D2 dopamine receptors and modulation of spontaneous acetylcholine (ACh) release from rat striatal synaptosomes

1. The effect of two D_3/2 dopamine receptor agonists, LY-171555 (quinpirole) and 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) on spontaneous [³H]-acetylcholine ([³H]-ACh) release were investigated in rat striatal synaptosomes.
2. Quinpirole and 7-OH-DPAT inhibited in a concentration-dependent manner the basal efflux of [³H]-ACh with similar E_max (maximal inhibitory effect) values (29.95±2.91% and 33.19±1.21%, respectively). Significant differences were obtained between the pEC₅₀ (−log of molar concentration) of quinpirole (7.87±0.12) and 7-OH-DPAT (7.21±0.17; P<0.01).
3. Different concentrations (0.3–10 nM) of haloperidol (D_2/3 dopamine receptor antagonist) shifted to the right the concentration-response curves elicited by quinpirole and 7-OH-DPAT, without modifications in the E_max.
4. Slopes of a Schild plot obtained with haloperidol in the presence of quinpirole and 7-OH-DPAT were not signficantly different from unity (0.85±0.05 and 1.17±0.11, respectively) and consequently haloperidol interacted with a homogeneous receptor population. The pK_B values of haloperidol obtained from Schild regression were 9.96±0.15 (in presence of quinpirole) and 9.90±0.09 (in presence of 7-OH-DPAT).
5. Specific binding of [³H]-YM-09151-2 to membranes of striatal synaptosomes and cells expressing D₂ and D₃ dopamine receptors was inhibited by haloperidol. Analysis of competition curves revealed the existence of a single population of receptors. There were no differences between the estimated pK_i (−log of molar concentration) values for synaptosomes (8.96±0.02) and cells expressing D₂ receptors (8.81±0.05), but the pK_i value from cells expressing D₃ dopamine receptors differed significantly (8.48±0.06; P<0.01).
6. In conclusion, the data obtained in the present study indicate that quinpirole and 7-OH-DPAT, two D_3/2 dopamine receptor agonists, inhibit the spontaneous [³H]-ACh efflux and this effect is competitively antagonized by haloperidol and probably mediated through dopamine D₂ receptors.

     

Cannabinoid receptor-mediated inhibition of acetylcholine release from hippocampal and cortical synaptosomes

In previous studies cannabinoid agonists have been found to inhibit and cannabinoid antagonists to enhance electrically-evoked [(3)H]-acetycholine (ACh) release in hippocampal slices. The present study was undertaken to determine if similar cannabinoid effects could be observed in synaptosomes. [(3)H]-ACh release was evoked by two methods, both sensitive to presynaptic receptor effects. The first involved the addition of 1.3 mM calcium following perfusion with calcium-free Krebs and the second the addition of 11 mM potassium following perfusion with normal Krebs. In hippocampal synaptosomes the 1.3 mM calcium-evoked release and the high potassium-evoked [(3)H]-ACh release were inhibited by the cannabinoid agonist, WIN 55212-2, by 59 and 39%, respectively, and with an EC(50) of approximately 1 nM. WIN 55212-2 produced a similar, although less potent, inhibition of [(3)H]-ACh release in cortical synaptosomes. No inhibitory effect of WIN 55212-2 on [(3)H]-ACh release in striatal synaptosomes was observed, supporting previous data collected in this area with brain slices. The cannabinoid antagonist, SR 141716A, produced a robust enhancement of 1.3 mM calcium-evoked [(3)H]-ACh release in hippocampal synaptosomes (EC(50)<0.3 nM) but had no effect on potassium-evoked release or on [(3)H]-ACh release in the cortex or striatum. In conclusion our data demonstrates the inhibitory effects of WIN 55212-2 observed on ACh release in brain slices can be observed in hippocampal and cortex synaptosomes, suggesting a direct action of these compounds on the synaptic terminals. The SR 141716A-induced enhancement of ACh release can similarly be observed in hippocampal synaptosomes and is probably due to an inverse agonist action at constitutively active receptors.     

Blockade of nicotinic receptor-mediated release of dopamine from striatal synaptosomes by chlorisondamine administered in vivo.

