Pathways of dopamine metabolism in the rabbit caudate nucleus in vitro

Summary Slices of rabbit caudate nucleus were preincubated with ³H-dopamine and then superfused. ³H-dopamine and its metabolites were separated by column chromatography.The basal outflow of tritium consisted of 68% DOPAC, 21% OMDA metabolites (most of which was HVA), 6% dopamine and 5% MTA. Except for an increase in ³H-dopamine, the basal outflow was little changed by nomifensine or cocaine. Amezinium reduced the outflow of ³H-DOPAC and ³H-OMDA metabolites and increased that of ³H-dopamine and ³H-MTA; its effect was antagonized by nomifensine. Haloperidol 10^–7 M selectively enhanced the outflow of ³H-DOPAC and ³H-OMDA metabolites. At haloperidol 10^–5M, a large increase in these two fractions was accompanied by a small increase in ³H-dopamine and ³H-MTA. Nomifensine diminished only slightly the outflow of ³H-DOPAC caused by haloperidol.The overflow of tritium elicited by electrical stimulation at 3 Hz consisted of 74% DOPAC, 15% OMDA metabolites, 10% dopamine and 0.4% MTA. Lowering the frequency reduced the overflow of total tritium and caused a decrease in the percentage of ³H-DOPAC and an increase in the percentage of ³H-dopamine. Nomifensine or cocaine greatly diminished the evoked overflow of ³H-DOPAC and ³H-OMDA compounds and increased the evoked overflow of ³H-dopamine and ³H-MTA. The effects of apomorphine and bromocriptine were similar to those caused by decreasing the stimulation frequency.The major pathways of the metabolism of previously taken up dopamine, under the conditions of our experiments, are as follows. When the neurones are at rest, dopamine metabolism is initiated by leakage of the amine into the axoplasm, where it is degraded through the aldehyde to DOPAC. Part of the DOPAC is methylated extraneuronally to HVA. Traces of MTA are formed by extraneuronal methylation of dopamine. When action potentials arrive, dopamine metabolism is initiated by exocytosis. The bulk of the extracellular dopamine is taken up back into the neurones. When the stimulation frequency is 3 Hz, most of the axoplasmic dopamine is subsequently transformed to DOPAC; little seems to be re-stored. HVA and MTA are generated essentially as during neuronal rest. When dopamine release is low (stimulation at low frequency; addition of apomorphine or bromocriptine), a larger portion seems to be re-stored, thus leading to a decrease in the percentage of DOPAC. Haloperidol, apart from its receptor blocking properties, acts on dopaminergic axons in a manner akin to the effect of reserpine.Abbreviations and Terminology AD Aldehyde dehydrogenase (E.C.1.2.1.3.) - COMT catechol-O-methyl transferase (E.C 2.1.1.6.) - DOPAC 3,4-dihydroxyphenylacetic acid - DOPEG 3,4-dihydroxyphenylglycol - DOPET 3,4-dihydroxyphenylethanol - HVA 3-methoxy-4-hydroxyphenylacetic acid - MAO monoamine oxidase (E.C. 1.4.3.4.) - MOPET 3-methoxy-4-hydroxyphenylethanol - MTA 3-methoxytyramine - OMDA metabolites O-methylated and deaminated metabolites. Intraneuronal refers to location in dopaminergic terminal axons, extraneuronal to location in other cells, neuronal as well as nonneuronal     

Cardiac electrophysiology of four neuroleptics: Melperone,haloperidol, thioridazine and chlorpromazine

