Rat Phrenic Nerve Research Articles

Tonic adenosine A 2A receptor activation modulates nicotinic autoreceptor function at the rat neuromuscular junction

The influence of the activation of presynaptic adenosine receptors on nicotinic autofacilitation of electrically evoked [ 3H]acetylcholine release from rat phrenic motor nerve terminals was investigated. Blocking the adenosine A 2A receptor with 3,7-dimethyl-1-propargylxanthine (DMPX, 10 μM0 greatly potentiated, whereas the adnosine A 1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 2.5 nM), partially prevented the facilitatory effect of the nicotinic receptor agonist. 1,1-dimethyl-4-phenylpiperazinium (DMPP, 1 μM, 3 min), on evoked [ 3H]acetylcholine release. The adenosine A 2A receptor agonist, 2-[ p-(2-carboxyethyl)phenethylaminol]-5′- N-ethylcarboxamideadenosine (CGS 21680C, 3 nM), but not the adenosine A 1 receptor agonist, R- N 6-phenylisopropyl adenosine (R-PIA, 300 nM), partially blocked the DMPP (1 μM) facilitation. Forskolin (3 μM) mimicked the attenuation caused by CGS 21680C; inhibition of adenylate cyclase with N-(as-2-phenylcyclopentyl)azacyclo-tridecan-2-imine hydrochloride (MDL 12,330A, 10 μM) markedly enhanced the facilitatory effect of DMPP (1 μM). Prolonged exposure to a high concentration of DMPP (10 μM, 15 min) decreased evoked tritium outflow. The decrease in evoked [ 3H]acetylcholine release following prolonged exposure to DMPP was augmented by pretreatment with CGS 21680C (3 nM) and forskolin (3 μM), and was abolished by inactivating endogenous adenosine with adenosine deaminase (0.5 U/ml). It is concluded that tonic adenosine A 2A receptor activation regulates nicotonic acetylcholine artofacilitation. This action is likely to be mediated through an adenylate cyclase/cyclic AMP-dependent mechanism.

Toxicological evaluation of Nerium oleander on isolated preparations

AbstractIn vitro toxicological evaluation of Nerium oleander was carried out using different isolated preparations. The methanolic leaf extract potentiated both spontaneous and electrically evoked contractions of vas deferens of rats and guinea‐pig ileum in a concentration dependent manner. The effect was not antagonized by the adrenergic blocker, tolazoline. The extract inhibited electrically stimulated neurogenic twitch responses of rat phrenic nerve diaphragm preparation. The dose dependent inhibition of twitch response in this preparation could not be reversed by the anticholinesterase drug, neostigmine. The extract evoked a persistent depolarizing effect on the phrenic nerve diaphragm preparation. The extract exhibited its most potent action on the isolated right atrial preparation of rat. It inhibited the rate of spontaneously beating atria in a concentration dependent manner. The negative chronotropic effect was not antagonized either by antimuscarinic drug or adrenergic agonist.

Isolation of a neuromuscular junctional blocking agent from Cleistanthus collinus plant leaf extract

AbstractA potent neuromuscular junctional blocking agent was isolated from chloroform extracts of poisonous Cleistanthus collinus leaf by a combined chromatographic—cholinesterase inhibition technique. An amorphous yellowish green extract was eluted from paper or micro thin layer chromatographic plates. This compound caused a 93% fade on nerve evoked muscle tension and a 95% decrement in nerve evoked muscle action potentials and least change in muscle evoked muscle action potentials and tension in a rat phrenic nerve—diaphragm preparation, suggesting the presence of a potent pharmacologically active ingredient causing neuromuscular blockade which is irreversible.

Potentiation by tonic A2a‐adenosine receptor activation of CGRP‐facilitated [3H]‐ACh release from rat motor nerve endings

1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors. Since both CGRP and A2a-adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.

Activation of internodal potassium conductance in rat myelinated axons.

