A NEW ELECTROPHYSIOLOGICAL TOOL TO INVESTIGATE THE SPATIAL NEURONAL PROJECTIONS WITHIN THE MYENTERIC ASCENDING REFLEX OF THE MOUSE COLON

Abstract

1. The intestinal peristaltic reflex is regulated by local microcircuits that, upon activation, result in an oral contraction and anal relaxation of the circular muscle. This contractile response is associated with typical electrophysiological changes in membrane potential resulting from excitatory and inhibitory myenteric pathways. 2. The aim of the present study was to investigate the influence of local electrical stimulation (ES; single pulses, 15 V, 0.3 msec duration) on the ascending gastrointestinal electrophysiological potentials of the mouse colon using a novel 12-channel stimulation electrode in a newly designed model of the ascending myenteric pathways with simultaneous intracellular recording. 3. Local myenteric reflex responses in the proximal colon were initiated by ES (12 bipolar stimulation electrodes (SE) 0.7 mm apart from each other) and excitatory and inhibitory junction potentials (EJP and IJP, respectively) were recorded from circular smooth muscle cells with intracellular recording techniques. In vivo colonic propulsion was determined by measuring the time to expulsion of a 3 mm glass bead inserted 2.5 cm into the distal colon of mouse. 4. Under basal conditions, circular smooth muscle cells displayed a stable membrane potential (-56.7 +/- 6.9 mV; n = 13). Electrical stimulation elicited a tetrodotoxin (3 micromol/L)-sensitive, neuronal-induced EJP (cholinergic; atropine (1 micromol/L) sensitive) and a biphasic IJP. Both the EJP and IJP showed characteristic responses dependent on the distance between stimulation and recording sites. The EJP could be recorded over long distances, resulting in a maximal EJP amplitude at a distance of 10 mm distance (represented by stimulation electrodes (SE) number 6/7) and a maximal projection distance of 18-20 mm. Both components of IJP were maximal during direct stimulation (at SE1; stimulation at the recording site) and gradually decreased to SE6/7 (10 mm). At distances greater than 10 mm apart, ES did not produce IJP. The ganglionic blocker hexamethonium (10-100 micromol/L) concentration dependently abolished all inhibitory junction potentials at distances greater than 10 mm and significantly reduced the amplitude of EJP for the first 10 mm. Colonic propulsion was decreased by hexamethonium (40 mg/kg) and atropine (0.7 mg/kg). 5. Neuronal circuits of the ascending myenteric reflex functionally project distances ranging up to 18-20 mm. Our newly designed setup allows simultaneous electrophysiological investigations of neuronal microcircuitry within the myenteric plexus over short and long distances and enables conclusions to be drawn regarding neuroneuronal and neuromuscular transmission.