Summary Effects of dopamine receptor agonists and antagonists on the release of dopamine were studied in the caudate nucleus of the rabbit. The nucleus contained 6.7 g/g of dopamine, but negligible levels of noradrenaline and dopamine--hydroxylase. No formation of ³H-noradrenaline was detected in caudate slices preincubated with ³H-dopamine, and more than 95% of the tritium content of the tissue consisted of ³H-dopamine.When caudate slices were preincubated with ³H-dopamine and then superfused with amine-free medium, there was a basal outflow of tritium that was not or only slightly changed by tetrodotoxin (10^–7 and 10^–6 M), apomorphine (up to 10^–5 M), bromocriptine (up to 10^–6 M), chlorpromazine (up to 10^–6 M), haloperidol (up to 10^–7 M), or omission of calcium. Electrical stimulation (3 Hz, 24 mA, 2 ms pulse duration, 2-min periods) greatly increased the outflow of tritium. The stimulation-evoked overflow was abolished by tetrodotoxin (10^–7 and 10^–6 M) and in calcium-free medium. Apomorphine (10^–8–10^–5 M) and bromocriptine (10^–8–10^–6 M) reduced, whereas chlorpromazine (10^–7 and 10^–6 M) and haloperidol (10^–8 and 10^–7 M) enhanced the evoked overflow. The inhibitory effect of apomorphine and bromocriptine was antagonized by chlorpromazine and haloperidol, but not by phentolamine.Silicone tubings that had been in contact with ³H-haloperidol retained tritiated material that was slowly eluted during perfusion with water or physiological salt solution. The material was identified as ³H-haloperidol. When silicone tubings pretreated with unlabelled haloperidol were used in subsequent dopamine release experiments, the inhibitory effect of apomorphine was not reproduced.It is concluded that, in the caudate nucleus of the rabbit, apomorphine and bromocriptine depress, whereas chlorpromazine and haloperidol facilitate action potential-evoked release of dopamine. The effects are mediated by specific receptors which may be located on the dopaminergic nerve terminals. The receptors appear to be normally activated by released dopamine itself, which thus inhibits its own further release. Part of the discrepancies in the literature concerning dopaminergic modulation of dopamine release may be due to retention of neuroleptic drugs in superfusion assemblies, followed by slow elution and interference with subsequent experiments.     

Interneurones are probably not involved in the presynaptic dopaminergic control of dopamine release in rabbit caudate nucleus

Summary Slices of rabbit caudate nucleus were preincubated with ³H-dopamine and then superfused. The influence of apomorphine and haloperidol on the overflow of tritium evoked by 20 mmol/l potassium was investigated in the presence and in the absence of tetrodotoxin. The potassium-evoked overflow was largely calcium-dependent and consisted mainly of ³H-dopamine. The dopamine receptor agonist apomorphine 0.01–1.0 mol/l reduced, whereas the antagonist haloperidol 0.1 mol/l enhanced the potassium-evoked overflow of tritium. The effects of apomorphine and haloperidol were as pronounced in the presence as in the absence of tetrodotoxin 0.3 mol/l. It is concluded that the presynaptic dopaminergic modulation of dopamine release is not mediated by a tetrodotoxin-sensitive interneuronal pathway.     

Transmitter release patterns of noradrenergic,dopaminergic and cholinergic axons in rabbit brain slices during short pulse trains,and the operation of presynaptic autoreceptors

Summary Slices of rabbit brain were field-stimulated either by single electrical pulses or by trains of 4 or 8 pulses at 1 or 100 Hz in order to study transmitter release patterns and the autoinhibition of transmitter release. The slices were preincubated with ³H-noradrenaline (cortex), ³H-dopamine (caudate nucleus) or ³H-choline (caudate nucleus).Slices preincubated with ³ H-noradrenaline were superfused with medium containing desipramine 1 gmol/l. The overflow of tritium elicited by single pulses amounted to 0 .19% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was similar, whereas that elicited by 4 pulses/100 Hz was 5.1-fold higher. Yohimbine 101000 nmol/l increased up to 2.5-fold the overflow evoked by 4 pulses/1 Hz but did not change the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-dopamine were superfused with medium containing nomifensine 1 mol/l. The overflow of tritium elicited by single pulses was 0.39% of the tritium content of the tissue. The overflow elicited by 4 pulses/1 Hz was 1.3-fold and the overflow elicited by 4 pulses/100 Hz 1.4-fold higher. Domperidone 1–100 nmol/l and sulpiride 10–1000 nmol/1 increased up to 2.4-fold the overflow evoked by 4 pulses/ 1 Hz but increased only slightly the overflow evoked by single pulses or 4 pulses/100 Hz. - Slices preincubated with ³ H-choline were superfused either with physostigmine-free medium or with medium containing physostigmine 1 mol/l. In physostigmine-free medium, atropine did not increase the evoked overflow of tritium at any stimulation condition. In physostigmine-containing medium, the overflow elicited by single pulses was 0.18% of the tritium content of the tissue. The overflow elicited by 8 pulses/1 Hz was 2.0-fold and the overflow elicited by 8 pulses/100 Hz 2.2-fold higher. Atropine 2–200 nmol/1 increased up to 2.4-fold the overflow evoked by 8 pulses/1 Hz but increased only slightly the overflow evoked bysingle pulses or 8 pulses/100 Hz. In physostigmine-free medium, sulpiride 10–1000 nmol/1 did not change the single-pulse-evoked overflow of tritium in the absence but increased it in the presence of nomifensine 1 mol/l.Single pulses elicit a large release of ³H-noradrenaline, ³H-dopamine and ³H-acetylcholine under the conditions of these experiments. Release elicited by single pulses is not subject to autoinhibition except for a small inhibition by spontaneously released transmitter in the case of dopaminergic and cholinergic axons. When 3 or 7 further pulses follow the first one at intervals of 1 s, they elicit much smaller release. At least a great part of the fall is due to autoreceptor mediated inhibition (for 3H-acetylcholine release in the presence of physostigmine only). When 3 or 7 further pulses follow at intervals of 10 ms, they elicit release that is either similar to that evoked by the first pulse (³H-noradrenaline) or much smaller (³H-dopamine, ³H-acetylcholine). However, the fall is not due to stimulation-dependent, auto-receptor-mediated inhibition; autoinhibition does not develop in these short high-frequency trains. Overall, the results are in accord with the autoreceptor theory. They demonstrate the role of autoinhibition in determining the transmitter release patterns of central noradrenergic, dopaminergic and cholinergic neurones. Send offprint requests to N. Limberger at the above address     