1. Voltage changes associated with currents crossing the internodal axolemma were monitored using a microelectrode inserted into the myelin sheath (peri-internodal region) of rat phrenic nerve fibres. This microelectrode was also used to change the potential and the ionic environment in the peri-internodal region. 2. Following stimulation of the proximal nerve trunk, the peri-internodal electrode recorded a positive-going action potential whose amplitude increased (up to 75 mV) with increasing depth of microelectrode penetration into the myelin. The resting potential recorded by the peri-internodal electrode remained within 4 mV of bath ground. 3. Confocal imaging of fibres injected peri-internodally with the fluorescent dye Lucifer Yellow revealed a staining pattern consistent with spread of dye throughout the myelin sheath of the injected internode. 4. After ionophoresis of K+ (but not Na+) into the peri-internodal region, the action potential was followed by a prolonged negative potential (PNP) lasting hundreds of milliseconds to several seconds. The duration of the PNP increased as the frequency of stimulation decreased. PNPs could also be evoked by sub-threshold depolarization of the internodal axolemma with peri-internodally applied current pulses. In the absence of action potentials or applied depolarization PNPs sometimes appeared spontaneously. 5. Peri-internodal application of Rb+ also produced evoked and spontaneous PNPs. These PNPs had longer durations (up to 20 s) than those recorded from K(+)-loaded internodes. 6. Spontaneous action potentials sometimes appeared during the onset of the PNP, suggesting that PNPs are associated with depolarization of the underlying axon. 7. Passage of current pulses during the PNP demonstrated that the PNP is associated with an increased conductance of the pathway linking the peri-internodal recording site to the bath. At least part of this conductance increase occurs across the internodal axolemma, since peri-internodally recorded action potentials evoked during the PNP had larger amplitudes than those evoked before or after the PNP. 8. PNPs were suppressed by tetraethylammonium (TEA, 10-20 mM) and by 4-aminopyridine (1 mM). 9. These results suggest that the PNPs recorded in K(+)- or Rb(+)-loaded myelin sheaths are produced by a regenerative K+ or Rb+ current that enters the internodal axolemma via K+ channels opened by action potentials or subthreshold depolarizations. 10. When normal extracellular [K+] was preserved (by using Na+ rather than K+ salts in the peri-internodal electrode), action potentials recorded within the myelin sheath were instead followed by a brief, positive after-potential that was inhibited by TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

Myelin-axon relationships in the rat phrenic nerve: longitudinal variation and lateral asymmetry.

It is known that the myelin sheath thickness-axon perimeter relationship varies between peripheral nerves. This study examines the possibility that that relationship may vary between levels along a given nerve or between corresponding levels of the right and left examples of the same nerve. The relationship is examined for large and small fibre classes at well separated upper and lower intrathoracic levels in the rat phrenic nerve. The study shows that the myelin-axon relationship differs between levels along the same nerve bundle in the same (intrathoracic) environment. Thus, for a given increase in the perimeter of large axons, sheath thickness increases significantly more at lower than at upper levels. In addition, myelin sheath thickness shows a statistically significant lateral asymmetry in favour of the left side for the large fibre class at the upper thoracic level. The setting of the myelin sheath thickness-axon perimeter relationship also differs between the large and small fibre classes at each level examined. Large fibres have proportionately thicker sheaths than small fibres and this difference is reflected in the significantly smaller g-ratio of the former. Systematic differences in the setting of the myelin sheath thickness-axon perimeter relationship between large and small fibre classes may be a widely occurring phenomenon. It may be concluded that the myelin-axon relationship varies significantly both within and between nerves and also between fibre classes. Accordingly, morphometric studies of normal or pathological nerves should take into account possible consistent longitudinal variation or lateral asymmetry in fibre parameters and myelin-axon relationships within a given nerve bundle or fibre class, in order to avoid introducing systematic bias and to minimize variance between samples.

