Here we report on the development of a high-resolution optical recording technique that is capable of detecting ATP release from single varicosities on the same terminal branch on an impulse-to-impulse basis.

Mice (Balb/C) were humanely killed. The Ca²⁺ indicator Oregon Green 488 BAPTA-1 10 kDa dextran was applied to the cut end of the isolated vas deferens to load nerve terminals orthogradely over 8-10 h (see Brain & Bennett, 1997) and here, a population of smooth muscle cells. Most preparations were examined using a Leica inverted confocal microscope, at a temporal resolution of either 200 or 400 ms per frame. In some experiments, Ca²⁺ transients were monitored with a Nipkow disc-scanning microscope (20 ms temporal resolution).

Within about 300 µm of the cut end of the vas deferens, fluorescence was detected in a small number of smooth muscle cells, consistent with the presence of Ca²⁺ indicator in the cytoplasm. In these cells, electrical field stimulation (p.w. 0.06 ms, 50 V), which induces action potentials in presynaptic terminals, elicited focal increases in [Ca²⁺] in individual smooth muscle cells, which we have termed neuroeffector Ca²⁺ transients (NCTs). Within a given cell, NCTs were spatially clustered in discrete regions (n_c = 114 cells, from n_p = 15 preparations). At 1 Hz, the probability of evoking a NCT from a cluster was 0.018 ± 0.002 (mean ± S.E.M.; Student’s t test; n_r = 111 regions; n_p = 10). Each NCT lasted about 400 ms. Spontaneous [Ca²⁺] transients were also occasionally detected (n_c = 20 cells; n_p = 12); in cells which were sampled for at least 150 s, the mean frequency of spontaneous [Ca²⁺] transients per cluster was 0.0012 Hz (n_r = 144; range 0-0.012 Hz). NCTs were not blocked by prazosin (100 nM; α1-blocker; change in probability, ▓Dgr│P = (-2.0 ± 1.3) Ω 10^-3), or nifedipine (1 µM; L-type Ca²⁺ channel blocker; ▓Dgr│P = (-1.2 ± 1.3) Ω 10^-3), but were abolished by α,β-methylene ATP (1 µM; n_r = 40; n_c = 5 cells; n_p = 3; P < 0.05), which desensitises P2X receptors, or the Na⁺ channel blocker saxitoxin (100 nM; n_c = 5 cells; n_p = 3; P < 0.05). In some preparations (n_c = 22 cells; n_p = 5) varicose nerve terminals labelled with the calcium indicator were found adjacent to labelled smooth muscle cells; these responded to every action potential. Under favourable recording conditions, NCT clusters were adjacent to a responding varicosity.

We hypothesise that NCTs are elicited by ATP released from varicose nerve terminals, which triggers Ca²⁺ influx through P2X receptors. The frequency of NCTs can be used to measure release probability from identified varicosities. The results confirm the intermittent nature of transmitter release in the mouse vas deferens (Cunnane & Stjèrne, 1984). A noradrenergic component has not been detected to date. This new approach complements well-established techniques to study transmitter release and has the additional advantage of providing greater spatial resolution.

Brain, K.L. & Bennett, M.R. (1997). J. Physiol. 502, 521-536. abstract

Cunnane, T.C. & Stjèrne, L. (1984). Neuroscience 13, 1-20.