Thyrotrophin releasing hormone stimulates release of [3H]-dopamine from slices of rat nucleus accumbens in vitro.

Thyrotrophin releasing hormone (TRH) (25 to 100 microM) was found to stimulate the efflux of [3Hu-dopamine from small slices of rat nucleus accumbens, but not from similar slices of rat caudate nucleus. Uptake inhibition was not responsible for this action, since at 10 and 50 microM TRH had no effect on the ability of small slices of nucleus accumbens to accumulate radioactivity when incubated with 10(-7) M [3H]-dopamine. In addition the hormone had no effect on basal or dopamine-stimulated adenylate cyclase, nor did it displace [3H]-spiperone binding, in membrane preparations from nucleus accumbens.     

The purported dopamine agonist DPI inhibits [3H]noradrenaline release from rat cortical slices but not [3H]dopamine and [14C]acetylcholine release from rat striatal slices in-vitro

The effects of the purported dopamine (DA) receptor agonist (3,4-dihydroxyphenylimino)-2-imidazolidine (DPI) upon the in-vitro K+-induced release of [3H]DA and [14C]acetylcholine from rat neostriatal slices, and of [3H]noradrenaline from rat neocortical slices have been investigated and compared with those of the DA receptor agonist TL-99 and the alpha-adrenoceptor agonist clonidine, respectively. The rapid decomposition of the catechol compounds DPI and TL-99 in the Krebs-Ringer bicarbonate superfusion medium was shown to be inhibited by both the chelating agent EDTA and the reducing agent ascorbic acid. The results suggest that in-vitro DPI is unable to stimulate striatal DA receptors, whereas it is effective in stimulating cortical alpha 2-adrenoceptors (EC50 = 61 nM). It is concluded that DPI should be considered as a mixed alpha 1/alpha 2-adrenoceptor agonist and that the designation of DPI as a DA receptor agonist should be abandoned.     

Presynaptic alpha-adrenoceptors and opiate receptors: inhibition of [3H]noradrenaline release from rat cerebral cortex slices by different mechanisms

The inhibitory effects of morphine and Cd2+ on electrically evoked [3H]noradrenaline release from superfused brain slices were unaffected when release was enhanced by increasing the pulse duration, while the inhibitory effect of noradrenaline and the enhancing effect of phentolamine were diminished. A similar enhancement of [3H]noradrenaline release by 4-aminopyridine reduced the modulatory effects of all drugs examined. Therefore there seem to be different mechanisms for the effect of presynaptic alpha-adrenoceptors and opiate receptors on the availability of Ca2+ for the stimulus-secretion coupling process in noradrenergic nerve terminals.     

Adrenergic receptors in the nucleus tractus solitarii of the rat

The nucleus tractus solitarii (NTS) is an area of the medulla in which baroreceptor afferent fibers mediating an inhibition of vasoconstriction terminate. The NTS also appears to be a site at which alpha-adrenergic receptors may be involved in blood pressure control. alpha- and beta-adrenergic agonists and antagonists were injected into the NTS via stainless steel cannulae and the effects on blood pressure determined. Norepinephrine (NE) and isoproterenol (ISO) caused dose-related decreases in mean arterial blood pressure (MABP). Phentolamine (PH) and propranolol (PR) caused dose-related increases in MABP. PH pretreatment in the NTS blocked the effects of ISO, but not NE. The effect of increased baroreceptor impulses to the NTS, produced by altering perfusion of the carotid sinus was blocked by propranolol. These results indicate the presence of beta- as well as alpha-receptors in the NTS, and also suggest the existence of presynaptic beta-receptors which facilitate neurotransmission.     

NMDA-induced hippocampal [3H]norepinephrine release is modulated by glycine

Kynurenic acid (KYN) non-competitively inhibited N-methyl-D-aspartate (NMDA)-induced [3H]norepinephrine ([3H]NE) release from rat hippocampal slices. At 100 microM KYN, the effect on release was primarily on the maximal obtainable response to NMDA. Glycine was able to completely block the inhibitory effects of 100 microM KYN on NMDA-evoked release. This ability to prevent KYN inhibition of release was shared by other amino acids with the following order of potency: glycine greater than D-serine greater than D-alanine much greater than L-serine greater than or equal to L-alanine. Neither isomer of valine or threonine was able to reverse KYN inhibition of NMDA-induced release. These potencies agreed with the relative abilities of these amino acids to displace strychnine-insensitive [3H]glycine binding to rat brain membranes. Glycine and D-serine had no effect on the inhibition of NMDA-stimulated [3H]NE release produced by D-2-amino-5-phosphonovaleric acid, MK-801 or Mg2+. Also, neither amino acid modified KYN inhibition of kainic acid-induced release. These data demonstrate that the glycine regulatory site associated with the NMDA receptor can be demonstrated in whole brain slices by using an antagonist to attenuate the influences of endogenous glycine.     

