Aspartate and glutamate as possible transmitters at the ‘slow’ and ‘fast’ neuromuscular junctions of the body wall muscles ofMusca larvae
Body wall muscles 6A and 7A ofMusca larvae are each dually innervated by a single fast and a single slow axon. Perfusion of 1 mM E-glutamate abolished the neurally evoked fast EPSP but not the slow EPSP, while 1 mM aspartate abolished the slow EPSP but not the fast EPSP.
Iontophoresis of L-glutamate or aspartate from single or multibarreled micropipettes produced potentials when ejected at localised sites on the muscle surface. These sites were identified as synaptic terminals by recording extracellular synaptic currents and the ability to evoke antidromic nerve impulses and EPSPs upon focal stimulation.
The site of action of iontophoretically applied E-glutamate corresponded to the fast neuromuscular junction, while that of aspartate corresponded to the slow neuromuscular junction. The conduction velocity of antidromic impulses evoked by focal stimulation was lower in slow axons than in fast axons. Sensitive sites could therefore be unequivocally identified as fast or slow synapses.
The two types of receptor (glutamate-preferring and aspartate-preferring) were agonist specific, although each agonist would act as an antagonist of the other receptor type.
The ionic fluxes of the fast EPSP were the same as for L-glutamate potentials, and the ionic fluxes of the slow EPSP were the same as for aspartate potentials. In conditions where comparisons were possible, there was no significant difference between the reversal potentials of iontophoretically applied L-glutamate and the neurally evoked fast EPSP. Similarly, there was no significant difference between iontophoretically applied aspartate and the neurally evoked slow EPSP. The reversal potentials of iontophoretically applied L-glutamate and aspartate were significantly different.
When axonal conduction was blocked with tetrodotoxin (TTX), graded focal stimulation of nerve terminals produced graded postsynaptic potentials. At identified fast terminals, the evoked graded potentials were similar in time course to iontophoretically applied L-glutamate potentials while at slow terminals, evoked potentials were similar to aspartate potentials.
When blunt iontophoretic micropipettes were positioned over fast synapses in the presence of TTX, the postsynaptic membrane became desensitised to both ejected L-glutamate and the natural transmitter. Dose-response curves of iontophoretically applied L-glutamate in the presence of the natural transmitter at the same terminal region indicated competitive interaction between L-glutamate and the natural transmitter.
Iontophoretic potentials could not be obtained in the presence of either 6 mM calcium or 20 mM magnesium, although large EPSPs could be recorded under these conditions. Iontophoretic potentials could be evoked in these conditions if 10 mM bicarbonate ions were present.
It is concluded the L-glutamate is a specific agonist at the fast transmitter receptor, while aspartate is a specific agonist at the slow transmitter receptor. However, in view of the different effects of calcium on the neurally evoked potential and the iontophoretic potentials, a better understanding of these synapses is needed before claiming a transmitter role for these amino acids.
KeywordsNeuromuscular Junction Reversal Potential Body Wall Muscle Specific Agonist Transmitter Receptor
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- Anwyl, R.: The effect of foreign cations, pH and pharmacological agents on the ionic permeability of an excitatory glutamate synapse. J. Physiol.273, 389–404 (1977)Google Scholar
- Atwood, H.L., Smyth, T., Johnston, H.S.: Neuromuscular synapses in the cockroach extensor tibiae muscles. J. Insect Physiol.15, 529–535 (1969)Google Scholar
- Balcar, V.J., Johnston, G.A.R.: The structural specificity of the high affinity uptake of L-glutamate and L-aspartate by rat brain slices. J. Neurochem.19, 2657–2666 (1972)Google Scholar
- Barker, J.L.: Divalent cations: effects on post-synaptic pharmacology of invertebrate synapses. Brain Res.92, 307–323 (1975)Google Scholar
- Brown, K.T., Flaming, D.G.: New microelectrode techniques for intracellular work with small cells. Neuroscience2, 813–827 (1977)Google Scholar
- Clements, A.N., May, T.E.: Studies on locust neuromuscular physiology in relation to glutamic acid. J. Exp. Biol.60, 673–705 (1974a)Google Scholar
