Fig. 1: ENS responses to mucosal high K⁺.

a, DiI was injected into a single villus tip (n = 3 preparations). b, Representative DiI-labelled villus. c, Overview of the mucosa in a full-thickness preparation with a DiI-labelled villus. d, DiI-labelled nerve fibres and submucosal (cyan) and myenteric (green) (GCaMP-expressing) neurons (arrowheads). The point of DiI application is marked with an x. e,i, High-K⁺ (75 mM) Krebs solution was perfused onto the mucosa at t = 10–20 s while imaging the underlying SMP (e) or MP (i). f,j, Submucosal (f) and myenteric (j) neurons responding (arrowheads) at t = 0 s (left panels), t = 25 s (f, right) and t = 19 s (j, right). g,k, Ca²⁺ transients in the indicated SMP (g) and MP (k) responders. h,l, Left, submucosal (h, green) and myenteric (l, cyan) responders. h, Middle, SMP immunolabelled for ChAT and VIP (n = 4 preparations). Right, responders included ChAT⁺ submucosal neurons. l, Middle, MP immunolabelled for calbindin and neuronal nitric oxide synthase (nNOS) (n = 4 preparations). Right, responders included calbindin⁺ myenteric neurons. m, Myenteric responses to mucosal high K⁺ in the full-thickness preparation (n = 3 preparations). n, Selected responders (arrowheads) at t = 0 s (left) and t = 19 s (right). o, Ca²⁺ transients in the indicated responders. p–r, The same preparation was peeled apart between the circular muscle and the SMP, severing connections between the MP and the mucosa (p). The separated layers were replaced, and the mucosa was depolarized. Responses in the same ganglion were abolished after peeling. q, Responders at t = 0 s (left) and t = 19 s (right). r, Ca²⁺ transients. s–u, The mucosal layer was removed to expose the MP (s). The same neurons were directly exposed to high K⁺ and depolarized. t, Responders at t = 0 s (left) and t = 14 s (right). u, Ca²⁺ transients. Scale bars, 1 mm (c), 100 μm (d) and 20 µm (f,h,j,l,n,q,t). Drawings in a,e,i,m,p and s are adapted from ref. ³⁰ (Wiley).