Desensitization of Nicotine-Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes

Potential desensitization of brain nicotinic receptors was studied using a [³H]dopamine release assay. Nicotine-stimulated [³H]dopamine release from mouse striatal synaptosomes was concentration-dependent with an EC₅₀ of 0.33 ± 0.13 μ M and a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC₅₀ and a half-time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration-dependent desensitization (EC₅₀=6.9 plusmn; 3.6 n M , t_1/2= 1.6-2.0 min, n _H=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [³H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time-dependent with half-times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [³H]nicotine to striatal membrane at 22°C included a K _Dof 3.7 ± 0.5 n M , B_max of 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half-times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half-time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [³H]-nicotine binding site could be the presynaptic receptor involved in [³H]dopamine release in mouse striatal synaptosomes.     

Release of [3H]Dopamine from Striatal and Cerebral Cortical Slices from Rats with Thioacetamide-Induced Hepatic Encephalopathy: Different Responses to Stimulation by Potassium Ions and Agonists of Ionotropic Glutamate Receptors

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [³H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Rapid Release of [3H]Dopamine from Median Eminence and Striatal Synaptosomes

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.     

Opiate Modulation of Striatal Dopamine and Hippocampal Norepinephrine Release Following Morphine Withdrawal

When opiates are abruptly withdrawn after chronic treatment, increases in hippocampal noradre-nergic function are observed which are accompanied by decreases in striatal dopamine release. The latter effects have to shown to persist for several weeks following the onset of opiate withdrawal. We examined the long-term effects of opiate withdrawal on 4-aminopyridine and potassium stimulated release of striatal dopamine and hippocampal norepinephrine. Tissue samples were obtained either from rats that had been exposed to opiate withdrawal following a seven day morphine infusion or sham treated control subjects. At 48 hours after the onset of withdrawal (cessation of morphine infusions), slices were loaded with [³H] neurotransmitter, washed extensively, and exposed to different drug treatments. 4-aminopyridine induced concentration related increases in striatal dopamine release, which was 36% calcium independent. Similar values for fractional release of striatal dopamine were obtained in morphine withdrawn and control subjects, for both potassium and 4-aminopyridine induced release. In addition, thresholds for 4-aminopyridine or potassium induced release of striatal dopamine did not differ between control and morphine withdrawn subjects. Treatment with 1.0 M morphine sulfate potentiated potassium evoked release of norepinephrine to an equal extent in both morphine withdrawn and sham treated hippocampal tissue. Exposure to a threshold concentration of potassium (8.0 mM), stimulated increased release of hippocampal norepinephrine in a significantly greater fraction of tissue samples obtained from morphine withdrawn animals. Although these results do not support changes in striatal dopamine release following opiate withdrawal, opiate mechanisms appear to be important determinants of in vitro hippocampal norepinephrine release.     

Reduction of Dopamine Synthesis Inhibition by Dopamine Autoreceptor Activation in Striatal Synaptosomes with In Vivo Reserpine Administration

Abstract: In an attempt to clarify the mechanisms by which dopamine (DA) autoreceptor activation inhibits DA synthesis, the efficacy and potency of the D₂ DA agonists bromocriptine, lisuride, and pergolide, and the D₁,-D₂ DA agonist apomorphine were studied in rat striatal synapto- somes, in which the rate of DA synthesis (formation of ¹⁴CO₂ from l -[1–¹⁴C]tyrosine) was increased 103% by treating the animals from which the synaptosomes were obtained with reserpine (5 mg/kg i.p. twice, 24 and 2 h before they were killed), using the striatal total homogenate as the standard synaptosomal preparation. The increase in DA synthesis evoked by reserpine was additive with that produced by treatment of the synaptosomes with dibutyryl cyclic AMP, suggesting that, not a cyclic AMP-dependent, but possibly a Ca²⁺-dependent mechanism was involved. The DA agonists showed a concentration-dependent inhibition of DA synthesis in the control synaptosomes, which was antagonized by the selective D₂ DA antagonist (-)-sulpiride. In the synaptosomes with increased rate of DA synthesis obtained from the rats treated with reserpine, the concentration-response curves of DA synthesis inhibition for the other DA agonists were shifted to the right, and the effect of bromocriptine was completely eliminated, whereas bromocriptine antagonized the effect of apomorphine. The increased rate of DA synthesis was not preserved in the striatal P₁+ P₂ fraction obtained from the reserpine-treated rats, but the effects of the DA agonists were still reduced to the same degree as those in the total homogenate. (-)-Sulpiride did not enhance DA synthesis in synaptosomes from the reserpine- treated rats. The results presented indicate that the reduced effect of the DA agonists in synaptosomes from the reserpine-treated rats was not due to endogenous DA occupying the DA autoreceptors. Because it is known from the literature that reserpine in vivo increases impulse activity in DA neurons and, as a result, increases the Ca²⁺ concentration, these results suggest that the effect of DA agonists was reduced because DA autoreceptors may normally control DA synthesis by decreasing the free intraneuronal Ca²⁺ concentration, and consequently, the Ca²⁺-dependent phosphorylation of tyrosine hydroxylase.     

