3.1. Rin and membrane potential
The effect of 18βGA (10 μM) on Rin was tested on both mouse and guinea pig vas deferens. A partially reversible increase in Rin was observed in guinea pig (by 354 ± 50%; control: 9.6 ± 1.4 MΩ, 18βGA: 43.6 ± 1.5 MΩ; nc = 26, np = 5, P < 0.001; a) and mouse (by 137 ± 17%, control: 24.1 ± 4.5 MΩ, 18βGA: 57.0 ± 11.3 MΩ; nc = 36, np = 6, P < 0.05; c) vas deferens. 18βGA also significantly depolarised cells in guinea pig (by 8 ± 1%, control: − 75.8 ± 2.1 mV, 18βGA: − 69.8 ± 2.6 mV; nc = 36, np = 6, P < 0.005; b) and mouse (by 11 ± 1%, control: − 79.4 ± 1.1 mV, 18βGA: − 70.9 ± 2.5 mV; nc = 35, np = 6, P < 0.01; d) vas deferens.
Fig. 1 The effect of 18βGA on cell input resistance and membrane potential. 18βGA (10 μM) increased both the input resistance (a, c) and resting membrane potential (b, d) in both guinea pig (n = 6; a, b) and mouse (more ...)
Unlike 18βGA (10 μM), heptanol (2 mM) in mice did not affect resting Em. The mean Em in control (− 79 mV) and with heptanol (− 78 mV) was not significantly different.
3.2. Spontaneous EJPs (sEJPs)
Changes in cell-to-cell coupling affecting the spread of current between cells should produce changes in sEJP shape and amplitude, as explained in the discussion. In the mouse, heptanol (2 mM) did not significantly change sEJP amplitude, frequency or time to fall from 90% to 50% of peak (F90–50; median amplitude 1.93 ± 0.32 to 1.96 ± 0.35 mV; frequency 0.188 ± 0.034 to 0.189 ± 0.024 Hz; fall time 21.0 ± 0.6 to 20.7 ± 0.7 ms; np = 6).
Similarly, 18βGA (10 μM) did not change sEJP characteristics significantly in the mouse (). However, in 5 of 6 preparations there was a significant increase in sEJP frequency (19 ± 3%, nc = 30, np = 5, P < 0.05). In guinea pig vas deferens 18βGA administration also increased sEJP amplitude (by 34 ± 4%, nc = 36, np = 6, P < 0.05; a). Associated with this change was an increase in high amplitude sEJPs (c) and a statistically significant increase in sEJP frequency (by 71 ± 25%; control: 0.07 ± 0.02 Hz, 18βGA: 0.12 ± 0.02 Hz; nc = 36, np = 6, P < 0.05; i). On the other hand, the time course of the sEJP, assessed as the F90–50, was not significantly affected (b).
The effects of 18βGA on sEJPs in mouse vas deferens. 18βGA (10 μM) had no effect on sEJP (a) amplitude, (b) frequency or (c) F90–50 in the mouse vas deferens.
Fig. 3 The effects of 18βGA on sEJPs in guinea pig vas deferens. The effects of 18βGA (10 μM) on sEJP (a) amplitude and (b) F90–50. (c) shows the amplitude histogram of sEJPs illustrating an increase in sEJP frequency (more ...)
Heptanol (2 mM) reversibly decreased average EJP amplitude in mouse vas deferens (by 60 ± 5%; control: 19.0 ± 5.1 mV, heptanol: 7.5 ± 2.4 mV, nc = 36, np = 6, P < 0.05; ). Paired-pulse facilitation was calculated as the ratio of the amplitude of the second stimulus to the first stimulus (no example is shown). No significant change in the facilitation was observed after heptanol. Further, the latency from stimulus to EJP peak was reversibly increased by heptanol (by 27 ± 5%, nc = 36, np = 6, P < 0.05). Although a consistent effect on the variability of EJP amplitude was not found, 4 of the 6 tissues did show a significant (P < 0.05) increase in variability of the first stimulus EJP amplitude using the F-test.
