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Notes: Abstract: The release of γ-aminobutyric acid (GABA) was studied in slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused. Aminooxyacetic acid was present throughout. Both the tritium in the slices and that in the superfusate consisted practically entirely of [3H]GABA. Stimulation for 2 min by electrical field pulses of 3 ms width and 9 V/cm voltage drop (36 mA current strength) at 5 or 20 Hz elicited an overflow of [3H]GABA that amounted to 0.23 or 0.47% of the tritium content of the tissue, respectively, and was diminished by 85% in the presence of tetrodotoxin. At higher current strength, less of the stimulation-evoked overflow was tetrodotoxin-sensitive. cis-1,3-Aminocyclohexane carboxylic acid diminished the uptake of [3H]GABA into the tissue but did not change the percentage released by electrical stimulation. Ca2+ withdrawal greatly accelerated basal [3H]GABA efflux and almost abolished the response to stimulation. Nipecotic acid 10–1,000 μM enhanced both the basal and (up to eightfold) the stimulation-evoked overflow. The method described allows us to elicit electrically a quasiphysiological, i.e., Ca2+-dependent and tetrodotoxin-sensitive, neuronal release of [3H]GABA. Nipecotic acid diverts released [3H]GABA from reuptake to overflow.

Notes: Abstract: Various putative striatal transmitters and related compounds were studied for their effects on the release of γ-aminobutyric acid (GABA) from slices of the head of the rabbit caudate nucleus. The slices were preincubated with [3H]GABA and then superfused and stimulated electrically at 5 or 20 Hz. Aminooxyacetic acid was present throughout. The main changes observed were the following. The basal and, less consistently, the electrically evoked overflow of [3H]GABA were enhanced by 3,4-dihydroxyphenylethylamine (dopamine), an effect not blocked by cis-flupentixol or domperidone and not mimicked by apomorphine and D1-selective agonists. The electrically evoked overflow was diminished by 5-hydroxytryptamine (serotonin); the inhibition was prevented by methiothepin. The basal but not the electrically evoked overflow was enhanced by carbachol; acetylcholine and nicotine also accelerated the basal out-flow whereas oxotremorine caused no consistent change; the effects of carbachol and acetylcholine were blocked by hexamethonium but not by atropine or by tetrodotoxin. These findings indicate that the GABA neurons in the caudate nucleus may be stimulated by dopamine, although the receptor type involved remains unclear; inhibited by serotonin; and stimulated by acetylcholine acting via a nicotine receptor. However, all drug effects observed were relatively small. No evidence was obtained for autoreceptors, α2-adrenoceptors or receptors for opioids, adenosine or substance P at the GABA neurons.

Notes: Summary Segments of the rabbit ear artery were preincubated with (−)-3H-noradrenaline and then perfused/superfused and stimulated by transmural electrical pulses. The outflow of 3H-noradrenaline and total tritium was determined. In the first series of experiments, stimulation periods of approximately constant length (50 s) were used (cocaine 5 μM present). Thirteen pulses (0.25 Hz) elicited an overflow of 3H-noradrenaline of 0.024% of tissue tritium; 26 pulses (0.5 Hz) elicited an overflow of 0.059%, and 52 pulses (1 Hz) of 0.166%. Rauwolscine 1 μM did not change the overflow evoked by 13 pulses, increased that evoked by 26 pulses and increased most markedly that evoked by 52 pulses. Phentolamine 1 μM decreased the overflow at 13, did not change the overflow at 26, and increased the overflow at 52 pulses. Corynanthine 1 μM decreased the overflow at 13, and did not change the overflow at 26 and 52 pulses. The effect of tetraethylammonium (TEA) 100 μM was opposite to that of rauwolscine; it increased most markedly the overflow evoked by 13 pulses, increased less that evoked by 26 pulses, and least the overflow at 52 pulses. In the second series of experiments, the frequency of stimulation was kept constant (2 Hz). In the absence of cocaine, 10 pulses elicited an overflow of 3H-noradrenaline of 0.023% of tissue tritium; 20 pulses elicited an overflow of 0.043%, and 40 pulses of 0.089%. Phentolamine 1 μM did not change the overflow evoked by 10 pulses, increased that evoked by 20 pulses, and increased most markedly that evoked by 40 pulses. TEA 100 μM increased the evoked overflow at all pulse numbers. Similar results were obtained in the presence of cocaine 5 μM. The results demonstrate that the enhancement by α-adrenoceptor antagonists of the release of noradrenaline depends on the biophase concentration of noradrenaline. Under the present conditions, graded increases in biophase noradrenaline concentration led to graded increases in the effect of the antagonists. A second prerequisite for the release-enhancing effect appears to be a sufficient length of the pulse train. Under the present conditions, graded increases in train length up to about 20s led to graded increases in the effect of the antagonists, even though the average biophase concentration of noradrenaline did not change with the pulse train length. This pattern of effects of the α-antagonists is not shared by at least one other release-enhancing drug, namely TEA.