Interacting Presynaptic k-Opioid and GABAA Receptors Modulate Dopamine Release from Rat Striatal Synaptosomes

Abstract— The presynaptic regulation of stimulated dopa-mine release from superfused rat striatal synaptosomes by opioids and γ-aminobutyric acid (GABA) was studied. It was found that in addition to dopamine D₂ autoreceptors, calcium-dependent K⁺-stimulated [³H]dopamine release was inhibited through activation of a homogeneous population of k -opioid receptors in view of the potent inhibitory effect of the k -selective agonist U69.593 (EC₅₀ 0.2 nM) and its antagonism by norbinaltorphimine. Neither μ-nor δ-selective receptor agonists affected release of [³H]-dopamine. In addition, GABA potently inhibited the evoked [³H]dopamine release (EC₅₀ 0.4 nM) through activation of GABA_A receptors in view of the GABA-mimicking effect of muscimol, the sensitivity of its inhibitory effect to picro-toxin and bicuculline, and the absence of an effect of the GABA_B receptor agonist baclofen. In the presence of a maximally effective concentration of GABA, U69,593 did not induce an additional release-inhibitory effect, indicating that these receptors and the presynaptic D₂ receptor are colocalized on the striatal dopaminergic nerve terminals. The excitatory amino acid agonists N-methyl-d -aspartate and kainate, as well as the cholinergic agonist carbachol, stimulated [³H]dopamine release, which was subject to k -opioid receptor-mediated inhibition. In conclusion, striatal dopamine release is under regulatory control of multiple excitatory and inhibitory neurotransmitter by activation of colocalized presynaptic receptors for excitatory amino acids, acetylcholine, dopamine, dynorphins, and GABA within the dopaminergic nerve terminals. Together, these receptors locally control ongoing dopamine neurotransmission.     

Voltage-Sensitive Ca2+ Channels Involved in Nicotinic Receptor-Mediated [3H]Dopamine Release from Rat Striatal Synaptosomes

Abstract: The potent nicotinic agonist anatoxin-a elicits mecamylamine-sensitive [³H]dopamine release from striatal synaptosomes, and this action is both Na⁺ and Ca²⁺ dependent and is blocked by Cd²⁺. This suggests that stimulation of presynaptic nicotinic receptors results in Na⁺ influx and local depolarisation that activates voltage-sensitive Ca²⁺ channels, which in turn provide the Ca²⁺ for exocytosis. Here we have investigated the subtypes of Ca²⁺ channels implicated in this mechanism. [³H]Dopamine release evoked by anatoxin-a (1 µM) was partially blocked by 20 µM nifedipine, whereas KCl-evoked release was insensitive to the dihydropyridine. However, a ⁸⁶Rb⁺ efflux assay of nicotinic receptor function suggested that nifedipine has a direct effect on the receptor, discrediting the involvement of L-type channels. The N-type Ca²⁺ channel blocker ω-conotoxin GVIA (1 µM) blocked anatoxin-a-evoked [³H]dopamine release by 60% but had no significant effect on ⁸⁶Rb⁺ efflux; release evoked by both 15 and 25 mM KCl was inhibited by only 30%. The P-type channel blocker ω-agatoxin IVA (90 nM) also inhibited KCl-evoked release by ～30%, whereas anatoxin-a-evoked release was insensitive. The Q-type channel blocker ω-conotoxin MVIIC (1 µM) had no effect on either stimulus. These results suggest that presynaptic nicotinic receptors on striatal nerve terminals promote [³H]dopamine release by activation of N-type Ca²⁺ channels. In contrast, KCl-evoked [³H]dopamine release appears to involve both N-type and P-type channels.     

