Single Nerve Impulse Research Articles

Compound Motor Action Potential Quantifies Recurrent Laryngeal Nerve Innervation in a Canine Model.

The compound motor action potential (CMAP) is the summated action potential from multiple muscle fibers activated by a single nerve impulse. The utility of laryngeal muscle CMAP for quantifying innervation following recurrent laryngeal nerve (RLN) injury was investigated. In a series of 21 canine hemi-laryngeal preparations, RLNs were exposed and a stimulating electrode placed. Maximum CMAP amplitudes and area under the curve from the thyroarytenoid (TA) muscles were obtained at baseline and at 6 months following injury to the RLN. Injury mechanisms included crush, stretch, cautery, and complete transection with microsuture repair. Prior to injury, baseline CMAP amplitudes and area under the curve were 15.81 mV and 15.49mVms, respectively. Six months following injury, CMAP amplitude and area under curve were 105.1% and 102.1% of baseline for stretch, 98.7% and 112.7% for crush, 93.3% and 114.3% for cautery. The CMAP amplitude and area under the curve in the transection/repair group had a 54.3% and 69.4% recovery, respectively, which were significantly different than baseline (P < .01, P < .05). These values were correlated with vocal fold motion. The CMAP is a measure of vocal fold innervation. The technique could be further developed for clinical and experimental applications.

Specific mechanism of use-dependent channel block of calcium-permeable AMPA receptors provides activity-dependent inhibition of glutamatergic neurotransmission

This study examined the blocking action of the selective channel blocker of calcium-permeable (CP) AMPA receptors, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925), on excitatory postsynaptic currents in rat neostriatal and cortical neurons and in fly neuromuscular junctions. In both preparations, the blocking of CP-AMPA receptor currents increased along with the stimulation frequency. The continuous presence of kainate, which activates AMPA receptors, in the external solution also caused an enhanced blocking effect. Likewise, decrease of the synaptic release by lowering calcium concentration resulted in significant reduction of the blocking action. The activity dependence of the block is explained using the guarded receptor model. The drug molecule can only bind if the channel is open. After the channel has closed, the drug molecule remains trapped inside. However, the trapped molecule slowly egresses from closed channels to the cytoplasm. The total block effect is determined by the equilibrium between accumulation of the drug in the open channels and relief from the closed channels. Therefore, the conditions that favour the open state result in enhanced inhibition. This significant finding reveals a new way to modulate CP-AMPAR-mediated transmission using a physiologically relevant approach. Moreover, it allows the involvement of CP-AMPARs in the physiological and pathological processes – such as high-frequency synaptic activity or increase of the steady-state glutamate concentration – to be examined.

Structural Factors Influencing the Efficacy of Neuromuscular Transmission

Neuromuscular junctions (NMJs) in different species share many features of structure and function. At the same time, important differences distinguish, for example, human NMJs from those in other species. An understanding of the biological context of the human NMJ helps in the interpretation of the effects of disease on the biophysical properties of neuromuscular transmission. Many NMJs consist of a number of spot-like synaptic regions 1-5 microm across. Usually only a few multimolecular "quanta" of transmitter are released from each presynaptic "bouton" by a single nerve impulse. The total number of quanta released from an NMJ is roughly proportional to its total area. For example, human NMJs are about 10-fold smaller than those in frogs and release about 20 quanta/impulse versus 100-200 in frog NMJ. Although human NMJs release relatively few quanta, the effect of the transmitter is amplified by the high density of voltage-gated sodium channels (Na(V)1.4) in the highly folded postsynaptic membrane. A genetic influence on NMJ size has recently been discovered in some patients with limb-girdle myasthenia (LGM). Mutations of the gene encoding Dok-7, an essential component of the agrin-muscle-specific kinase pathway that controls postsynaptic differentiation at the mammalian NMJ, results in impaired transmission because the NMJs are abnormally small and have reduced folding but have a normal local density of normal acetylcholine receptors. This condition emphasizes the importance of structural features in achieving reliability of neuromuscular transmission.