Comparison of the effects of mercuric chloride and methylmercuric chloride on the isolated phrenic nerve and on the phrenic nerve-diaphragm preparation from the rat

This study compared the effects of HgCl 2 and CH 3HgCl on the compound action potential of the isolated rat phrenic nerve and the twitch contractions of rat phrenic nerve-diaphragm preparations during direct and indirect stimulation. Both HgCl 2 and CH 3HgCl, at a concentration of 3.7 × 10 −5 m, inhibited the twitches during indirect stimulation. CH 3HgCl was slightly more potent than HgCl 2. The inhibition might be explained by a simultaneous inhibition of the phrenic nerve. CH 3HgCl caused an additional curare-like inhibition at the neuromuscular junction. HgCl 2 potentiated the indirectly elicited twitches before the inhibitory effect ensued. The twitch potentiation was inhibited by low temperature (20°C), high Ca 2+ (10.8 m m) and d-tubocurarine (7.3 × 10 −7 m). It was probably caused by multiple excitation of some of the nerve terminals. Both agents were less efficient at inhibiting the preparations during direct stimulation than during indirect stimulation. However, during direct stimulation, HgCl 2 was a considerably more potent inhibitor than CH 3HgCl. Both mercury compounds caused a slow increase in the baseline tension. An additional rapid initial phase with HgCl 2 could be partly inhibited by dantrolene (1.5 × 10 −5 m), high Ca 2+ (10.8 × 10 −3 m) or low temperature (20°C), indicating that it was a depolarization contracture. The slow phase was not affected by dantrolene, and may be characterized as a rigor. The effects of HgCl 2 and CH 3HgCl were irreversible on washing, but they could be partly antagonized by washing plus exposure to dithiothreitol, to keep SH groups in the reduced state.

Effects of hexamethonium on transmitter release from the rat phrenic nerve.

Hexamethonium (HEX) was applied to isolated transected diaphragm-phrenic nerve preparations of the rat in order to further elucidate the functional role of the presynaptic nicotinic autoreceptors. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the neuromuscular junctions in the presence and absence of HEX to determine the relative effect of this nicotinic antagonist on end-plate sensitivity and evoked release. In this study we show that HEX enhances transmitter release for the first few stimuli, but this action is not maintained during a train-of-six stimulation. While these results support the hypothesis that transmitter released from the nerve terminal normally has a negative feedback effect by depressing transmitter release it is proposed that HEX also has secondary actions on the neuromuscular junction that are unrelated to autoreceptor blockage. The results with HEX suggests that the presynaptic receptors may differ pharmacologically from the postsynaptic receptors.

The effect of potential therapeutics on ciguatoxin inhibition of the rat phrenic nerve: A. W. Wong Hoy and R. J. Lewis (Southern Fisheries Centre, Queensland Department of Primary Industries, Deception Bay, Queensland 4508, Australia).

The effect of potential therapeutics on ciguatoxin inhibition of the rat phrenic nerve: A. W. Wong Hoy and R. J. Lewis (Southern Fisheries Centre, Queensland Department of Primary Industries, Deception Bay, Queensland 4508, Australia).

Neurotoxic effect of two root canal sealers with calcium hydroxide on rat phrenic nerve in vitro

Root canal sealers placed into contact with the inferior alveolar nerve can cause paraesthesia in the area innervated by this nerve. The paraesthesia is due to neurotoxic effects of the sealers. In this study we examined the effect on nerve conduction in vitro of two calcium hydroxide-containing root canal sealers, Sealapex and CRCS. An isolated rat phrenic nerve was placed between two suction electrodes in a bath containing Tyrode solution. The sealers were brought into contact with the nerve, and the evoked compound action potentials (cAP) were recorded before and after contact with the materials. Both Sealapex and CRCS induced a fast and complete inhibition of the cAP in this model. The degree of reversibility of the inhibition varied from experiment to experiment. Both materials exhibited reversible and irreversible block of the nerve conduction after 90s and 5 min exposure. However, after 30 min of contact, the conduction of the cAP was irreversibly blocked for both materials.