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.     

Effects of Metabotropic Glutamate Receptor Agonists and Antagonists on D-Aspartate Release from Mouse Cerebral Cortical and Striatal Slices

The cytosolic release of L-glutamate has been held to be responsible for the increase in extracellular glutamate to toxic levels in the brain. The mechanism and regulation of this release was now studied in cerebral cortical and striatal slices with D-[³H]aspartate, a non-metabolized analogue of L-glutamate and a poor substrate for vesicular uptake. L-Glutamate and D-aspartate strongly stimulated the release in a concentration-dependent manner. Of the ionotropic glutamate receptor agonists, only kainate enhanced the basal release in the striatum. Of the metabotropic glutamate receptor ligands, the group I agonist (S)-3,5-dihydroxyphenylglycine (S-DHPG) failed to affect the basal release but inhibited the D-aspartate-evoked release in the striatum. The group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) had no effect on the basal release in either preparation but enhanced the L-glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum, not however in the cerebral cortex. The group II agonist (2S,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG IV) and the group II antagonist (2S)-2-ethylglutamate (EGLU) were without effect on the basal, D-aspartate- and L-glutamate-evoked releases of D-[³H]aspartate in either preparation. The group III agonist L-serine-O-phosphate (L-SOP) failed to affect the basal release but reduced the D-aspartate-evoked release in the striatum. The group III antagonist (RS)-methylserine-O-phosphate (MSOP) failed to affect the basal release but increased the glutamate-evoked release and inhibited the D-aspartate-evoked release in the striatum. Both L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC) and (2S, 1S, 2R)-2-carboxycyclopropyl)glycine (L-CCG-III), transportable inhibitors of the high-affinity glutamate uptake, enhanced the basal release, more strongly in the striatum than in the cerebral cortex. L-CCG-III also increased the L-glutamate-evoked release in the striatum. Nontransportable dihydrokainate enhanced the basal release much less and failed to affect the glutamate-evoked release. The results indicate that the release of glutamate from cytosolic pools is carrier-mediated via homoexchange. This process is regulated in the striatum by metabotropic group I and group III receptors in a manner different from the regulation of the vesicular release of glutamate from presynaptic terminals.     

Glutamatergic Control of Dopamine Release in the Rat Striatum: Evidence for Presynaptic N-Methyl-D-Aspartate Receptors on Dopaminergic Nerve Terminals

The N-methyl-D-aspartate (NMDA) receptor-mediated regulation of the release of newly synthesized [3H]dopamine [( 3H]DA) was studied in vitro, both on rat striatal slices using a new microsuperfusion device and on rat striatal synaptosomes. Under Mg2(+)-free medium conditions, the NMDA (5 X 10(-5) M)-evoked release of [3H]DA from slices was found to be partly insensitive to tetrodotoxin (TTX). This TTX-resistant stimulatory effect of NMDA was blocked by either Mg2+ (10(-3) M) or the noncompetitive antagonist MK-801 (10(-6) M). In addition, the TTX-resistant NMDA-evoked response could be potentiated by glycine (10(-6) M) in the presence of strychnine (10(-6) M). The coapplication of NMDA (5 X 10(-5) M) and glycine (10(-6) M) stimulated the release of [3H]DA from striatal synaptosomes. This effect was blocked by Mg2+ (10(-3) M) or MK-801 (10(-5) M). These results indicate that some of the NMDA receptors involved in the facilitation of DA release are located on DA nerve terminals. These presynaptic receptors exhibit pharmacological properties similar to those described in electrophysiological studies for postsynaptic NMDA receptors.     

Differential Effect of Stress on In Vivo Dopamine Release in Striatum, Nucleus Accumbens, and Medial Frontal Cortex

Microdialysis was used to assess extracellular dopamine in striatum, nucleus accumbens, and medial frontal cortex of unanesthetized rats both under resting conditions and in response to intermittent tail-shock stress. The dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid also were measured. The resting extracellular concentration of dopamine was estimated to be approximately 10 nM in striatum, 11 nM in nucleus accumbens, and 3 nM in medial frontal cortex. In contrast, the resting extracellular levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid were in the low micromolar range. Intermittent tail-shock stress increased extracellular dopamine relative to baseline by 25% in striatum, 39% in nucleus accumbens, and 95% in medial frontal cortex. 3,4-Dihydroxyphenylacetic acid and homovanillic acid also were generally increased by stress, although there was a great deal of variability in these responses. These data provide direct in vivo evidence for the global activation of dopaminergic systems by stress and support the concept that there exist regional variations in the regulation of dopamine release.     