Clathrin‐mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis (tau = 15 s) operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.

Clathrin-Mediated Endocytosis Is the Dominant Mechanism of Vesicle Retrieval at Hippocampal Synapses

The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.

Release probability modulates short-term plasticity at a rat giant terminal.

1. Modulation of release probability is a major factor underlying short-term synaptic plasticity in the central nervous system. We have investigated the relationship between release probability ((Pr) and paired-pulse modulation at a large auditory calyceal synapse containing many transmitter release sites. Whole-cell patch electrode recordings were made of excitatory postsynaptic currents (EPSCs), evoked by stimulation of auditory nerve fibres giving rise to the endbulbs of Held. 2. Quantitative estimates of Pr and quantal amplitude were obtained using the recently developed variance-mean analysis technique. Release probability conditions were modulated by bath application of cadmium, elevated calcium and protein kinase C activation by phorbol esters. 3. Our results show that, under physiological conditions, most sites released neurotransmitter following a single presynaptic nerve impulse, with a mean Pr of 0.6. The mean quantal amplitude was 44 pA, which was consistent with the mean amplitude of miniature EPSCs (47 pA). 4. Under high release probability conditions with elevated calcium or phorbol esters, Pr at all sites approached 1.0. At these high Pr values, variance-mean analysis indicated a significant postsynaptic contribution to paired-pulse depression. The miniature EPSC amplitudes were decreased following stimulation in elevated calcium, confirming a postsynaptic component of paired-pulse depression at this glutamatergic connection. 5. A notable feature was the large variability between neurons in the relationship between paired-pulse ratio and Pr. Based on current models of vesicle release and ultrastructural evidence, we suggest that this variability may be partly due to morphological differences between endbulb specializations, particularly in the ratio of fusion-ready to reserve populations of vesicles at endbulb release sites.

Quantal acetylcholine release: vesicle fusion or intramembrane particles?

Images of vesicle openings in the presynaptic membrane have regularly been shown to increase in number after stimulation of cholinergic nerves. However, with a very few exceptions, the occurrence of vesicle openings is delayed in time with respect to the precise moment of transmitter release. In contrast, a transient change in the size and distribution of intramembrane particles (IMPs) has constantly been found as a characteristic change affecting the presynaptic membrane in a strict time coincidence with the release of acetylcholine quanta. This is illustrated here in a rapid-freezing experiment performed on small specimens of the Torpedo electric organ during transmission of a single nerve impulse. A marked change affected IMPs in the presynaptic membrane for 3-4 ms, i.e., a population of IMPs larger than 10 nm momentarily occurred in coincidence with the passage of the impulse. The nicotinic receptors, abundantly visible in the postsynaptic membranes, also underwent very fleeting structural changes during synaptic transmission. In conclusion, for rapidly operating neurotransmitters like acetylcholine, a characteristic IMP change was regularly found to coincide in the presynaptic membrane with the production of neurotransmitter quanta, whereas images of vesicles fusion were either delayed or even dissociated from the release process. This is discussed in connection to the different modes of release recently described for other secreting systems.

Physiological and behavioral studies of escape responses in calanoid copepods

Electrophysiological techniques have been applied to monitoring sensory discharges from the first antennae of calanoid copepods. Extracellular nerve impulse traffic from both mechanoreceptors and putative chemore‐ceptors has been recorded. The first antennae of some, but not all, calanoid groups possess “giant” mechano receptive axons generating very large (mV) extracellular signals. There are two such giant antennal mechano receptors (GAMs) innervating setae of each distal tip. These are sensitive to small (< 10 nm) controlled hydro‐dynamic disturbances, including abrupt displacements and sinusoidal vibrations with frequencies up to and exceeding 2 kHz. Behavioral studies show that escape “jumps"can be triggered in Labidocera madurae by the same types of disturbances. Sensitivities as low as 4 nm were observed at frequencies of ca. 900 Hz. Behavioral sensitivities are similar to those measured physiologically and suggest that firing of the GAMs is capable of triggering escape behavior, perhaps even with a single nerve impulse.

