Excitatory Neuromuscular Transmission Research Articles

Avermectin B1a, a paralyzing anthelmintic that affects interneurons and inhibitory motoneurons in Ascaris.

Avermectin B1a (AVM) is an antiparasitic agent that paralyzes nematodes without causing hypercontraction or flaccid paralysis. Using selective stimulation techniques, we have shown that AVM blocks transmission between interneuron(s) and excitatory motorneurons in the ventral nerve cord of Ascaris. It also inhibits transmissin between inhibitory motoneurons and muscle but has little effect on excitatory neuromuscular transmission. Picrotoxin can reverse the AVM-induced block of interneuron-excitatory motoneuron transmission but has no effect on the inhibitory motoneuronal synapse in either the presence or absence of AVM. Our results provide an explanation of how AVM may cause paralysis of nematodes.

The pharmacology of insect visceral muscle

1. 1. At least five sites-of-action must be considered in most visceral nerve-muscle preparations. 2. 2. L-Glutamate is perhaps the excitatory neuromuscular transmitter in some organs of the viscera but not others. Proctolin is probably a neuromuscular transmitter in some organs of the viscera defined criteria for rapid chemical transmission. 3. 3. Although 5-HT and certain catecholamines are present in insects and have a pronounced effect on visceral muscle no functional roles has been established for them. 4. 4. Various peptides released from neuroendocrine organs probably regulate visceral muscle function. Moreover such hormonal actions seem to be mediated by cyclic AMP in some cases. 5. 5. Recent developments in methods for detecting peptides and prospective transmitters have increased the possibility of rapid progress in insect nerve-muscle pharmacology.

Evidence for identified dopamine motor neurons to the gill of Aplysia

Dopamine (DA) is present in the gill of Aplysia in considerable quantity. We have investigated the possibility that DA may be an excitatory neuromuscular transmitter. Perfusion of DA through the gill elicits contraction of the same muscles as are activated by the identified motor neurons L 7 and L 9. The cholinergic motor neuron LDG 1 activates different muscles. The contractions induced by DA and three L 9 neurons but not that of L 7 are blocked by ergometrine. These results are consistent with the possibility that the neurons L 9, L 9-A and L 9-P are dopaminergic motor neurons.

A study of excitatory neuromuscular transmission in the bovine trachea

1. The excitatory innervation of bovine tracheal smooth muscle has been studied with the sucrose-gap apparatus.2. Single 2 ms electrical stimuli applied to the whole tissue excited intrinsic nerves, and produced a small transient depolarization of the smooth muscle, the excitatory junction potential (e.j.p.). The e.j.p. caused a twitch-type contraction; twitches and e.j.p.s summated during repetitive stimulation but facilitation was not observed, and action potentials were never elicited.3. The effects of electrical stimulation could be abolished by atropine (5 x 10(-7) mol/l) and augmented by neostigmine (4 x 10(-6) mol/l), and were mimicked by exogenous acetylcholine (1.0 mug/ml).4. With the electron microscope, the density of innervation was found to be low (one axon per ninety smooth muscle cells). Axons were found in small groups in the clefts between bundles of cells, but no axons penetrated within the muscle bundles. Naked axon varicosities containing agranular vesicles were seen, but no axon approached within 200 nm of a smooth muscle cell.5. It is difficult to reconcile the sparsity of innervation with the dependence of the tissue on nerve excitation to initiate activity.

Effect of colchicine and vinblastine on crayfish neuromuscular junction

1. 1. The effect of the microtubule disrupting agents colchicine and vinblastine was studied on crayfish excitatory neuromuscular transmission. 2. 2. Colchicine, in concentrations of 1–3 mM, brought about a decrease in the amplitudes of intracellularly recorded excitatory junctional potentials (ejps). Vinblastine, in concentrations of 0.08–0.32 mM, increased the amplitudes of ejps. 3. 3. Drug-induced alterations in ejp amplitude resulted from changes in quantal content of the excitatory nerve as determined by extracellular recording. The effect could not be explained by changes in quantal size. 4. 4. The effect of colchicine and vinblastine does not appear to be related to an action on microtubules.

Acetylcholine as an excitatory neuromuscular transmitter in the stomatogastric system of the lobster.