1. The chronic nicotinic blockade produced following in vivo administration of chlorisondamine was investigated in vitro. Nicotine-induced [3H]-dopamine release from striatal synaptosomes was used as a measure of central nicotinic receptor function. 2. In synaptosomal preparations from rats pretreated with a single administration of chlorisondamine (10 mg kg-1, s.c.), 1, 7, 21, 42, 63 or 84 days before they were killed, responses to (-)-nicotine (10(-6) M) were blocked. 3. In vivo administration of chlorisondamine (10 mg kg-1, s.c.), 7 days before rats were killed, produced a nicotinic blockade in vitro that was insurmountable even with a high concentration of (-)-nicotine (10(-4) M). 4. Both in vitro and in vivo administration of chlorisondamine blocked nicotinic responses to acetylcholine (10(-4) M). In contrast, neither in vitro nor in vivo administration of chlorisondamine reduced [3H]-dopamine release induced by high K+ (20 x 10(-3) M) or (+)-amphetamine (10(-6) M). 5. Nicotinic blockade resulting from in vitro administration of chlorisondamine (10(-5) M) recovered partially after 60 min wash-out, and completely by 90 min. In contrast, no recovery was seen in synaptosomes prepared from rats pretreated with chlorisondamine (10 mg kg-1, s.c.) in vivo. 6. Thus, in vivo treatment with chlorisondamine results in a quasi-irreversible, insurmountable block of CNS nicotinic receptors. The persistence of this block ex vivo indicates that physical trapping by the blood brain barrier is not solely responsible for the persistent blockade seen in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes.

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.

Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).

In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.

Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Cannabinoid CB1 receptor-mediated inhibition of glutamate release from rat hippocampal synaptosomes

Cannabinoid receptors are widely expressed in the brain and have been shown to regulate synaptic transmission through a presynaptic mechanism. Using synaptosomal preparation, I show here that 2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)-pyrrolo-1,4-benzoxazin-6-yl-1-naphthalenylmethanone (WIN 55212-2) strongly depressed 4-aminopyridine-evoked glutamate release in a concentration-dependent manner, and this effect was reversed by the selective cannabinoid CB(1) receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM 281). The inhibitory modulation by WIN 55212-2 was not due to a decrease in synaptosomal excitability or a direct effect on the release machinery because WIN 55212-2 did not alter 4-aminopyridine-mediated depolarization and ionomycin-induced glutamate release. In addition, the WIN 55212-2-mediated inhibition of glutamate release was blocked by the G(i)/G(o) protein inhibitor pertussis toxin, but not by the protein kinase A inhibitor 2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo-benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720). Furthermore, this inhibitory effect was associated with a decrease in 4-aminopyridine-evoked Ca(2+) influx, which could be completely prevented in synaptosomes pretreated with the N- and P/Q-type Ca(2+) channel blockers. Together, these observations indicate that activation of cannabinoid CB(1) receptors inhibit 4-aminopyridie-evoked glutamate release from hippocampal synaptosomes through a inhibitory G protein to suppress N- and P/Q-type Ca(2+) channel activity.     

Nicotine-evoked [3H]5-hydroxytryptamine release from rat striatal synaptosomes

The aim of this study was to characterize the pharmacology of presynaptic nicotinic cholinoceptors (nAChRs) that modulate release of 5-hydroxytryptamine (5-HT) from superfused rat brain synaptosomes preloaded with [3H]5-HT. Nicotine increased 5-HT release from striatal synaptosomes (maximally by 15-30%) but not from cerebral cortex or hippocampal synaptosomes. Release of striatal 5-HT was increased in a concentration-dependent manner by nicotine, epibatidine, cytisine, and ACh (with added esterase inhibitor and muscarinic antagonist). Respective EC50 values were: 0.5, 0.003, 0.1 and 0.7 microM. The maximal effect of each agonist was virtually completely blocked by a high concentration of the insurmountable nicotinic antagonist mecamylamine; at a higher concentration of epibatidine (3 microM), a mecamylamine-insensitive effect was revealed. Nicotine, ACh and epibatidine appeared equally efficacious, whereas cytisine was of lower efficacy (60-70% of ACh). Release evoked by a half-maximal concentration of nicotine was inhibited by the nicotinic antagonists dihydro-beta-erythroidine (IC50 0.04 microM) and methyllycaconitine (IC50 0.06 microM). Nicotine-evoked 5-HT release was not reduced by tetrodotoxin given in a concentration that blocked veratridine-evoked release. These findings provide functional evidence for a direct action of nicotine on 5-HT neurons in the brain. The presynaptic nAChRs that modulate striatal 5-HT release appear to possess a novel pharmacological profile.     

Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes

Summary The effects of oxymetazoline and noradrenaline (in the presence of desipramine) on the release of ³H-noradrenaline from rat brain cortex synaptosomes were studied using a superfusion technique. Both drugs (at 1M concentrations) were found to reduce the depolarization-induced (15 mM K⁺) release of ³H-noradrenaline. The release-modulating effect of noradrenaline was antagonized by phentolamine and yohimbine.The data provide direct evidence for the hypothesis that -receptors modulating the release of noradrenaline are localized on varicosities of central noradrenergic neurones.     

Presynaptic noradrenergic alpha-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes.

The depolarization (15 mM K+)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of alpha-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K+ stimulation greatly enhanced the sensitivity of 3H-NA release to alpha-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the alpha-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons, Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less senstive to alpha-receptor-mediated presynaptic inhibition than those in the cortex.     

Blockade of nicotinic receptor-mediated release of dopamine from striatal synaptosomes by chlorisondamine and other nicotinic antagonists administered in vitro.

1. Central nicotinic receptor function examined in vitro, by measuring nicotine-induced [3H]-dopamine release from rat striatal synaptosomes. 2. The agonists (-)-nicotine, acetylcholine, 1,1-dimethyl-4-phenylpiperazinium (DMPP) and cytisine (10(-7)-10(-4) M) all increased [3H]-dopamine release in a concentration-dependent manner. Cytisine did not produce a full agonist response, compared to the other agonists. 3. The actions of nicotine, acetylcholine and cytisine were largely dependent on external Ca2+. In contrast, DMPP (10(-5) and 10(-4) M) evoked a marked release of [3H]-dopamine even in the absence of Ca2+. Nevertheless, in the presence of external Ca2+, responses to DMPP were completely blocked by the nicotinic antagonists chlorisondamine and mecamylamine (5 x 10(-5) M); in the absence of external Ca2+, blockade was only partial. 4. Chlorisondamine, mecamylamine and dihydro-beta-erythroidine (10(-8)-10(-4) M) produced a concentration-dependent block of responses to nicotine (10(-6) M). Approximate IC50 values were 1.6, 0.3 and 0.2 x 10(-6), respectively. Chlorisondamine and mecamylamine blocked responses to nicotine (10(-7)-10(-4) M) insurmountably, whereas dihydro-beta-erythroidine behaved in a surmountable fashion. 5. The occurrence of use-dependent block was tested by briefly pre-exposing the synaptosomes to nicotine during superfusion with antagonist, and determining the response to a subsequent nicotine application. Consistent with a possible channel blocking action, brief pre-exposure to agonist increased the antagonist potency of chlorisondamine (approximately 25 fold). No significant use-dependent block was detected with dihydro-beta-erythroidine.     

Involvement of protein kinase C in the presynaptic nicotinic modulation of [(3)H]-dopamine release from rat striatal synaptosomes.

1. Presynaptic nicotinic ACh receptors modulate transmitter release in the brain. Here we report their interactions with protein kinase C (PKC) with respect to [(3)H]-dopamine release from rat striatal synaptosomes, monitored by superfusion. 2. Two specific PKC inhibitors, Ro 31-8220 (1 microM) and D-erythro-sphingosine (10 microM) significantly reduced (by 51 and 26% respectively) [(3)H]-dopamine release stimulated by anatoxin-a (AnTx), a potent and selective agonist of nicotinic ACh receptors. The inactive structural analogue of Ro 31-8220, bisindolylmaleimide V (1 microM) had no effect. 3. Two phorbol esters, PDBu (1 microM) and PMA (1 microM) potentiated AnTx-evoked [(3)H]-dopamine release by 50 - 80%. This was Ca(2+)-dependent and prevented by PKC inhibitors. In the absence of nicotinic agonist, phorbol esters enhanced basal release through a PKC-independent mechanism. 4. A (86)Rb(+) efflux assay of nicotinic ACh receptor function confirmed that Ro 31-8220 has no nonspecific effect on presynaptic nicotinic ACh receptors. 5. These results suggest that PKC is activated by nicotinic ACh receptor stimulation and mediates a component of AnTx-evoked [(3)H]-dopamine release. In addition, independent activation of PKC can further amplify the response, offering a potential mechanism for receptor crosstalk.     

