Studies on Locust Neuromuscular Physiology in Relation to Glutamic Acid

1. Two nerve-muscle preparations were used to investigate the physiology of the locust retractor unguis muscle in relation to L-glutamic acid. These were an ‘isolated preparation’, in which the muscle and its nerve were dissected out, and a ‘perfusedfemur preparation’, in which the muscle suffered no mechanical disturbance. 2. Exposure of the nerve--muscle preparations to glutamate caused a variety of responses, some of which were shown to be abnormal and due to the experimental conditions. 3. When locust femora were perfused with saline or haemolymph the retractor unguis muscles were much more severely affected by glutamate if the hydrostatic pressure was slightly raised. At raised pressures the perfused-femur preparations were particularly prone to give repetitive and spontaneous contractions. 4. Analysis of haemolymph from adult male locusts showed that it contained, on average, 0-2 mmol/1 L-glutamate, 45 mol/1 total non-peptide amino acids, 5-0 mmol/1 calcium, and 11-6 mmol/1 magnesium. It was calculated that approximately 50% of the calcium and 75% of the magnesium ions are bound to amino acids, and that approximately 25% of the glutamic acid is bound to divalent metal ions. 5. The isolated preparations were severely affected by glutamate at the concentration at which it occurs in haemolymph, and it was concluded that in the intact locust some mechanism must protect the neuromuscular synapses from haemolymphg lutamate. No evidence could be obtained of the sequestration of glutamate by haemocytes, or of binding of glutamate to haemolymph proteins. 6. Calcium and magnesium ions reduced the sensitivity of nerve-muscle preparations to glutamate to a greater extent than could be accounted for by the formation of amino acid-metal complexes. This suggests that the protection afforded by calcium and magnesium involves an interaction of the metal ions with the neuromuscular system itself. 7. The retractor unguis muscle was much less sensitive to glutamate when it was contained within an undissected femur than in an isolated preparation. It was concluded that the muscle is normally protected from haemolymph glutamate by a diffusion barrier which is damaged on dissection. 8. Comparison of the fine structure of retractor unguis muscles, fixed either after dissection or while still contained within the femur, showed that dissection normally caused a partial separation of muscle fibres and damage to the connective tissue sheath, with the resultant exposure of some nerve endings. The connective tissue sheath may constitute the postulated diffusion barrier. 9. The excitatory synapses of the locust retractor unguis muscle are believed to be isolated from haemolymph glutamate by a diffusion barrier, which is tentatively identified with the connective tissue sheath that binds the muscle fibres together. Calcium and magnesium ions reduce the sensitivity of nerve-muscle preparations to glutamate, and may have such a role in the living insect.