Reduced Expression of the Vesicular Acetylcholine Transporter and Neurotransmitter Content Affects Synaptic Vesicle Distribution and Shape in Mouse Neuromuscular Junction

Hermann A Rodrigues,Wallace L Camargo,Marco A M Prado,Matheus De C Fonseca,Vânia F Prado,Cristina Guatimosim,Patrícia M Martinelli,Patrícia M A Lima,Lígia A Naves,William Phillips

doi:10.1371/journal.pone.0078342

Hermann A Rodrigues, Wallace L Camargo + Show 8 more

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https://doi.org/10.1371/journal.pone.0078342

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Abstract

In vertebrates, nerve muscle communication is mediated by the release of the neurotransmitter acetylcholine packed inside synaptic vesicles by a specific vesicular acetylcholine transporter (VAChT). Here we used a mouse model (VAChT KDHOM) with 70% reduction in the expression of VAChT to investigate the morphological and functional consequences of a decreased acetylcholine uptake and release in neuromuscular synapses. Upon hypertonic stimulation, VAChT KDHOM mice presented a reduction in the amplitude and frequency of miniature endplate potentials, FM 1–43 staining intensity, total number of synaptic vesicles and altered distribution of vesicles within the synaptic terminal. In contrast, under electrical stimulation or no stimulation, VAChT KDHOM neuromuscular junctions did not differ from WT on total number of vesicles but showed altered distribution. Additionally, motor nerve terminals in VAChT KDHOM exhibited small and flattened synaptic vesicles similar to that observed in WT mice treated with vesamicol that blocks acetylcholine uptake. Based on these results, we propose that decreased VAChT levels affect synaptic vesicle biogenesis and distribution whereas a lower ACh content affects vesicles shape.

Highlights

Acetylcholine (ACh) plays an important role during nervous system development [1,2,3]
Previous studies from our research group showed that internalization of FM1-43 by motor terminals of vesicular acetylcholine transporter (VAChT) KDHOM mice and WT controls in response to electrical stimulation is very similar, suggesting that endocytosis is not affected in VAChT KDHOM mice [9]
We investigated the impact of reduced expression of VAChT on the morphology of neuromuscular junction (NMJ) from the diaphragm muscle of VAChT KDHOM adult mice

Summary

Introduction

Acetylcholine (ACh) plays an important role during nervous system development [1,2,3]. VAChT knockout (VAChTdel/del) mice appear to have normal SV recycling, but they are unable to store or release sufficient ACh in response to neural activity. As a consequence, they do not survive more than few minutes after birth [3]. To investigate whether decreased levels of VAChT, and reduced ACh storage, can regulate any aspect of the SV cycle, studies using the NMJ are ideal, due to the homogenous cholinergic nature of this synapse and its accessibility to imaging and electron microscopy. In the present study we characterized, at the ultrastructure level, the morphology of synaptic nerve terminals from diaphragm muscles of VAChT KDHOM mice. Our results demonstrate a link between ACh storage and regulation of SV recycling

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