William Van der Kloot

2-(4-phenylpiperidino) cyclohexanol (AH5183) decreases quantal size at the frog neuromuscular junction

AH5183 was studied because it inhibits acetylcholine transport into synaptic vesicles. The drug apparently is a slow-acting anti-cholinesterase, so further experiments were performed with this enzyme inhibited. Soaking for hours in AH5183 in Ringer does not decrease quantal size. However, a few minutes of tetanic nerve stimulation results in a marked decrease in quantal size. Quantal size also decreased after hours in a hypertonic Ringer containing the drug.

Vesicle size and transmitter release at the frog neuromuscular junction when quantal acetylcholine content is increased or decreased

We investigated whether the synaptic vesicles at the neuromuscular junction change size when their acetylcholine (ACh) content is altered. The size of the miniature endplate potential (MEPP) increased 3‐ or 4‐fold in preparations pre‐treated in a hypertonic solution in which the anion was gluconate. We measured the dimensions of synaptic vesicles in such preparations and in controls. The size of the vesicles and size distribution were indistinguishable. Quanta contained about half of the usual amount of ACh in preparations stimulated in the presence of hemicholinium‐3, an inhibitor of choline uptake, or in NH4+, which diminishes the proton gradient for ACh uptake into the vesicles. Neither treatment changed the size of the synaptic vesicles. ACh content and vesicle size were both decreased in preparations stimulated in (‐)‐vesamicol, an inhibitor of ACh uptake in vesicles. Since the other inhibitors decreased ACh content by a similar amount without altering vesicle size, (‐)‐vesamicol may decrease vesicle size by acting on another target. We also found that a hypertonic solution in which the anion was aspartate increased quantal size similar to gluconate. Both anions have high hydration energy and a large volume. When these treatments increased quantal size the mean 20‐80 % rise time of MEPPs recorded with an extracellular electrode was 170 μs. In the controls it was 97 μs. Perhaps some of the added ACh is bound within the vesicles, which slows the rise. Our major conclusion is that ACh content can change notably without any change in the size of the synaptic vesicles.

Recycling and refilling of transmitter quanta at the frog neuromuscular junction.

Fluorescent dyes have been used at the frog neuromuscular junction to label synaptic vesicular membrane. Retrieved membrane is reformed into vesicles, which are released along with pre‐existing vesicles. Consequently, if vesicular refilling with acetylcholine (ACh) is depressed by inhibitors, two sizes of quanta should be released: normal and smaller. As recycling continues the fraction of smaller size quanta should increase exponentially. We enhanced the rate of quantal release by elevating the K+ concentration. The principal inhibitors were (‐)‐vesamicol (VES), hemicholinium‐3 (HC3), and NH4+. Quantal size measurements were fitted to one and to two cumulative lognormal probability distribution functions. When two fitted better, the statistical significance assessment took into account the three additional parameters used in calculating the fit. After recycling in the presence of inhibitor, many sets were fitted better by two lognormal functions. As recycling continued, the fraction of the miniature endplate potential voltage‐time integrals (∫MEPPs) in the larger sub‐population decreased exponentially. The size of the releasable pool was estimated by counting the quanta released by carbonyl cyanide m‐chlorophenylhydrazone (CCCP). This was compared to pool sizes calculated from the inhibitor experiments. The two estimates of pool size were indistinguishable, with mean values ranging from about 170 000 to 270 000. With all of the treatments tested, the means of the sizes in the smaller sub‐population of ∫MEPPs were about 1/3 those of the larger sub‐populations. Recycling synaptic vesicles appear to be incorporated into the releasable pool from which they have roughly the same probability of release as the pre‐existing vesicles.