Control of tachykinin-evoked acetylcholine release from rat striatal slices by dopaminergic neurons

Summary The regulation of tachykinin-evoked acetylcholine release by the dopaminergic system in the neostriatum was examined. We studied the effect of selective and potent tachykinin agonists for each subtype of receptor ([Sar⁹,Met(O₂)¹¹]-Substance P for NK₁; [Nle¹⁰]-Neurokinin A_4–10 for NK₂; and senktide for NK₃) on endogenous acetylcholine release from rat striatal slices where the dopaminergic system was modified either by 6-hydroxydopamine lesion or by dopamine receptor antagonists. Unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway induced a decrease in senktide-evoked acetylcholine release and an increase in the effect of [Nle¹⁰]-Neurokinin A_4–10 The same results were obtained after chronic haloperidol treatment, whereas SCH-23390 or clozapine treatment had no effect on tachykinin-evoked acetylcholine release, suggesting an involvement of D₂ receptors. 6-hydroxydopamine lesion induced a diminution in the density of NK₃ receptor, which could be related to the reduction in senktide-evoked acetylcholine release. Correspondence to: J. Marsal at the above address     

Characterization of nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated rat stomach

We characterized nicotinic acetylcholine receptor-mediated noradrenaline release from the isolated, vascularly perfused rat stomach. The stomach was perfused via the coeliac artery with Krebs-Ringer solution at a constant flow rate of 4 ml per minute. Endogenous noradrenaline released into the perfusate was electrochemically measured using high-performance liquid chromatography. Nicotinic receptor agonists were applied once into the perfusion medium for 2 min and nicotinic receptor antagonists were administered throughout the experiments. The (-)-nicotine (3x10(-5) M)-induced noradrenaline release was abolished by tetrodotoxin and hexamethonium and partially blocked by dihydro-beta-erythroidine (up to 10(-5) M) (a relatively selective antagonist of alpha4beta2 nicotinic receptors) and abolished by mecamylamine (10(-5) M) (a relatively selective antagonist of alpha3beta4 nicotinic receptors), but not influenced by alpha-bungarotoxin (3x10(-7) M) or alpha-conotoxin ImI (10(-6) M) (antagonists of alpha7 nicotinic receptors). (+/-)-Epibatidine (3x10(-7) M) (a very potent, but non-selective agonist) and (-)-cytisine (3x10(-4) M) (an agonist of beta4 nicotinic receptors) effectively evoked the release of noradrenaline, while (E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine (RJR-2403) (up to 10(-4) M) (an agonist of alpha4beta2 nicotinic receptors) had no effect. The potency of these agonists was as followed; (+/-)-epibatidine>(-)-nicotine>(-)-cytisine>RJR -2403. These results are compatible with the published view that alpha3beta4 nicotinic receptors are predominant in other parts of the autonomic nervous system. These receptors (probably located on the gastric sympathetic ganglia) are involved in the release of noradrenaline from the rat stomach.     

Specificity of action of beta-bungarotoxin on acetylcholine release from synaptosomes.

Presynaptically acting phospholipase A2 (PLA2) neurotoxins such as beta-bungarotoxin (beta-BuTX) specifically modify the release of acetylcholine (ACh) in the periphery, whereas in the central nervous system (CNS) the release of other neurotransmitters such as norepinephrine (NE) and serotonin (5-HT) are also modified. In addition, ACh release in the periphery is modified in a triphasic manner (decrease, then increase, then block), while in the CNS only the increase has been demonstrated. To determine the specificity of the central effects of beta-BuTX we compared the effects of beta-BuTX and N. n. atra PLA2 on the release from rat cerebrocortical synaptosomes of ACh, NE, and 5-HT. We also measured the leakage of lactate dehydrogenase (LDH) in order to determine whether membrane permeablization was responsible for neurotransmitter leakage. Both the PLA2 neurotoxin (5.0 nM) and the non-neurotoxic enzyme (0.5 nM) stimulated the loss of NE and 5-HT, but only at concentrations which induced leakage of LDH. Conversely, beta-BuTX stimulated the release of ACh at a concentration (0.5 nM) which caused no leakage of LDH, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release. beta-Bungarotoxin thus exerts a specific effect on cholinergic nerve terminals, while the leakage of NE and 5-HT induced by beta-BuTX and N. n. atra PLA2 correlates with membrane disruption due to their PLA2 activities. Within 20 min, 0.5 nM beta-BuTX increased the resting release of ACh and decreased the stimulated release induced by depolarization with 4-aminopyridine, while N. n. atra PLA2 (0.5 nM) did not stimulate ACh release and required 45 min to exert an inhibitory effect. beta-BuTX (5.0 nM) also exerted an inhibitory effect on ACh release stimulated by veratridine, but not by high KCl. It is concluded that in low concentrations that do not disrupt membrane permeability, beta-BuTX acts specifically on cholinergic terminals in rat synaptosomes, where it exerts both stimulatory and inhibitory effects.     

