Ca²⁺ imaging of activity in ICC-MY during local mucosal reflexes and the colonic migrating motor complex in the murine large intestine

Colonic migrating motor complexes (CMMCs) are neurally mediated, cyclical contractile and electrical events, which typically propagate along the colon every 2-3 min in the mouse. We examined the interactions between myenteric neurons, interstitial cells of Cajal in the myenteric region (ICC-MY) and smooth muscle cells during CMMCs using Ca²⁺ imaging. CMMCs occurred spontaneously or were evoked by stimulating the mucosa locally, or by brushing it at either end of the colon. Between CMMCs, most ICC-MY were often quiescent; their lack of activity was correlated with ongoing Ca²⁺ transients in varicosities on the axons of presumably inhibitory motor neurons that were on or surrounded ICC-MY. Ca²⁺ transients in other varicosities initiated intracellular Ca²⁺ waves in adjacent ICC-MY, which were blocked by atropine, suggesting they were on the axons of excitatory motor neurons. Following TTX (1 μm), or blockade of inhibitory neurotransmission with N^ω-nitro-l-arginine (l-NA, a NO synthesis inhibitor, 10 μm) and MRS 2500 (a P2Y₁ antagonist, 1 μm), ongoing spark/puff like activity and rhythmic intracellular Ca²⁺ waves (38.1 ± 2.9 cycles min^-1) were observed, yet this activity was uncoupled, even between ICC-MY in close apposition. During spontaneous or evoked CMMCs there was an increase in the frequency (62.9 ± 1.4 cycles min^-1) and amplitude of Ca²⁺ transients in ICC-MY and muscle, which often had synchronized activity. At the same time, activity in varicosites along excitatory and inhibitory motor nerve fibres increased and decreased respectively, leading to an overall excitation of ICC-MY. Atropine (1 μm) reduced the evoked responses in ICC-MY, and subsequent addition of an NK1 antagonist (RP 67580, 500 nm) completely blocked the responses to stimulation, as did applying these drugs in reverse order. An NKII antagonist (MEN 10,376, 500 nm) had no effect on the evoked responses in ICC-MY. Following TTX application, carbachol (1 μm), substance P (1 μm) and an NKI agonist (GR73632, 100 nm) produced the fast oscillations superimposed on a slow increase in Ca²⁺ in ICC-MY, whereas SNP (an NO donor, 10 μm) abolished all activity in ICC-MY. In conclusion, ICC-MY, which are under tonic inhibition, are pacemakers whose activity can be synchronized by excitatory nerves to couple the longitudinal and circular muscles during the CMMC. ICC-MY receive excitatory input from motor neurons that release acetylcholine and tachykinins acting on muscarinic and NK1 receptors, respectively. The movements of the large intestine are regulated by the enteric nervous system and pacemaker cells called interstitial cells of Cajal (ICC). It is unknown whether particular ICC that form a network near the myenteric nerve plexus (ICC-MY) have pacemaker activity and are controlled by nerves. We studied the activation of ICC-MY during the colonic migrating motor complex (CMMC), which is a neurally mediated motor behaviour that propagates as a contractile wave down the colon, using calcium imaging. We show, for the first time, that ICC-MY in the mouse colon are activated by nerves during the CMMC, and appear to be responsible for exciting the longitudinal and circular muscle layers to produce contraction.