The effect of 3,3-dipyridylmethyl-1-phenyl-2-indolinone on the neuromuscular transmission in the rodent skeletal muscles.

The effects of a cognition enhancer, 3,3-dipyridylmethyl-1-phenyl-2-indolinone (DPMPI) (21.5-645 microM), on neuromuscular transmission were studied electrophysiologically on diaphragms of mouse and rat and the soleus muscle of rat. The drug DPMPI (21.5-645 microM) increased both direct and indirect twitch tension of mouse diaphragm. It also increased (a) the frequency of miniature endplate potentials and (b) the quantal content of endplate potential. However, DPMPI (64.5 microM) affected neither the amplitude of the directly elicited action potential of soleus muscle in the rat nor the magnitude of the resting membrane potential of mouse diaphragm, although DPMPI (215 microM) decreased the amplitude of the compound action potential of phrenic nerve. Based on these results, it is concluded that DPMPI had several effects on neuromuscular transmission, i.e. it (a) facilitated the transmitter releasing process of the motor nerve terminal, (b) decreased the conduction in the phrenic nerve and (c) increased the directly elicited twitch tension.     

Effects of the sulphydryl inhibitor N-ethyl-maleimide on the phrenic nerve and diaphragm muscle of the rat

N-Ethyl-maleimide (NEM, 2.5 x 10(-5) M) inhibited the compound action potential of the phrenic nerve and increased the spontaneous release of transmitter from the nerve terminals, recorded as miniature endplate potentials. The first effect was the cause of a blockade of the phrenic nerve diaphragm preparation, during indirect stimulation. The left phrenic nerve was more susceptible to inhibition than the right. An increase of the threshold was observed during the progression of the inhibition. The inhibition was not use-dependent and there was no synergistic interaction with the local anaesthetic drug, tetracaine. The inhibition was partly antagonized by di-thio-threitol (3.0 x 10(-3) M). The increase of spontaneous release of transmitter was not accompanied by an increase of the stimulus-evoked release since the amplitude of the endplate potential was not increased and partial inhibition caused by d-tubocurarine or magnesium chloride was not antagonized. When the concentration of NEM was increased to 2.75 x 10(-4) M, the directly-elicited twitches were inhibited, and the baseline tension was increased. This increase of tension was slightly reduced in a preparation depolarized with potassium chloride; a small depolarization could partly explain this effect. It was not reduced by dantrolene or in a calcium-free solution. The inhibition of the twitch and the increased baseline tension (probably a rigor) might be caused by a reduced sensitivity of the contractile proteins for calcium ions and an inhibition of the myosin ATPase activity, respectively.     

A selective inhibitor of cAMP-specific phosphodiesterase, Ro 20-1724, has no effect on the quantal release of acetylcholine from the mouse phrenic nerve

The indirect twitch response of the mouse isolated phrenic nerve-diaphragm preparation, partially paralysed by tubocurarine, was restored only by about 10% by Ro 20-1724 at 2 to 280 microM. The solvent vehicle, dimethylsulphoxide, also showed the same effect to a similar extent. Intracellular recordings with glass microelectrodes revealed that Ro 20-1724 (40 microM) affected neither the resting membrane potential, the amplitude and frequency of miniature endplate potentials nor the amplitude of nerve-impulse evoked endplate potentials recorded in curarized preparations. The result indicates that Ro 20-1724 at a concentration four times the IC50 of phosphodiesterase inhibition has no effect on the quantal release of acetylcholine from a mammalian motor nerve and suggests that cAMP has no modulatory effect on the transmitter release.     

A pharmacological study of the effect of carbamazepine on neuromuscular transmission in the rat diaphragm

The effects of carbamazepine (0.042-0.42 mM) on neuromuscular transmission were studied on the isolated rat phrenic nerve diaphragm preparation using standard pharmacological and electrophysiological methods. Carbamazepine decreased (1) the antidromic activity of the phrenic nerve, (2) the amplitude of the endplate potential (EPP) and miniature endplate potential (MEPP), (3) the quantal content of the endplate potential, (4) the indirectly-elicited twitch tension, (5) the muscle contracture in chronically denervated muscle induced by acetylcholine (ACh) and (6) the amplitude of the compound phrenic nerve action potential, in a concentration-dependent manner. The antidromic activity of the phrenic nerve was the most affected, while the phrenic nerve compound action potential was least affected. However, the IC50 for carbamazepine (the concentration of carbamazepine that inhibited 50% of the response) was in the same order of concentration, i.e. 0.11-0.3 mM. Compared with the effect of carbamazepine on the indirectly-elicited twitch tension with its actions described above, it is concluded that carbamazepine interfered with the neuromuscular activity by inhibiting pre- and postsynaptic process and conduction in the phrenic nerve.     

