Simultaneous intracellular recordings from longitudinal and circular muscle during the peristaltic reflex in guinea-pig distal colon

Abstract

1. Simultaneous intracellular recordings were made from longitudinal muscle (LM) and circular muscle (CM) cells of guinea-pig distal colon during the peristaltic reflex. 2. Spontaneous rhythmical depolarizations with superimposed action potentials (mean amplitude: 19 +/- 2 mV) were regularly recorded from the LM (mean interval: 7 +/- 1 s). In contrast, in the CM layer, spontaneous action potentials occurred with an irregular frequency. Although spontaneous action potentials in LM were rarely correlated in time with those in CM, spontaneous inhibitory junction potentials (sIJPs) were found to occur synchronously in both muscles (5 out of 27 animals; 19 %). 3. Graded inflation of an intra-luminal balloon or mucosal stimulation oral to the recording electrodes elicited gradeable compound IJPs synchronously in both LM (mean amplitude: 6 +/- 1 mV) and CM (mean amplitude: 9 +/- 1 mV) (descending inhibitory reflex). Evoked IJPs were often followed by action potentials in both muscle layers. 4. Mucosal stimuli applied anal to the recording electrodes elicited compound excitatory junction potentials (EJPs) synchronously in both muscles layers that were often associated with the generation of action potentials. In the LM, evoked EJP amplitudes ranged from 3 mV (subthreshold) to 31 mV (including the action potential) and in the CM from 4 mV (subthreshold) to 44 mV (including the action potential). 5. Apamin (500 nM) reduced the evoked IJP in the CM by 55 % (from 11 +/- 2 to 5 +/- 1 mV), but caused no significant reduction in the LM layer (from 8 +/- 1 to 6 +/- 1 mV). Apamin-resistant IJPs in both muscle layers were likely to be due to nitric oxide, since they were abolished by L-NA (100 microM). 6. Atropine (1 microM) abolished the ascending excitatory reflex in both muscles. 7. Injection of neurobiotin into the LM and CM confirmed that simultaneous intracellular recordings were made from different muscle layers. 8. In conclusion, during the peristaltic reflex, the LM and CM layers receive synchronous inhibitory neuromuscular inputs during descending inhibition and synchronous excitatory neuromuscular inputs during ascending excitation. No evidence was found to support reciprocal innervation.

Figure 1. Preparation used for simultaneous intracellular recording from longitudinal muscle (LM) and circular muscle (CM) during reflex activation of the colon

A, photograph of the preparation used to simultaneously record from LM and CM layers. Left side of photo shows the thicker undissected CM and the microelectrode (1) positioned above the CM cells. Right side of photo shows the myenteric plexus upon the underlying thinner LM following removal of a strip of CM. The position of the microelectrode (2) is located directly above the LM. B, a schematic representation of the colonic preparation. An intraluminal balloon was used for colonic distensions. Distension volume was monitored with a sliding potentiometer. A fine artists’ paint brush was also used for mucosal stimulation, applied oral or anal to the microelectrodes.

Figure 2. Spontaneous intracellular electrical activity recorded simultaneously from the LM and CM

In the LM, spontaneous rhythmic depolarizations are shown. In the CM, there is an irregular discharge of spontaneous action potentials. Note the absence of electrotonic conduction of action potentials, since activity in one muscle layer does not propagate into the neighbouring muscle layer.

Figure 3. Spontaneous electrical activity when recorded simultaneously from the LM and CM layers

A, in the LM, spontaneous depolarizations occur with superimposed action potentials. In contrast, in the neighbouring CM, spontaneous IJPs occur, with no electrotonic conduction of action potentials from the LM to the CM. A single spontaneous action potential is shown in the CM. B, in another animal, spontaneous IJPs occur synchronously in both the LM and CM layers.

Figure 4. Effects of balloon distension on intracellular electrical activity recorded simultaneously from the LM and CM layers

Upon distension of an intra-luminal balloon (500 μl; lower trace) applied oral to the recording electrodes, IJPs were evoked simultaneously in both muscle layers with an identical latency (descending inhibitory reflex). Following the evoked IJPs, there was a burst of action potentials in both muscles. Note, spontaneous and evoked action potentials in the LM do not propagate into the neighbouring CM and vice versa.

Figure 5. Effects of grading the distension stimulus intensity on evoked inhibitory junction potentials in the LM and CM

A, graded increases in the balloon distension volume elicited similar graded increases in the amplitude of IJPs evoked in both the LM and CM. B, graphical representation of the effects of increasing distension stimuli on evoked IJP amplitudes in the LM (•) and CM layers (□) (from n = 5–9 animals). C, effects of mucosal stimulation on electrical activities of the LM and CM. A single brush stroke applied orally elicited synchronous IJPs in both LM and CM with an identical latency (see dotted line). In the LM layer, a single action potential occurred on repolarization of the IJP.

Figure 6. Effects of intra-luminal balloon distension on electrical activity recorded simultaneously from two CM cells

Despite the two electrodes being separated by 5.5 mm in the circumferential axis, spontaneous action potentials can be seen to occur synchronously. When the balloon is inflated oral to the two electrodes, IJPs are evoked synchronously at the two distant recording sites. Following the IJPs, there is a brief burst of action potentials, which are phase locked at the two recording sites. The lower trace is a monitor of the distension stimulus.

Figure 7. Effects of apamin and a nitric oxide synthesis inhibitor on the descending inhibitory reflex

A, control electrical responses in the LM and CM following intraluminal balloon distension. B, apamin (500 nm) attenuated the amplitude of the ‘fast’ IJP in the CM layer, without any pronounced effect on the LM layer. C, the ‘slow’ apamin-resistant IJP in both muscle layers is abolished by the further addition of l-NA (100 μm).

Figure 8. Simultaneous intracellular recordings from LM and CM during the ascending excitatory reflex

A, mucosal stimuli (1 brush stroke, see arrow), applied anal to the electrodes, elicited synchronous EJPs and an accompanying action potential in both the LM and CM. B, in another preparation, single anal mucosal stimuli applied on four separate occasions (see arrows) elicited an EJP in the CM that did not reach action potential threshold, but in the LM an EJP and action potential were evoked.

Figure 9. Effects of atropine on the ascending excitatory reflex

A, a single brush stroke applied to the mucosa (see arrow), anal to the recording electrodes, evoked a compound EJP and associated action potentials synchronously in the LM and CM layers. Spontaneous depolarizations and action potentials can be observed in the LM prior to and following the stimulus. B, in the presence of atropine (1 μm), the evoked EJP was abolished in both the LM and CM and a small IJP was evoked in both muscles synchronously.

Figure 10. Confocal micrograph (Z-series 15 stacks) showing neurobiotin injection into a single LM cell and CM cell

After the neurobiotin was conjugated with streptavidin FITC and viewed under the appropriate fluorescence filters, a single LM cell (B) and CM cell (A) were observed. LM cells could be clearly identified over CM cells, since they were at 90 deg to each other. The scale bar in each panel represents 50 μm.