STRUCTURE AND FUNCTION OF NEUROMUSCULAR JUNCTIONS IN THE VASTUS LATERALIS OF MAN

The properties of neuromuscular junctions (NMJs) in the vastus lateralis of man have been studied in motor point biopsy samples and compared with those reported for lower vertebrates (frogs and mice). The patients studied had no convincing evidence of a primary disturbance of neuromuscular transmission or other neurogenic component. Morphological studies were made using a variety of methods at the light- and electron-microscope levels. The size of the presynaptic nerve terminal and the area of postsynaptic specialization were smaller, relative to the size of the muscle fibres, than in the lower vertebrates. In contrast, the extent of postsynaptic folding was greater. Intracellular recordings from single muscle fibres showed that the duration of synaptic currents was longer than in most other vertebrates so far studied and that the number of transmitter 'quanta' released by a single nerve impulse, about 20, was lower, probably reflecting the small size of the presynaptic terminals. The hypothesis is discussed that in man, a relatively weak effect of transmitter on the muscle fibre surface is amplified by voltage-dependent sodium channels which have been shown in the rat to be concentrated in the depths of the synaptic folds. The implications of this hypothesis for the interpretation of pathological findings in myasthenic syndromes are also discussed.

Noradrenaline and ATP as cotransmitters in sympathetic nerves

Evidence is presented in support of the view that adenosine 5?-triphosphate (ATP) is a cotransmitter with noradrenaline (NA) in sympathetic nerves supplying the vas deferens and a number of blood vessels. ATP is responsible for the excitatory junctional potentials (EJPs) in response to single nerve impulses and the initial twitch responses of the smooth muscle, while NA produces the longer-lasting tonic contractions. The proportions of ATP to NA vary between different sympathetic nerves; they also change during development and in some pathological conditions. Prejunctional neuromodulation of release of the two cotransmitters appears to involve independent mechanisms and is frequency dependent; this raises the question of whether ATP and NA are stored in separate vesicles or whether there are subpopulations of sympathetic nerves with a predominance of either ATP or NA. ATP and NA have synergistic postjunctional actions, whether excitatory (as in the vas deferens and most blood vessels) or inhibitory (as in rabbit coronary vessels). It is suggested that use of the term "adrenergic nerves" as a synonym for sympathetic nerves is no longer appropriate, although "adrenergic transmission" or "purinergic transmission" are still useful terms.

Momentary alteration of the postsynaptic membrane during transmission of a single nerve impulse.

Transmission of a nerve impulse at neuromuscular and other synapses is an extremely brief event. By using rapid-freezing and cryofracture techniques in the electric organ of Torpedo, synaptic transmission was found to be accompanied by significant changes affecting the postsynaptic membrane for a few milliseconds. In the replicas, the protoplasmic leaflet of this membrane was seen to contain intramembrane particles (IMPs) of two different forms, globular and elongated. Globular IMPs had a mean diameter of 8.8 nm; they were the most frequently found (80% in unstimulated specimens). Elongated IMPs had a major diameter of 17.9 nm, about twice that of globular IMPs. Transmission of a single nerve impulse was accompanied by a marked decrease in the number of globular IMPs and by an increase in the number of elongated IMPs, as if there were a coalescence of two adjacent round particles to form an elongated one. These changes started soon after the electrical stimulus and lasted for approximately 3 ms. IMPs in the postsynaptic protoplasmic face are thought to correspond to a certain proportion of nicotinic acetylcholine receptors that were extracted with this leaflet during the fracture process. The phenomenon described here reflects an abrupt change in the membrane, probably linked to activation of the acetylcholine nicotinic receptors.

