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.

The sympathetic nerve terminals of the mouse vas deferens were loaded with the calcium indicator Oregon Green 488 BAPTA‐1 by orthograde transport along the postganglionic nerves. Changes in the calcium concentration in the varicosity (Δ[Ca²⁺]_v) were determined following single impulses, and short (5‐impulse) and long (200‐impulse) trains at 5 Hz. All varicosities showed a significant Δ[Ca²⁺]_v in response to every single impulse. The elevated Δ[Ca²⁺]_v declined in two phases with similar kinetics for all varicosities: a fast phase (time constant, 0.42 ± 0.05 s) and a moderate phase (3.6 ± 0.4 s). Line scanning confocal microscopy revealed that the Δ[Ca²⁺] of a single terminal following single impulses was smaller for the intervaricose regions than for the varicosities. Blockade of the voltage‐sensitive calcium channels with Cd²⁺ (in calcium‐free solution) completely blocked the Δ[Ca²⁺]_v on stimulation. The addition of either nifedipine (10 μm), ω‐conotoxin GVIA (100 nM) or ω‐agatoxin TK (100 nm) showed that 47 ± 6% of the evoked response was mediated by N‐type calcium channels. Ryanodine (10 μm) did not significantly change the amplitude of Δ[Ca²⁺]_v in response to short trains. Spontaneous increases in Δ[Ca²⁺]_v were observed in individual varicosities, with coupling in the increase of Δ[Ca²⁺]_v between varicosities. The presynaptic α₂‐receptor antagonist yohimbine (10 μm) increased the amplitude of Δ[Ca²⁺]_v in response to five impulses (5 Hz) by 54 ± 14%, while the α₂‐receptor agonist clonidine (1 μm) decreased the Δ[Ca²⁺]_v by 55 ± 4%. These results are discussed in terms of the hypotheses that the increased probability for secretion at sympathetic nerve terminals which accompanies facilitation and augmentation is due to the residual Δ[Ca²⁺]_v remaining after the calcium influx following impulses and that noradrenaline acts presynaptically to decrease the probability of secretion by modifying calcium influx.