Ca2+

Reportedly, stimulation of D-2 dopamine receptors inhibits the depolarization-induced release of acetylcholine from the neostriatum in a cyclic AMP-independent manner. In the present study, we investigated the role of K+ and Ca2+ in the D-2 receptor-mediated inhibition of evoked [3H]acetylcholine release from rat striatal tissue slices. It is shown that the D-2 receptor-mediated decrease of K+-evoked [3H]acetylcholine release is not influenced by the extracellular Ca2+ concentration. However, increasing extracellular K+, in the presence and absence of Ca2+, markedly attenuates the effect of D-2 stimulation on the K+-evoked [3H]acetylcholine release. Furthermore, it is shown that activation of D-2 receptors in the absence of Ca2+ also inhibits the veratrine-evoked release of [3H]acetylcholine from rat striatum. These results suggest that the D-2 dopamine receptor mediates the decrease of depolarization-induced [3H]acetylcholine release from rat striatum primarily by stimulation of K+ efflux (opening of K+ channels) and inhibition of intracellular Ca2+ mobilization.     

Characterization of [(125) I]epibatidine binding and nicotinic agonist-mediated (86) Rb(+) efflux in interpeduncular nucleus and inferior colliculus of beta2 null mutant mice

The beta2 nicotinic acetylcholine receptor subunit null mutation eliminated most high affinity [(3) H]epibatidine binding in mouse brain, but significant binding remained in accessory olfactory nucleus, medial habenula, inferior colliculus and interpeduncular nucleus. Residual [(125) I]epibatidine binding sites in the inferior colliculus and interpeduncular nucleus were subsequently characterized. Inhibition of [(125) I]epibatidine binding by 12 agonists and six antagonists was very similar in these regions. Most acetylcholine-stimulated (86) Rb(+) efflux is eliminated in thalamus and superior colliculus of beta2 null mutants, but significant activity remained in inferior colliculus and interpeduncular nucleus. This residual activity was subsequently characterized. The 12 nicotinic agonists tested elicited concentration-dependent (86) Rb(+) efflux. Epibatidine was the most potent agonist. Cytisine was also potent and efficacious. EC(50) values for quaternary agonists were relatively high. Cytisine-stimulated (86) Rb(+) efflux was inhibited by six classical nicotinic antagonists. Mecamylamine and D-tubocurarine were most potent, while decamethonium was the least potent. Agonists and antagonists exhibited similar potency in both brain regions. Alpha-bungarotoxin (100 nm) did not significantly inhibit cytisine-stimulated (86) Rb(+) efflux, while the alpha3beta4 selective antagonist, alphaConotoxinAuIB, inhibited a significant fraction of the response in both brain regions. Thus, beta2 null mutant mice express residual nicotinic activity with properties resembling those of alpha3beta4*-nAChR.     

Pregnenolone sulfate induces NMDA receptor dependent release of dopamine from synaptic terminals in the striatum

Neuromodulators that alter the balance between lower-frequency glutamate-mediated excitatory and higher-frequency GABA-mediated inhibitory synaptic transmission are likely to participate in core mechanisms for CNS function and may contribute to the pathophysiology of neurological disorders such as schizophrenia and Alzheimer's disease. Pregnenolone sulfate (PS) modulates both ionotropic glutamate and GABA(A) receptor mediated synaptic transmission. The enzymes necessary for PS synthesis and degradation are found in brain tissue of several species including human and rat, and up to 5 nM PS has been detected in extracts of postmortem human brain. Here, we ask whether PS could modulate transmitter release from nerve terminals located in the striatum. Superfusion of a preparation of striatal nerve terminals comprised of mixed synaptosomes and synaptoneurosomes with brief-duration (2 min) pulses of 25 nM PS demonstrates that PS increases the release of newly accumulated [3H]dopamine ([3H]DA), but not [14C]glutamate or [3H]GABA, whereas pregnenolone is without effect. PS does not affect dopamine transporter (DAT) mediated uptake of [3H]DA, demonstrating that it specifically affects the transmitter release mechanism. The PS-induced [3H]DA release occurs via an NMDA receptor (NMDAR) dependent mechanism as it is blocked by D-2-amino-5-phosphonovaleric acid. PS modulates DA release with very high potency, significantly increasing [3H]DA release at PS concentrations as low as 25 pM. This first report of a selective direct enhancement of synaptosomal dopamine release by PS at picomolar concentrations via an NMDAR dependent mechanism raises the possibility that dopaminergic axon terminals may be a site of action for this neurosteroid.     

Differential Inhibition by α-Conotoxin-MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α-conotoxin-MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)-anatoxin-a-evoked release of [³H]dopamine from rat striatal synaptosomes and slices. α-Conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC₅₀ = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα-conotoxin-MII. On perfused striatal synaptosomes and slices, α-conotoxin-MII dose-dependently inhibited [³H]dopamine release evoked by 1 µM (±)-anatoxin-a with IC₅₀ values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α-conotoxin-MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)-anatoxin-a-evoked [³H]dopamine release by α-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α-conotoxin-MII-insensitive nAChR.     

Identification of Dopamine "D3' and "D4' Binding Sites, Labelled with [3H]2-Amino-6,7-Dihydroxy- 1,2,3,4- Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively

On the basis of affinity differences for spiperone, two binding sites for [3H](+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: "D3" with a low and "D4" with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well-known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H-agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4-dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.     

Stress-Induced Enhancement of Suppression of [3H]GABA Release from Striatal Slices by Presynaptic Autoreceptor

The effect of cold and immobilization stress on presynaptic GABAergic autoreceptors was examined using the release of [3H]GABA (gamma-aminobutyric acid) from slices of rat striatum. It was found that in vitro addition of delta-aminolevulinic acid, as well as GABA agonists such as muscimol and imidazoleacetic acid, exhibited a significant suppression of the striatal release of [3H]GABA evoked by the addition of high potassium, whereas delta-aminovaleric acid had no significant effects on the evoked release. These suppressive actions were antagonized invariably by the GABA antagonists, bicuculline and picrotoxin, but not by the glycine antagonist, strychnine. Cholinergic agonists, such as pilocarpine and tetramethylammonium, also attenuated significantly the evoked release of [3H]GABA from striatal slices, while none of its antagonists, including atropine, hexamethonium and d-tubocurarine, affected the release. On the other hand, in vitro addition of dopamine receptor agents such as dopamine, apomorphine, and haloperidol, or the inhibitory amino acids, glycine, beta-alanine, and taurine failed to influence the evoked release of [3H]GABA from striatal slices. Application of a cold and immobilization stress for 3 h was found to induce a significant enhancement of the suppressive effects by muscimol and delta-aminolevulinic acid on the evoked release of [3H]GABA, without affecting that by pilocarpine and tetramethylammonium. These results suggest that the release of GABA from striatal GABA neurons may be regulated by presynaptic autoreceptors for this neuroactive amino acid, and may play a significant functional role in the exhibition of various symptoms induced by stress.