Aggregates of acetylcholine receptors are not observed under anti-agrin staining Schwann cell processes at the frog neuromuscular junction

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.

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Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3

Werle

VanSaun

2003

J Neurocytol

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Agrin is a heparan sulfate proteoglycan, which plays an essential role in the development and maintenance of the neuromuscular junction. Agrin is a stable component of the synaptic basal lamina and strong evidence supports the hypothesis that agrin directs the formation of the postsynaptic apparatus, including aggregates of AChRs, and junctional folds. Changes in the distribution of agrin during synaptic remodeling, denervation and reinnervation reveal that agrin can be quickly and efficiently removed from the synaptic basal lamina in a regulated manner. In order to fully understand this mechanism we sought to identify those molecules that were responsible for the removal of agrin. Matrix Metalloproteinases (MMPs) were the most likely molecules since MMPs are involved in the regulation of the pericellular space, including the cleavage of matrix proteins. In particular, MMP3 has been shown to be effective in cleaving heparan sulfate proteoglycans. Antibodies to MMP3 recognize molecules concentrated in the extracellular matrix of perisynaptic Schwann cells. MMP3 specific phylogenic compounds reveal that active MMP3 is localized to the neuromuscular junction. Purified recombinant MMP3 can directly cleave agrin, and it can also remove agrin from synaptic basal lamina. MMP3 activity is itself regulated as activation of MMP3 is lost in denervated muscles. MMP3 null mutant mice have altered neuromuscular junction structure and function, with increased AChRs, junctional folds and agrin immunoreactivity. Altogether these results support the hypothesis that synaptic activity induces the activation of MMP3, and the activated MMP3 removes agrin from the synaptic basal lamina.

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Aggregates of acetylcholine receptors are not observed under anti-agrin staining Schwann cell processes at the frog neuromuscular junction

Cited by 8 publications

References 34 publications

Matrix metalloproteinase-3 removes agrin from synaptic basal lamina

Matrix metalloproteinase-3 removes agrin from synaptic basal lamina

Seasonal comparison of the neuromuscular junction morphology of Bufo marinus

Activity dependent removal of agrin from synaptic basal lamina by matrix metalloproteinase 3

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