All procedures used for the acquisition of physiological data from isolated tissues were approved by the Animal Experimentation Ethics Committee at the Australian National University. Guinea-pigs of either sex were stunned, exsanguinated and the stomach removed. In the initial experiments the stomach was first cut along the greater curvature and then along the lesser curvature; one hemi-stomach was immersed in oxygenated physiological saline (composition, mm): NaCl, 120; NaHCO3, 25; NaH2PO4, 1.0; KCl, 5; MgCl2, 2; CaCl2, 2.5; and glucose, 11; bubbled with 95% O2–5% CO2. The fundus and lower pylorus were discarded; the mucosa, followed by the serosa, was dissected away from the remaining tissue. Preparations were pinned, serosal surface uppermost, in a recording chamber with a base consisting of a microscope cover-slip coated with Sylgard silicone resin (Dow Corning Corp., Midland, MI, USA) and viewed with an inverted compound microscope. The antrum was impaled with two independently mounted sharp electrodes, with resistances of 100–140 MΩ, filled with 0.5 m KCl. The orientation and separation between electrodes were noted. Membrane potential changes (and when appropriate membrane currents) were amplified using an Axoclamp-2B amplifier (Axon Instruments), low-pass filtered (cut-off frequency, 1000 Hz), digitized and stored on computer for later analysis. In some experiments, similar preparations were made but the longitudinal and adhering ICCMY layers were dissected away from most of the preparation, leaving a 3 mm wide band of longitudinal muscle, with the ICCMY network intact, along the greater curvature: regions of tissue devoid of ICCMY were impaled with two independently mounted sharp electrodes, care being taken to impale the same bundle of circular muscle. The separation between electrodes was again determined.
The conduction velocities of pacemaker potentials in the anal and circumferential directions were determined using preparations of longitudinal muscle, with ICCMY attached (see Hirst & Edwards, 2001). Preparations were pinned over a bar stimulating electrode, let into the base of the chamber; a second parallel bar electrode was placed above the tissue (Hirst et al. 2002b). The preparations were orientated so that conduction from the plane of stimulation, with a pulse width of 5 ms and with a stimulus strength adjusted to 110% threshold value, was either in an anal or in a circumferential direction. Intracellular recordings were made using two electrodes, with the separation between recording electrodes being determined. The arrival times of a pacemaker potential at the two points in the ICCMY network was determined from measurements of the 10% rise point of the potential change produced in the longitudinal muscle layer by a pacemaker potential. The conduction velocity was determined by dividing the separation between electrodes by the difference in arrival times. Atropine (1 µm) was added to the physiological saline to abolish the effects of concurrent stimulation of excitatory nerves (see Hirst & Edwards, 2001).
The electrical properties of the circular layer were determined using individual bundles of circular muscle (diameter 60–150 µm, length 2.0–3.5 mm; see Suzuki & Hirst, 1999 for details). Preparations were again impaled with two independently mounted sharp electrodes: one was positioned at each end of the single bundle of circular muscle. Current pulses were passed through one electrode and the other was used to measure the resulting electrotonic potentials. The electrotonic potentials could be simulated when the bundle was modelled as an electrically short cable with sealed ends. Jack & Redman (1971) have derived the Laplace transform of the voltage response at the centre of such a cable (length 2 L) to a step of current injected at distance X from the recording electrode (see eqn (A20) (Jack & Redman)). Multiplying this expression by 2 and setting X to L gives the Laplace transform for the voltage response at one end of a cable (length L) to current injection at the other end. Applying substitutions that were used to solve the case for an infinite cable (eqn 3.24 in Jack et al. 1975) and taking the inverse Laplace transform yields the following equation:
where Em(T) represents membrane potential, I injected current, ra axial resistance, λ length constant, L cable length measured in length constants and T time measured in time constants. This expression was verified by the method of reflections (see p. 69 in Jack et al. 1975). To fit this expression to a physiologically acquired response, values of equivalent membrane resistance, axial resistance and membrane time constant were varied according to a Simplex algorithm until the best least-squares fit was obtained (Matlab 6.5.1 Release 13; The MathWorks, Natick, MA, USA). During each optimization step, the series of infinite cable responses was summed to account for only the first 20 reflections, as adding further reflection components did not affect the solution. For each bundle the electrical length constant was determined from the values of membrane resistance and axial resistance (eqn 3.10 of Jack et al. 1975), and the asymptotic value of electrotonic conduction velocity in the analogous infinite cable was calculated (p. 34 of Jack et al. 1975). The membrane time constant, electrical length constant and electrotonic conduction velocity of each bundle was determined.