Effect of in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes

The effect of inorganic lead in vitro in several aspects of [3H]dopamine release from superfused rat striatal synaptosomes was examined. Under conditions of spontaneous release, lead (1-30 microM) induced dopamine release in a concentration-dependent manner. The onset of the lead-induced release was delayed by approximately 15-30 sec. The magnitude of dopamine release induced by lead was increased when calcium was removed from the superfusing buffer. Lead-induced release was unaffected in the presence of putative calcium, sodium, and/or potassium channel blockers (nickel, tetrodotoxin, tetraethylammonium, respectively). Depolarization-evoked dopamine release, produced by a 1-sec exposure to 61 mM potassium, was diminished at calcium concentrations below 0.254 mM. The onset of depolarization-evoked release was essentially immediate following exposure of the synaptosomes to high potassium. The combination of lead (3 or 10 microM) with high potassium reduced the magnitude of depolarization-evoked dopamine release. This depression of depolarization-evoked release by lead was greater in the presence of 0.25 mM than 2.54 mM calcium in the superfusing buffer. These findings demonstrate multiple actions of lead on synaptosomal dopamine release. Lead can induce dopamine release by yet unidentified neuronal mechanisms independent of external calcium. Lead can also reduce depolarization-evoked dopamine release by apparent competition with calcium influx at the neuronal membrane calcium channel.     

Phospholipid-induced activation of tyrosine hydroxylase from rat brain striatal synaptosomes.

Lysolecithin and phosphatidylserine stimulate rat striatal tyrosine hydroxylase, partially purified from the crude synaptosomal fraction. The stimulatory effect is associated with a 3- to 4-fold decrease in K_m for 6-methyl-tetrahydropterin or tetrahydrobiopterin without an alteration in the K_m for l-tyrosine or in the V_max. In addition, the K_i for dopamine inhibition is increased approximately 3-fold. Centrifugation of the enzyme on linear sucrose gradients gave a sedimentation coefficient (S_{20, w}) of 8.6, either in the absence or presence of lysolecithin, indicating that no significant changes in the molecular weight are caused by the phospholipid. The enzyme obtained after high speed centrifugation of whole striatal tissue was activated by a combination of lysolecithin plus a cyclic AMP-ATP mixture to a greater extent than that obtained by either activating condition alone. The data presented suggest a potential regulatory function of phospholipids in the control of striatal neurotransmitter synthesis.     

Noradrenergic modulation of 4-aminopyridine-induced acetylcholine release from rat cerebral cortex

The noradrenergic influence on cortical acetylcholine (ACh) release was investigated by the cortical cup technique in urethane anaestetized rats treated with 4-aminopyridine (4-AP). The following results were obtained: 1) The increase in ACh release induced by 4-AP (3 mg/kg i.p.) was strongly potentiated by pretreatment with -methyl-p-tyrosine (alpha-MPT) which inhibits catecholamine biosynthesis or by N-(2-chloroethyl)-N-ethyl-bromobenzylamine (DSP4) bringing about a selective degeneration of noradrenergic fibres. Neither pretreatment enhanced the spontaneous ACh output. 2) Pretreatment with p-chlorophenylalanine (PCPA), an inhibitor of serotonin synthesis, did not modify 4-AP effect on ACh output. 3) The alpha blockers, yohimbine (1 mg/kg i.p.) and prazosin (4 mg/kg i.p.), did not enhance the 4-AP effect on ACh release but only delayed its onset. 4) Yohimbine (7 mg/kg i.p.) completely reversed 4-AP effect on ACh release which was significantly decreased. It is concluded therefore that pretreatments with alpha-MPT and DSP4 remove an inhibitory noradrenergic control on cortical ACh release. On the other hand, the alpha blockers might interfere with the ionic mechanisms underlaying the 4-AP effect thus, masking the removal of the noradrenergic control, due to an alpha blockade.     