Variability in the initiation of diffuse nerve-net impulses in the mangrove jellyfish Cassiopea xamachana (Coelenterate: Scyphozoa)

Abstract 1. Single electric stimuli given anywhere on the subumbrellar epiderm of the rhizostome Cassiopea xamachana cause initiation of discrete distinguishable single nerve impulses in each of two nerve-nets, the giant-fiber nerve-net (GFNN) and the diffuse nerve-net (DNN). 2. DNN pulse arrival times to remote recording sites are very variable, from trial to trial, but highly correlated when measured to separate recording sites along different pathways after a single stimulus. Less than 10% of the total variability is due to short-term random variation in conduction. 3. Evidence is presented in support of a model of long and variable time required for DNN pulse initiation by stimuli but relatively constant pulse conduction. The two nerve-nets are contrasted in these regards.

Adaptation in Helix pomatia neurons

1. 1. Spike frequency adaptation has been studied on neurons of Helix pomatia subesophageal ganglia and interpreted by means of a behavioural model describing the phenomenon in neurons either silent or autorhythmic at rest. 2. 2. At low stimulating currents the initial discharge frequency F(0) is linearly related to the current strength G. 3. 3. In the linearity range F(0)/ G each neuron was characterized by means of four model parameters: the proportionality constant between F(0) and G, the decay constant of the frequency, the inhibitory current from a single nerve impulse and the decay time constant of the inhibitory current. 4. 4. The four parameters varied in different cells with a range of 0.18–4.98 Hz/nA, 1.02–3.85 sec, 0.05–0.95 nA and 1.74–22.33 see, respectively. 5. 5. Experimental results have been analyzed considering inhibitory current, electrogenie sodium pump and other proposed adaptation parameters.

Brief occurrence of a population of presynaptic intramembrane particles coincides with transmission of a nerve impulse.

Small pieces of Torpedo electric organ were cryofixed at 1-ms time intervals in a liquid medium at -190 degrees C before, during, and after the passage of a single nerve impulse. In contrast to studies using this or other preparations, these experiments were done without 4-aminopyridine or other drugs that potentiate transmitter release. Freeze-fracture replicas were made from the most superficial layers of the tissue, where the rate of cooling was rapid enough to retain ultrastructure in the absence of chemical fixation. We found that the transmission of an impulse was accompanied by the momentary appearance of a population of large intramembrane particles in both the protoplasmic (P) and the external (E) leaflets of the presynaptic plasma membrane. The change was very brief, appearing soon after the stimulus artifact. It lasted for 2-3 ms. Large pits denoting vesicle openings at the presynaptic membrane were found in a small proportion of nerve terminals; their number did not increase during transmission of the nerve impulse. Reducing the temperature from 16 to 5 degrees C slowed the time course of both the electrophysiological response and the change in intramembrane particles. The number of large particles did not increase when stimulation was applied in a low-Ca medium, a condition where the nerve terminals were still depolarized by the action potential but did not release the neurotransmitter. From these and other observations, we conclude that this transient change of intramembrane particles is closely linked to the mechanism of acetylcholine release at the nerve-electroplaque junction.

Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve-electroplaque junction

Small pieces of Torpedo electric organ were treated with 4-aminopyridine. a drug which greatly increases the duration of transmitter release in a single nerve impulse, transforming the normally brief electroplaque potential to a giant discharge. Specimens of tissue were cryofixed by rapid freezing using liquid coolants at precise time intervals during transmission of a single giant discharge, and then examined by freeze fracture. In each experiment, we monitored the electrical response of one specimen during the freezing run to check the physiological responsiveness of the tissue and to determine the precise time of contact with the cryogenic liquid. The general appearance of nerve terminals after cryofixation was similar to that of terminals from chemically fixed and cryoprotected tissue. The major morphological change observed during the time course of the giant discharge was a marked increase in the density of intramembrane particles larger than 10 nm on both the protoplasmic and external faces of the presynaptic membrane. This change appeared in specimens frozen within the first few milliseconds after the stimulus, that is, at a time corresponding to the onset of the rising phase of the potential (3 ms). At the end of the giant discharge, the particle density returned to control values with the same time course as the potential trace. Pits of 20 nm or larger, probably due to vesicle-membrane interaction, were found in a small proportion of nerve terminals. Their occurrence increased only at 120–150 ms after the stimulus, that is a long time after the beginning of the giant potential and of the change in intramembrane particles. The size distribution of particles was also determined in the membrane of synaptic vesicles exposed by cross fracture of terminal boutons; it was found to be similar to that of the unstimulated presynaptic membrane and it did not change during the giant discharge. Stimulation experiments were also earned out in a modified solution containing no added calcium. 20 mM magnesium and 4-aminopyridine. The propagation of impulses along the nerves to the electric organ was not inhibited in the modified solution but acetylcholine release was prevented and no increase in particle density was found on the presynaptic membrane. These and previous biochemical experiments on this tissue suggest that the release of the neuro. transmitter acetylcholine is associated with a transient occurrence of large intramembrane particles on the two fracture faces of the presynaptic membrane. At variance from the results obtained on the frog neuromuscular junction [Heuser et al. (1979) J. Cell Biol. 81, 275] the present changes of intramembrane particles apparently did not follow vesicle fusion. The possible explanations for the discrepancy between the two sets of results have been discussed.

Chemo-electrical transduction in insect olfactory receptors.

Insect behavior can be controlled by a single odor or by a blend of a few components in an exact ratio of concentrations. These odor compounds or odor mixtures are called pheromones if used for intraspecific communication. Each of the pheromone components is perceived by a particular type of receptor cell that is specifically tuned to its key compound and may respond to single odor molecules by firing single nerve impulses. The entire response range of a receptor cell may cover many decades of stimulus intensities. Each olfactory receptor cell is a bipolar neuron. Its dendrite innervates a specialized cuticular structure, usually a hollow cuticular hair that captures the stimulus molecules. The axon runs directly to the deutocerebrum-the first synaptic relay station of the central nervous system. Chemo-electrical transduction in these organs comprises the processes leading from the adsorption of odor molecules on the cuticular surface via stimulus transport to the generation of receptor potentials and nerve impulses and, eventually, to the inactivation of the stimulus molecules. The interconnections of these processes and their influences on the ultimate impulse response of the receptor cells are largely unknown. This review covers electrophysiological investigations of cellular response charac­ teristics as well as biophysical and biochemical work, much of which is based on radiolabeled pheromone compounds. These studies employed a few species of insects, mainly of moths with olfactory organs that are relatively large and accessible for experimental work. The olfactory hairs of

Different nerve responses in consecutive sections of the arterial system.

Isometric responses to nerve stimulation and to applied noradrenaline were compared in isolated ring preparations of three consecutive types of rat arteries, viz. the aorta, the superior mesenteric artery and 200-micron branches of the latter. Intramural nerves were activated by graded electrical field stimulation; obtained responses were blocked by tetrodotoxin, phentolamine or prazosin. Also direct muscle activation could be accomplished, using impulses of long duration. In the aorta responses to nerve stimulation were sluggish, could reach only 30-40% of the maximal noradrenaline response and single impulses were ineffective. In the small resistance arteries, neurogenic responses matched the maximal noradrenaline responses and distinct, rapid contractions occurred to single nerve impulses. The superior mesenteric artery was intermediate in these respects. By contrast, on direct muscle activation all vessels responded to single impulses with rapid contractions that differed little in velocity. Pharmacological inhibition of transmitter reuptake increased noradrenaline sensitivity most in the small arteries, but nerve responses were most enhanced in the superior mesenteric artery. These regional differences, probably reflecting decreasing neuromuscular distance with decreasing vessel size, imply that generalizations concerning the relation between responses to nerve stimulation and to exogenous noradrenaline cannot be made from one vascular model only.

How do changes in diameter at the precapillary level affect cardiovascular function?