Ca2+-Dependent and Ca2+-Independent [3H]GABA Release from Rat Brain Synaptosomes: the Effect of Temperature

The main inhibitory neurotransmitter GABA in the mammalian brain is distributed in the nerve terminals between two pools, vesicular (synaptic vesicles) and cytosolic. GABA is released from these pools by different mechanisms; there are calcium-activated exocytotic release and calcium-independent sodium-dependent release from the cytosolic pool (resulting from the membrane GABA transporter reversal). We investigated the influence of temperature on [³H]GABA release from rat brain synaptosomes, which was induced by stimulation of both these processes. In addition, we used -latrotoxin as a stimulant of [³H]GABA release. Synaptosomes from the rat brain were used in the experiments. 4-Aminopyridine (4-AP) and high [KCl] were applied to stimulate calcium-activated and calcium-independent [³H]GABA release, respectively. 4-AP-evoked [³H]GABA release was of the same intensity at 37 and 25°C (10.1 ± 1.2 and 10.1 ± 0.8% of total [³H]GABA incorporated into the synaptosomes, respectively). The effect of 4-AP on the ⁴⁵Ca²⁺ influx into synaptosomes was also temperature-independent: 0.775 ± 0.075 and 0.725 ± 0.100 nmol/min/mg of protein at 37 and 25°C, respectively. A drop in the effect of 4-AP was observed only at 15°C. When synaptosomes were depolarized with 50 mM KCl, a temperature decrease from 37°C to 25°C resulted in a twofold drop in the [³H]GABA release, from 20.5 ± 1.4 to 10.3 ± 0.7%; at 15°C [³H]GABA release dropped to less than one-third of the norm (6.0 ± 0.5%). -Latrotoxin-stimulated [³H]GABA release was diminished from 32.5 ± 2.5 at 37°C to 17.2 ± 1.3 at 25°C and 5.9 ± 0.4% at 15°C and was not affected by the presence or absence of calcium in the medium. It seems likely that the observed effect of temperature can be interpreted as based on the temperature dependence of the -latrotoxin insertion into the membrane. It is suggested that the pattern of the temperature sensitivity of GABA release from the synaptosomes can be used as a criterion for identification of the mode of neurotransmitter release.     

Effects of Arachidonic Acid on Dopamine Synthesis, Spontaneous Release, and Uptake in Striatal Synaptosomes from the Rat

Abstract: Arachidonic acid (AA) markedly stimulated, in a dose-dependent manner, the spontaneous release of [³H]dopamine ([³H]DA) continuously synthesized from [³H]tyrosine in purified synaptosomes from the rat striatum. As estimated by simultaneous measurement of the rate of [³H]H₂O formation (an index of [³H]tyrosine conversion into [³H]DOPA), the AA response was associated with a progressive and dose-dependent reduction of [³H]DA synthesis. In contrast to AA, arachidic acid, oleic acid, and the methyl ester of AA (all at 10⁻⁴ M ) did not modify [³H]DA release. The AA (3 × 10⁻⁵ M )-evoked release of [³H]DA was not affected by inhibiting AA metabolism, with either 5,8,11,14-eicosatetraynoic acid or metyrapone, suggesting that AA acts directly and not through one of its metabolites. AA also inhibited in a dose-dependent manner [³H]DA uptake into synaptosomes, with a complete blockade observed at 10⁻⁴ M . However, AA (10⁻⁴ M ) still stimulated [³H]DA spontaneous release in the presence of either nomifensine or other DA uptake inhibitors, indicating that AA both inhibits DA reuptake and facilitates its release process. Finally, the AA (10⁻⁴ M )-evoked release of [³H]DA was not affected by protein kinase A inhibitors (H-89 or Rp -8-Br-cAMPS) but was markedly reduced in the presence of protein kinase C inhibitors (Ro 31-7549 or chelerythrine).     