Branching patterns of the rat phrenic nerve during development and reinnervation

Previous studies have shown that the phrenic motor nucleus in the rat projects onto the diaphragm muscle, forming an orderly topographic map. Moreover, this topography is partially restored upon reinnervation. This orderly map is expressed prior to birth, suggesting that early contacts between nerve and muscle are topographically appropriate. The phrenic divides during embryonic development into rostral and caudal branches, and motor axons preferentially enter the appropriate branch. In an effort to understand the mechanisms that underlie the choices growing phrenic neurons make in selecting their appropriate muscle targets, we examined the patterns of branching displayed by the phrenic nerve during development and reinnervation. In all muscles studied the phrenic nerve splits into three primary branches, rostral, caudal, and crural. At a coarse level the pattern of branching of the phrenic is remarkably consistent from animal to animal and at all ages of development. At a finer level of resolution, however, there is an asymmetry between right and left hemidiaphragms. Moreover, the precise emergence of any particular branch is unpredictable, resulting in an overall incongruence in branching architecture from animal to animal. The hemidiaphragm muscle grows unevenly, particularly on the right side, resulting in greater muscle fiber elongation medially. Upon reinnervation, the same coarse pattern of branching is reestablished, but the higher order pattern is much simpler and muscle growth is slower than in controls. These results suggest that very early in development primary branches of the phrenic funnel axons into three well-defined zones in the muscle. These observations taken together with previous physiological studies indicate that an orderly topographic map can emerge in spite of considerable variation in the pattern of branches.

Backfiring of the isolated rat phrenic nerve does not collide with impulse propagation following repetitive nerve stimulation at 1?50 Hz

Acetylcholinesterase inhibition with neostigmine in the isolated rat phrenic nerve-hemidiaphragm preparation induced axonal backfiring and repetitive compound muscle action potentials following single nerve stimulation. The duration of backfiring and the repetitive compound muscle action potentials did not exceed 55 ms. With repetitive nerve stimulation at frequencies ranging from 1 to 50 Hz, backfiring was present only with the first stimulus and the amplitude of the second compound muscle action potential was maximally reduced, while the subsequent responses recovered gradually. However, the amplitudes of the concomitant antidromic nerve action potentials remained unchanged during the entire train of stimulation. Lack of nerve action potential amplitude changes and the short duration of backfiring of the first nerve action potential exclude a collision phenomenon of backfiring with the nerve action potential induced by the second stimulus. Moreover, the duration of the repetitive compound muscle action potentials did not exceed the duration of backfiring. Therefore, the prolongation of the muscle membrane refractory period by reexcitation following backfiring cannot explain the decrement of the second compound muscle action potential.

STIMULATION OF β1‐ADRENOCEPTORS ENHANCES ELECTRICALLY EVOKED [3H]‐ACETYLCHOLINE RELEASE FROM RAT PHRENIC NERVE

1. The effects of isoprenaline, noradrenaline and fenoterol on the electrically evoked release of [3H]-acetylcholine from the rat phrenic nerve were investigated. 2. Isoprenaline (0.1 mumol/L) and noradrenaline (1 mumol/L) enhanced evoked [3H]-acetylcholine release by about 90%, an effect which was abolished by CGP 20712A (0.1 mumol/L), a specific antagonist at beta 1-adrenoceptors. Noradrenaline still enhanced [3H]-acetylcholine release in the presence of phentolamine (1 mumol/L). 3. The enhancing effect of both isoprenaline and noradrenaline decreased at prolonged exposure times (24-32 min). A pre-exposure of the tissue to a low concentration (0.01 mumol/L) of isoprenaline prevented the enhancing effect of 0.1 mumol/L isoprenaline. 4. Fenoterol, a specific agonist at beta 2-adrenoceptors, did not modify evoked [3H]-acetylcholine release. 5. The present results indicate the existence of facilitatory beta 1-adrenoceptors on the motor nerve. These receptors appear to be desensitized either by a high concentration of, or by a long exposure to, agonists. Under the present conditions noradrenaline enhances the release of newly synthesized transmitter mainly by stimulation of beta-adrenoceptors.