Differential control of Ca2+-dependent [3H]noradrenaline release from rat brain slices through presynaptic opiate receptors and alpha-adrenoceptors

The inhibitory actions of the Ca2+ antagonist Cd2+, morphine and noradrenaline (exogenously added + endogenously released) on electrically evoked release of [3H]noradrenaline from superfused rat neocortical slices were strongly reduced when release was enhanced by 4-aminopyridine. In the presence of 4-aminopyridine the release inhibiting effects of these drugs were restored by lowering the extracellular Ca2+ concentration. When release was enhanced by prolonging the pulse duration, only the release inhibiting effect of noradrenaline was reduced but the effects of Cd2+ and morphine were unchanged. Irrespective of the pulse duration, blockade of presynaptic alpha-adrenoceptors with phentolamine did not affect the release inhibiting effects of Cd2+ and morphine. The inhibitory effects of morphine and noradrenaline remained unchanged in Cl--free medium. Furthermore, these drugs strongly reduced the [3H]noradrenaline release induced by 20 mM K+ in the presence of tetrodotoxin. The results suggest that activation of presynaptic opiate-receptors inhibits Ca2+ entry through voltage-sensitive Ca2+ channels, whereas presynaptic alpha-adrenoceptors affect a step in the secretory process subsequent to Ca2+ influx. Moreover, the involvement of (direct) changes in Na+, K+ or Cl- permeability appears unlikely for both receptor systems.     

Presynaptic GABAB receptors and the regulation of [3H]noradrenaline release from rat anococcygeus muscle

The release of preaccumulated [3H]noradrenaline from the isolated rat anococcygeus muscle, a tissue endowed with a rich noradrenergic innervation, was investigated. Field stimulation produced a marked, calcium-dependent enhancement in release, which could be substantially inhibited by the inclusion of low concentrations of GABA or (-)-baclofen (but not the (+)-isomer) in the bathing medium. Classical GABAA site agonists such as muscimol or 3-aminopropanesulphonic acid were inactive, or only weakly so. Picrotoxin and bicuculline were unable to antagonise the effects of GABA or baclofen on [3H]noradrenaline release, although 5-aminovalerate was effective. These data provide further evidence for the existence of GABAB receptors associated with presynaptic noradrenergic terminals in the rat anococcygeus muscle.     

Inhibitory muscarinic receptors modulate the potassium-evoked release of [3H]serotonin from rat hypothalamic slices

The effects of oxotremorine, acetylcholine and nicotine have been investigated on the potassium-evoked release of [³H]serotonin from slices of rat hypothalamus. Oxotremorine and acetylcholine in the presence of physostigmine inhibited potassium-evoked tritium release without affecting the spontaneous release. Nicotine had no effect. The response to oxotremorine was unaffected by tetrodotoxin but was blocked by atropine and hyoscine suggesting that the muscarinic receptor mediating the response to oxotremorine was located on the serotonergic nerve terminal.     

Activation of dopamine D4 receptors modulates [3H]GABA release in slices of the rat thalamic reticular nucleus

The thalamic reticular nucleus (nRt) is innervated by dopaminergic projections from the sustantia nigra compacta (SNc) and is rich in dopamine D4 receptors, however, the functional effects of dopamine on this structure are unknown. We examined whether the D1 receptor agonist SKF 38393, or the D2 class receptor agonist quinpirole, modify depolarization evoked Ca(2+)-dependent [3H]GABA release. SKF 38393 was without effects, whereas quinpirole inhibited [3H]GABA release with an IC50 of 81 +/- 33 nM. Dose-dependence determinations of agonists (quinpirole and PD 168, 077) and antagonists (L-745,870, U-101958, clozapine and raclopride) with different affinities for different D2 class subtype receptors showed that a D4 receptor mediates quinpirole inhibition. We used methylphenidate, an agent that acts by increasing interstitial dopamine, to determine whether endogenous dopamine modulates [3H]GABA release. Methylphenidate inhibited [3H]GABA release showing that the nRt contains sufficient endogenous dopamine to activate D4 receptors. This inhibition was completely reversed by selectively blocking D4 receptors with L-745,870 or U-101958 indicating that the catecholamine receptors that modulate GABA release are D4 receptors. Given the importance of the nRt in the control of attention, sensory processing and the generation of rhythmic activity during slow wave sleep, it is possible that abnormal nRt function may generate some of the manifestations of the disorders of dopaminergic transmission.     

