Figure 5.

GDNF and NRTN acutely influence GCaMP activity of largely nonoverlapping adult distal colon myenteric neuron populations. (A–C) Feature plots show Gfra1 primarily in Nos1/Vip/Gal neurons (A), Gfra2 in Chat neurons (B), and Ret in almost all neurons except Chat3 (C). (D) Whole mount immunohistochemistry shows GFP (green) in most NOS1+ (red) neurons of Gfra1^Gfp/wt distal colon. White arrowheads show GFP+NOS1+ neurons. Yellow arrowhead shows GFP-NOS1+ neuron. (E) Most NOS1+ neurons are GFP+. Most NOS1– neurons are GFP-, consistent with RNA-seq (P < .0001, Student’s t test, n = 3 mice (Gfra1^Gfp/wt)). (F) Whole mount immunohistochemistry using Gfra1^Gfp/wt distal colon shows colocalization of GFP (green) with S100B+ (red) glia and HuC/D (blue) neurons. Scale bar = 100 μm. (G, H) Time-lapse images (top) (pixels were assigned color based on transients timing; color = activity) and traces (bottom) of GCaMP6s activity from regions of interest on myenteric neurons during baseline (left) and after adding 10 nM GDNF (G) or 10 nM NRTN (H) (right). (I) Sample traces from neurons with activity increased (top) or decreased (bottom) by GDNF (red) or NRTN (blue). Baseline (gray) and percent neurons (in parentheses) with increased or decreased activity (>2 SD change). (J) Percent neurons responding to only GDNF (red), only NRTN (blue), or both (gray) (P < .05, 1-way analysis of variance, Tukey’s multiple comparisons test). (K) Iris plot of GDNF and NRTN responsive myenteric neurons (n = 260 of 523 [49.7%] of total). GDNF is shown in the outer circle (red), NRTN is shown in the inner circle (blue) (n = 5 mice), and gray indicates no ligand-induced activity change. Light shades of red and blue indicate decreased activity after ligand addition. Most responsive neurons are affected by either GDNF or NRTN, not both. (L) NADPH diaphorase stained colon identifies nitric oxide–producing neurons. (M) Corresponding GCaMP6s imaging field. (N) GCaMP6s imaging field shown in M, where yellow arrowheads identify putative nitrergic myenteric neurons and asterisks indicate putative NADPH diaphorase positive neurons with low GCaMP6s signal. (O) Quantification of GDNF- and NRTN-responsive nitrergic neurons (n = 3 fields from separate experiments, P = .0298, Fisher exact test, 2 × 2 contingency table [NOS+/NOS– and GDNF response/NRTN response]). (P, Q) Quantitative data for GCaMP6s imaging fields stained post hoc for NADPH diaphorase, indicating response to GDNF and NRTN. (P) Responsive nitrergic neurons. (Q) Responsive non-nitrergic neurons. (E, J) Mean ± SD. (D, F, G, H) Scale bar = 100 μm. (L–Q) n = 3 fields of view from separate mice. ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001.