Figure 4.

Electrically- and DMPP-evoked [Ca²⁺]_i responses in submucosal neurons are increased following CT-exposure. (A–C) A submucosal ganglion from a CT-incubated preparation responding to a train of stimuli (20 pulse). Cholinergic neurons were identified with post-hoc labeling for choline acetyltransferase (ChAT, red); non-cholinergic neurons were vasoactive intestinal peptide (VIP)-immunoreactive (white) (C). Arrows indicate cholinergic ChAT⁺ neurons and arrowheads indicate VIP⁺ neurons. Scale bar = 20 μm. In each histogram (D,E,G) the amplitude (ΔFi/F0) of “Total” neurons that responded was presented (left) then divided into those of ChAT⁻ (middle) and ChAT⁺ (right) groups. Electrically-evoked responses (D single pulse and E 20 pulse) of submucosal neurons after CT-incubation had a higher amplitude compared to that of controls (single pulse: ^**P < 0.01, control: n = 43 neurons, CT: n = 39 neurons; 20 pulse: ^***P < 0.001, control: n = 92 neurons, CT: n = 64 neurons), and this was specifically observed in cholinergic neurons (^***P < 0.001). (F) Averaged traces of responses to 20 pulse stimulation (applied at 10 s) in ChAT⁺ (black trace) vs. ChAT⁻ neurons (gray trace; mean ± SEM) in a CT-treated preparation, as depicted in (A–C). (G) Overall responses to DMPP were significantly higher after CT-treatment (^*P < 0.05; control: n = 91 neurons, CT: n = 60 neurons), and in particular, responses in cholinergic (ChAT⁺) submucosal neurons were significantly increased (^*P < 0.05; control: n = 36 neurons, CT: n = 22 neurons).