Cholinoceptive properties of respiratory neurones in the rat medulla.

The effects of iontophoretically applied acetylcholine, the acetylcholine agonists nicotine and muscarine, and the antagonists atropine, dihydro-beta-erythroidine (DH beta E) and mecamylamine, together with the excitatory amino acids, glutamate and D,L-homocysteic acid (DLH) were examined on the activity of respiratory-related neurones in the rat medulla and were compared with effects on non-respiratory brain stem neurones. Most neurones were excited by acetylcholine and no inhibitory responses were seen. Glutamate and DLH also excited but there was a trend for the phasic activity of respiratory neurones to be converted to a tonic discharge. Nicotine also excited most neurones to which it was applied and these responses were blocked by DH beta E but not by atropine. Muscarine also caused excitation and these responses were blocked by atropine but not by DH beta E. Both antagonists blocked acetylcholine-induced excitation but had no effect on responses to glutamate or DLH. Mecamylamine was without effect. It is concluded that the proportion of cholinoceptive respiratory neurones in the rat brain stem is similar to that for non-respiratory neurones. It seems likely that both nicotinic and muscarinic receptors are present on the majority of respiratory neurones and that both contribute to the response produced by iontophoretically-applied acetylcholine.

Electrophysiological evidence for kappa-agonist activity of dynorphin in rat brain.

Dynorphin (1-13) and the mu-agonist, FK 33,824, have been applied microiontophoretically to single neurones in the hippocampus of the rat. Whereas FK 33,824 predominantly caused an increase in neuronal firing rates, dynorphin (1-13) decreased activity. Both effects were blocked by the opiate antagonist naloxone, whereas the effects induced by GABA and glutamate were not. This action of dynorphin (1-13) is comparable to that of other kappa-agonists in this brain region and suggests that dynorphin (1-13) may be a ligand for the kappa opiate receptor.

Histamine-induced excitation of spontaneously active medullary neurones in the rat brain is mediated by H2-receptors. A microiontophoretic study using H1- and H2-agonists and antagonists.

The effects of histamine, applied by microiontophoresis onto spontaneously-active medullary neurones were investigated in the rat. Histamine caused current-dependent excitation of these neurones, an action that is at variance with previous studies in the cat. The nature of the receptor mediating these effects was examined using a number of agonists with differing potencies at peripheral H1- and H2-receptors. The precursor of histamine, L-histidine and the metabolite, N-telemethylhistamine did not mimic the effects of histamine while the H2-agonist, 4-methylhistamine caused similar but weaker excitation. The extent of excitations produced by the H1-agonists, 2-pyridylethylamine, 2-methylhistamine and 2-thiazolylethylamine could be related to their activity at H2-receptors. Metiamide was ineffective in antagonising responses to histamine and related agonists as was mepyramine. The H2-antagonist ranitidine, however, proved a good antagonist of responses to histamine and the H1- and H2-agonists, despite an unrelated excitatory action which may be linked to inhibition of cholinesterase. It is concluded that the excitatory effects of microiontophoretically-applied histamine and the agonists on medullary neurones in the rat is probably a result of activation of H2-receptors.

Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine.

As a result of controversy in the literature regarding the classification and nomenclature of functional receptors for 5-hydroxytryptamine (5-HT), a framework for classification is proposed. The formulation of these proposals has only been made possible by the recent advent of new drug tools. It is considered that there are three main types of 5-HT receptor, two of which have been well characterised pharmacologically, using selective antagonists, and which it is proposed to name 5-HT2 and 5-HT3. These two groups broadly encompass the "D" and "M" receptors, respectively, which Gaddum identified in the guinea-pig ileum (Gaddum and Picarelli, 1957). The 5-HT2 receptor, which mediates a variety of actions of 5-HT, has been definitively shown to correlate with the 5-HT2 binding site in the brain. No binding studies in brain tissue have yet been published with radiolabelled ligands specific for 5-HT3 receptors. A number of other actions of 5-HT appear to be mediated via receptors distinct from 5-HT2 or 5-HT3 receptors. Since selective antagonists are not yet available, these receptors cannot be definitively characterised, although in many cases they do have some similarities with 5-HT1 binding sites, which are a heterogeneous entity. Criteria are proposed for tentatively classifying these receptors as "5-HT1-like" (Table 1). Definitive characterisation of these receptors will await the identification of specific antagonists. This classification of 5-HT receptors into three main groups (Table 1) is based largely, but not exclusively, on data from studies in isolated peripheral tissues where definitive classification is possible. However, it is believed that this working classification will be relevant to functional responses to 5-HT in the central nervous system.

