Seasonal regulation of quantal neurotransmitter release from amphibian neuromuscular junctions

Abstract

Background: At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to severe degeneration. By contrast, amphibian NMJs do not show such severe degeneration even though they can remain inactive for many years of drought imposed inactivity. Neurotransmitter release from nerve terminals was shown to be a key factor in maintenance of NMJ structure in both mammals and amphibians. Amphibian NMJs are composed of hundreds of neurotransmitter release sites which exhibit a high level of non-uniformity in the probability of neurotransmitter release which undergoes seasonal variation. Neurotransmitter release from these release sites is highly dependent on Ca 2+ as well as on primed vesicle availability. Understanding how the probability of neurotransmitter release is regulated in the short and long term is of fundamental importance to neural function. In the present project, I have chosen to examine the functional and morphological changes of the amphibian NMJ which has been shown by previous studies to be regulated by seasonal factor(s). I first examined the extent of the variability in neurotransmitter release and sensitivity of NMJs to calcium and dynorphin-A, during the dry and wet seasons. Short-term changes in the probability of neurotransmitter release can easily be achieved by modulation of voltage gated calcium channels (VGCCs) thus regulating Ca 2+ entry. Opiates, which were previously discovered in the skin, brain and circulating system of amphibians have been shown to inhibit neurotransmitter release by a Ca 2+ and K + dependent mechanism. In this study, I examined the sensitivity of the NMJs to dynorphin-A during the wet and dry seasons. Furthermore, I then compared the pre-and postsynaptic morphology of toad Bufo marinus NMJs obtained from the wild during the wet (January to April) and dry (August to November) southern hemisphere seasons.Methods: Iliofibularis muscles were isolated from both wet and dry season animals obtained from the wild, 2-kilometre radius from the University of Queensland. Electrophysiological recordings: toad NMJs were visualised using DiOC2(5)-fluorescence and focal loose patch extracellular or intracellular recordings were used to record the end-plate currents (EPCs) from small groups of release sites or endplate potentials (EPPs). Miniature EPCs (MEPCs) and miniature EPPs (MEPPs) were also recorded. Quantal content (m ), average probability of release (p) and the number of active release sites (n) involved in neurotransmitter release were determined for different extracellular calcium concentrations ([Ca 2+ ]o). Also the quantal size and frequency of MEPPs/MEPCs was estimated. The effect of dynorphin-A on neurotransmitter release was also examined and compared between the wet and dry season NMJs. Morphological studies: iliofibularis muscles were isolated, and prepared for immuno-staining with anti-SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2 in the nerve terminal. The muscles were also stained for the location of postsynaptic acetylcholine ...