A confocal Ca2+ imaging technique has been used to detect ATP release from individual sympathetic varicosities on the same nerve terminal branch. Varicose nerve terminals and smooth muscle cells in mouse vas deferens were loaded with the Ca2+ indicator Oregon Green 488 BAPTA‐1. Field (nerve) stimulation evoked discrete, focal increases in [Ca2+] in smooth muscle cells adjacent to identified varicosities. These focal increases in [Ca2+] have been termed ‘neuroeffector Ca2+ transients’ (NCTs). NCTs were abolished by α,β‐methylene ATP (1 μM), but not by nifedipine (1 μM) or prazosin (100 nm), suggesting that NCTs are generated by Ca2+ influx through P2X receptors without a detectable contribution from L‐type Ca2+ channels or α1‐adrenoceptor‐mediated pathways. Action potential‐evoked ATP release was highly intermittent (mean probability 0.019 ± 0.002; range 0.001‐0.10) at 1 Hz stimulation, even though there was no failure of action potential propagation in the nerve terminals. Twenty‐eight per cent of varicosities failed to release transmitter following more than 500 stimuli. Spontaneous ATP release was very infrequent (0.0014 Hz). No Ca2+ transient attributable to noradrenaline release was detected even in response to 5 Hz stimulation. There was evidence of local noradrenaline release as the α2‐adrenoceptor antagonist yohimbine increased the probability of occurrence of NCTs by 55 ± 21 % during trains of stimuli at 1 Hz. Frequency‐dependent facilitation preferentially occurred at low probability release sites. The monitoring of NCTs now allows transmitter release to be detected simultaneously from each functional varicosity on an identified nerve terminal branch on an impulse‐to‐impulse basis.

AbstractöWhile nicotine is known to act at neuronal nicotinic acetylcholine receptors (nAChRs) to facilitate neurotransmitter release, the mechanisms underlying this action are poorly understood. Some of its e¡ects are known to be mediated by presynaptic receptors. In the mouse vas deferens nicotine (10^30 WM) transiently increased the force of neurogenic contraction by 135 þ 25%, increased the amplitude of excitatory junction potentials by 74 þ 6% and increased the frequency of spontaneous excitatory junction potentials in four out of six preparations. Confocal microscopy and the calcium indicator Oregon Green 488 BAPTA-1 dextran were used to measure calcium concentration changes in the nerve terminals. Nicotine did not a¡ect the action potential-evoked calcium transient but instead triggered small, random £uctuations (`calcium spikes') in intra-varicosity calcium concentrations at an average frequency of 0.09 þ 0.02 Hz. These were insensitive to tetrodotoxin at a concentration that blocked action-potential evoked calcium transients (300 nM). They were abolished by the nAChR blocker hexamethonium (100 WM) and by both ryanodine (100 WM) and ca¡eine (3 mM), agents that modify calcium release from intracellular stores.We propose a novel mechanism whereby nicotine's action at nAChRs triggers calcium-induced calcium release from a ryanodine-sensitive calcium store in nerve terminals. This primes neurotransmitter release mechanisms and enhances both spontaneous and action potential-evoked neurotransmitter release. ß

1. Action potential‐evoked Ca2+ transients in postganglionic sympathetic axon bundles in mouse vas deferens have been characterized using confocal microscopy and Ca2+ imaging. Axonal Ca2+ transients were tetrodotoxin sensitive. The amplitude depended on both the frequency of stimulation and the number of stimuli in a train. Removal of extracellular Ca2+ abolished the Ca2+ transient. Cd2+ (100 μm) inhibited the Ca2+ transient by 78 ± 10 %. The N‐type Ca2+ channel blocker ω‐conotoxin GVIA (0.1 μm) reduced the amplitude by −35 ± 4 %, whereas nifedipine (10 μm; L‐type) and ω‐conotoxin MVIIC (0.1 μm; P/Q type) were ineffective. Caffeine (10 mm), ryanodine (10 μm), cyclopiazonic acid (30 μm) or CCCP (10 μm) had no detectable effects. Blockade of large and small conductance Ca2+‐dependent K+ channels with iberiotoxin (0.1 μm) and apamin (1 μm), respectively, or Ca2+‐dependent Cl− channels by niflumic acid (100 μm) did not alter Ca2+ transients. In contrast, the non‐specific K+ channel blockers tetraethylammonium (10 mm) and 4‐aminopyridine (10 mm) markedly increased the amplitude of the Ca2+ transient. Blockade of delayed rectifiers and A‐like K+ channels, by tityustoxin‐K (α) (0.1 μm) and pandinustoxin‐K (α) (10 nm), respectively, also increased the Ca2+ transient amplitude. Thus, Ca2+ transients are evoked by Na+‐dependent action potentials in axons. These transients originate mainly from Ca2+ entry through voltage‐dependent Ca2+ channels (80 % Cd2+ sensitive of which 40 % was attributable to N‐type). Twenty per cent of the Ca2+ transient was not due to Ca2+ entry through voltage‐gated Ca2+ channels. Intracellular stores and mitochondria were not involved in the generation of the transient. Ca2+ transients are modulated by A‐like K+ channels and delayed rectifiers (possibly KV1.2) but not by Ca2+‐activated ion channels.

Action potential-evoked calcium transients in varicosities in mouse vas deferens were monitored using laser scanning confocal microscopy. Their signi®cance was examined by comparison with excitatory junction potentials (EJPs) and neurogenic contractions, both indirect measurements of transmitter release. Bretylium abolished EJPs, as well as the ATP and NA-mediated phases of contraction. However, bretylium revealed a prominent late component of contraction that was atropine-sensitive. Bretylium abolished calcium transients in 21%, enhanced in 16% and had no e ect in 63% of varicosities examined. Pre-treatment with 6-OHDA reduced NA levels to below detectable levels but many strings of varicosities still responded to nerve impulses with`normal' calcium transients. Varicosities in which calcium transients were abolished by these agents were sympathetic. The identity of those varicosities in which calcium transients were resistant to bretylium (sympathetic but no uptake-1 sites, parasympathetic, sensory) remains to be established.

The strength of synaptic transmission is highly variable between different synapses. The present study examined some factors that may contribute to this variation in the strength of neurotransmission in sympathetic varicosities of the mouse vas deferens. Transmitter release was measured using a focal macropatch electrode placed over pairs of visualised varicosities. By regulating the calcium concentration of the solutions inside the recording electrode and in the bath independently of each other, transmitter release was restricted to one or two surface varicosities at each recording site. Using this technique, transmitter release probability was shown to be highly variable, even between adjacent varicosities on single axon branches. Very little variation was observed in the calcium influx following single impulse nerve stimulation between adjacent Oregon Green BAPTA-1 loaded varicosities. However, the staining intensities of three vesicular proteins, SV2, synaptophysin, and synaptotagmin 1, showed considerable variation between adjacent varicosities on single axon branches. This variation in staining intensity may be partly explained by variation in the density of synaptic vesicles. However, double staining experiments using two vesicular antigens showed some varicosities staining for one vesicular antigen, but not for the second, suggesting that the expression of these release machinery proteins is regulated locally within the varicosities. The results of the present study strengthen suggestions that synaptic strength is at least in part, regulated by variation in the expression of vesicular proteins.