Differential blockade by nifedipine and ω-conotoxin GVIA of α1- and β1-adrenoceptor-controlled calcium channels on motor nerve terminals of the rat

Electrically evoked release of [ 3H]acetylcholine ([ 3H]ACh) from the rat phrenic nerve and its facilitation by stimulation of presynaptic α 1- and β 1-adrenoceptors were investigated in the absence and presence of nifedipine and ω-conotoxin GVIA. Both calcium channel antagonists did not modify electrically evoked [ 3H]ACh release, but selectively blocked the effect triggered by both facilitatory adrenergic receptors. The increase in [ 3H]ACh release mediated via β 1-adrenoceptor activation was abolished by low concentrations (1 nM) of ω-conotoxin GVIA, whereas nifedipine (100 nM) abolished the facilitatory effect mediated via α 1-adrenoceptor stimulation. Therefore, the β 1-adrenoceptor is apparently coupled to a calcium channel that can be regarded as of the N-type, and the α 1-adrenoceptor is apparently coupled to a calcium channel that appears as a subtype of the L-type which is not sensitive to ω-conotoxin GVIA.

Dose-response inhibition of rat compound nerve action potential by dextropropoxyphene and codeine compared to morphine and cocaine in vitro

1. The effects of dextropropoxyphene hydrochloride (dextropropoxyphene) codeine phosphate (codeine), morphine hydrochloride (morphine), and cocaine hydrochloride (cocaine) on the compound action potentials (cAP) of the rat phrenic nerve were studied in vitro. 2. Dextropropoxyphene, cocaine, codeine and morphine depressed the cAP in a reversible, dose-dependent way. After 60 min exposure to the drugs, dextropropoxyphene, inhibited the cAP between 0.025 and 0.5 mM, cocaine between 0.01 and 5 mM, codeine between 0.1 and 10 mM and morphine between 0.5 and 70 mM. The inhibition caused by 5 mM morphine could not be reversed by naloxone. 3. Dextropropoxyphene demonstrated the most potent cAP inhibition compared to the other drugs tested. The observed dose range for the inhibition of the cAP of rat phrenic nerve includes that observed after intoxications with dextropropoxyphene (0.004–0.06 mM) in the clinic. 4. For all other drugs tested the present observed dose ranges are much greater than the plasma concentrations previously reported after therapeutic use and intoxication.

Stimulation of α 1-adrenoceptors increases electrically evoked [ 3H]acetylcholine release from the rat phrenic nerve

The modulation by sympathomimetic amines of the electrically evoked [ 3H]acetylcholine release from the motor nerve was investigated. Phenylephrine (10 μmol/1), α-methylnoradrenaline (10 μmol/1) and adrenaline (1 μmol/1) enhanced the electrically evoked [ 3H]acetylcholine release from the rat phrenic nerve. The enhancing effect of phenylephrine was completely prevented by low concentrations of prazosin (0.1 or 0.01 μmol/1) whereas a high concentration of yohimbine (1 μmol/1) was necessary to abolish the effect of phenylephrine. The simultaneous blockade of α- and β-adrenoceptors, i.e. propranolol (0.1 μmol/1) together with prazosin (0.01 μmol/1) or yohimbine (1 μmol/1), was required to abolish the enhancing effect of α-methylnoradrenaline. Likewise, the enhancing effect of adrenaline could be abolished only by a combination of two antagonists (propranolol together with yohimbine or prazosin). Neither clonidine nor oxymetazoline (1 or 10 μmol/1) modulated the evoked [ 3H]acetylcholine release. The experiments showed the existence of α-adrenoceptors which are present on the motor nerve and whose stimulation mediates an increase in evoked transmitter release. The different potencies found for prazosin and yohimbine indicate that an α 1-subtype of the receptors was involved. Increased sympathetic activity may facilitate neuromuscular transmission by stimulation of presynaptic α- and β-adrenoceptors.

Pertussis toxin prevents the inhibitory effect of adenosine and unmasks adenosine-induced excitation of mammalian motor nerve endings.

Pertussis toxin (PTX), which blocks certain classes of guanine nucleotide binding proteins (G proteins), consistently blocked the inhibitory effects of adenosine (100 microM-250 microM) on quantal acetylcholine (ACh) secretion in rat phrenic nerve hemidiaphragm preparations. PTX pretreatment also highlighted long-lasting increases in evoked ACh release elicited by adenosine. The results suggest that specific G proteins are involved in mediating the inhibitory effects of adenosine at motor nerve endings.