Effects of butanedione monoxime on neuromuscular transmission.

1. The amplitude of endplate potentials was increased by concentrations of butanedione monoxime (BDM, 5-20 mM) that typically caused muscle paralysis. 2. Although BDM slowed the decay of spontaneous miniature endplate currents, the effect was insufficient to explain most of the large increase in amplitude of endplate potentials. 3. The quantal content of endplate potentials was increased by BDM in a reversible, concentration-dependent manner. 4. The frequency of miniature endplate potentials was not changed by 10 mM BDM in the presence of normal or raised potassium concentrations, indicating that BDM does not change quantal content by a direct effect on calcium channels or on steady-state intracellular calcium concentration. 5. A change in the time course of the extracellularly recorded nerve terminal action potential caused by BDM was similar to the change produced by 4-aminopyridine (4-AP). 6. The increase in quantal content produced by BDM was only slightly reduced in the presence of 1 mM tetraethylammonium (TEA) but was significantly reduced in the presence of 0.5 to 1 mM 4-AP. 7. It was concluded that BDM blocks a 4-AP-sensitive potassium conductance in motor nerve terminals and, by increasing the duration of the action potential in this way, increases evoked transmitter release.     

Inhibition of quantal release from motor nerve by wortmannin.

1. The effects of wortmannin, an inhibitor of phosphatidylinositol (PI) kinases and myosin light chain kinase, on the quantal release of neurotransmitter from mouse phrenic nerve were investigated. 2. Wortmannin (10 - 100 microM) initially enhanced, thereafter progressively depressed spontaneous quantal discharge (miniature endplate potential, mepp). The mean amplitude and the amplitude distribution of mepp were not altered. 3. The compound inhibited and prevented the intensive quantal release evoked by high KC1 solution as well as the mepp burst induced by alpha-latrotoxin, a polypeptide toxin that possesses Ca2+-independent synaptic action to trigger quantal release. The inhibitory actions of wortmannin were partially reversible. 4. Wortmannin depressed the amplitude of endplate potentials (epps) and increased the coefficient of variance of epps. The profile of epps in response to high frequency nerve stimulation exhibited fluctuations between run-down and run-up. The phenomenon is thus different from the consistency of run-up characteristic as the motor nerve Ca2+ channel is blocked by omega-agatoxin IVA. 5. LY294002, another inhibitor of PI 3-kinase, raised mepp frequency without causing late phase suppressions. The compound did not inhibit KC1-, alpha-latrotoxin- or nerve stimulation-evoked quantal release. 6. The results suggest that wortmannin could depress quantal release beyond the step of Ca2+ channel blockade, probably by interfering with the exocytotic cascade.     

The effect of carbamazepine on the post-tetanic twitch tension in the diaphragm of the mouse

The effects of carbamazepine (0.084-0.25 mM) on the post-tetanic potentiation of the twitch tension, were studied on the isolated phrenic nerve diaphragm preparation of the mouse. Carbamazepine decreased the post-tetanic potentiation of the twitch tension. The maximal depressant effect was found after higher frequencies and longer durations of stimulation. After repetitive stimulation, the amplitude of the endplate potential and the frequency of miniature endplate potentials were potentiated. Carbamazepine decreased the post-tetanic potentiation of the amplitude of endplate potential and the frequency of miniature endplate potentials. The directly-elicited muscle action potential was not affected when post-tetanic potentiation of the twitch tension was decreased. It is concluded that carbamazepine suppressed the post-tetanic potentiation of the indirectly-elicited twitch tension, mainly due to its pre-synaptic inhibitory effect.     