Influence of fentanyl,levorphanol and pethidine on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of fentanyl, levorphanol and pethidine on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by low, and was accelerated by high concentrations of the drugs. The overflow of tritium evoked by electrical stimulation at 3 Hz was diminished by 10^–8–10^–7 M fentanyl and by 10^–7–10^–6 M levorphanol, but was augmented by 10^–5 M levorphanol. Naloxone prevented the inhibitory effect of fentanyl and levorphanol. In contrast to fentanyl and levorphanol, pethidine did not decrease, but at concentrations of 10^–6–10^–5 M greatly increased the stimulation-induced overflow of tritium. However, the increase was abolished, and the stimulation-evoked overflow slightly reduced, after the re-uptake of noradrenaline had been blocked by cocaine. It is concluded that fentanyl, levorphanol and pethidine share with morphine the ability to inhibit the release of transmitter from cerebrocortical noradrenaline neurones evoked by nerve impulses.     

Influence of morphine and naloxone on the release of noradrenaline from rat brain cortex slices

Summary In slices of rat brain cortex preincubated with (–)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow of tritium was not changed by 10^–7–10^–5 M morphine and by 10^–6–10^–4 M naloxone, but was accelerated by 10^–4 M morphine. Electrical field stimulation augmented tritium outflow. The overflow evoked per ppulse decreased as the frequency of stimulation was increased from 0.3 to 3 Hz, but remained approximately constant when it was further increased to 10 Hz. At frequencies of 0.3, 1, and 3 Hz, but not at 10 Hz, morphine in concentrations of 10^–7–10^–5 M depressed the stimulation-induced overflow of tritium. 10^–4 M morphine did not influence the overflow induced by stimulation at 0.3 and 1 Hz and increased that evoked by stimulation at 10 Hz. Naloxone (10^–6–10^–4 M) did not change the response to stimulation. In the presence of 10^–4 M naloxone, 10^–6 M morphine did not diminish, and 10^–5 M morphine even enhanced the stimulation-induced overflow of tritium. The inhibitory effect of 10^–6 M morphine was not reduced, after tyrosine hydroxylase had been blocked by -methyltyrosine-methylester. It is concluded that morphine through an action on specific opiate receptors inhibits the release of transmitter from cerebrocortical noradrenergic neurones evoked by nerve impulses. By an action unrelated to opiate receptors, morphine at high concentrations increases the stimulation-induced overflow of noradrenaline, presumably by inhibiting its re-uptake into nerve endings.     

Acetylcholine release in rat nucleus accumbens is regulated through dopamine D2-receptors