- Constanti, A., Nistri, A.A.: A study of the interactions between glutamate and aspartate at the lobster neuromuscular junction. Br. J. Pharmacol.62, 495–506 (1978)Google Scholar
- Constanti, A., Nistri, A.A.: Further observations on the interaction between glutamate and aspartate on lobster muscle. Br. J. Pharmacol.65, 287–301 (1979)Google Scholar
- Crawford, A.C., McBurney, R.N.: The post-synaptic action of some putative excitatory transmitter substances. Proc. R. Soc. (London) Ser. B192, 481–489 (1976)Google Scholar
- Crawford, A.C., McBurney, R.N.: The synergistic action of L-glutamate and L-aspartate at the crustacean excitatory neuromuscular junction. J. Physiol.268, 697–709 (1977a)Google Scholar
- Crawford, A.C., McBurney, R.N.: Termination of transmitter action at the crustacean neuromuscular junction. J. Physiol.268, 711–729 (1977b)Google Scholar
- D'Aniello, A., Giuditta, A.: Identification of D-aspartate in the brain ofOctopus vulgaris Lam. J. Neurochem.29, 1053–1057 (1977)Google Scholar
- D'Aniello, A., Giuditta, A.: Presence of D-aspartate in squid axoplasm and in other regions of the cephalopod nervous system. J. Neurochem.31, 1107–1108 (1978)Google Scholar
- Davies, L.P., Johnston, G.A.R.: Uptake and release of D- and L-aspartate by rat brain slices. J. Neurochem.26, 1007–1014 (1976)Google Scholar
- Dudel, J.: Aspartate and other inhibitors of excitatory synaptic transmission in crayfish muscle. Pflügers Arch.369, 7–16 (1977)Google Scholar
- Eusebi, F., Palmieri, P., Picardo, M.: Action of glutamic acid and some glutamate analogues on the molluscan central neurones. Experientia34, 867–868 (1978)Google Scholar
- Evans, P.D.: The uptake of L-glutamate by the peripheral nerves of crabCarcinus maenas (L.). Biochim. Biophys. Acta311, 302–313 (1973)Google Scholar
- Evans, P.D., O'Shea, M.: The identification of an octopaminergic neurone and the modulation of a myogenic rhythm in locust. J. Exp. Biol.73, 235–260 (1978)Google Scholar
- Florey, F., Woodcock, B.: Presynaptic excitatory action of glutamate applied to crab neuromuscular preparation. Comp. Biochem. Physiol.26, 651–661 (1968)Google Scholar
- Florkin, M., Jeuniaux, C.: Haemolymph: Composition. In: The physiology of insecta, Vol. V. Rockstein, M. (ed.), pp. 256–308. New York, London: Academic Press 1974Google Scholar
- Freeman, A.R.: Polyfunctional role of glutamic acid in excitatory synaptic transmission. Prog. Neurobiol.6, 137–153 (1976)Google Scholar
- Gent, J.P., Morgan, R., Wolstencroft, J.H.: Determination of the relative potency of two excitant amino acids. Neuropharmacology13, 441–447 (1974)Google Scholar
- Hall, J.G., McLennan, H., Wheal, H.V.: The actions of certain amino acids as neuronal excitants. J. Physiol.272, 52P (1977)Google Scholar
- Hall, J.G., Hicks, T.P., McLennan, H., Richardson, T.L., Wheal, H.V.: The excitation of mammalian central neurones by amino acids. J. Physiol.286, 29–40 (1979)Google Scholar
- Hardie, J.: Studies on supercontracting cross-striated muscles in insects. Ph. D. thesis, University of Birmingham, England (1975)Google Scholar
- Hardie, J.: Motor innervation of the supercontracting longitudinal ventro-lateral muscles of the blowfly larva. J. Insect Physiol.22, 661–668 (1976)Google Scholar
- Hardie, J., Osborne, M.P.: The electrical and mechanical properties of supercontracting body-wall muscles of the blowfly larva,Calliphora erythrocephala (Meig.). Comp. Biochem. Physiol.57 A, 59–66 (1977)Google Scholar
- Harris, E.J.: The importance of carbon dioxide for calcium uptake by some mitochondria. Nature274, 820–821 (1978)Google Scholar
- Haycock, J.W., Levy, W.G., Derner, L.A., Cotman, C.W.: Effects of elevated potassium on the release of neurotransmitters from cortical synaptosomes, efflux or secretion. J. Neurochem.30, 1113–1125 (1978)Google Scholar
- Hoyle, G.: Neural control of skeletal muscle. In: The physiology of insecta, Vol. 4. Rockstein, M. (ed.), pp. 176–236. New York, London: Academic Press 1974Google Scholar
- Hubbard, J.E., Llinás, R., Quastel, D.M.J.: Analysis of subsynaptic events. In: Electrophysiological analysis of synaptic transmission, pp. 174–211. London: Arnold 1969Google Scholar
- Irving, S.N.: Studies on the fine structure, pharmacology and neurophysiology of the insect neuromuscular junction with special reference to the larvae ofLucilia sericata. Ph. D. thesis, University of Birmingham, England (1977)Google Scholar