A microiontophoretic study of the actions of mu-, delta- and kappa-opiate receptor agonists in the rat brain.

The actions of mu-, delta- and kappa-opiate receptor agonists have been compared on the activity of single neurones in the brain stem, caudate nucleus and hippocampus of the rat, using the technique of microiontophoresis. In the brain stem and caudate nucleus the predominant effect of all the opiate agonists tested was depression of neuronal activity which was antagonized by naloxone. The selectivity of naloxone as an opiate receptor antagonist was indicated by its lack of effect on gamma-aminobutyric acid (GABA)-induced responses. In the hippocampus both mu- and delta-agonists mainly caused an increase in neuronal firing rates, though some neurones were depressed. In contrast, all the kappa-agonists, including the proposed endogenous ligand for the kappa-receptor, dynorphin, caused depression of neuronal activity. All of these effects were antagonized by naloxone. There was a clear distinction in the areas within the hippocampus in which the mu- and delta-agonists produced different effects. Neurones in the pyramidal cell layer were always excited by these drugs, whereas neurones in the granule cell layer of the dentate gyrus were always depressed by the same drug.

The effects of microiontophoretically applied morphine and transmitter substances in rats during chronic treatment and after withdrawl from morphine.

The effects of microiontophoretically applied acetylcholine, noradrenaline, 5-hydroxytryptamine and morphine were studied on single brain stem neurones of rats during chronic morphine pretreatment and 24 h after its withdrawal. No significant changes were observed in the initial spontaneous neuronal firing rate or in the qualitative or quantitative effects of acetylcholine, noradrenaline or 5-hydroxytryptamine. However, in chronically treated animals there was a significant decrease in the number of neurones excited by morphine or showing tachyphylaxis to morphine on repeated microiontophoretic applications.We suggest that some of the cellular central nervous system changes which occur during chronic morphine treatment are reflected by the decrease in sensitivity of neurones to morphine excitation.

Further studies on the mode of action of psychotomimetic drugs: antagonism of the excitatory actions of 5-hydroxytryptamine by methylated derivatives of tryptamine.

1 The actions of 5-methoxytryptamine (5-MeOT), N,N-dimethyltryptamine (DMT), 5-hydroxy-N,N-dimethyltryptamine (bufotenine, 5-HODMT) and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), and their interactions with 5-hydroxytryptamine (5-HT), acetylcholine, (-)-noradrenaline, and glutamate were studied by microiontophoresis on single neurones in the brain stem of rats anaesthetized with urethane or decerebrate cats.2 Like D-lysergic acid diethylamide (LSD 25) the three psychotomimetic derivatives (DMT, 5-HODMT, 5-MeODMT) specifically antagonized 5-HT excitations of single neurones, but the non-psychotomimetic 5-MeOT had no antagonistic effects.3 In contrast to LSD 25, the psychotomimetic tryptamines only rarely antagonized glutamate effects, indicating that the excitatory 5-HT receptors and the glutamate receptors on the same neurones may be closely related spatially, but are separate.4 The methylated tryptamine derivatives were able to mimic the actions of 5-HT on neurones. The non-psychotomimetic 5-MeOT was most potent in this respect, while the other three derivatives which are psychotomimetic, were less active.5 The 5-HT mimicking actions of 5-MeOT were the same in rats pretreated with p-chlorophenylalanine or reserpine as in untreated rats. It therefore seems that the 5-HT mimicking actions are unlikely to be due to release of 5-HT, but are due to direct actions on 5-HT receptors.6 The evidence presented supports the hypothesis that LSD-like psychotomimetics act by an antagonism of 5-HT in the lower brain stem, and is not compatible with the suggestion that the psychotomimetic action of these drugs is related to 5-HT receptor stimulation.

Morphine and neurotransmitter substances: Microiontophoretic study in the rat brain stem.

1 The effects of microiontophoretically applied morphine and its interactions with the effects of microiontophoretic applications of either acetylcholine, (-)-noradrenaline or 5-hydroxytryptamine have been studied on single neurones in the brain stem of rats anaesthetized with urethane.2 Morphine excited or inhibited most neurones tested and the effects, especially excitation, were often extremely powerful. However, the time course of the excitatory and inhibitory effects were somewhat different.3 Desensitization to the excitation produced by morphine was seen after repeated or prolonged applications and it is suggested that this phenomenon may be related to the tolerance which develops after chronic administration of morphine. No desensitization was observed to inhibition of neuronal activity by morphine.4 Morphine usually reduced the excitation of neurones by acetylcholine, noradrenaline or 5-hydroxytryptamine but sometimes potentiated the effect, although not always on the same neurones. Inhibition of neuronal activity by these compounds was never modified by morphine and neither were the effects of glutamate or D,L-homocysteic acid when used as control agonists.5 The in vitro release of morphine from six micropipettes was determined and the transport number was calculated to be 0.051 (s.d. 0.021).6 The implications of these observations in explaining the pharmacological actions of morphine are discussed.