Summary Experiments in slices of rat nucleus accumbens were carried out in order to investigate whether the release of acetylcholine in this tissue is modulated through dopamine receptors. The slices were preincubated with ³H-choline and then superfused and stimulated electrically twice for 2 min each at a frequency of 3 Hz.The electrically evoked overflow of tritium averaged 2.9–3.9% of the tritium content of the tissue in the various groups. The D₂-selective agonist quinpirole (0.01–1 mol/l) reduced the evoked overflow of tritium by maximally 56%, an effect antagonized by the D₂-selective antagonist (–)-sulpiride (1 mol/l). The D₁-selective agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF 38393) caused a slight decrease only at the high concentration of 10 mol/l. (–)-Sulpiride (0.1–10 mol/l) moderately increased the evoked overflow of tritium when given alone. The dopamine uptake inhibitor nomifensine (10 mol/l) caused a decrease, and in its presence the increase produced by (–)-sulpiride became much more marked, amounting to maximally 149%. (+)-Sulpiride (0.1–1 mol/l) failed to change the evoked overflow of tritium in the presence of nomifensine. The dopamine-releasing agent (±)-amphetamine (1 mol/l) also reduced the evoked overflow, an effect abolished by (–)-sulpiride. Finally, bretylium (1 mmol/l), which blocks the release of dopamine, increased the evoked overflow. (–)-Sulpiride (1 mol/l) lost its facilitatory effect in slices treated with bretylium.We conclude that the release of acetylcholine in rat nucleus accumbens, like its release in the nucleus caudatusputamen, is modulated through dopamine D₂-receptors. The receptors are activated by endogenous dopamine under the conditions of these experiments. Send offprint requests to K. Starke at the above address     

Effects of verapamil,diltiazem and ryosidine on the release of dopamine and acetylcholine in rabbit caudate nucleus slices

Summary Slices of the rabbit caudate nucleus were preincubated with ³H-dopamine or ³H-choline and then superfused with label-free medium. Release of ³H-dopamine and ³H-acetylcholine was elicited by either electrical stimulation at 8 (in one series 2) Hz, or an increase in the K⁺ concentration by 50 mmol/l, or addition of L-glutamate 1 mmol/l. Verapamil 1 mol/l, diltiazem 1 and 10 mol/l, and ryosidine 1 mol/l failed to the reduce the electrically-, K⁺- and glutamate-evoked overflow of tritium. Verapamil 1 mol/l and diltiazem 10 mol/l also failed to reduce the electricallyevoked overflow (2 Hz) when dopamine receptors, neuronal dopamine uptake, and neuronal choline uptake were blocked by domperidone, nomifensine and hemicholinium, respectively. Inhibition of the evoked overflow of tritium was only obtained when concentrations were increased to verapamil 10 mol/l, diltiazem 100 mol/l and ryosidine 10 mol/l. The inhibition was generally small. It was more evident for slices preincubated with ³H-choline than for those preincubated with ³H-dopamine, because in the latter verapamil, diltiazem and (much less) ryosidine accelerated the basal efflux of tritium. The inhibition of the K⁺-evoked overflow of tritium was probably due to blockade of Ca²⁺ channels because this overflow was Ca²⁺-dependent but tetrodotoxin-resistant. In contrast, the inhibition of the electrically- and glutamateevoked overflow possibly involved blockade of Na⁺ channels as well. The results indicate that three calcium antagonists from different chemical classes are very weak inhibitors of Ca²⁺ entry into, and hence transmitter release from, the terminal axons of central dopaminergic and cholinergic neurones. The function of the high affinity calcium antagonist binding sites that have been identified in brain remains unknown.     

Cannabinoid CB1 receptor-mediated inhibition of NMDA- and kainate-stimulated noradrenaline and dopamine release in the brain

Guinea-pig hippocampal slices preincubated with [³H]noradrenaline were superfused with medium containing desipramine and rauwolscine and rat striatal slices preincubated with [³H]dopamine were superfused with medium containing nomifensine; the effect of cannabinoid receptor ligands on tritium overflow stimulated by NMDA or kainate was examined. Furthermore, the affinity of the drugs for cannabinoid CB₁ receptors was determined in rat brain cortex membranes using [³H]SR 141716. In guinea-pig hippocampal slices preincubated with [³H]noradrenaline, tritium overflow stimulated by NMDA 100 μM and 1000 μM and by kainate 1000 μM was inhibited by the cannabinoid receptor agonists CP-55,940 and/or WIN 55,212-2. The CB₁ receptor antagonist SR 141716 increased the NMDA (1000 μM)-stimulated tritium overflow but did not affect tritium overflow stimulated by NMDA 100 μM or kainate 1000 μM. The inhibitory effect of WIN 55,212-2 on the NMDA (100 μM)- and kainate (1000 μM)-evoked tritium overflow was antagonized by SR 141716. In rat striatal slices preincubated with [³H]dopamine, WIN 55,212-2 inhibited the NMDA (1000 μM)-stimulated tritium overflow. SR 141716, which, by itself, did not affect tritium overflow, counteracted the inhibitory effect of WIN 55,212-2. [ ³ H]SR 141716 binding to rat cortical membranes was inhibited by SR 141716, CP-55,940 and WIN 55,212-2 (pK _i 8.53, 7.34 and 5.93, respectively) but not affected by desipramine, rauwolscine and nomifensine (pK _i < 5). In conclusion, activation of CB₁ receptors inhibits the NMDA- and kainate-stimulated noradrenaline release in guinea-pig hippocampus and the NMDA-stimulated dopamine release in rat striatum. The explanation for the facilitatory effect of SR 141716 might be that it acts as an inverse agonist at CB₁ receptors or that these receptors are activated by endogenous cannabinoids. Received: 25 February 1999 / Accepted: 12 April 1999     