Dopamine D(1) receptor-mediated control of striatal acetylcholine release by endogenous dopamine

The role of dopamine D(1) and D(2) receptors in the control of acetylcholine release in the dorsal striatum by endogenous dopamine was investigated by monitoring with microdialysis the effect of the separate or combined administration of the dopamine D(1) receptor antagonist, SCH 39166 ?(-)-trans-6,7,7a,8,9, 13b-exahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]-nap hto-[2, 1b]-azepine hydrochloride? (50 microg/kg subcutaneous (s.c.)), of the dopamine D(2)/D(3) receptor agonist, quinpirole (trans-(-)-4aR, 4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo-(3,4-g)-quinoline hydrochloride) (5 and 10 microg/kg s.c.), and of the D(3) receptor selective agonist, PD 128,907 [S(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin -9-ol hydrochloride] (50 microg/kg s.c.), on in vivo dopamine and acetylcholine release. Microdialysis was performed with a Ringer containing low concentrations (0.01 microM) of the acetylcholinesterase inhibitor, neostigmine. Quinpirole (10 microg/kg s.c.) decreased striatal dopamine and acetylcholine release. Administration of PD 128,907 (50 microg/kg) decreased dopamine but failed to affect acetylcholine release. SCH 39166 (50 microg/kg s.c.) stimulated dopamine release and reduced acetylcholine release. Pretreatment with quinpirole reduced (5 microg/kg s.c.) or completely prevented (10 microg/kg s.c.) the stimulation of dopamine release elicited by SCH 39166 (50 microg/kg s.c.); on the other hand, pretreatment with quinpirole (5 and 10 microg/kg) potentiated the reduction of striatal acetylcholine release induced by SCH 39166 (50 microg/kg s.c.). Similarly, pretreatment with PD 128,907 (50 microg/kg) which prevented the increase of dopamine release induced by SCH 39166 (50 microg/kg), potentiated the reduction of striatal acetylcholine transmission elicited by SCH 39166. Thus, pretreatment with low doses of quinpirole or PD 128,907 influences in opposite manner the effect of SCH 39166 on striatal dopamine and acetylcholine release, counteracting the increase of dopamine release and potentiating the decrease in acetylcholine release. These results provide further evidence for the existence of a tonic stimulatory input of endogenous dopamine on striatal acetylcholine transmission mediated by dopamine D(1) receptors.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Adenosine receptor-mediated control of in vitro release of pain-related neuropeptides from the rat spinal cord

Although it is well established that adenosine exerts antinociceptive effects at the spinal level in various species including human, the mechanisms responsible for such effects are still a matter of debate. We presently investigated whether adenosine-induced antinociception might possibly be related to an inhibitory influence of this neuromodulator on the spinal release of neuropeptides implicated in the transfer and/or control of nociceptive signals. For this purpose, the K(+)-evoked overflow of substance P-, calcitonin gene-related peptide (CGRP)- and cholecystokinin-like materials was measured from slices of the dorsal half of the rat lumbar enlargement superfused with an artificial cerebrospinal fluid supplemented with increasing concentrations of various adenosine receptor ligands. The data showed that stimulation of adenosine A(1) and (possibly) A(3) receptors, but not A(2A) receptors, exerted an inhibitory influence on the spinal release of CGRP-like material. In contrast, none of the adenosine A(1), A(2A) and A(3) receptor agonists tested within relevant ranges of concentrations significantly affected the release of substance P- and cholecystokinin-like materials. These results support the idea that adenosine-induced antinociception at the spinal level might possibly be caused, at least partly, by the stimulation of inhibitory adenosine A(1) receptors located presynaptically on primary afferent fibres containing CGRP but not substance P.     

Evoked release of endogenous amino acids from rat striatal slices and its modulation

The release of endogenous gamma-aminobutyric acid (GABA), glutamate and aspartate stimulated by high K+ was studied by superfusing rat striatal slices. Stimulation with 5 min of 30 mM K+ was applied twice (S1 and S2) at a 20 min interval. The maximum release of GABA following stimulation was 40 (at S1) and 26 (at S2) times greater than the basal release. S1 and S2 each produced a maximum release of almost same magnitude for both glutamate and aspartate (about 2.5 times basal release). The removal of Ca2+ from the perfusion medium reduced the maximum release of these amino acids by more than 80% without affecting basal release significantly. Striatal slices were next stimulated in the same way after the addition of apomorphine or haloperidol to the perfusion medium. Apomorphine, 10 or 100 microM, reduced the K+-evoked release of GABA (by 24% at S1 and 35% at S2 with 10 microM; by 37% at S1 and 47% at S2 with 100 microM) but failed to affect the simultaneous release of glutamate and aspartate. Haloperidol (1 microM) reduced the S1-induced release of GABA but had no significant effect on the subsequent S2-induced release. The evoked release of aspartate or glutamate did not respond significantly to haloperidol, but there was a tendency to a decrease in aspartate release similar to the decrease seen for GABA release, particularly with S1. Based on these results, we discuss the property of GABA, aspartate and glutamate as neurotransmitters and the possible dopaminergic regulation of the release of these amino acids.