The effects of tetraethylammonium during twitch and tetanic stimulation of the phrenic nerve diaphragm preparation in the rat

Tetraethylammonium (TEA) (2.6 x 10(-3) M) potentiated the twitches of the indirectly- or directly-stimulated phrenic nerve diaphragm of the rat at 37 degrees C by prolonging the action potential of the sarcolemma, due to an inhibition of the repolarizing K+ current. With indirect stimulation, TEA caused a use-dependent inhibition of tetanic contractions, induced every 10 min by 10 sec of 50 Hz stimulation, and a post-tetanic depression of the twitches was observed after about 40 min. Recording of the electromyogram (EMG) and compound action potentials of the phrenic nerve, localized the two inhibitory effects to the neuromuscular junction. They were caused by different mechanisms of action. Choline (3.6 x 10(-4) M) antagonized the depression of the twitch but not the use-dependent inhibition. Lowering the temperature to 20 degrees C reduced the depression of the twitch, whereas the use-dependent inhibition was enhanced. The release of transmitter was probably normal during tetanic stimulation; a post-synaptic desensitization of acetylcholine (ACh) receptors caused the inhibition. Microelectrode recordings of endplate potentials supported this conclusion. The depression of the twitch was due to a presynaptic depletion of transmitter. This was confirmed by inducing an additional depletion and depression of the twitch with N-ethyl-maleimide (2.5 x 10(-5) M). Since the depression of the twitch was antagonized by choline, the depletion was probably due to an inhibited uptake of choline into the nerve terminals.     

An anti-curare effect of hexamethonium at the mammalian neuromuscular junction

1. Experiments were performed on the isolated phrenic nerve and diaphragm preparation of the rat.2. In preparations partly blocked with (+)-tubocurarine, the twitch amplitude increased after hexamethonium. This enhancement was not seen in preparations partly blocked with Mg(++) or with gallamine. High concentrations of hexamethonium produced failure of contraction.3. Extracellular endplate potentials were recorded from blocked preparations. The administration of hexamethonium resulted in an increased amplitude of these potentials only in curarized muscle.4. Hexamethonium had no anticholinesterase activity nor did it depolarize muscle cells or increase the quantal release of transmitter.5. It is concluded that hexamethonium exerts a specific anti-curare action. Experiments on the recovery of the twitch after washing out antagonists indicate that this process is limited by diffusion. The difference in rates of diffusion of hexamethonium and (+)-tubocurarine does not account for their interaction. The basis of the anti-curare action of hexamethonium is discussed.     

Reduction of quantal size and inhibition of neuromuscular transmission by bafilomycin A

The energy for uphill transport of neurotransmitters into synaptic vesicles is created by bafilomycin A- and concanamycin A-sensitive vacuolar H(+)-ATPase (V-ATPase). Both blockers (at 0.1-5 microM) depressed twitch tension and induced tetanic fade of mouse diaphragm on stimulation of the phrenic nerve. Axonal impulse conduction and depolarization of motor endplate by exogenous acetylcholine were not inhibited. The IC(50)s for bafilomycin A and concanamycin A were 1.1+/-0.2 and 0.7+/-0.1 microM, respectively. Contractile response evoked by stimulation of diaphragm, muscle resting membrane potential and membrane resistance were not altered. V-ATPase blockers decreased quantal size and shifted the distribution of miniature endplate potentials (mepps) to low amplitude direction. The increase of mepp events in high KCl medium was suppressed slightly. The blockers depressed endplate potentials (epps) with IC(50)s of 0.7+/-0.2 microM (bafilomycin A) and 0.4+/-0.1 microM (concanamycin A). On high frequency stimulation, the coefficient of variance and run-down of epps were increased. The inhibitory effects on mepps and epps were irreversible and augmented by nerve stimulation. The results suggest that inhibition of V-ATPase reduces the acetylcholine content of synaptic vesicles, leading to suppression of neuromuscular transmission.     

Muscle paralyzing effect of the juice from the trunk of the banana tree

Y. N. Sinsgh and W. F. Dryden. Muscle paralyzing effect of the juice from the trunk of the banana tree. Toxicon23, 973–981, 1985. — The effect of an extract from the trunk of the banana tree (Musa sapientum) was investigated in isolated skeletal muscle preparations from the chick, mouse and frog using twitch tension and intracellular recording techniques. The extract produced, in the same concentration range and after an initial period of twitch augmentation, paralysis of skeletal muscle in both directly and indirectly stimulated preparations. It also had a dosedependent stimulant effect on the muscle causing a contracture. The neuromuscular blockade was reversed by calcium, but only when added before complete paralysis of the muscle. On the other hand, neostigmine usually hastened the blockade and aggravated the contracture. The frequency of the miniature endplate potential in the mouse phrenic nerve — diaphragm preparation greatly increased initially, declining to an elevated plateau. Effects on quantal content of endplate potentials e.p.p.s were studied in the transected mouse phrenic nerve — hemidiaphragm using trains of e.p.p.s. In the presence of the extract, only a few e.p.p. trains could normally be evoked, probably due to nerve terminal block. When quantal content could be measured at low concentrations of the extract, an increase was usually obtained. Muscle action potentials in the frog sartorius muscle were decreased in amplitude until no further potentials could be generated. The results suggest that the nature of the block produced by the extract resembles that of a potent local anaesthetic with an initial atypical labilizing effect on cell calcium rather than a conventional curariform block.     