Repetitive nerve stimulation decreases the acetylcholine content of quanta at the frog neuromuscular junction

1 We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation‐contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents (∫MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 μm neostigmine (NEO) 3000 stimuli reduced ∫MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, ∫MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size (∫MEPP) followed repetitive stimulation of contracting preparations. 2 The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. 3 Following 3000 stimuli at 10 Hz the sizes of the ∫MEPCs increased back to pre‐stimulus values with a half‐time of 8–10 min. Recovery was blocked by (–)‐vesamicol (VES), by hemicholinium‐3 (HC3) and by nicotinic cholinergic agonists ‐ all of which inhibit ACh loading into synaptic vesicles. 4 The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m‐chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. 5 We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.

Anandamide, a naturally-occurring agonist of the cannabinoid receptor, blocks adenylate cyclase at the frog neuromuscular junction

Abstract Anandamide (arachydonylethanolamide) is a naturally-occurring ligand of the cannabinoid receptor. When anandamide binds to its receptor, adenylate cyclase is inhibited. At the frog neuromuscular junction, anandamide lessened the increase in quantal size produced by pretreatment in hypertonic solution. It did not alter the increases in quantal size produced by insulin or by a permeable agonist of cAMP. It was known that hypertonic treatment increases quantal size by way of the cAMP-protein kinase A pathway. Anandamide had no effect on miniature endplate potential frequency (f mepp ) in untreated preparations. After f mepp was increased in the presence of a permeable cAMP agonist, anandamide brought f mepp back to resting levels. The conclusions are that the motor nerve terminal has a cannabinoid receptor. The binding of anandamide to this receptor seems to block adenylate cyclase.

Calcium And Transmitter Release

Publisher Summary This chapter reviews the experiments on the relation between [Ca 2+ ] in and transmitter release at synapses, with an emphasis on the neuromuscular junction. Then, the quantitive models of calcium action, starting with the simplest and adding variables stepwise are discussed, until a model with the minimum complexity consistent with the data is reached. The models are compared to the data for transmitter release at synapses. In the end, the implications of the model for the control of spontaneous quantal release and for synaptic facilitation are considered. The simplest realistic model for Ca 2+ action in triggering the quantal release of neurotransmitter takes into account the following: the Ca 2+ that enters following stimulation adds to [Ca 2+ ] in already present in the resting terminal. Then there is a cooperative interaction in release, n Ca 2+ binds to a receptor to trigger the release of a quantum, and there are a finite number of release sites. There are several reasons to believe that the mechanism of Ca 2+ action in eliciting secretion is substantially different at the neuromuscular junction and the adrenal medulla.

The timing of channel opening during miniature end-plate currents

Many chemical transmitters act by opening channels with exponentially distributed life-times. We present a way to analyze a synaptic current in terms of its component channels. We can estimate the numbers and times of channel opening within the synaptic current. This approach is used to study miniature end-plate currents (m.e.p.c.s) at the frog neuromuscular junction. The results support the idea that some transmitter rebinds after dissociation from post-synaptic receptors, and suggest that the time of channel closing is related to the time at which acetylcholine leaves the receptor.

Potassium‐stimulated respiration and intracellular calcium release in frog skeletal muscle

1. In 1931 Fenn showed that the respiration of frog twitch muscles increases when [K+]o is raised. The present paper is a further study of potassium‐stimulated respiration. Stimulation depends on membrane potential, since respiration is also stimulated by elevated [Rb+]o or [Cs+]o in direct relation to their ability to depolarize.

The timing of channel opening during miniature endplate currents at the frog and mouse neuromuscular junctions: effects of fasciculin-2, other anti-cholinesterases and vesamicol

Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the “tail” that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 μM (−)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (−)-vesamicol.

Calcitonin gene‐related peptide acts presynaptically to increase quantal size and output at frog neuromuscular junctions

1 Calcitonin gene‐related peptide (CGRP) is found in dense‐cored vesicles in the motor nerve terminal. 2 Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (−)‐vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3 The hCGRP effect was not blocked by fragment 8–37, an antagonist of one class of CGRP receptor. 4 hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5 CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.