- Irving, S.N., Miller, T.A.: Ionic differences in ‘fast’ and ‘slow’ neuromuscular transmission in body wall muscles ofMusca domestica larvae. J. Comp. Physiol.135, 291–298 (1980)Google Scholar
- Irving, S.N., Osborne, M.P., Wilson, R.G.: Virtual absence of L-glutamate from the haemoplasm of arthropod blood. Nature263, 431–433 (1976)Google Scholar
- Irving, S.N., Osborne, M.P., Wilson, R.G.: Studies on L-glutamate in insect haemolymph. (1) Effect of injected L-glutamate. Physiol. Entomol.4, 139–146 (1979a)Google Scholar
- Irving, S.N., Wilson, R.G., Osborne, M.P.: Studies on L-glutamate in insect haemolymph. (2) Distribution and metabolism of radiolabelled amino acids injected into the haemolymph. Physiol. Entomol. (in press) (1979b)Google Scholar
- Irving, S.N., Wilson, R.G., Osborne, M.P.: Studies on L-glutamate in insect haemolymph. (3) Amino acid analyses of the haemolymph of various arthropods. Physiol. Entomol. (in press) (1979c)Google Scholar
- Kawai, N., Niwa, A.: Hyperpolarisation of the excitatory nerve terminals by inhibitory nerve stimulation in lobster. Brain Res.137, 375–368 (1977)Google Scholar
- Kravitz, E.A., Slater, C.R., Takahashi, K., Bounds, M.D.: Excitatory transmission in invertebrates: glutamate as a potential neuromuscular transmitter compound. In: Excitatory synaptic mechanism. Andersen, P., Jansen, J.K.S. (eds.), pp. 85–93. Universitetsforlaget 1970Google Scholar
- Longenecker, H.E., Hurlbut, W.P., Mauro, A., Clark, A.W.: Effects of black widow spider venom on the frog neuromuscular junction. Nature (London)225, 701–703 (1970)Google Scholar
- May, T.E., Brown, B.E., Clements, A.N.: Experimental studies on a bundle of tonic fibres in the locust extensor tibiae muscle. J. Insect Physiol.25, 169–182 (1979)Google Scholar
- McCreery, M.J., Carpenter, D.O.: Synergistic action of L-glutamate and L-aspartate inAplysia. Fed. Proc.37, Abs. 1941 (1978)Google Scholar
- Miller, T., Rees, D.: Excitatory transmission in insect neuromuscular systems. Am. Zool.13, 299–313 (1973)Google Scholar
- Onodera, K., Takeuchi, A.: Permeability changes produced by L-glutamate at the excitatory post-synaptic membranes of the crayfish muscle. J. Physiol.255, 669–685 (1976)Google Scholar
- Osborne, M.P.: The fine structure of neuromuscular junctions in the segmental muscles of the blowfly larva. J. Insect Physiol.13, 827–835 (1967)Google Scholar
- Roskoski, R.: Net uptake of aspartate by a high-affinity rat cortical synaptosomal transport system. Brain Res.160, 83–93 (1979)Google Scholar
- Shinozaki, H., Ishida, M.: Pharmacological distinction between the excitatory junctional potential and the glutamate potential revealed by concanavalin A at the crayfish neuromuscular junction. Brain Res.161, 493–501 (1979)Google Scholar
- Smyth, T.J., Geer, M.H., Griffiths, D.J.G.: Insect neuromuscular synapses. Am. Zool.13, 315–319 (1973)Google Scholar
- Takagaki, G.: Sodium and potassium ions and accumulation of labelled D-aspartate and GABA in crude synaptosomal fraction from rat cerebral cortex. J. Neurochem.30, 47–56 (1978)Google Scholar
- Takeuchi, A.: Excitatory and inhibitory transmitter actions at the crayfish neuromuscular junction. In: Motor innervation of muscle. Thesleff, S. (ed.), pp. 231–262. London, New York, San Francisco: Academic Press 1976Google Scholar
- Thieffry, M., Bruner, J.: Direct evidence for a presynaptic action of glutamate at a crayfish neuromuscular junction. Brain Res.156, 402–406 (1978a)Google Scholar
- Thieffry, M., Bruner, J.: Sensibilité des fibres musculaires d'Ecrevisse au glutamate et au médiateur naturel dans des solutions pauvres en calcium. C. R. Acad. Sci. Paris286, 1813–1816 (1978b)Google Scholar
- Titmus, M.J., Hoyle, G.: Morphology of identified neuromuscular junctions in locust jumping muscle. Neurosci. Abstr.3, 595 (1977)Google Scholar
- Usherwood, P.N.R.: Nerve-muscle transmission. In: Insect neurobiology. Treherne, J.E. (ed.), pp. 245–306. Amsterdam, Oxford: North-Holland Publishing Company 1974Google Scholar
- Usherwood, P.N.R.: Neuromuscular transmission in insects. In: Identified neurons and behavior of arthropods. Hoyle, G. (ed.), pp. 31–48. New York, London: Plenum Press 1977Google Scholar
- Usherwood, P.N.R., Cull-Candy, S.G.: Pharmacology of somatic nerve-muscle synapses. In: Insect muscle. Usherwood, P.N.R. (ed.), pp. 207–280. London, New York, San Francisco: Academic Press 1975Google Scholar
- Zucker, R.S.: Crayfish neuromuscular facilitation activated by constant presynaptic action potentials and depolarising pulses. J. Physiol.241, 69–89 (1974)Google Scholar