1. The relationship between the nerve terminal action potential and transmitter release from sympathetic postganglionic nerve terminals has been studied in vitro by focal extracellular recording. 2. In the absence of stimulation, 'spontaneous excitatory junction currents' (SEJCs) were recorded with amplitudes up to 500 microV, durations of 50-80 ms and frequencies of occurrence of 0.3-0.05 Hz; SEJCs of unusually long time course were also observed. The SEJCs were not recorded in tissues pre-treated with 6-hydroxydopamine to destroy sympathetic nerves, were unaffected by tetrodotoxin (TTX), the competitive alpha-adrenoceptor antagonists, prazosin and phentolamine, the irreversible alpha-adrenoceptor antagonist benextramine but were blocked by alpha,beta-methylene ATP which desensitizes P2-purinoceptors. 3. During trains of supramaximal stimuli at 0.1-4 Hz stimulus locked 'excitatory junction currents' (EJCs) were evoked intermittently from the population of varicosities located under the suction electrode with a probability of occurrence of 0.005-0.8. Although EJCs occurred intermittently, they were always preceded by an associated, non-intermittent, nerve impulse (delay less than or equal to 3 ms). 4. The EJCs reflect transmitter release from nerves because they were abolished by TTX, removal of calcium from the bathing medium, exposure to alpha-beta-methylene ATP and exhibited frequency-dependent facilitation. 5. Amplitude distributions of SEJCs and EJCs recorded in the same attachment were similar and skewed towards low-amplitude events. Individual SEJCs and EJCs could be found which were identical in amplitude and time course. 6. Locally applied TTX blocked impulse propagation and transmitter release in the terminal region; electrotonic invasion of the terminals from the point of block did not activate the transmitter release process. 7. These studies indicate that (1) intermittence of transmitter release is caused by a low probability of release in the invaded varicosity and is not caused by conduction failure in the terminal regions, (2) only a single quantum is normally secreted when the release mechanism of a varicosity is activated by the nerve impulse and (3) active invasion of the terminals is necessary for transmitter release to occur.

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.

At the skeletal neuromuscular junction, electrophysiological methods have provided much useful information about the mechanisms involved in the release of transmitter. At the autonomic neuroeffector junction it has not been possible to carry out similar studies. Here we report a method of extracellular recording which allows simultaneous measurement of both the nerve action potential and transmitter release from postganglionic sympathetic nerve terminals. We have confirmed that release is intermittent, but the importance of this new approach is that the relationship between the nerve terminal action potential and transmitter release can be studied unambiguously for the first time. Thus we are able to show unequivocally that intermittence is caused by a low probability of release in the invaded varicosity and not by failure of the action potential to invade the varicosity.

Spontaneous purinergic neurotransmission was characterized in the mouse urinary bladder, a model for the pathological or ageing human bladder. Intracellular electrophysiological recording from smooth muscle cells of the detrusor muscle revealed spontaneous depolarizations, distinguishable from spontaneous action potentials (sAPs) by their amplitude (< 40 mV) and insensitivity to the L-type Ca 2+ channel blocker nifedipine (1 μm) (100 ± 29%). Spontaneous depolarizations were abolished by the P2X 1 receptor antagonist NF449 (10 μm) (frequency 8.5 ± 8.5% of controls), insensitive to the muscarinic acetylcholine receptor antagonist atropine (1 μm) (103.4 ± 3.0%), and became more frequent in latrotoxin (LTX; 1 nm) (438 ± 95%), suggesting that they are spontaneous excitatory junction potentials (sEJPs). Such sEJPs were correlated, in amplitude and timing, with focal Ca 2+ transients in smooth muscle cells (measured using confocal microscopy), suggesting a common origin: ATP binding to P2X 1 receptors. sAPs were abolished by NF449, insensitive to atropine (126 ± 39%) and increased in frequency by LTX (930 ± 450%) suggesting a neurogenic, purinergic origin, in common with sEJPs. By comparing the kinetics of sAPs and sEJPs, we demonstrated that sAPs occur when sufficient cation influx through P2X 1 receptors triggers L-type Ca 2+ channels; the first peak of the differentiated rising phase of depolarizations -attributed to the influx of cations through the P2X 1 receptor -is of larger amplitude for sAPs (2248 mV s −1 ) than sEJPs (439 mV s −1 ). Surprisingly, sAPs in the mouse urinary bladder, unlike those from other species, are triggered by stochastic ATP release from parasympathetic nerve terminals rather than being myogenic.

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