Pattern of catecholamine release in the nucleus tractus solitarii of the cat

Summary The release of endogenous dopamine, noradrenaline and adrenaline was studied in the nucleus tractus solitarii (subnucleus medialis) of anaesthetized cats. Two push-pull cannulae were inserted into the right and left nuclei tractus solitarii which were simultaneously superfused with artificial cerebrospinal fluid. The release of the neurotransmitters was determined in the superfusates which were continuously collected in 10 min or 2.5 min time periods for 6 h or 2 h, respectively. Collection of the superfusates in time periods of 10 min showed that the release rates of endogenous dopamine, noradrenaline and adrenaline varied rhythmically according to an ultradian rhythm, the time interval between 2 adjacent phases of high release rate being approximately 1 h. Some of the phases of high release rates of the three endogenous catecholamines timely coincided with each other. Collection of the perfusates in time periods of 2.5 min revealed the existence of additional oscillations with a frequency of about 1 cycle/10 min for dopamine, noradrenaline and adrenaline. The findings suggest that catecholamines are released in the nucleus tractus solitarii according to an ultradian rhythm with low (1 cycle per h) and high (1 cycle per 10 min) frequencies, which might reflect the activity of catecholaminergic neurons in this brain structure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung (P5750) Send offprint requests to A. Philippu at the above address     

The release of labelled acetylcholine and choline from cerebral cortical slices stimulated electrically

1 In order to establish the origin of the increased efflux of radioactivity caused by electrical stimulation of cerebral cortical slices which had been incubated with [(3)H]-choline, labelled choline and acetylcholine (ACh) collected by superfusion were separated by gold precipitation.2 In the presence of physostigmine electrical stimulation (1 Hz, 10 min) increased the release of only [(3)H]-ACh which was greatly enhanced by the addition of atropine.3 Continuous stimulation in the presence of physostigmine resulted in an evoked release of [(3)H]-ACh which declined asymptotically. This evoked release appeared to follow first-order kinetics with a rate constant which remained stable over the course of prolonged stimulation.4 The rate constant for the evoked release of [(3)H]-ACh with 1 Hz stimulation was three times greater in the presence of physostigmine and atropine than in the presence of physostigmine alone, while the size of the store from which [(3)H]-ACh was released was nearly identical under these two conditions.5 In the absence of physostigmine and atropine, stimulation caused the appearance of only [(3)H]-choline in the samples.6 Reduction of [(3)H]-ACh stores before the application of physostigmine resulted in a reduced evoked release of total radioactivity, both in the absence or presence of physostigmine and atropine, and decreased the evoked release of [(3)H]-ACh without affecting the release of [(3)H]-choline.7 Results suggest that electrical stimulation of cortical slices which had been incubated with [(3)H]-choline causes the release of only [(3)H]-ACh, both in the presence or absence of an anticholinesterase. The evoked increase in the efflux of total radioactivity is therefore a good measure of the release of [(3)H]-ACh.     

Effect of an intracellular calcium chelator on the regulation of electrically evoked [3H]-noradrenaline release from rat hippocampal slices.

1. The electrically (3 Hz, 5 min) evoked [3H]-noradrenaline ([3H]-NA) release from rat hippocampal slices was reduced by prior treatment of the slices with 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetomethylester (BAPTA/AM) in a concentration-(10 to 500 microM) dependent manner (40% at 30 microM). Reduction of medium calcium from 1.3 to 0.5 mM caused a larger (70%) decrease. BAPTA free acid (100 mM), a non-permeable Ca(2+)-chelator had no significant effect. 2. Basal [3H]-noradrenaline release was reduced by BAPTA/AM in a concentration-dependent manner (50% at 30 microM), but reduction of external Ca2+ from 1.3 to 0.5 mM did not alter basal release. 3. About 10% of total [3H]-NA in the slices was released at 3 Hz stimulation in 1.3 mM Ca2+ buffer. Addition of the alpha 2-adrenoceptor antagonist, idazoxan (1 microM), increased electrically evoked [3H]-NA release to 26% but stimulated release was not altered by the adenosine A1-receptor antagonist, 8-cyclopentyl theophylline (8-CPT) (1 microM). 4. Evoked release was reduced by the alpha 2-receptor agonist, UK 14,304, in a concentration-dependent manner in the presence of 8-CPT (1 microM). The magnitude of this effect was not altered by the treatment of slices with 30 microM BAPTA/AM. 5. The adenosine A1 receptor agonist, N6-cyclohexyl adenosine (CHA) (1 microM) inhibited electrically evoked [3H]-NA release by about 40% in the presence of idazoxan (1 microM). The effect of CHA was not significantly altered by treatment of slices with BAPTA/AM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of [3 H]dopamine from guinea pig striatal slices is modulated by sigma1 receptor agonists