Notes: Summary Experiments were carried out in rabbit cerebrocortical slices in order to find out whether the attenuation by presynaptic α2-autoreceptors of effects mediated by presynaptic opioid κ- and adenosine A1-receptors requires activation of the α2-receptors. The slices were preincubated with 3H-noradrenaline and then superfused with medium containing desipramine 1 μmol/l. They were stimulated electrically either with single pulses or with trains of 32 pulses at 1 Hz. The overflow of tritium elicited by a single pulse amounted to 0.21% of the tritium content of the tissue. It was Ca2+-dependent and tetrodotoxin-sensitive and not changed by rauwolscine 1 μmol/l or yohimbine 0.3 μmol/l. Ethylketocyclazocine (EK; 0.1–10 nmol/l) and R-(−)-N6-phenylisopropyladenosine (PIA; 1–1,000 nmol/1) potently inhibited the overflow evoked by a single pulse, and their effects were not changed by yohimbine. — The overflow of tritium elicited by trains of 32 pulses at 1 Hz amounted to 0.92% of the tritium content of the tissue and was increased approximately fourfold by yohimbine 0.3 μmol/l. EK and PIA were less potent inhibitors than in the one pulse experiments. Yohimbine greatly enhanced the effects of EK and PIA. The enhancement was even more pronounced when the Ca2+ concentration in the medium was reduced in order to obtain a control tritium overflow similar to that evoked by 32 pulses in the absence of yohimbine. The results demonstrate that there is no α2-adrenergic autoinhibition when noradrenaline release is elicited by a single pulse. Under these conditions, the non-activated presynaptic α2-adrenoceptor does not interfere with presynaptic opioid κ- and adenosine A1-receptor mechanisms. It is only when the autoreceptor is activated by released noradrenaline that it attenuates neighbouring presynaptic receptor mechanisms, and this attenuation is removed by α2-adrenoceptor antagonists.

Notes: 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 μmol/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 μmol/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 μmol/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.

Notes: Summary The pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with 3H-serotonin and then superfused with medium containing fluvoxamine 3 μmol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC50 values and maximal effects and antagonist K B values. Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 〉 serotonin 〉 8-OH-DPAT in the rat and 5-CT 〉 serotonin 〉 RU 24969 〉 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the K B values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 μmol/1 did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (−)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species. The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT1D. The serotonin axons of rat brain cortex may possess 5-1D in addition to 5-HT1B autoreceptors. In many previous studies agonist potencies at, and antagonist affinities for, presynaptic serotonin autoreceptors have been underestimated due to the use of too intense stimuli to elicit serotonin release.

Notes: Summary The interaction between presynaptic, release-inhibiting α 2-adrenoceptors and opioid receptors was studied in slices of the parieto-occipital cortex of rabbits. The slices were preincubated with 3H-noradrenaline and then superfused with 3H-noradrenaline-free medium and stimulated electrically (3 or 7 Hz, 2 or 5 V/cm voltage drop between the electrodes). Clonidine and ethylketocyclazocine (EK) depressed, whereas yohimbine increased the electrically evoked overflow of tritium. When clonidine was administered first and retained in the medium for the rest of the experiment, the overflow-inhibiting effect of EK was reduced. When yohimbine was administered first and kept for the rest of the experiment, the effect of EK was enhanced. When, finally, EK was adminstered first and clonidine as the second drug, the overflow-inhibiting effect of clonidine was attenuated. The changes in the effect of EK (by clonidine or yohimbine) and clonidine (by EK) were not due to the changes in release per se produced by the drugs that were given first. Naloxone shifted the concentration-response curve of EK to the right; the dissociation constant of the naloxone-receptor complex, calculated from the shift, was 13 nmol/l. It is concluded that there is an interaction between presynaptic α 2-adrenoceptors and opioid к-receptors, either at the level of the receptors themselves or of the post-receptor reaction chains. Activation of one kind of receptor blunts the inhibition of release produced by activation of the other kind of receptor.