Inhibition of the Electrically Evoked Release of [3H]Acetylcholine in Rat Striatal Slices: An Experimental Model for Drugs that Enhance Dopaminergic Neurotransmission

The activation by endogenous dopamine of the inhibitory 3,4-dihydroxyphenylethylamine (dopamine) receptors modulating the electrically evoked release of [3H]acetylcholine [( 3H]ACh) and [3H]dopamine in rat striatal slices is a function of the concentration of dopamine accumulated in the synaptic cleft during electrical stimulation. When the release of 3H-neurotransmitters was elicited with a 2-min period of stimulation at a frequency of 1 Hz, neither dopamine autoreceptors nor dopamine receptors modulating [3H]ACh were activated by endogenously released dopamine. On the other hand, exposure to (S)-sulpiride facilitated the release of [3H]dopamine and [3H]ACh elicited when the 2-min stimulation was carried out at a frequency of 3 Hz but this effect was not observed at a lower frequency of stimulation (1 Hz). In the presence of amphetamine the dopamine receptors modulating the electrically evoked release of [3H]ACh can be activated by endogenous dopamine even at the lower frequency of stimulation (1 Hz). Similar effects can be obtained if the neuronal uptake of dopamine is inhibited by cocaine or nomifensine. The inhibition by amphetamine of the release of [3H]ACh elicited by electrical stimulation at 1 Hz involves dopamine receptors and can be fully antagonized by clozapine, haloperidol, chlorpromazine, or pimozide. The stereoselectivity of this antagonism can be demonstrated with the optical enantiomers of sulpiride and butaclamol. This inhibitory effect of amphetamine on cholinergic neurotransmission appears to be the result of the stimulation of dopamine receptors of the D2 subtype, as they were resistant to blockade by the preferential D1 receptor antagonist SCH 23390.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Inhibition by Anandamide and Synthetic Cannabimimetics of the Release of [3H]d-Aspartate and [3H]GABA from Synaptosomes Isolated from the Rat Hippocampus

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K(+)-evoked release of [3H]D-aspartate ([3H]D-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]D-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2'-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]D-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.     

Stereoselective Nicotine-Induced Release of Dopamine from Striatal Synaptosomes: Concentration Dependence and Repetitive Stimulation

Using a sensitive perfusion system we have studied the nicotine-induced release of [3H]dopamine ([( 3H]DA) from striatal synaptosomes. Nicotine-evoked release was concentration dependent with an EC50 of 3.8 microM. The response to 1 microM nicotine was comparable to that to 16 mM K+; 10 microM veratridine evoked a larger response. All three stimuli were Ca2+ dependent but only the response to veratridine was blocked by tetrodotoxin. Repetitive stimulations by 1 microM (-)-nicotine (100 microliters) at 30-min intervals resulted in similar levels of [3H]DA release; higher concentrations of (-)-nicotine resulted in an attenuation of the response particularly following the third stimulation. This may reflect desensitisation or tachyphylaxis of the presynaptic nicotinic receptor. The action of nicotine was markedly stereoselective: a 100-fold higher concentration of (+)-nicotine was necessary to evoke the same level of response as 1 microM (-)-nicotine. It is proposed that these presynaptic nicotinic receptors on striatal terminals are equivalent to high-affinity nicotine binding sites described in mammalian brain.     

Stimulation of [3H]Dopamine Release by Nicotine in Rat Nucleus Accumbens

Abstract: The mesolimbic system of the brain has been shown to be involved in the reward properties of a number of agents. It is possible that release of monoamines by nicotine in this brain area could be related to the pleasurable aspects related to cigarette smoking. In this investigation, the effect of nicotine on the release of [³H]dopamine in the nucleus accumbens of the rat was studied. It was shown that nicotine produced a concentration-dependent increase in [³H]dopamine release at concentrations of 0.1 μ M and above. The increase in release was found to be almost completely calcium dependent. The nicotine-induced release was only partially blocked by the nicotinic antagonists hexamethonium and d -tubocurarine. A number of cholinergic agonists, as well as other compounds, were tested for their capacity to mimic the effect of nicotine. At equimolar concentrations there was, at most, only 50% of the activity of nicotine. The results of this study demonstrate that nicotine stimulates the release of dopamine in the nucleus accumbens at concentrations similar to those in the blood of cigarette smokers. This suggests that the release of mono-amines in specific nuclei of the mesolimbic system may be an important determinant of the desire to smoke cigarettes.