Influence of morphine and naloxone on the release of noradrenaline from rat cerebellar cortex slices

Summary In slices of rat cerebellar cortex preincubated with (-)-³H-noradrenaline, the influence of morphine and naloxone on the efflux of tritium was investigated. The spontaneous outflow was not changed by 10^–5 M of either morphine or naloxone. On the other hand, morphine caused a concentration-dependent decrease of the overflow, of tritium evoked by electrical field stimulation. Naloxone did not change the stimulation-induced overflow, but prevented its inhibition by morphine. It is concluded that morphine, through an action on opiate receptors located on cerebellar noradrenergic neurones, inhibits the secretion of the transmitter in response to nerve impulses.     

Phencyclidine and ketamine: Comparison with the effect of cocaine on the noradrenergic neurones of the rat brain cortex

Summary In slices of the rat occipital cortex, the influence of phencyclidine and ketamine on the accumulation of ³H-noradrenaline and the subsequent outflow of tritium was investigated, and was compared with the effect of cocaine.-All three drugs inhibited the accumulation of tritium during incubation of the slices with ³H-noradrenaline. Phencyclidine was slightly, whereas ketamine was much less effective than cocaine.-All three drugs accelerated the spontaneous outflow of tritium from slices preincubated with ³H-noradrenaline. The acceleration caused by low concentrations probably reflects an inhibition of the re-uptake of spontaneously released ³H-noradrenaline; in addition, high concentrations (10^–4M phencyclidine, 3×10^–4–10^–3M cocaine and 10^–3–3×10^–3M ketamine) appear to release tritiated compounds from the neurones. The distance between uptakeinhibiting and releasing concentrations was much greater for cocaine than for phencyclidine and ketamine.-All three drugs enhanced the overflow of tritium evoked by electrical field stimulation. The increase probably reflects an inhibition of the re-uptake of released ³H-noradrenaline; in addition, phencyclidine appears to enhance the release of noradrenaline per pulse.-The actions of phencyclidine and ketamine on central noradrenergic neurones may contribute to the characteristic psychotropic side-effects of these general anaesthetics.     

Presynaptic serotonin receptors regulate the release of 3H-serotonin in hypothalamic slices of the rabbit

Summary Hypothalamic slices of the rabbit brain were incubated with 10^–7 M of ³H-serotonin (³H-5HT). After the incubation and an initial washout period, a nearly constant basal efflux of tritium was detected. This basal efflux was not significantly altered by Ca²⁺-free solution or by the 5HT-antagonist metitepin (10^–5 M), but was augmented by chlorimipramine (10^–5 M) and by unlabelled 5HT (10^–6 M); the acceleration caused by unlabelled 5HT was absent in presence of chlorimipramine (10^–5 M). Both electrical stimulation (4 Hz, 50 mA, 2 min) and high K⁺ (50 mM) induced an overflow of ³H. This overflow was nearly abolished in Ca²⁺-free solution. In presence of chlorimipramine (10^–5 M) both the tritium overflow evoked by electrical stimulation and that evoked by high K⁺ were augmented by metitepin (10^–5 M) and decreased in a concentration dependent manner by unlabelled serotonin (10^–8–10^–6 M); the latter effect was antagonized by metitepin (10^–6 M and 10^–5 M). These experiments suggest that in rabbit hypothalamic slices, the release of ³H-5HT is controlled by a negative feedback mechanism acting via presynaptic serotonin receptors.     

