Excitatory cholinergic responses in mouse colon intramuscular interstitial cells of Cajal are due to enhanced Ca ²⁺ release via M ₃ receptor activation

Abstract

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca²⁺ release in ICC-IM activates Ano1, a Ca²⁺ -activated Cl^- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca²⁺ imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M₂ , M₃ ), and NK₁ receptors were enriched in ICC-IM. NK₁ receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca²⁺ transients. These effects were reversed by DAU 5884 (M₃ receptor antagonist) but not AF-DX 116 (M₂ receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca²⁺ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca²⁺ stores with cyclopiazonic acid and reduced by inhibiting Ca²⁺ influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884, and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M₃ receptor activation. Enhancing ICC-IM Ca²⁺ release and Ano1 activation contributes to excitatory responses of colonic muscles.