Seasonal comparison of the neuromuscular junction morphology of Bufo marinus

Abstract

At mammalian neuromuscular junctions (NMJs), prolonged inactivity leads to muscle denervation and atrophy. By contrast, amphibian NMJs do not show such degeneration even though they can remain in a state of drought‐imposed dormancy (hibernation) for many years. We have previously reported that during the dry season, toad (Bufo marinus) NMJs display decreased sensitivity to extracellular calcium‐dependent neurotransmitter release, which leads to minimal neuromuscular transmission. In the present study, we examined and compared NMJ morphology of toads obtained from the wild during the wet season (February–March) when these toads are active, to toads obtained from dry season (October–November) when toads are inactive. Iliofibularis muscles were isolated and prepared for immunostaining with anti‐SV2, a monoclonal antibody that labels synaptic vesicle glycoprotein SV2. The corresponding postsynaptic acetylcholine receptors were stained using Alexa Fluro‐555 conjugated α‐bungarotoxin. Confocal microscopy and three‐dimensional reconstructions were then used to examine the pre‐and postsynaptic morphology of toads NMJs from the dry (inactive) and wet (active) seasons. Total axon branch number, the percentage of axon branches with discontinuous distributions of synaptic vesicles, and further the Pearson value of colocalization of pre and postsynaptic elements in each NMJs from both the dry and wet season were compared. While our previous studies on dry toads revealed a significant reduction in evoked neurotransmission, our present findings show that the structure of the NMJs suffered limited level of remodeling, suggesting a mechanism utilized by NMJs in dry season toads to support quick recover from their dormant state after the heavy rain in wet season.