Sigma receptors are found in motor and limbic areas in the brains of humans, non-human primates, and rodents. The most extensive pharmacological studies of ligand binding to sigma receptors have utilized brain tissue from guinea pigs, where two subtypes of sigma receptor, designated sigma₁ and sigma₂, have been identified. Few functional roles for sigma receptors have been described. Their location in guinea pig striatum, a terminal field of dopaminergic projections arising from the substantia nigra, suggested that this tissue would be a logical choice in which to examine physiological properties of sigma receptor activation. We found that sigma₁ receptor agonists inhibited N-methyl-d-aspartate-stimulated [³H]dopamine release from guinea pig striatal slices in a concentration-dependent manner. The inhibition by sigma₁ receptor agonists was reversed by a selective sigma₁ receptor antagonist, as well as by a non-subtype-selective sigma receptor antagonist. The ability of agonists working through sigma₁ receptors, but not through sigma₂ receptors, to inhibit the stimulated release of catecholamines appears to be a unique characteristic of guinea pig striatum. We have previously reported that in rat striatum and hippocampus, as well as in guinea pig nucleus accumbens, prefrontal cortex, and hippocampus, activation of either sigma receptor subtype inhibits such release. Stimulated release of [³H]dopamine from guinea pig striatum was also inhibited by the phencyclidine receptor agonist dizocilpine, but this inhibition was not reversed by the sigma receptor antagonists. Therefore, the inhibition produced by sigma receptor agonists was not mediated via the phencyclidine binding site within the N-methyl-d-aspartate-operated cation channel. Our findings support the hypothesis that sigma receptor activation provides a mechanism of modulating dopamine release from striatum, and that striatal tissue from guinea pigs appears to be an appropriate model for characterizing sigma₁ receptor-mediated effects. Received: 18 March 1997 / Accepted: 9 June 1997     

alpha-bungarotoxin-sensitive nicotinic receptors indirectly modulate [(3)H]dopamine release in rat striatal slices via glutamate release

Nicotinic agonists elicit the release of dopamine from striatal synaptosomes by acting on presynaptic nicotinic acetylcholine receptors (nAChRs) on dopamine nerve terminals. Both alpha3beta2* and alpha4beta2 nAChR subtypes (but not alpha7* nAChRs) have been implicated. Here, we compared nAChR-evoked [(3)H]dopamine release from rat striatal synaptosome and slice preparations by using the nicotinic agonist anatoxin-a. In the more integral slice preparation, the concentration-response curve for anatoxin-a-evoked [(3)H]dopamine release was best fitted to a two-site model, giving EC(50) values of 241 nM and 5.1 microM, whereas only the higher-affinity component was observed in synaptosome preparations (EC(50) = 134 nM). Responses to a high concentration of anatoxin-a (25 microM) in slices (but not in synaptosomes) were partially blocked by ionotropic glutamate receptor antagonists (kynurenic acid, 6,7-dinitroquinoxaline-2,3-dione) and by alpha7*-selective nAChR antagonists (alpha-bungarotoxin, alpha-conotoxin-ImI, methyllycaconitine) in a nonadditive manner. In contrast, the alpha3beta2-selective nAChR antagonist alpha-conotoxin-MII partially inhibited [(3)H]dopamine release from both slice and synaptosome preparations, stimulated with both low (1 microM) and high (25 microM) concentrations of anatoxin-a. Antagonism by alpha-conotoxin-MII was additive with that of alpha7*-selective antagonists. These data support a model in which alpha7* nAChRs on striatal glutamate terminals elicit glutamate release, which in turn acts at ionotropic glutamate receptors on dopamine terminals to stimulate dopamine release. In addition, non-alpha7* nAChRs on dopamine terminals also stimulate dopamine release. These observations have implications for the complex cholinergic modulation of inputs onto the major efferent neurons of the striatum.     

Effects of pentazocine and other opioids on the potassium-evoked release of [3H]noradrenaline from guinea pig cortical slices

Noradrenaline release and its modulation via presynaptic opioid receptors were examined in guinea pig cortical slices. Slices preloaded with [3H]noradrenaline were superfused in the presence of desipramine (1 microM) and were stimulated by 16 mM K+. 1-Pentazocine inhibited the K+-evoked release of [3H]noradrenaline in a dose-dependent manner (3 x 10(-7)-10(-5) M), while d-pentazocine did not inhibit. This inhibitory effect of 1-pentazocine was antagonized by Mr 2266 (10(-6) M) but not by naloxone (10(-6) M). Among other opioids, dynorphin A-(1-13) and ethylketocyclazocine (kappa agonists) decreased the K+-evoked release of [3H]noradrenaline. Tyr-D-Ala-Gly-NMe-Phe-Gly-ol (DAGO, mu agonist) also inhibited [3H]noradrenaline release but was less potent than the kappa agonists. [D-Pen2,D-Pen5]enkephalin (DPDPE, delta agonist) and phencyclidine (sigma agonist) had no effects on the stimulated release of [3H]noradrenaline. Thus, it was shown that kappa receptors are the major subtype of opioid receptor involved in modulation of noradrenaline release in guinea pig cortex, and that 1-pentazocine inhibits the K+-evoked release of noradrenaline through activation of these receptors.