The neuromuscular blocking action of spectinomycin on the mouse hemidiaphragm preparation

1. The mechanism of action of the muscle paralysing property of the antibiotic spectinomycin has been investigated on the isolated mouse phrenic nerve-hemi-diaphragm preparation. 2. Spectinomycin produced a neuromuscular blockade that was poorly reversed by neostigmine (3 μmol/l) but well reversed by both calcium (5 mmol/1) and 3,4-diaminopyridine (0.1 mmol/1). 3. Intracellular techniques were used to record endplate potentials and miniature endplate potentials from hemidiaphragms paralysed by spectinomycin. Miniature endplate potentials were reduced both in frequency and in amplitude. The quantal content of the endplate potential during spectinomycin paralysis calculated by the method of variance was 14, a value similar to that found in preparations paralysed by the prejunctionally active magnesium. 4. Thus spectinomycin possesses weak neuromuscular blocking activity by a predominant action on acetylcholine release.     

Blocking effects of hypaconitine and aconitine on nerve action potentials in phrenic nerve-diaphragm muscles of mice

The mechanisms of neuromuscular blockade by hypaconitine and aconitine were investigated electrophysiologically in isolated phrenic nerve-diaphragm muscles of mice. Hypaconitine (0.08-2 microM) and aconitine (0.3-2 microM) depressed the nerve-evoked twitch tension, without affecting the contraction evoked by stimulation of the muscle. At the concentrations of hypaconitine (up to 5 microM) and aconitine (up to 2 microM) that depressed the nerve-evoked twitch tension, the resting membrane potential of the muscle cells was unchanged. Hypaconitine (0.1-2 microM) and aconitine (2 microM) blocked the end-plate potential (epp), without affecting the amplitude of the miniature epp (mepp). The quantal content of end-plate potentials was decreased by these agents in parallel with the decrement in amplitude. The nerve compound action potential was inhibited by hypaconitine (5 microM) and aconitine (2-10 microM), as well as by 1 microM tetrodotoxin (TTX). When the nerve compound action potential was completely blocked by 2 microM aconitine, the muscle action potential was unaffected, although 1 microM TTX suppressed both potentials to the same degree. These results indicate the neuromuscular blockade produced by hypaconitine and aconitine were caused by reducing the evoked quantal release. The mechanism of this effect was attributed mainly to blocking of the nerve compound action potential.     

Effects of the sea anemone Anemonia sulcata toxin II on skeletal muscle and on neuromuscular transmission

Effects of anemone toxin II (ATX II) have been analysed on the neuromuscular junction of the frog and different twitch muscles. Amplitudes of evoked endplate potentials and endplate currents are increased by ATX II, without effects on the amplitudes of miniature endplate potentials and endplate currents resulting from ionophoretically applied transmitter. The increase in evoked transmitter release is due to an increase in quantal content caused by an effect of the toxin on the presynaptic action potentials. ATX II is also effective on muscle fibers. The action potentials of frog twitch muscles are reversibly prolonged by ATX II. Their rate of rise and amplitudes are increased, while there is no effect on resting membrane potential. Similarly, action potentials of fast twitch muscle (extensor digitorum longus, EDL) of the mouse are reversibly prolonged by ATX II. In slow twitch muscle (soleus, SOL) of the mouse the toxin induces repetitive action potentials following the generation of a single action potential. Tetrodotoxin resistant action potentials of both denervated EDL and SOL are greatly and irreversibly prolonged by ATX II. The effects on muscle are due to a Na+ channel specific action of ATX II. Na+ current inactivation is slowed with the time constant tau h increasing towards positive membrane potentials. The steady state inactivation curve hoo was shifted to more positive potentials and its slope reduced.     

Relative potencies of metal ions on transmitter release at mouse motor nerve terminals.

