Abstract
In recent years, considerable evidence is accumulated pointing to participation of gamma-aminobutyric acid (GABA) in intercellular signaling in the peripheral nervous system, including, in particular, neuromuscular transmission. However, where in the neuromuscular synapse GABA is synthesized remains not quite clear. We used histochemical methods to detect GABA and l-glutamate decarboxylase (GAD) in developing skeletal muscle fibers and in cultured motor neurons. We found that GABA can be detected already in myocytes, but with further muscle maturation, GABA synthesis gradually attenuates and completely ceases in early postnatal development. We found also that formation of GABA in muscle tissue does not depend on activity of GAD, but presumably proceeds through some other, alternative pathways. In motor neurons, GABA and GAD can be detected at the early stage of development (prior to synapse formation). Our data support the hypothesis that GABA and GAD, which are detectable in adult neuromuscular junctions, have neuronal origin. The mechanism of GABA production and its role in developing muscle tissue need further clarification.
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Acknowledgments
The authors are grateful to Dr. Victor I. Ilyin, Ph.D., (Kazan (Volga Region) Federal University, Kazan, Russia) for critical reading of the manuscript and for helpful comments and discussion. The study was carried out on the equipment of the CSF-SAC FRC KSC RAS.
Funding
This study was financially supported by a grant from the Russian Science Foundation (17-15-01279).
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Experiments were performed in accordance with the guidelines for the use of laboratory animals of the Kazan (Volga Region) Federal University and the Kazan Medical University, in compliance with the NIH Guide for the Care and Use of Laboratory Animals. The experimental protocol met the requirement of the European Communities Council Directive (86/609/EEC).
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Fig. S1
Positive and negative controls for antibody specificity. Positive controls: GABA (top panel) and GAD (bottom panel) immunoreactivity in cortex area of rat (P8) brain. Negative control: preincubation of the primary anti-GABA antibody with 50 mM GABA and anti-GAD antibody with corresponding blocking peptide completely abolished the staining of cultured motor neurons. Scale bar 50 μm (PNG 829 kb)
ESM_2 Video
Immunoreactivity of cultured motor neurons on GABA (green) and GAD (red) antibodies. Video was made from a set of images taken at different focus levels (“Z-stack”). Depending on the focus change, the presence of only one of the two antibodies or both antibodies together in the cell can be observed. The XYZt mode of the confocal microscope was used for Z-stacks obtaining. Stack is composed by N = 71 slices, Size-Depth is 11,75 μm, StepSize is 0,17 μm. Scale bar 50 μm (AVI 80068 kb)
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Sibgatullina, G.V., Malomouzh, A.I. GABA in developing rat skeletal muscle and motor neurons. Protoplasma 257, 1009–1015 (2020). https://doi.org/10.1007/s00709-020-01485-1
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DOI: https://doi.org/10.1007/s00709-020-01485-1