Iontophoretic release of acetylcholine, noradrenaline, 5-hydroxytryptamine and D-lysergic acid diethylamide from micropipettes.

1. The in vitro iontophoretic release of tritium-labelled acetylcholine and 5-hydroxytryptamine from large and small micropipettes and noradrenaline and D-lysergic acid diethylamide from small micropipettes was determined by liquid scintillation counting.2. The release was directly proportional to the electrical charge passed in the range normally used in the iontophoretic study of these compounds. The transport numbers obtained for the large micropipettes were approximately double those with the small micropipettes. A very low transport number was found for D-lysergic acid diethylamide.3. The spontaneous leakage was small and did not vary appreciably with time.4. The iontophoretic release of acetylcholine in vitro agreed with the in vitro measurements.5. The brain-stem tissue concentration of D-lysergic acid diethylamide after intravenous injection into intact and decerebrate cats was determined.

Observations on the pharmacology of cholinoceptive neurones in the rat brain stem.

The pharmacology of spontaneously active cholinoceptive neurones in the brain stem of rats anaesthetized with urethane has been investigated using microiontophoresis to administer muscarinic and nicotinic agonists and antagonists. 2. Acetylcholine (ACh) excited most cells but occasionally depressed their activity. Muscarine, and the muscarinic agonists methacholine and bethanechol produced prolonged excitation or inhibition of cells whereas nicotine produced prolonged excitations but no inhibitions. 3 Atropine selectively antagonized ACh excitations and both excitation and inhibition of neuronal activity produced by muscarine and muscarinic agonists, but not the excitations produced by nicotine, glutamate or DL-homocysteic acid. 4 Dihydro-beta-erythroidine (DHBE) and tubocurarine antagonized both ACh and nicotine excitations but not those induced by glutamate or DL-homocysteic acid. Inhibitions by ACh or muscarine were not affected. 5 It is concluded that excitations of cholinoceptive neurones in the rat brain stem may be mediated by activation of both muscarinic and nicotinic receptors whereas inhibitions are mediated by activation of a muscarinic receptor.

Short-latency excitation of brain stem neurones in the rat by acetylcholine.

A fast excitatory response to acetylcholine (ACh) which has not previously been reported, has been found in the rat brain stem. Micro-iontophoretic applications of ACh to single brain stem neurones in unanaesthetized rats excited 81% and inhibited 3% of neurones studied. Two types of excitatory response were distinguished by their time course. Type I ACh excitation of neurones was of long latency resembling that previously reported in various parts of the brain. Type II excitation was of short latency, similar to that of micro-iontophoretically applied glutamate ions and to ACh excitation of Renshaw cells.

Modification of the responses of brain stem neurones to transmitter substances by anaesthetic agents.

1. The effects of microiontophoretic applications of acetylcholine (ACh), (-)-noradrenaline ((-)-NA) and 5-hydroxytryptamine (5-HT) have been investigated on spontaneously active brain stem neurones in decerebrate unanaesthetized rats and in rats anaesthetized with either tribromoethanol, urethane or pentobarbitone.2. Four types of responses to both (-)-NA and 5-HT were seen. These were: simple excitation; excitation preceded by a short-lasting inhibition; short-lasting inhibition and prolonged inhibition. Three types of responses to ACh were seen: an excitation with long latency of onset; excitation with short latency of onset, resembling the response to an excitant amino acid, and a short-lasting inhibitory response.3. The types of responses to microiontophoretically applied ACh, (-)-NA or 5-HT in anaesthetized and unanaesthetized animals were similar.4. The number of ACh excitatory responses with short latency of onset were significantly reduced in the pentobarbitone-anaesthetized group and a small but significant increase in the number of 5-HT inhibitory effects were observed in each anaesthetized group of animals.5. A significantly greater proportion of slower firing neurones (less than 10 spikes/s) were found in the pentobarbitone-anaesthetized animals.6. The effects of microiontophoretically applied and i.v. administered pentobarbitone were studied on spontaneously active neurones which responded consistently to ACh and a control agonist.7. Pentobarbitone administered by either route reduced the firing rate of most neurones studied and was shown to antagonize specifically the excitation of neurones by exogenously applied ACh.8. It is suggested that postsynaptic antagonism of endogenously released ACh may be a contributing factor in the mechanism of action of pentobarbitone.