Histamine H3 receptor-mediated inhibition of noradrenaline release in pig retina discs

Summary Discs of pig retina were preincubated with ³H-noradrenaline, ³H-dopamine or ³H-serotonin and then superfused. Electrical field stimulation increased the outflow of tritium from discs preincubated with ³H-noradrenaline or ³H-dopamine, but no from discs preincubated with ³H-serotonin. The tritium content at the end of superfusion was similar in discs preincubated with ³H-noradrenaline or ³H-dopamine but about tenfold lower in discs preincubated with ³H-serotonin. The tritium content in discs preincubated with ³H-noradrenaline was markedly reduced when desipramine was present during preincubation but was not affected by selective inhibitors of dopamine and serotonin uptake. The tritium content in discs preincubated with 3Hdopamine and ³H-serotonin, in contrast, was reduced or tended to be reduced by a selective dopamine and serotonin uptake inhibitor, respectively.The electrically evoked overflow of tritium from discs preincubated with ³H-noradrenaline was abolished by tetrodotoxin or omission of Ca²⁺. In discs superfused with desipramine, the electrically evoked overflow was enhanced by phentolamine but not affected by histamine. When both desipramine and phentolamine were present in the superfusion medium, histamine inhibited the evoked overflow (pIC₁₅ 6.85). This effect was mimicked by the histamine H₃ receptor agonist R-(–)--methylhistamine as well as by its S-(+)-enantiomer (pIC₁₅ 7.85 and 5.30, respectively) but not by the H₁ receptor agonist 2-(2-thiazolyl)ethylamine and the H₂ receptor agonist dimaprit (each 10 mol/l). The inhibitory effect of histamine was abolished by the H₃ receptor antagonist thioperamide 0.32 mol/l and attenuated by impromidine 3.2 mol/l but not affected by the H₁ receptor antagonist dimetindene 3.2 mol/l and the H₂ receptor antagonist ranitidine 10 mol/l.The results suggest that, in the pig retina, noradrenaline is taken up into, and released from, noradrenergic neurones (most likely vascular postganglionic sympathetic nerve fibres, less probably tissue-specific noradrenergic neurones of the retina) and that noradrenaline release is subject to modulation via H₃ receptors and probably also a-adrenoceptors.Send offprint requests to E. Schlicker at the above address     

Phenoxybenzamine blocks dopamine autoreceptors irreversibly: Implications for multiple dopamine receptor hypotheses

Phenoxybenzamine in μM concentrations increased the electrically evoked overflow of recently taken up [³H]dopamine from superfused slices of cat caudate nucleus, an effect which is also observed for dopamine receptor antagonists. The magnitude of the increase in electrically evoked [³H]dopamine release caused by 1 μM phenoxybenzamine was equal to that elicited by maximally effective concentrations of the specific dopamine receptor antagonist, S-sulpiride. Phenoxybenzamine (1 μM) completely antagonized the inhibition of [³H]dopamine release caused by the dopamine receptor agonist pergolide (10 nM). The α-adrenoceptor antagonist phentolamine (1 μM) had no effect on the electrically evoked overflow of [³H]dopamine, ruling out the possibility that the effect of phenoxybenzamine could be attributed to α-adrenoceptor blockade. A 20 min exposure to 1 μM phenoxybenzamine increased the electrically evoked [³H]dopamine overflow even after the tissue had been washed for two and a half hours. When the caudate slices were exposed for 30 min to the reversible dopamine receptor antagonist S-sulpiride (1 μM) and washed for two and a half hours, no similar increase in stimulation-evoked [³H]dopamine overflow was observed. When sulpiride (1 μM) was present during the exposure to phenoxybenzamine (1 μM), no increase in electrically evoked [³H]dopamine overflow was observed after the washout period. Thus phenoxybenzamine at 1 μM appears to block irreversibly the dopamine autoreceptor in the caudate nucleus. Phenoxybenzamine has been previously reported to block irreversibly dopamine-stimulated adenylate cyclase (D₁) and neuroleptic receptor binding (D₂). The present demonstration that phenoxybenzamine also blocks the dopamine autoreceptor irreversibly thus supports the view that all currently well-established dopamine receptors are sensitive to phenoxybenzamine.     