1. The effects of a range of metal ions were systematically studied at the mouse neuromuscular junction in order to investigate the type of calcium channel present at the nerve terminal. 2. Endplate potentials and miniature endplate potentials were recorded from the phrenic nerve diaphragm muscle preparation with glass microelectrodes. 3. Endplate potential amplitudes and quantal contents were reduced by manganese (IC50 220 microM), cadmium (IC50 11 microM), cobalt (IC50 350 microM), and nickel (IC50 420 microM). Miniature endplate potentials were not affected by these ions at concentrations equal to the IC50s. Gadolinium did not reduce endplate potentials up to 100 microM. 4. Comparisons made with known channel types in neuroblastoma cell lines suggest that the calcium channels at the motor nerve terminal are different from those types studied in the cell lines, although most similarity is shown to the high-voltage activated calcium channel types.     

Neuromuscular actions of sodium selenite on chick biventer cervicis nerve-muscle preparation.

Sodium selenite was found to be toxic to chicks, with an LD50 of 8.5 micrograms/g, which was increased to 16.3 micrograms/g by NaCN. The major symptoms of chicks, treated with selenite, were sedation and then dyspnea and paralysis. The cause of death by selenite was apparently due to the respiratory failure. The possible mechanism of toxicity was explored in the isolated chick biventer cervicis nerve-muscle preparation. Selenite initially increased the amplitude of the twitch, reversed the suppression of the twitch caused by d-tubocurarine, Mg2+, Cd2+ or Mn2+ and significantly increased the quantal content and amplitude of endplate potentials. Subsequently, selenite depressed the amplitude of the twitch, blocked the axonal conduction and inhibited excitatory postsynaptic potentials. Both NH4+ and K+ enhanced the action of selenite in depressing the twitches. In addition, selenite induced a sustained contracture of the muscle, which was partially inhibited by removal of external Ca2+ and markedly blocked by EGTA. Entry of Ca2+ and release of the internal Ca2+ were considered to be responsible for inducing contracture by selenite. Pretreatment with trypsin, glutathione (GSH) and cyanide profoundly inhibited the effects of selenite, indicating that the site of action of selenite was on the outer membrane and the binding of selenite to the sulfhydryl groups of membrane proteins was proposed to be an essential step for selenite-induced contracture and neuromuscular action. These findings suggest that neuromuscular blockade and tetanic spasm, produced by selenite in chicks, may play a role in causing respiratory failure in vivo.     

Effects of tacrine, velnacrine (HP029), suronacrine (HP128), and 3,4-diaminopyridine on skeletal neuromuscular transmission in vitro.

1. The effects of tacrine (9-amino-1,2,3,4-tetrahydroacridine), velnacrine (HP029, 9-amino-1,2,3,4-tetrahydroacridin-1-ol maleate), suronacrine (HP128, 9-benzylamino-1,2,3,4-tetrahydroacridin-1-ol maleate), and 3,4-diaminopyridine on neuromuscular transmission were compared on isolated nerve-muscle preparations. 2. Tacrine, HP029, and 3,4-diaminopyridine augmented responses of chick biventer cervicis preparations to nerve stimulation, with tacrine and HP029 increasing responses to exogenously applied acetylcholine. HP128 blocked responses to nerve stimulation and to carbachol, but increased responses to acetylcholine. 3. In mouse diaphragm preparations that were partially paralysed by tubocurarine or low calcium solutions, tacrine, HP029, and 3,4-diaminopyridine reversed the twitch block. HP128 deepened the block. 4. In mouse triangularis sterni preparations, tacrine and HP029 prolonged the decay phase of endplate potentials and miniature endplate potentials, but had no effect on quantal content at 36 degrees C; above 10 microM, they reduced endplate potential amplitude. 3,4-Diaminopyridine increased quantal content without affecting the time course of the endplate potentials. HP128 (1-10 microM) had no effect on amplitude or time course of endplate potentials, but reduced their amplitude at higher concentrations. 5. Extracellular recording of nerve terminal currents from triangularis sterni preparations revealed that 3,4-diaminopyridine and HP128 had a selective blocking action on the waveform associated with K+ currents, tacrine reduced and prolonged the K(+)-related waveform, and HP029 had nonselective blocking actions only seen at high concentrations. 6. Tacrine and HP029 behave predominantly as anticholinesterase agents, while HP128 has weaker anticholinesterase actions that are masked by cholinoceptor blockade. Tacrine and HP128, but not HP029, have some blocking actions on K+ currents of mouse motor nerve terminals.     

