Acetylcholine-Induced Inhibition of Presynaptic Calcium Signals and Transmitter Release in the Frog Neuromuscular Junction

Eduard Khaziev; Dmitry Samigullin; Nikita Zhilyakov; Nijaz Fatikhov; Ellya Bukharaeva; Alexei Verkhratsky; Evgeny Nikolsky

doi:10.3389/fphys.2016.00621

Acetylcholine-Induced Inhibition of Presynaptic Calcium Signals and Transmitter Release in the Frog Neuromuscular Junction

Front Physiol. 2016 Dec 12:7:621. doi: 10.3389/fphys.2016.00621. eCollection 2016.

Authors

Eduard Khaziev¹, Dmitry Samigullin¹, Nikita Zhilyakov², Nijaz Fatikhov³, Ellya Bukharaeva², Alexei Verkhratsky⁴, Evgeny Nikolsky⁵

Affiliations

¹ Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of SciencesKazan, Russia; Open Laboratory of Neuropharmacology, Kazan (Volga Region) Federal UniversityKazan, Russia; Institute of Applied Electrodynamics, Photonics and Living Systems, A.N. Tupolev Kazan National Research Technical UniversityKazan, Russia.
² Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of SciencesKazan, Russia; Open Laboratory of Neuropharmacology, Kazan (Volga Region) Federal UniversityKazan, Russia.
³ Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of Sciences Kazan, Russia.
⁴ Faculty of Life Sciences, University of Manchester Manchester, UK.
⁵ Laboratory of Biophysics of Synaptic Processes, Kazan Institute of Biochemistry and Biophysics, Kazan Scientific Center of the Russian Academy of SciencesKazan, Russia; Open Laboratory of Neuropharmacology, Kazan (Volga Region) Federal UniversityKazan, Russia; Department of Biophysics, Kazan State Medical UniversityKazan, Russia.

Abstract

Acetylcholine (ACh), released from axonal terminals of motor neurons in neuromuscular junctions regulates the efficacy of neurotransmission through activation of presynaptic nicotinic and muscarinic autoreceptors. Receptor-mediated presynaptic regulation could reflect either direct action on exocytotic machinery or modulation of Ca²⁺ entry and resulting intra-terminal Ca²⁺ dynamics. We have measured free intra-terminal cytosolic Ca²⁺ ([Ca²⁺]_i) using Oregon-Green 488 microfluorimetry, in parallel with voltage-clamp recordings of spontaneous (mEPC) and evoked (EPC) postsynaptic currents in post-junctional skeletal muscle fiber. Activation of presynaptic muscarinic and nicotinic receptors with exogenous acetylcholine and its non-hydrolized analog carbachol reduced amplitude of the intra-terminal [Ca²⁺]_i transients and decreased quantal content (calculated by dividing the area under EPC curve by the area under mEPC curve). Pharmacological analysis revealed the role of muscarinic receptors of M₂ subtype as well as d-tubocurarine-sensitive nicotinic receptor in presynaptic modulation of [Ca²⁺]_i transients. Modulation of synaptic transmission efficacy by ACh receptors was completely eliminated by pharmacological inhibition of N-type Ca²⁺ channels. We conclude that ACh receptor-mediated reduction of Ca²⁺ entry into the nerve terminal through N-type Ca²⁺ channels represents one of possible mechanism of presynaptic modulation in frog neuromuscular junction.

Keywords: N-type Ca channels; calcium transient; muscarinic receptors; neuromuscular synapse; nicotinic receptors; presynaptic acetylcholine receptors; quantum secretion of acetylcholine.