Notes: Abstract The effect of neuropeptide Y [NPY(1–36)] and related peptides on the voltage-dependent currents and the nicotinic acetylcholine receptor (nAChR) currents (IACh) of bovine adrenal chromafptn cells was investigated using the whole-cell patch clamp technique. Catecholamine release from single chromaffin cells was measured by means of fast cyclic voltammetry. The potency order of these peptides in inhibiting IACh evoked by nicotine was NPY(1–36), NPY (16–36) 〉 peptide YY(PYY) 〉 [Leu31, Pro34] NPY. NPY(16–36) produced a similar degree of inhibition, irrespective of whether nicotine or an equipotent concentration of acetylcholine was used to evoke IACh. NPY(16–36) failed to alter voltage-dependent inward or outward currents. Intracellular cAMP, and extracellular dibutyryl-cAMP, produced a slowly developing increase in IACh. Intracellular cAMP, extracellular 8-Br-cAMP or dibutyryl-cAMP, and an inhibitor of cyclic nucleotide phosphodiesterases 3-isobutyl-l-methylxanthine (IBMX), decreased the inhibitory effect of NPY(16–36) on lACh. Although the intracellular application of the cAMP-dependent protein kinase A inhibitor [PKI(14–24)amide] alone did not alter IACh, it potentiated the effect of NPY(16–36) in interaction experiments. While the NPY(16–36)-induced inhibition of IACh was reversed on washout of the peptide, the slightly shorter C-terminal fragment NPY(18–36) caused a long-lasting depression of both IAch and catecholamine secretion evoked by nicotine. This depression was smaller in the presence of extracellular 8-Br-cAMP than in its absence. NPY(18–36) did not alter the secretory activity induced by a high concentration of potassium. It appears that, by activating Y3-receptors, NPY inhibits nAChR-current and the resulting secretion of catecholamines from bovine chromaffin cells. This process may involve a G protein-mediated decrease in intracellular cAMP with a subsequent decrease in the degree of phosphorylation of the nAChR-channel.

Notes: Summary An attempt was made to determinepA2 values of antagonists at the presynaptic, release-inhibiting α2-autoreceptorsof rabbit and rat brain cortex under conditions when there was very little released noradrenaline in the autoreceptor biophase and, hence,pA2 values were not distorted by endogenous autoinhibition. Cortex slices were preincubated with3H-noradrenaline and then superfused and stimulated by trains of 4 pulses delivered at 100 Hz or, in a few cases, by trains of 36 pulses at 3 Hz. The α-adrenoceptor agonists clonidine, noradrenaline, and α-methylnoradren-aline concentration-dependently decreased the stimulation-evoked overflow of tritium. The a-adrenoceptor antagonists yohimbine, rauwolscine and idazoxan did not increase the overflow of tritium elicited by 4 pulses/100 Hz in rabbit brain slices and increased it only slightly in rat brain slices. In contrast, the antagonists increased markedly the overflow at 36 pulses/3 Hz. All antagonists caused parallel shifts to the right of the concentration-response curves of clonidine, noradrenaline, and α-methylnoradrenaline.pA2 values were calculated either from linear regression of log [agonist concentration ratio − 1] on log [antagonist concentration] or from sigmoid curve fitting. The slopes of the linear regression lines were close to unity, and thepA2 values calculated by the two methods agreed well. There was no consistent preferential antagonism of any antagonist to any agonist.pA2 values determined with stimulation by 4 pulses/100 Hz were by 0.53–0.80 log units higher than those determined with stimulation by 36 pulses/3 Hz. ThepA2 values (4 pulses/100 Hz) of yohimbine and rauwolscine in rabbit brain slices (approximately 7.9 and 8.2, respectively), were slightly higher than in rat brain slices (approximately 7.6 and 7.7, respectively), whereas thepA2 value of idazoxan in the rabbit. (about 7.1) was lower than itspA2 value in the rat (about 8.0). The experiments confirm thatpA2 values determined under conditions of autoinhibition are too low. Stimulation with short (30 ms) bursts of pulses permits the estimation ofpA2 values at presynaptic a2-autoreceptors without (rabbit) or almost without (rat) the complication of autoinhibition. The values suggest that α2-adrenoceptors in rabbit brain cortex differ slightly from those in rat brain cortex.

Notes: Summary The mechanism of the attenuation, by serotonin uptake blockers, of the release-inhibiting effect of exogenous serotonin autoreceptor agonists was studied in rabbit brain cortex and rat hypothalamus slices. The slices were preincubated with 3H-serotonin and then superfused and stimulated electrically. In rabbit brain slices stimulated by trains of 4 pulses at 100 Hz, 5-carboxamidotryptamine and 5-methoxytryptamine reduced the evoked overflow of tritium, and their concentration-response curves were not changed by any of three serotonin uptake inhibitors, namely citalopram, fluvoxamine and 6-nitroquipazine. In contrast, when the slices were stimulated by trains of 10 pulses at 0.033 Hz, fluvoxamine shifted the concentration-response curve of 5-methoxytryptamine to the right. Experiments with the autoreceptor antagonist metitepine indicated that little, if any, endogenous autoinhibitory tone developed in the course of trains of 4 pulses/100 Hz, irrespective of the absence or presence of uptake inhibitors, as well as during trains of 10 pulses/0.033 Hz in the absence of uptake inhibitors, whereas marked autoinhibition developed when 10 pulses/0.033 Hz were applied in the presence of fluvoxamine. In rat hypothalamic slices stimulated by trains of 4 pulses at 100 Hz, citalopram also failed to change the concentration-response curve of 5-methoxytryptamine. These results indicate that serotonin uptake blockers attenuate the effect of exogenous autoreceptor agonists by an increase in the biophase concentration of released serotonin and, hence, in endogenous autoinhibitory tone, and not by some direct “molecular link” unrelated to the biophase concentration of released serotonin.