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.
Highlights
Acetylcholine (ACh) when released from nerve endings upon action potential-driven depolarization, triggers postsynaptic response in muscle cell, and regulates its own secretion from presynaptic terminal (Ciani and Edwards, 1963; Parnas et al, 2000; Tomas et al, 2014)
Voltage-Gated Ca2+ Channels Involved in Carbachol Modulation of [Ca2+]i dynamics. Since both acetylcholine and carbachol reduce [Ca2+]i transient and quantum content of endplate currents, we suggested that cholinergic modulation of neurotransmitter release could result from attenuated Ca2+ entry into motor nerve endings (Khaziev et al, 2012)
Neurotransmitter release from frog nerve endings is modulated by cholinomimetics: by acting through nicotinic receptors, they change kinetics of secretion and its quantum content, while by activation of muscarinic receptors they contribute only to regulation of quantum content (Ciani and Edwards, 1963; Nikolsky et al, 2004)
Summary
Acetylcholine (ACh) when released from nerve endings upon action potential-driven depolarization, triggers postsynaptic response in muscle cell, and regulates its own secretion from presynaptic terminal (Ciani and Edwards, 1963; Parnas et al, 2000; Tomas et al, 2014). Muscarinic receptors can be activated in the absence of stimulation during spontaneous neurotransmitter release (Kupchik et al, 2008) This phenomenon has received a significant research attention in the past (Nikol’skiı and Giniatullin, 1979; Wessler, 1989; Macleod et al, 1994; Van der Kloot et al, 1997; Nikolsky et al, 2004), fine details of ACh-dependent presynaptic regulation remain obscure. Both direct action of ACh on exocytotic machinery (Linial et al, 1997) and inhibition of presynaptic Ca2+ entry (Wu and Saggau, 1997; Parnas et al, 2000; Khaziev et al, 2012) have been suggested. The present study was designed to investigate the role of presynaptic calcium influx in autoregulation of ACh secretion by presynaptic receptors in the frog neuromuscular junction
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