Cholinergic regulation of the evoked quantal release at frog neuromuscular junction

Abstract

The effects of cholinergic drugs on the quantal contents of the nerve-evoked endplate currents (EPCs) and the parameters of the time course of quantal release (minimal synaptic latency, main modal value of latency histogram and variability of synaptic latencies) were studied at proximal, central and distal regions of the frog neuromuscular synapse. Acetylcholine (ACh, 5 x 10(-4) M), carbachol (CCh, 1 x 10(-5) M) or nicotine (5 x 10(-6) M) increased the numbers of EPCs with long release latencies mainly in the distal region of the endplate (90-120 microm from the last node of Ranvier), where the synchronization of transmitter release was the most pronounced. The parameters of focally recorded motor nerve action potentials were not changed by either ACh or CCh. The effects of CCh and nicotine on quantal dispersion were reduced substantially by 5 x 10(-7) M (+)tubocurarine (TC). The muscarinic agonists, oxotremorine and the propargyl ester of arecaidine, as well as antagonists such as pirenzepine, AF-DX 116 and methoctramine, alone or in combination, did not affect the dispersion of the release. Muscarinic antagonists did not block the dispersion action of CCh. Cholinergic drugs either decreased the quantal content m(o) (muscarinic agonist, oxotremorine M, and nicotinic antagonist, TC), or decreased m(o) and dispersed the release (ACh, CCh and nicotine). The effects on m(o) were not related either to the endplate region or to the initial level of release dispersion. It follows that the mechanisms regulating the amount and the time course of transmitter release are different and that, among other factors, they are altered by presynaptic nicotinic receptors.

Figure 1

Presynaptic nerve action potentials (NAPs) recorded extracellularly from the proximal, central and distal regions of a synapse before (Control, continuous line) and in the presence of 1 × 10⁻⁵ m carbachol (CCh, dashed line). Selected NAPs not followed by the endplate currents (failures) were superimposed (35 in each case). The amplitudes of the three-phase nerve spike decreased along the nerve ending but were not affected by CCh.

Figure 2

Latencies of quantal release in proximal, central and distal regions of the synapse before (Control), in the presence of 5 × 10⁻⁴ m acetylcholine (ACh) and 60 min after washout (Wash). Recordings (9–11) were superimposed, showing extracellularly recorded presynaptic nerve action potentials (NAPs), individual endplate currents (EPCs) and stimulus artifacts (SA). The time intervals between the peak of the inward presynaptic Na⁺ currents of the nerve spike (downward deflection) and the times at which the rising phases of each EPC reached 20% of maximum was defined as the release latency. Note the most pronounced increase in latency dispersion in the distal region induced by ACh.

Figure 3

Latency histograms and cumulative plots of latencies of the uniquantal EPCs recorded simultaneously by three microelectrodes located in proximal, central and distal regions of the same endplate before ACh (Control: open circles and columns) and after ACh application (filled columns and asterisks). Left, non-corrected latency histograms (values of minimal synaptic latencies were not subtracted) of the uniquantal EPCs (data from a single experiment). The bin size was 0.05 ms. Right, the cumulative plots of latencies for the same data, corrected for minimal synaptic delays. The vertical dotted lines indicate the times when 90% of the quanta have been released (P₉₀) in the proximal, central and distal regions.

Figure 4

Effects of 5 × 10⁻⁴ m acetylcholine (ACh) and 1 × 10⁻⁵ m carbachol (CCh) on the quantal contents, minimal latencies, mean modal values of the latency histograms and the P₉₀ parameter in the proximal, central and distal regions of the synapse. The values (means ± s.e.m.) from 5–6 endplates are normalized to the controls before application of the drugs (1.0). Asterisks indicate statistically significant differences at P < 0.05.

Figure 5

Effects of 1 × 10⁻⁵ m carbachol (CCh) and 5 × 10⁻⁶ m nicotine before (upper row) and after (lower row) application of 5 × 10⁻⁷ m (+)tubocurarine (TC) on the quantal contents, minimal latencies, mean modal values of the latency histograms and the P₉₀ parameter in proximal, central and distal regions of the synapse. Values (means ± s.e.m.) from 5–7 endplates were normalized to controls before application of the drugs (1.0). Asterisks indicate statistically significant differences at P < 0.05.

Figure 6

Effects of 1 × 10⁻⁶ m oxotremorine M (OXO-M), 1 × 10⁻⁶ m propargyl ester of arecaidine (APET), 1 × 10⁻⁵ m pirenzepine alone and in combination with 1 × 10⁻⁵ m carbachol (CCh), 5 × 10⁻⁶ m AF-DX 116 (AFDX) alone and in combination with 1 × 10⁻⁵ m carbachol on the quantal contents, minimal latencies, mean modal values of the latency histograms and the P₉₀ parameter in proximal, central and distal regions of the synapse. Values (means ± s.e.m.) from 4–6 endplates were normalized to controls before application of the drugs (1.0). Asterisks indicate the statistically significant differences at P < 0.05.

Figure 7

Effects of 5 × 10⁻⁶ m methoctramine on pre- and postsynaptic responses. A, seven superimposed extracellular recordings from the distal region of a single endplate before (Control) and after application of methoctramine. B, the normalized (to the control = 1.0) values (means ± s.e.m.) from 6 endplates of quantal contents, minimal latencies, mean modal values of the latency histograms and the P₉₀ parameter in proximal, central and distal regions of the synapse in the presence of methoctramine. C, same as in B but in the presence of a combination of 1 × 10⁻⁵ m pirenzepine with 5 × 10⁻⁶ m methoctramine. D, histograms of uniquantal postsynaptic current amplitudes in control and in the presence of a combination of methoctramine and pirenzepine.