THERE is much physiological evidence that L-glutamate acts as an excitatory transmitter at many arthropod neuromuscular synapses1–3. The only suggestion that acetylcholine (ACh) has such a function is due to Futamachi4. He showed that the nerve terminal region innervated by the largest crayfish slow flexor excitatory motor neurone is depolarised by iontophoretic applications of ACh, but not L-glutamate. I now have biochemical and physiological evidence that ACh is the neurotransmitter at some, but not all, of the excitatory neuromuscular synapses of the lobster stomatogastric system.

5-Hydroxytryptamine and Dopamine in Biomphalaria glabrata

5-hydroxytryptamine (5-HT) and dopamine (DA) have been isolated and identified in the tissues of the snail, Biomphalaria glabrata. The brain contained approximately 15 tAg, the cephalopedal region 2 ,ug, and the mantle region 1 jug of 5-HT per gram of fresh weight. The concentrations of DA in the brain and the cephalopedal region were of the same order of magnitude as those of 5-HT, and 5 times lower in the mantle region. Neither 5-HT nor DA were detectable in the hepatopancreas-ovotestis region. The distribution of these 2 biogenic amines was also studied by the histochemical fluorescence technique of Falck and Owman (1965). Only green (catecholamine) fluorescent neurons were observed in the buccal ganglia; they are tentatively considered as DA neurons. Neuropile areas probably contain a mixture of 5-HT and DA. The nerve fibers emanating from the ganglia predominantly contain DA. With the exception of the buccal ganglia, 5-HT neurons were more numerous and comparatively large in the other ganglia. Green catecholamine fluorescence was detected in areas nearest to the epithelium of the foot. The presence of two biogenic amines, 5hydroxytryptamine (5-HT) and dopamine (DA), has been demonstrated in a variety of gastropods (Carpenter et al., 1971; Cottrell and Maser, 1967; Dahl et al., 1966; Gerschenfeld, 1971; Sweeney, 1963; Twarog, 1968; Walker et al., 1968; Kerkut and Cottrell, 1963; Kerkut et al., 1966). These amines are localized predominantly in the nervous system of this class of animals, and evidence for their function as neural transmitters has been reported (Carpenter et al., 1971; Dahl et al., 1966; Gerschenfeld, 1971; Gerschenfeld and Stefani, 1968; Sweeney, 1963; Twarog, 1968). The gastropod Biomphalaria glabrata is an intermediate host of the parasitic trematode, Schistosoma mansoni. 5-HT and norepinephrine have been identified as constituents of the adult form of this parasite (Bennett et al., 1969; Bennett and Bueding, 1971). Furthermore, recent evidence indicates the presence of dopamine receptors in the longitudinal muscle of adult S. mansoni, a function of 5-HT as an excitatory neuromuscular transmitter in the parasite. (T. Tomosky et al., 1974), and a high affinity uptake Received for publication 2 November 1973. * This investigation was supported by NIH training grant AI-149, and research grants AI-03515 and AI-08022. t Present address: National Institute of Child Health and Human Development, Gerontology Research Center, Baltimore City Hospitals, Baltimore, Maryland 21224. mechanism for 5-HT providing a neurotransmitter which the adult worm cannot synthesize de novo. The question arises whether biogenic amines play a role in the development of the larval stages of schistosomes in their intermediate host; this, in turn, requires information about the distribution of these amines in B. glabrata. The present study is concerned with this problem. MATERIALS AND METHODS Biomphalaria glabrata with shell diameters of 9 to 14 mm were used. Determination of biogenic amines The tissues were dissected, quickly frozen in liquid nitrogen, and homogenized in a solution containing perchloric acid (0.4 M), ascorbic acid (10 mM), and EDTA (5.4 mM) (Anden and Magnusson, 1967) in a motor-driven all-glass homogenizer at 4 C. One ml of solution was used per 100 mg tissue. The mixture was transferred into a centrifuge tube and, after washing of the container with a measured volume of the same solution, was centrifuged at 10,000 g for 15 min at 4 C. The pH of the supernatant solution was adjusted to 5 to 6 by addition of 0.5 ml of 2 M potassium phosphate buffer (pH 6.5) and a measured volume of 2 N KOH. After centrifugation (10,000 g, 15 min, 4 C) the supernatant solution was used for the determinations of 5-HT and of DA.

Transmitter substances in insects: A review

1. 1. The most probable insect CNS excitatory transmitter in ACh. GABA is probably an inhibitory CNS transmitter. 2. 2. Other possible central transmitters in insects include dopamine, adrenaline, noradrenaline, and 5-HT. 3. 3. Glutamate is almost certainly the excitatory neuromuscular transmitter at somatic muscle. It may also be involved at some nerve-visceral muscle junctions. GABA appears to be the inhibitory transmitter at somatic muscle. 4. 4. Biogenic amines may be involved in neurohormonally mediated cardio-acceleration and neural excitation in the firefly light organ. 5. 5. Peptide neurohormones may mediate actions in several tissues including heart, gut, and nerve-cord.