Interactions of (+)-amphetamine and chlorpromazine on neurones in the lower brain stem of the rat.

1 The ability of chlorpromazine to antagonize the effects of iontophoretic application of (+)-amphetamine to single neurones in the medulla and lower pons of anaesthetized rats has been studied. 2 Chlorpromazine, administered systemically or iontophoretically, consistently and specifically antagonized the excitatory actions of (+)-amphetamine, but not those of noradrenaline on the same neurone. 3 It is concluded that chlorpromazine reduces the effect of (+)-amphetamine by a presynaptic mechanism. 4 (+)-Amphetamine did not mimic the prolonged inhibitory response of some neurones to noradrenaline but often excited these neurones and chlorpromazine blocked these excitatory responses to (+)-amphetamine.

Tryptamine-induced vasoconstrictor responses in rat caudal arteries are mediated predominantly via 5-hydroxytryptamine receptors.

It has been suggested that tryptamine can stimulate specific receptors distinct from those for 5-hydroxytryptamine (5-HT). We have examined this possibility in the rat isolated caudal artery, paying particular attention to the involvement of monoamine oxidase metabolism and alpha-adrenoceptors, two factors that can complicate the quantification of antagonist potencies at 5-HT receptors. 5-HT and tryptamine were agonists over the concentration-ranges 3.0 X 10(-8) - 3.0 X 10(-5) mol l-1 and 1.0 X 10(-6) - 3.0 X 10(-4) mol l-1 respectively. The sensitivity of the caudal artery to tryptamine was increased by about 44 fold in the presence of iproniazid (5.0 X 10(-5) mol l-1) and about 17 fold in the presence of pargyline (1.0 X 10(-5) mol l-1), while responses to 5-HT and methoxamine were unaffected. In the absence of iproniazid, ketanserin and methysergide were potent antagonists of responses to 5-HT with pA2 values of 9.08 and 9.11 and slopes of the Schild regressions of 1.15 and 1.00 respectively. However, against tryptamine the antagonists were weaker such that pA2 values were similar to those against 5-HT but the slopes of the Schild regressions were 0.47 and 0.47. In the presence of iproniazid (or pargyline), the 5-HT antagonists were more potent against tryptamine such that the pA2 values and the slopes of the Schild regressions were not significantly different from those against 5-HT. Phentolamine was a weak antagonist of responses to both 5-HT and tryptamine in the presence of iproniazid. 5 The findings in this study suggest that the contractile action of tryptamine in rat caudal artery is mediated predominantly by the same receptor as 5-HT and that the differential inactivation of tryptamine by monoamine oxidase enzymes largely accounts for the different susceptibilities of 5-HT and tryptamine to the antagonists examined.

Evidence for the existence of 5-hydroxytryptamine receptors, which are not of the 5-HT2 type, mediating contraction of rabbit isolated basilar artery.

In the rabbit isolated basilar artery the contractile action of 5-hydroxytryptamine (5-HT) was little affected by high concentrations of ketanserin (1.0 X 10(-6) M) indicating that 5-HT-receptors other than those of the 5-HT2-type were involved. The contractile action of 5-HT was mimicked by methysergide and 5-carboxamidotryptamine (5-CT) with equipotent concentration ratios (5-HT = 1) of about 22 and 0.6 respectively. This profile is characteristic of that in the dog saphenous vein which contains a 5-HT receptor type that may be described as '5-HT1-like'.

Antagonism of 5-hydroxytryptamine by LSD 25 in the central nervous system: a possible neuronal basis for the actions of LSD 25.

1. 5-Hydroxytryptamine (5-HT), acetylcholine (ACh), noradrenaline (NA), glutamate, D,L-homocysteic acid (DLH), glycine and gamma-aminobutyric acid (GABA) were applied to single neurones in the brain stem of decerebrate cats by microiontophoresis. The abilities of D-lysergic acid diethylamide tartrate (LSD 25), methysergide maleate (UML 491) and 2-bromo-lysergic acid diethylamide (BOL 148) to antagonize the actions of these compounds were studied.2. LSD 25 antagonized 5-HT excitation of single neurones when applied iontophoretically or administered intravenously. LSD 25 also antagonized glutamate excitation of neurones which could be excited by 5-HT. Inhibitory effects of 5-HT, the action of glutamate on neurones which could be inhibited by 5-HT and the actions of all the other compounds tested were unaffected by LSD 25.3. Iontophoretically applied UML 491 was also a specific antagonist to 5-HT and glutamate excitation but was less potent than LSD 25, and BOL 148 rarely exhibited antagonism.4. It is suggested that antagonism to 5-HT and glutamate excitation of brain stem neurones may be the basis of the psychotomimetic action of LSD 25. It is also suggested that there may be similarities in the mechanisms by which 5-HT and glutamate produce excitation where they act on the same neurone.