A survey is given of the various high-pressure precapillary vascular sections and of how they affect local and overall cardiovascular functions, with special emphasis put on the important "precapillary resistance vessels." The complex interactions between hemodynamic effects dependent on (a) vessel design, (b) transmural pressure, (c) "passive" wall distensibility, and (d) "active" smooth muscle responses are outlined in principle and experimentally illustrated with respect to systemic resistance control. Particular attention is devoted to the influence of the sympathetic vasoconstrictor fibers on precapillary vascular functions, concerning aspects such as speed, precision, range, and differentiation of the neurogenic effects, because these fibers represent the most powerful and widespread of the vascular control mechanisms involved in cardiovascular homeostasis. How these fibers in well-innervated vascular circuits can command up to the maximum contractile capacity of both the precapillary resistance and postcapillary capacitance vessels is illustrated, as well as the way in which these sets of vessels respond to even single nerve impulses with twitchlike, rapid contractions.

Effects of hypertonic solutions on quantal transmitter release at the crayfish neuromuscular junction.

1. The effects of a hypertonic bathing medium, containing either NaCl or melezitose, on the average number (m) and the time course of quantal release following an action potential were studied using focal extracellular recording methods at synaptic sites on the opener muscle of the crayfish leg.2. After the application of hypertonic saline, the rate of spontaneous quantal release increased but m decreased to a new level within 1 min; the extent of depression depended on the magnitude of the increase in tonicity until the osmolarity was 50% greater than the normal value of 0.43 osmol/l (Osm), but greater increases in tonicity exerted little further effect.3. The synaptic delays were increased and distributed over a longer range of time in hypertonic solutions; also, the latency between the first and second quantal releases in a multiple response to a single nerve impulse was also increased.4. Hypertonicity had no significant effect on the conduction velocity of the action potential, the independence of successive quantal releases in the same response, or the uniformity of the rise and fall of the probability of quanta release following an action potential, alpha(t).5. The time course of alpha(t) is prolonged in hypertonic solutions; this was manifested as an increase in the time constant of the exponential decline in alpha(t) from its peak value following the nerve impulse.5. When the potentiating agent 5-hydroxytryptamine (5-HT) was added to the hypertonic saline, m was increased, and the time course of alpha(t) was prolonged further than in the hypertonic solution alone; 5-HT produced no change in the time course of alpha(t) when it was added to normal saline, although m was increased.6. It is concluded that in this preparation hypertonicity decreases the rate of release of each quantum from the nerve terminal following a nerve impulse.

Effects of calcium and strontium in the process of acetylcholine release from motor nerve endings

1. The effects of Ca and Sr ions on synchronous acetyleholine (ACh) secretion (the impulsive, physiologically functional form of secretion which produces an end-plate potential in response to a single nerve impulse) and on asynchronous ACh secretion (the delayed, residual increase in miniature end-plate potential frequency evoked by repetitive nerve impulses or by accumulation of intracellular divalent cations) were studied at frog neuromuscular junctions.2. In a comparison of their extracellular effects, Ca was far more effective than Sr in supporting synchronous ACh secretion but less effective than Sr in mediating asynchronous release evoked by repetitive nerve impulses.3. In studies of their intracellular effects, Sr and Ca were delivered to the nerve terminal cytoplasm using liposomes as a vehicle. Ca-containing liposomes, although producing effects on asynchronous ACh secretion that were indistinguishable from those of equimolar Sr-containing liposomes, were more effective than Sr-containing liposomes in increasing synchronous release.4. Extracellular Ca behaved as a potent competitve inhibitor of asynchronous, neurally evoked release mediated by Sr. In contrast, intracellular Ca (i.e. liposomal Ca), whilst increasing synchronous ACh release, failed to antagonize evoked asynchronous release.5. The results demonstrate that synchronous and asynchronous secretion have different sensitivities to alterations in intracellular divalent cation concentrations. It is suggested that selectivity for Ca over Sr may occur at intraterminal sites responsible for synchronous ACh secretion but not at sites responsible for asynchronous ACh release. Furthermore, Ca appears to bind with high affinity as an antagonist at the external surface of the nerve ending. These results are discussed in conjunction with current theories of depolarization-secretion coupling.