Fig. 4 The effect of heptanol on EJPs in guinea pig vas deferens. There was a decrease in EJP amplitude on exposure to heptanol (2 mM) in mice. (a) shows representative EJPs before and after heptanol exposure. (b) shows that the mean EJP amplitude decreased (more ...)
Similarly, 18βGA (10 μM) decreased EJP amplitude in guinea pig (by 44 ± 3%, control: 5.7 ± 1.2 mV, 18βGA: 3.2 ± 1.0 mV; nc = 36, np = 6, P < 0.005; a, b) and mouse (by 32 ± 3%, control: 25.6 ± 1.9 mV, 18βGA: 17.3 ± 2.5 mV; nc = 36, np = 6, P < 0.005; c, d) vas deferens. In the guinea pig the EJP was almost completely abolished upon stimulation in several cells. Upon washout the effect of 18βGA was not reversed and EJP amplitude remained significantly smaller than in control in both mouse (by 25 ± 3%, P < 0.05) and guinea pig (by 34 ± 7%, P < 0.05) vas deferens. Analysis of facilitation and the stimulus peak latency showed no significant change in either species. A significant, irreversible decrease in the time constant of decay of the EJP was found in guinea pig vas deferens (by 35 ± 2% P < 0.005). In mouse (4/6 tissues), and guinea pig (5/6 tissues) vas deferens, there was a significant increase in the variability of the EJP amplitude.
Fig. 5 Decreased EJP amplitude in 18βGA from guinea pig and mouse vas deferens. Sample traces of EJP amplitude before and after 18βGA in (a) guinea pig and (b) mouse vas deferens. Mean EJP amplitude decreased in (c) guinea pig (n = 6) (more ...)
3.4. Nerve terminal Ca2+ imaging
Potential prejunctional effects of heptanol (2 mM) and 18βGA (10 μM) were further investigated by imaging the Ca2+ concentration in the nerve terminals of the mouse vas deferens. The fluorescent signal from each nerve terminal (F; see A for an example of the region sampled) increased on each field (nerve) stimulus (B; ‘EFS’). The value of F immediately after the stimulus was compared to the trough immediately before it (Fo); their difference (ΔF) was normalised (ΔF/Fo) to give a relative measure of the change in Ca2+ concentration in a nerve terminal ([Ca2+]t). Heptanol (2 mM) either abolished the Ca2+ transient (13 of 25 terminals from 3 of 4 vasa deferentia; 52%), or caused intermittent evoked Ca2+ transients (12 of 25 terminals from 4 of 4 vasa deferentia; 48%). The Ca2+ transients did not return (nor become less variable) by increasing the amplitude of the field stimulus to 50 V, suggesting that the stimulus threshold for initiating nerve terminal action potentials had not changed, at least over the range tested. In those terminals still intermittently responding, the mean probability of response per stimulus was 0.42 ± 0.06 (n = 12 terminals) and the amplitude of such responses was 78 ± 5% of the control amplitude (paired two-tailed t-test; P < 0.05). After washing out the bath for 10 min the Ca2+ transients partially returned (except in one terminal) with a mean probability of response per stimulus of 0.72 ± 0.06 and such responses were of the same amplitude as the control (103 ± 5%; n = 24 terminals; paired t-test, P = 0.46).
Fig. 6 The effect of heptanol on nerve terminal Ca2+. a, shows a field of OGB-1 loaded nerve terminals in the mouse vas deferens. A sample ROI has been drawn in white around a terminal in the upper part of the figure. The scale bar is 20 μm. (more ...)
Using the same exposure protocol, 18βGA (10 μM) had no significant effect on the [Ca2+
in response to field stimuli. The Ca2+
transients did not become intermittent and the amplitude remained unchanged at 106 ± 3% of the control amplitude (n
= 21 terminals from 4 vas deferens; P
= 0.11 with a Wilcoxon signed rank test). However, in the presence of 18βGA 4 of these terminals (all from 1 preparation; see Supplementary Movie
) showed spontaneous whole-terminal Ca2+
transients of similar amplitude to those observed following field stimulation. Such spontaneous transients were never observed during the control recording (for any experimental protocol).