In the experiments where the connectivity between nearby bundles of smooth muscle was characterized, segments of circular layer, with the longitudinal and ICCMY layers removed, were isolated. The segments had lengths of 1–1.2 mm and were four or five bundles wide. A bundle at one edge of the preparation was first impaled with two electrodes. Current pulses were passed through one electrode and the resulting electrotonic potentials were recorded using the second electrode: the time course and amplitude was determined from an average of 20 successive electrotonic potentials. Subsequently the recording electrode was withdrawn and the adjacent bundle was impaled: current pulses of the same amplitude were passed through the current-passing electrode and electrotonic potentials were recorded from the adjacent bundle with the second electrode. The process was repeated with the recording electrode being inserted in the next more distant bundle. The connectivity between bundles, termed transfer ratio, was quantified by determining the ratio of steady state amplitudes of electrotonic potentials, produced by injecting constant intensity current pulses into the same and the adjacent muscle bundle.
During each experiment, preparations were constantly superfused with physiological saline solution warmed to 37°C; nifedipine (1 µm) was added to the physiological saline to reduce the amplitudes of the contractions associated with each slow wave or regenerative potential. When the electrical properties of single bundles of smooth muscle and the coupling between nearby bundles of smooth muscle were being determined, caffeine (1 mm) was added to the physiological saline to inhibit the occurrence of unitary potentials and regenerative potentials (Edwards et al. 1999), to ensure only passive cable properties were measured. After caffeine wash-out the frequency of regenerative potentials was increased for up to 30 min; no attempt was made to allow a complete return to control conditions as this meant that the impalements had to be maintained for excessive times. Thus, some of the recordings made from paired bundles of circular muscle give a misleading impression of the natural frequency of generation of regenerative potentials (Fig. 5).
Figure 5. Electrical coupling between adjacent bundles circular muscle in the guinea-pig antrum The upper pair of traces (Aa and Ab) shows simultaneous recordings from the same bundle of circular muscle. Note that recorded membrane potential changes were very similar. When a current pulse was passed through one electrode it produced an electronic potential with a steady state amplitude of about 11 mV (C). The lower pair of traces (Ba and Bb) shows simultaneous recordings from adjacent muscle bundles; although the regenerative potentials occurred synchronously, their shapes differed in detail. Current passed through one electrode evoked an electrotonic potential in the second bundle with a steady state amplitude of about 5 mV (C). The peak negative membrane potential of both muscle bundles, recorded in the presence of caffeine (1 mm), was −65 mV; each electrotonic potential is an average of 20 successive responses. The upper time and voltage calibration bars apply to the upper four traces. The lower time calibration bar applies to the lower voltage and current traces.
Download figure to PowerPoint
To determine the distribution of ICCMY in living preparations, preparations were first washed with warmed physiological saline for 30 min. The preparations were then incubated for 15 min with an antibody to CD 117 (rat anti-mouse CD 117 C-kit, Cymbus Biotechnology, Chadlers Ford, UK), diluted 1 in 500 in physiological saline. Subsequently the preparations were washed in warmed physiological saline for 15 min and then incubated in Alexa Fluor 488 (goat-anti-rat; Molecular Probe, Eugene, OR, USA), again diluted 1 in 500 in physiological saline, for 15 min. Preparations were washed with warmed solution and viewed with a confocal microscope, illumination wavelength 488 nm, emission wavelength above 505 nm. The serosal surface of the preparation was first viewed and the microscope focus moved down until ICC were visualized. In control preparations, ICCMY were first detected; subsequently ICCIM were detected in a lower plane of focus. When the longitudinal layer was removed, ICCMY were not detected but ICCIM were apparent.