Ca antagonistic and non-specific effects of diltiazem on the rat phrenic nerve diaphragm preparation

The calcium antagonist diltiazem (2.8 × 10 −4 M) blocked the twitches of a rat phrenic nerve diaphragm preparation after a period of twitch potentiation. Its ability to block twitches was greater during indirect than direct stimulation. Experiments on the isolated phrenic nerve indicated that the excitability of the nerve was blocked. Diltiazem (2.3–9.0 × 10 −5 M) caused a similar inhibition of indirectly and directly elicited tetanic contractions and EMG. Experiments with d-tubocurarine and lowered temperature disclosed a separate inhibition at the neuromuscular junction. High Ca 2+ did not reverse the diltiazem-affected twitch or tetanic contractions, which suggests that they are non-specific effects. KCl (100 mM)-induced contractures were antagonized at low (2.3–4.5 × 10 −5 M) but not at high (1 mM) concentrations of diltiazem. Diltiazem depressed the initial phase of the two-phasic caffeine (10 mM) contracture and increased and accelerated the slow phase. Diltiazem greatly reduced the amplitude and duration of the caffeine-potentiated KCl contracture, and reduced and delayed the slow phase of the KCl-potentiated caffeine contracture. The effects on the combined contractures (caffeine-induced, KCl-potentiated) were partly antagonized by a high Ca 2+ (2.2 × 10 −5 M) solution, which suggests that diltiazem has calcium antagonistic effects.

Fatigue during continuous 20 Hz stimulation of the rat phrenic nerve diaphragm preparation.

The site and mechanism of action of fatigue was investigated in the isolated rat phrenic nerve diaphragm preparation, with indirect and direct stimulation at 20 Hz and recording of tension and EMG. An equal decay of the subtetanic tension during indirect and direct stimulation, and a parallel decay of tension and EMG, suggested a mechanism of fatigue localized to structures that were 'seen' by the EMG electrodes. A comparison of the responses to sub- and supra-maximum direct stimulation did not show increased fatigue at sub-maximum stimulation. Therefore, the fatigue was probably not caused by an increased threshold of the excitability of the sarcolemma. However, prolongation of the stimulus pulse during direct stimulation from 0.5 ms to 5 ms in the fatigued preparation caused a two-phasic recovery of tension. The initial phase, but not the slow phase, was inhibited by tetrodotoxin (TTX). Thus a recovery of sarcolemma action potentials could explain the initial phase. The slow phase was probably caused by a mechanism localized at more distal potential-dependent sites, probably in the T tubules.

Facilitatory and inhibitory muscarine receptors on the rat phrenic nerve: effects of pirenzepine and dicyclomine.

Neuronal transmitter stores of the rat phrenic nerve were labelled by an incubation with [3H]choline. Release of [3H]acetylcholine was elicited either by a short (100 pulses, 5 Hz) or by a long (1500 pulses, 5 or 25 Hz) period of electrical nerve stimulation. Pirenzepine and dicyclomine enhanced transmitter release evoked by the short stimulation period. Both antagonists reduced transmitter release evoked by the long stimulation period. Pirenzepine reduced transmitter release at low concentrations (1 nmol/l) whereas a higher concentration was necessary for the enhancing effect; the opposite pattern was found for dicyclomine. A low concentration of oxotremorine (10 nmol/l) enhanced and a high concentration (1 mumol/l) reduced transmitter release evoked by the short stimulation period. Both effects could be prevented by a low concentration of pirenzepine (10 nmol/l). It is concluded that facilitatory and inhibitory muscarine receptors are present on the motor nerve. A short stimulation period activates predominantly the negative muscarinic feedback, whereas during a long period of continuous nerve stimulation the positive muscarinic feedback mechanism is additionally activated. Both the facilitatory and inhibitory receptors might be regarded as M1-receptors but differences in the pharmacological properties between both receptor populations appear possible.