Mode of neuromuscular blocking action of a toxic phospholipase A2 from Pseudocerastes fieldi (Field's horned viper) snake venom

The effects of a toxic phospholipase A2 (Fr.Cb) isolated from the venom of Pseudocerastes fieldi were studied on the chick biventer cervicis muscle and the mouse phrenic nerve--diaphragm preparations. In the chick muscle, Fr.Cb (10 micrograms/ml) caused complete neuromuscular blockade without producing contracture or affecting the response of the muscle to acetylcholine. In the mouse diaphragm, Fr.Cb blocked the indirectly elicited contraction without affecting that evoked directly. In a low calcium medium (0.5 mM), Fr.Cb produced a triphasic change of the indirectly elicited contractions. The frequency of miniature endplate potentials (m.e.p.p.s) in the mouse diaphragm was first increased 3--4 fold 40 min after toxin (10 micrograms/ml) application, then gradually decreased, while the amplitude of m.e.p.p.s. was not decreased, even after the evoked release of transmitter had failed. Giant m.e.p.p.s were frequently observed. The quantal content first increased and then decreased gradually. The resting membrane potential and the compound phrenic nerve action potential were not significantly affected by the toxin at 10 micrograms/ml after 2 hr of incubation. The motor nerve terminals in the Fr.Cb intoxicated mouse diaphragm showed swelling and vacuolization of both synaptic vesicles and mitochondria. It is concluded that the toxin produces a neuromuscular blockade by acting selectively on the presynaptic site.     

Neuromuscular blocking profile of the vecuronium analogue, Org-9487, in the rat isolated hemidiaphragm preparation.

1. The neuromuscular effects of the short-acting aminosteroid muscle relaxant Org-9487 have been studied in the in vitro rat phrenic nerve/hemidiaphragm preparation by use of twitch tension and electrophysiological recording techniques. 2. Org-9487 (5-100 microM) produced a concentration-dependent decrease in the amplitude of twitches (0.1 Hz) and tetanic contractions (50 Hz) evoked by motor nerve stimulation. The compound produced fade of force during both 50 Hz stimulation and train-of-four stimulation at 2 Hz, indicating a prejunctional component of action. 3. Anticholinesterases only partially reversed the effect of Org-9487 on twitch responses. This was possibly because, at the concentrations required to block twitches in the rat, Org-9487 itself was found to possess significant anticholinesterase activity. 4. Org-9487 (3 microM) increased the rundown of endplate current amplitudes during a 2 s train of 50 Hz nerve stimulation. This was because Org-9487 increased the quantal content of the first endplate current in the train without affecting acetylcholine release towards the latter part of the train. 5. Org-9487 (10 microM) produced a voltage-dependent decrease in the time constant of decay of endplate currents at 32 degrees C and 0.5 Hz, indicative of a block of endplate ion channels. The blocking rate constant increased with membrane hyperpolarization.     

Do motor-nerve terminals have gamma-aminobutyric acid receptors?

1 gamma-Aminobutyric acid (GABA, 0.1 to 1 mM) had no significant effect on the amplitude, rise time, half decay time or frequency of miniature endplate potentials (m.e.p.ps) at the frog or mouse neuromuscular junctions in vitro. 2 Addition of GABA (1 mM) to preparations previously treated with 11 mM K+-Ringer did not cause any further increase in m.e.pp. frequency. GABA also failed to increase the m.e.p.p. frequency in a low Cl--Ringer. 3 GABA (0.1 to 1 mM) did not reduce the high m.e.p.p. frequency induced by veratrine (20 to 40 mg/l). 4 GABA (0.5 to 1 mM) did not affect the amplitude of the extracellularly-recorded nerve terminal spike, whereas 15 mM [K+] reduced the spike. 5 The quantal content (m) of the evoked endplate potential was not significantly altered by GABA; 9 mM [K+] significantly increased m. 6 When external d.c. potential differences were recorded in a three-chambered bath, GABA (0.1 to 1 mM) produced a very small depolarization if applied to the phrenic nerve trunk, but not if applied to the pre-terminal axon/motor nerve terminal region. Carbachol (0.3 to 1 mM) evoked a small depolarization when applied to the nerve terminal chamber. 7 These results fail to provide evidence for the existence of GABA receptors on motor nerve terminals.