Fig. 5: The PL^Glu perceives gastric pain signals.

A, B Representative images of co-expressed c-Fos (red), glutamatergic neurons or GABA neurons (green), and DAPI (blue) in the PL (left). The percentage of co-expression in the PL (right, n = 4 mice). C Schematic of AAV-vglut2-GCaMP6s injection and fiber implantation in the PL (left). Expression of GCaMP6s in the PL (right). D Heatmap and average Ca²⁺ transients of the PL^Glu receiving GD stimulation. E Averaged peak ΔF/F of calcium activity of the PL^Glu (right, n = 6 mice, P = 0.0001 in 40 mmHg, 60 mmHg, and 80 mmHg). F Experimental procedure of optogenetic manipulation of the PL^Glu (left). Schematic of the EMG recording setup (right). G Representative images expression of virus in the PL. Similar results were obtained in six independent experiments and the data shown were from one representative experiment. H Representative EMG traces and statistical graph of EMG recordings AUC responses to GD under normal conditions or upon ChR2-stimulation (n = 6 mice, P = 0.0004 in 40 mmHg, P = 0.0001 in 60 mmHg and 80 mmHg). I Representative EMG traces and statistical graph of EMG recordings AUC responses to GD under normal conditions or upon eNpHR-stimulation (n = 6 mice, P = 0.0235 in 40 mmHg, P = 0.0001 in 60 mmHg and 80 mmHg). J Heatmap and average Ca²⁺ transients of the PL^Glu in pre and post-receiving SDx in GD stimulation, respectively (left). Averaged peak ΔF/F of calcium activity of the PL^Glu in pre and post-receiving SDx in GD stimulation (right, n = 6 mice, P = 0.0058). K Heatmap and average Ca²⁺ transients and averaged peak ΔF/F of calcium activity of the PL^Glu in pre and post-receiving T5x in GD stimulation (n = 6 mice). L Schematic of AAV injection in the NTS (left). Decreased c-Fos expression in the PL after CNO injection compared with the saline group (middle). Statistical graph of c-Fos⁺ cells number (right, n = 3 mice, P = 0.0177). M Schematic of AAV injection in the NTS, AAV, and optic fiber implantation in the PL (left). Heatmap of the PL^Glu in receiving saline or CNO in GD stimulation (middle) and averaged peak ΔF/F of calcium activity of the PL^Glu (right, n = 6 mice, P = 0.0002). AM acromiotrapezius muscle. Scale bar = 100 μm in (A–C, G, and L), scale bar = 20 μm in zoomed image in (A, B, and G). Significance was assessed by two-way repeated-measure ANOVA in (E, H, and I), and two-sided Student’s t-test in (J–M). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, n.s. not significant. All data are presented as mean values ± SEM.