Correlation of prostaglandin release from the cerebral cortex of cats with the electrocorticogram, following stimulation of the reticular formation.

1. Prostaglandin-like material has been found in superfusates of cerebral cortex in unanaesthetized encéphale isolé cat preparations.2. The material was assayed on the isolated rat uterus and identified by thin-layer chromatography.3. The level of spontaneous release of prostaglandin-like material was greater than that which had been found in anaesthetized preparations and it increased further with electrical stimulation of the reticular formation which induced electrocortical arousal.4. Chlorpromazine (1.0-8.0 mg/kg) not only depressed the spontaneous release but blocked the increase evoked by stimulation concomitantly with blocking electrocortical arousal. Increasing the stimulating voltage to restore the arousal response also restored the evoked release of prostaglandins.5. Most of the prostaglandin-like material released spontaneously was represented by E type compounds, but the increase with stimulation was mainly of F compounds.

A neuronal basis for the alerting action of (+)-amphetamine.

1. (+)-Amphetamine mimicked the excitatory and inhibitory actions of (-)-noradrenaline on single neurones in the brain stem of acute halothaneanaesthetized rats when these compounds were applied by iontophoresis. (+)-Amphetamine had no actions on neurones unaffected by (-)-noradrenaline.2. These mimicking actions of (+)-amphetamine could not be observed 20 h after treatment of the animals with reserpine 5 mg/kg.3. The enzyme inhibitors alpha-methyl-p-tyrosine and FLA 63 also greatly reduced the number of (-)-noradrenaline-mimicking responses to (+)-amphetamine.4. In animals pretreated with alpha-methyl-p-tyrosine, but not in those pretreated with FLA 63, excitatory actions of (+)-amphetamine on neurones excited by (-)-noradrenaline could be elicited 45-90 min after systemic injection of L-DOPA.5. These results indicate that (+)-amphetamine can release noradrenaline from presynaptic sites in the brain stem, which may be a basis for its alerting actions.

Actions of noradrenaline, other sympathomimetic amines and antagonists on neurones in the brain stem of the cat.

1. The effects of (-)-noradrenaline ((-)-NA) and related compounds on brain stem neurones in decerebrate unanaesthetized cats have been investigated using the technique of iontophoretic application from micropipettes.2. Four types of response to (-)-NA have been described. These were short lasting inhibition, long lasting inhibition, excitation, and a biphasic response consisting of short lasting inhibition followed by excitation. A variable amount of desensitization of the excitatory response, but not of inhibitory responses, was observed.3. Experiments in which small currents were used to pass (-)-NA from pipettes with smaller tips did not lead to any appreciable change in the proportions of neurones excited or inhibited.4. A variety of sympathomimetic agonists was tested. Short lasting inhibition was less sensitive than excitation to changes in molecular structure. Long lasting inhibition was more sensitive to molecular change and was not mimicked by some of the agonists which mimicked short lasting inhibition.5. Although agonists without one ring hydroxyl had weaker effects than those with both, compounds in which both ring hydroxyl groups were absent (beta-hydroxyphenylethylamine, ephedrine and amphetamine) mimicked excitation strongly. It is possible that the compounds without both ring hydroxyl groups had some effect other than simple agonistic activity.6. A dissociation was observed between responses to dopamine and (-)-NA. p-Tyramine mimicked dopamine, rather than (-)-NA.7. Neither the alpha-agonist, phenylephrine nor the beta-agonist, isoprenaline mimicked neuronal responses to (-)-NA. The alpha-antagonists phentolamine and phenoxybenzamine and the beta-antagonists dichloroisoprenaline, propranolol and D(-)-INPEA and combinations of propranolol with phentolamine or phenoxybenzamine were ineffective in blocking either excitation or inhibition. Thus, the central receptors appear to be different from peripheral alpha- and beta-receptors.8. The most effective antagonist of excitation was (-)-alpha-methylnoradrenaline. Metaraminol and dihydroergotamine also had some antagonistic activity. None of the compounds tested blocked inhibition. The effects of (-)-alpha-methylnoradrenaline have been discussed in relation to the hypotensive action of alpha-methyldopa.