Glutamate uptake by a stimulated insect nerve muscle preparation.

Recent reports suggest that glutamate may be the excitatory neuromuscular transmitter in insects. In this study, glutamate uptake by isolated cockroach nerve muscle preparations was investigated by means of chemical and electron microscope radioautographic techniques. We found that the preparation had a high affinity for glutamate and that nerve stimulation enhanced glutamate uptake. Chemical studies showed that the average tissue concentration of glutamate bound during a 1 hr incubation period in 10(-5)M glutamate-(3)H after nerve stimulation was 2.8 x 10(-5)M. Less than 1% of the radioactivity was present in the perchloric acid-precipitated protein fraction. Using electron microscope radioautography, we observed that sheath cells showed the highest glutamate concentration of all cellular compartments. Uptake was greater at neuromuscular junctions than in other regions of the tissue. The data suggest a possible mechanism for transmitter inactivation and protection of synapses from high blood glutamate.

Responses of perfused isolated leg preparations of the cockroach, Gromphadorhina portentosa, to L-glutamate, gaba, picrotoxin, strychnine and chlorpromazine.

AbstractA perfused leg preparation of Gromphadorhina portentosa (Orthoptera) was developed for use in pharmacological studies and bioassay procedures. Study of tibial flexion with increasing stimulation intensity at low frequency revealed five fast motor units. Chlorpromazine (2.5 × 10−4 M) progressively blocked these motor units. GABA generally failed to affect fast system responses, even at high concentration (2.5 × 10−4 M). A high concentration (2.5 × 10−4 M), picrotoxin produced effects consistent with the interpretation that motor axon excitability was affected. Strychnine (2.5 × 10−4 M) produced no effect on isolated leg preparations. L‐Gultamate (pH 7.2) produced contractures; the threshold for this response was 2.5 × 10−4 M. Contractures were produced after application to resting muscle, and were also observed in preparations receiving low frequency indirect stimulation. The response to glutamate under the latter condition closely resembled incomplete tetanus. L‐Glutamate (2.5 × 10−4 M) produced a reversible depression of kick force in locusts. These responses are compatible with the hypothesis that L‐glutamate is an excitatory neuromuscular transmitter in insects.

Effect of calcium on excitatory neuromuscular transmission in the crayfish

1. The effects of varying the external Ca concentration from 1.8 to 30 mM/l. ((1/8)-2 times normal) have been studied at the in vitro crayfish excitatory neuromuscular junction. Electrophysiological techniques were used to record transmembrane junctional potentials from muscle fibres and extracellular junctional currents from the vicinity of nerve terminals.2. The excitatory junctional potential amplitude was proportional to [Ca](0) (n), where n varied between 0.68 and 0.94 (mean 0.82) when [Ca](0) was varied from 1.8 to 15 mM/l.3. The increase in junctional potential amplitude on raising [Ca](0) resulted primarily from an increase in the average number of quanta of excitatory transmitter released from the presynaptic nerve terminal by the nerve impulse.4. The size of the quanta, synaptic delay, presynaptic potential and electrical properties of the muscle membrane were little affected by changes in calcium concentration in the range studied.

Neuromuscular Transmitter Substance in Insect Visceral Muscle

Stimulation of the nerves innervating the proctodeum (hindgut) of the cockroach Periplaneta americana (L.) causes a slow-type, graded contraction of the longitudinal muscles. An unidentified substance, or substances, present in the foregut and hindgut, the specific activity of which is highest in the nerves innervating these organs, effects a similar contraction. This "gut-factor" is depleted from the hindgut after surgical section of the proctodeal nerves. None of Factors P(1) and P(2), 5-hydroxytryptamine, acetylcholine, adrenaline, noradrenaline, alpha-ami-nobutyric acid or glutamate duplicates the pharmacological behavior of this substance. The active factor is associated with subcellular particles that require centrifugal forces of approximately 1,000,000 g-min for sedimentation. The substance is inactivated in homogenates of gut tissue in the absence of suitable precautions. It is proposed that the "gut-factor" functions as an excitatory neuromuscular transmitter substance in insect visceral muscle.