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

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.

Figure 1

Acetylcholine and carbachol reduce [Ca²⁺]_i transients and the EPC quantal content. (A) Representative EPCs in control conditions and in the presence of 10 μM carbachol. (B) Quantal content in the presence of 10 μM carbachol or 100 μM ACh. (C) [Ca²⁺]_i transients in control and in the presence of 10 μM carbachol. (D) Amplitude of [Ca²⁺]_i transients in the presence of carbachol and acetylcholine as % of control (control set as100%). Abbreviations: Contr, control; CCh, carbachol; ACh, acetylcholine. ^*P < 0.05 vs. control saline; n = 4–6.

Figure 2

Role of Ca²⁺ entry in generation of [Ca²⁺]_i transients and regulation of quantal release. (A) [Ca²⁺]_i transients at two different Ca²⁺ concentrations in extracellular solution. Amplitude of [Ca²⁺]_i transients at 0.6 mM Ca²⁺ is set as 100%. (B) Average quantum content of EPC at two different concentrations of extracellular Ca²⁺. ^*P < 0.05 vs. control saline; n = 3.

Figure 3

Modulation of [Ca²⁺]_i transient by muscarine and nicotine. (A) [Ca²⁺]_i transient in the presence of carbachol (10 μM) after pre-treatment with the mixture of atropine (1 μM) and d-tubocurarine (10 μM). (B) Average amplitudes of [Ca²⁺]_i transient normalized to the control in the presence of nicotine (10 μM); in the presence of nicotine after pre-treatment with d-tubocurarine (10 μM); in the presence of muscarine (10 μM); in the presence of muscarine after pre-treatment with atropine (1 μM), in the presence of carbachol (10 μM) after pre-treatment with d-tubocurarine (10 μM), in the presence of carbachol (10 μM) after pre-treatment with atropine (1 μM) and in the presence of carbachol after pre-treatment with the mixture of atropine (1 μM) and d-tubocurarine (10 μM). Abbreviations: d-TC, d-tubocurarine; Atr, atropine; CCh, carbachol; Nic, nicotine (10 μM); Musc, muscarine (10 μM). ^*P < 0.05 vs. control saline; n = 4–7.

Figure 4

Specific effects of activation of muscarinic and nicotinic receptors on [Ca²⁺]_i transients. (A) [Ca²⁺]_i transients in the presence of muscarine (10 μM) after blockade of nicotinic receptors by d-tubocurarine (10 μM). (B) Average amplitude of [Ca²⁺]_i transient in the presence of muscarine (10 μM) after blockade of nicotinic receptors by tubocurarine (10 μM). (C) Amplitude of [Ca²⁺]_i transient in the presence of nicotine (10 μM) after blockade of muscarinic receptors by atropine (1 μM). (D) Average amplitude of [Ca²⁺]_i transient in presence of nicotine when muscarinic receptors are blocked by atropine. Abbreviations: d-TC, d-tubocurarine; Atr, atropine; Nic, nicotine; Musc, muscarine. ^*P < 0.05; n = 4–6.

Figure 5

Identification of muscarinic receptor subtypes mediating effects of cholinomimetics. (A) [Ca²⁺]_i transients in the presence of muscarine (10 μM) and pirenzepine (100 nM). (B) Mean values of amplitude of [Ca²⁺]_i transients in the presence of muscarine and pirenzepine. (C) [Ca²⁺]_i transients in the presence of methoctramine (10 nM) and muscarine (10 μM). (D) Mean values of amplitude of [Ca²⁺]_i transients in the presence of methoctramine (10 nM) and muscarine (10 μM). Abbreviations: Pirenz, pirenzepine; Musc, muscarine; Methoct, methoctramine. ^*P < 0.05 vs. control; n = 5.

Figure 6

Identification of nicotinic receptor subtypes mediating effects of cholinomimetics. Mean values of magnitude of [Ca²⁺]_i transients in the presence of different concentrations (they are indicated in brackets) of mecamylamine blocking various subtypes of nicotinic receptors and after addition of nicotine (10 μM); in the presence of metillikakonitin (10 nM) and after addition of nicotine (10 μM). Abbreviations: Nic, nicotine; Mecam, mecamylamine. MLA, metillikakonitin. Control corresponds to 100%. ^*P < 0.05 vs. control saline; n = 3–6.

Figure 7

The role of voltage-gated Ca²⁺-channels in the effects of carbachol on [Ca²⁺]_i dynamics. (A) [Ca²⁺]_i transients in the presence of specific blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA (300 nM). (B) Mean values of amplitudes of [Ca²⁺]_i transients normalized to control. (C) Effect of carbachol (10 μM) on [Ca²⁺]_i transients in the presence of ω-conotoxin GVIA. (D) Mean values of amplitudes of [Ca²⁺]_i transients. Abbreviations: Contr, control; ω-CTx GVIA, ω-conotoxin GVIA; CCh, carbachol. ^*P < 0.05; n = 5.

Figure 8

Action of antagonists of nicotinic and muscarinic acetylcholine receptors on [Ca²⁺]_i-dynamics in the absence of exogenous cholinomimetics. (A) [Ca²⁺]_i transients in control and in the presence of d-tubocurarine (10 μM); (B) mean values of amplitudes of [Ca²⁺]_i transients in the presence of d-tubocurarine; (C) [Ca²⁺]_i transients in control and in the presence of atropine (1 μM); (D) mean values of amplitudes of [Ca²⁺]_i transients in the presence of atropine. Abbreviations: Contr, control; d-TC, d-tubocurarine; Atr, atropine. ^*P < 0.05 vs. control; n = 7–15.

Figure 9

Effects of acetylcholinesterase inhibitor neostigmine on [Ca²⁺]_i-dynamics. (A) [Ca²⁺]_i transients in control and in the presence of neostigmine (1 μM). (B) Mean values of amplitude of [Ca²⁺]_i transient in the presence of acetylcholine (100 μM); in the presence of neostigmine alone and after pre-treatment by d-tubocurarine (10 μM) and atropine (1 μM). Mean values of [Ca²⁺]_i transient amplitude normalized to control. Effect of exogenous acetylcholine is presented for comparison reasons. Mixture of d-tubocurarine and atropine, completely eliminated neostigmine effects on [Ca²⁺]_i transients. Abbreviations: Contr, control; Neost, neostigmine; ACh, acetylcholine; Atr, atropine (1 μM), d-TC, d-tubocurarine (10 μM). ^*P < 0.05 vs. control saline; n = 5.