Alpha-receptor-mediated modulation of transmitter release from central noradrenergic neurones

Summary Slices of rat cerebral cortex were preincubated with 10^–7 M (-)-³H-noradrenaline, and the outflow of tritium was determined. Oxymetazoline, phentolamine and cocaine did not change the spontaneous efflux. The overflow of tritium evoked by electrical field stimulation was decreased by oxymetazoline, and enhanced by phentolamine, phenoxybenzamine, and cocaine. Oxymetazoline did not counteract the increase of the stimulation-induced overflow caused by cocaine, but strongly antagonized the increase caused by phentolamine and phenoxybenzamine. When the stimulation-induced overflow was large under control conditions (high frequency of stimulation, addition of cocaine), the inhibitory effect of oxymetazoline was diminished. The results indicate that an -receptor-mediated feed-back control of noradrenaline release, previously demonstrated in postganglionic sympathetic nerves, also operates in central noradrenergic neurones.     

GABA-mediated potentiation of amine release from nigrostriatal dopamine neurones in vitro

The uptake and release of ³H-dopamine was studied in slices of corpus striatum and substantia nigra in the presence of nialamide. High potassium triggered the outflow of tritium in both brain structures and this release was potentiated by GABA in a dose related fashion, whereas the spontaneous overflow of radioactivity was unchanged. This action of GABA was mimicked by the GABA- antagonist aminooxyyanetic acid ethanolamine-O-sulphate, but not by the GABA analogues muscimol, 3-aminopropanesulphonic acid, γ-hydroxybutyrate or β-(p-chlorophenyl)-GABA. The responses to GABA was not blocked by picrotoxin, which itself facilitated the evoked release of ³H-dopamine, nor by bicuculline or the omission of calcium ions from the bathing medium. GABA facilitation of K⁺-evoked ³H-dopamine release was increased significantly on reducing tissue thickness and following prolonged incubation with GABA. GABA also potentiated the depolarization induced outflow of ³H-noradrenaline, ³H-5-hydroxytryptamine and ³H-histamine without affecting their initial accumulation. Veratridine, amphetamine and cold dopamine also raised the output of ³H-dopamine, but none of these releases was altered by GABA. The uptake of ³H-dopamine, but not that of ¹⁴C-GABA, was considerably attenuated in 6-hydroxydopamine lesioned corpora striata. The possible mechanism(s) of this stimulatory action of GABA is discussed.     

Cannabinoid receptor-mediated inhibition of dopamine release in the retina

The possible occurrence of cannabinoid (CB) receptors was studied on superfused guinea-pig retinal discs preincubated with [3H]dopamine or [³H]noradrenaline. Tritium overflow was evoked either electrically (3 Hz) or by re-introduction of Ca²⁺, 1.3 mM after superfusion with Ca²⁺-free medium containing K⁺ 30 rnM. The accumulation of [³H]dopamine ([³H]DA) and [³H]noradrenaline ([³H]NA) was inhibited by the selective inhibitor of the neuronal dopamine transporter GBR-12909 (pIC_50% 7.29 and 7.41, respectively) but not by the selective inhibitor of the neuronal noradrenaline transporter desipramine (1 M). The electrically or Ca²⁺-evoked tritium overflow in retinal discs preincubated with [³H]DA or [³H]NA was reduced by the CB receptor agonists CP-55,940 and WIN 55,212-2 (pIC_50% in discs preincubated with [³H]NA, electrical stimulation: 7.03 and 6.70, respectively) but not affected by the inactive S(–)enantiomer of the latter, WIN 55,212-3 (up to 10 M). The concentration-response curve of WIN 55,212-2 was shifted to the right by the CB₁ receptor antagonist SR 141716 (apparent pA₂: 8.29) which, by itself, increased the evoked overflow. The facilitatory effect of SR 141716 was not affected by GBR-12909 and the dopamine receptor antagonist haloperidol. In conclusion, the dopaminergic neurones of the guinea-pig retina can be labelled by both [³H]DA and [³H]NA. Transmitter release from the dopaminergic neurones is inhibited by activation of cannabinoid receptors of the CB₁ type, which appear to be tonically activated by an endogenous CB receptor ligand.     

