Figure 4. Abdominal sympathetic ganglia recruit Nos1+ gastric neurons to mediate ileal GLP-1 effects.

(A) After gastric infections with the retrograde construct AAVrg-Cre, the polysynaptic, Cre-dependent anterograde HSV strain HSV-LSL-TK-Tomato was injected into CG. Recombination of HSV-LSL-TK-Tomato in CG^Stom reveals that CG preferentially targets Nos1+ (Nos1^Stom) gastric neurons.

(B) Cre-dependent rabies constructs injected into the stomach of Nos1^Cre mice labels CG^Stom and parasympathetic dorsal motor vagal (DMV) neurons.

(C) HSV-LSL-TK-Tomato recombining in CG^Stom reveals that CG does not target ChAT-gastric neurons (ChAT^Stom).

(D) Cre-dependent rabies constructs injected into the stomach of ChAT-ires-Cre mice labels DMV neurons but not CG^Stom.

(E) Whereas sympathetic CG^Stom innervates only Nos1^Stom, DMV innervates both Nos1^Stom and ChAT^Stom.

(F) GLP-1ⁱⁱ-induced strong and specific Fos in Nos1^Stom, an effect abolished by DβH-sap.

(G) Left: injections of the Cre-dependent construct AAV9-syn-FLEX-jGCaMP8m into the stomach of Nos1^Cre mice allowed the expression of the genetic calcium sensor GCaMP6f specifically in Nos1^Stom. Right: upon implanting the abdominal glass window, intravital imaging revealed that GLP-1ⁱⁱ induces fast and sustained calcium transients in Nos1^Stom. Heatmap of Z scores calculated with respect to baseline. T = 0 min: GLP-1ⁱⁱ infusion.

(H) Much less pronounced effects were observed upon similar imaging of ChAT^Stom in ChAT-ires-Cre×Ai148D mice.

(I) Cell-specific ablation (AAV1-flex-Casp3) of Nos1^Stom abolishes GLP-1ⁱⁱ-induced gastric expansion, T(8) = 2.709,* p = 0.027.

(J) Same effects for GLP-1ⁱⁱ-induced appetite suppression, two-way mixed RM-ANOVA, infusate × group F(2,18) = 18.790,* p < 0.001.

(K) Cell-specific ablation of Nos1^Stom abolishes truncal vagotomy (VGx)-induced gastric bloating, one-way ANOVA F(3,16) = 43.610, p < 0.001; AAV1-DIO-EYFP+VGx versus AAV1-flex-Casp3+VGx Bonferroni * p < 0.001.

(L) Chemogenetic activation of Nos1^Stom (Nos1-Gs). Left: representative traces of EMG activity from stomach corpus following either saline (top trace) or CNO (lower trace). Blue insert traces show 1 s EMG, individual spikes shown were counted for plots. Right: aggregate data, average frequency EMG spikes, 20 min following saline or CNO, paired t test T(2) = 8.164, * p = 0.014.

(M) Chemogenetic Nos1^Stom activation induces marked stomach distension. Left: representative dissected stomachs; Right: volume of dissected stomachs, 2-sample t test T(8) = 6.960, * p < 0.001.

(N) Chemogenetic Nos1^Stom activation reduces 1 h-food intake in Nos1-Gs but not Nos1-GFP, two-way mixed RM-ANOVA, infusate × group F(2,16) = 10.806, * p = 0.001.

(O) Same as (M) and (N) but for chemogenetic activation of ChAT^Stom in ChAT-ires-Cre mice. CNO tended to increase frequency although effect was not significant, T(2) = 0.463, * p = 0.688.

(P) Chemogenetic activation of ChAT^Stom abolishes GLP-1ⁱⁱ-induced gastric bloating, one-way RM-ANOVA, F(2,12) = 66.773, p < 0.001, ChAT-Gs(GLP-1ⁱⁱ) versus ChAT-GFP(GLP-1ⁱⁱ) Bonferroni * p < 0.001.

(Q) Chemogenetic activation of ChAT^Stom abolishes GLP-1ⁱⁱ-induced anorexia, two-way mixed RM-ANOVA, treatment (Salineⁱⁱ versus GLP-1ⁱⁱ) × group F(2,24) = 9.704, p < 0.001; ChAT-Gs(GLP-1ⁱⁱ) versus ChAT-GFP(GLP-1ⁱⁱ) Bonferroni *p = 0.014.

(R–S) Gastric balloon inflation induces Fos in ChAT^Stom, one-way RM-ANOVA, F(3,16) = 7.521, p = 0.002, inflated-GFP versus inflated-Gs, Bonferroni * p = 0.003.

(T) ChAT^Stom activation entirely abolished gastric balloon-induced anorexia, (Ta) Chow intake: two-way mixed RM-ANOVA, inflation 3 group F(1,8)= 8.160, * p = 0.021, and group effect F(1,8) = 10.761, ** p = 0.011. (Tb) Normalized intake reduction upon balloon inflation, T(8) = 3.172, * p = 0.013.

(U) Cell-specific ablation (AAV1-flex-Casp3) of ChAT^Stom causes extreme gastric bloating irrespective of CGx, one-way ANOVA F(3,16) = 30.119, * p < 0.001, AAV1-flex-Casp3 versus AAV1-DIO-EYFP Bonferroni * p < 0.001. Data are represented as mean ± SEM in all figures. Scale bars, 100 μM.

See also Figure S4 and Video S1.