Role of dopamine receptors in the modulation of acetylcholine release in the rabbit hippocampus

Summary Modulation of acetylcholine release was studied in slices of the rabbit hippocampus preincubated with ³H-choline and then continuously superfused with a medium containing 10 mol/l hemicholinium-3. Electrical field stimulation of the superfused slices elicited an increase in tritium outflow, which was tetrodotoxin-sensitive and largely calcium-dependent. Stimulus-evoked acetylcholine release in the rabbit hippocampal slices was modulated by presynaptic muscarinic autoreceptors, as has been shown previously for the rat hippocampus. Drugs with affinity for - and or -adrenoceptors did not affect the evoked overflow of tritium from rabbit hippocampal slices. In contrast, the dopamine receptor agonist apomorphine (0.1 or 1 mol/l) and exogenous dopamine (1 or 10 mol/l) significantly reduced the evoked outflow by about 10 or 20%, respectively. This effect was antagonized by haloperidol (0.01 mol/l) but not by phentolamine (1 mol/l). Attempts to enhance (using nomifensine 10 mol/l) or reduce (using haloperidol, up to 1 mol/l; or bretylium, 1 mmol/l for 5 min) endogenous dopaminergic transmission in the hippocampal slices did not affect stimulation evoked acetylcholine release. In conclusion, presynaptic dopamine receptors modulating acetylcholine release are present in the rabbit hippocampus, but they seem not to be of physiological significance.     

Uptake inhibition of biogenic amines by newer antidepressant drugs: Relevance to the dopamine hypothesis of depression

The dopamine theory of depression was studied by assessing the effect of antidepressant drugs on uptake of dopamine, noradrenaline, and serotonin in synaptosomes from rat brain. Five newer drugs—butriptyline, maprotiline, trimipramine, iprindole, and mianserine—exhibited rather potent inhibition of ³H-dopamine uptake in corpus striatum, as their IC₅₀ values, which were in the order of 10^-6–10^-5 M, were only about 50 times higher than for nomifensine (IC₅₀=10^-7 M). The five drugs were weak, compared to chlorimipramine, on ¹⁴C-serotonin uptake in the whole forebrain, as their IC₅₀ were about 10^-5 M. Butriptyline, trimipramine, and iprindole were very weak uptake inhibitors of ³H-noradrenaline in the occipital cortex. Their IC₅₀ values were about 10^-6 M, which is almost 1000 times higher than for desmethylimipramine. These results are discussed in relation to comprehensive recent literature as further indicating a link between dopamine and depression.     

Differential effects of the stereoisomers of 3PPP in dopaminergic and cholinergic neurotransmission in superfused slices of the corpus striatum

Summary The two enantiomers of 3PPP were tested on the spontaneous and electrically-evoked release of ³H-dopamine from slices of the rabbit caudate nucleus and of ³H-acetylcholine (³H-ACh) from slices of the rat caudate nucleus.In caudate slices labelled with ³H-dopamine, exposure to (+)3PPP (0.1–1 M) facilitated the spontaneous outflow of radioactivity with a concomitant inhibition of the electrically-evoked release of ³H-dopamine. In the presence of cocaine 10 M, exposure to (+)3PPP (1 M) inhibited the electrically evoked release of ³H-dopamine without modifying the spontaneous outflow of radioactivity. This inhibitory effect was not significantly antagonized by S-sulpiride 0.01 M.Exposure to (+)3PPP 1 M inhibited the electrically-evoked release of ³H-ACh, and this effect was not modified by pretreatment with reserpine alone, or in combination with -methyl-p-tyrosine (-MT).In contrast to the (+) enantiomer, exposure to (-)3PPP (0.1–1 M) facilitated the electrically-evoked release of ³H-dopamine without affecting the spontaneous outflow of radioactivity. (-)3PPP antagonized the inhibitory effect of apomorphine on the electrically-evoked release of ³H-dopamine.Exposure to (-)3PPP 1 M did not modify the spontaneous or the electrically-evoked release of ³H-ACh. Yet, this concentration of (-)3PPP antagonized significantly the inhibitory effect of 0.03 M apomorphine, 1 M d-amphetamine, and 1 M (+)3PPP on the electrically-evoked release of ³H-ACh (-)3PPP (0.1–1 M) was about 100 times less potent than S-sulpiride at antagonizing the inhibitory effect of apomorphine on the electrically-evoked release of ³H-ACh.It is concluded that under in vitro conditions at the level of the dopamine receptor modulating the release of ³H-ACh from the cholinergic interneuron in the striatum, (+)3PPP behaves as a dopamine receptor agonist while (-)3PPP possesses dopamine receptor-antagonist properties. At the level of the dopaminergic nerve terminal, (-)3PPP facilitates the release of ³H-dopamine probably through the blockade of the dopamine autoreceptors. The dopamine autoreceptor agonists properties of (+)3PPP are difficult to establish in our model because of the dopamine releasing action of this enantiomer.Some of the results described in this publication have been presented at the British Pharmacological Society Meeting (Arbilla and Langer 1983).