Fig 2: Human enteric ganglia quadruple immunostaining for calbindin (CALB: a,b,c), calretinin (CALR: a’,b’,c’), somatostatin (SOM: a’’,b’’,c’’) and vasoactive intestinal peptide (VIP: a’’’,b’’’,c’’’). (a) Myenteric ganglion. Arrows point at three representative neurons displaying different combinations of immunolabeling. Horizontal arrows indicate a neuron positive for CALB and VIP but negative for CALR and SOM; vertical arrows show a neuron positive for CALB only; oblique arrows point at a neuron positive for CALR and SOM but negative for CALB and VIP. (b) External submucosal ganglion. Arrowheads point at two neurons positive for both VIP and CALR but negative for SOM, one neuron is positive for CALB (horizontal, filled arrowhead in (b), the other negative (vertical, empty arrowhead in (b). (c) Internal submucosal ganglion. Arrows point at two neurons positive for SOM but negative for VIP and CALR, one neuron is positive for CALB (horizontal, filled arrow in (c), the other one negative (vertical, empty arrow in (c). Patients data: (a) 69 years, ascending colon, female; (b,c) 76 years, duodenum, male.

Fig 3: Morphine upregulates the expression of Rac1 and Arhgef6 in SST-INs via a DOR-dependent mechanism. Cell type-specific RNA-seq reveals morphine upregulates Rac1 pathway in SST-INs. a Breeding scheme of SST-Cre::RPL22-HA mice and PV-Cre::RPL22-HA mice. b Schematic procedure showing ribotag immunoprecipitation (IP) and RNA-seq of the ribosome-associated transcripts in PV-INs or SST-INs in PrL after saline or 10 mg/kg morphine injection. c The heat map of hierarchical clustering of normalized level of ribotag-isolated transcripts in SST-INs (five mice/group). Each row corresponds to a single gene. d Representation of the morphine-regulated signaling network enrichment analysis including all modules and contributing genes in SST-INs. e–f Single-molecule fluorescence ISH for Rac1 or Arhgef6 transcript in SST-INs 1 h or 12 h after saline or morphine (10 mg/kg, i.p.) injection. Quantification of the fluorescent intensity of Rac1 or Arhgef6 transcripts in SST-INs. Three mice/group: Rac1: Sal, 247 cells, Mor 1 h, 150 cells, Mor 12 h, 192 cells; Arhgef6: Sal, 448 cells, Mor 1 h, 355 cells, Mor 12 h, 294 cells. g, h smFISH for Rac1 or Arhgef6 in SST-INs expressing DOR-shRNA, MOR-shRNA, or Scramble-shRNA 12 h after morphine (10 mg/kg, i.p.) injection. Quantification of the fluorescent intensity of Rac1 or Arhgef6 transcripts in SST-INs expressing shRNA. 4 mice/group; Rac1: Scramble, 172 cells, DOR-shRNA, 185 cells, 154 cells; Arhgef6: Scramble, 125 cells, DOR-shRNA, 258 cells, MOR-shRNA, 106 cells. One-way ANOVA by the Bonferroni’s post-hoc test for intensity (e: F(2,586) = 12.12, P < 0.0001; f: F(2,1094) = 12.45, P < 0.0001; g: F(2,508) = 52.05, P < 0.0001; h: F(2,486) = 13.57, P < 0.0001). Two-sample Kolmogorov–Smirnov test for cumulative frequency. Data are presented as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001

Fig 4: Effects of T2D on Type 2 diabetes (T2D) on stroke-induced Somatostatin (SOM)+ cells density (δc) increase 6 weeks after stroke discriminating between peri-infarct and infarct area. Representation of the areas in which striatum was divided for cell density analysis (δc) (a). SOM+ cell density (δc) in peri-infarct area in ipsilateral striatum compared to contralateral peri-infarct corresponding area in Standard Diet (SD) fed mice 6 weeks after stroke (b). SOM+ cell density (δc) in infarct area in ipsilateral striatum compared to contralateral infarct corresponding area (adjusted for infarct volume shrinking) in SD-fed mice 6 weeks after stroke (c). SOM+ cell density (δc) in peri-infarct area in ipsilateral striatum compared to contralateral peri-infarct corresponding area in High Fat Diet (HFD) fed mice 6 weeks after stroke (d). SOM+ cell density (δc) in infarct area in ipsilateral striatum compared to contralateral infarct corresponding area (adjusted for infarct volume shrinking) in HFD-fed mice 6 weeks after stroke (e). Stroke-induced SOM+ cell increase in peri-infarct and infarct areas in ipsilateral striatum compared to contralateral corresponding area (dotted line) in SD and HFD-fed mice 6 weeks after stroke (f). Unpaired t test with Welch’s correction, means ± SEM *P < 0.05

Fig 5: DOR and Rac1 in SST-INs are required for the enhancement of inhibitory transmission to FS PV-INs and the disinhibition of pyramidal neurons in PrL after morphine exposure. a–h AAV-Flex-DOR-shRNA-EGFP or AAV-Flex-Scramble-shRNA was co-injected with AAV-DIO-hChR2(H134R)-mCherry into the PrL of LhX6-EGFP/SST-Cre mice. a Schematic of the recording strategy and the spiking responses to intracellular current injection in FS PV-INs in acute slice 12 h after morphine exposure (10 mg/kg, i.p.). b Representative traces of the light-evoked response from SST-INs onto a FS PV-IN. c Quantification of the responsive probability from SST-INs onto a FS PV-IN in slice (n = 22–26 cells/5 mice in each group; χ2 test). d Quantitation of responsive IPSC amplitudes from SST-INs onto FS PV-INs (n = 19–23 cells from 5 mice/group; Mann–Whitney U test). e Schematic of the recording strategy and spiking responses to intracellular current injection in pyramidal neurons (PYR) in the same slice. f Representative traces of light-evoked response from SST-INs onto a pyramidal neuron. g Quantification of the responsive probability from SST-INs onto a pyramidal neuron in slice (n = 23–28 cells/5 mice in each group; χ2 test). h Quantitation of responsive IPSC amplitudes from SST-INs onto pyramidal neurons (n = 21–25 cells/5 mice in each group; Mann–Whitney U test). i–p AAV-DIO-Rac1-DN-mCherry or AAV-DIO-mCherry was co-injected with AAV-DIO-hChR2(H134R)-mCherry into the PrL of LhX6-EGFP/SST-Cre mice. i Schematic of the recording strategy in FS PV-INs in acute slice. j Representative traces of light-evoked response from SST-INs onto a FS PV-IN. k Quantification of the responsive probability from SST-INs onto a FS PV-IN in acute slice (n = 30–31 cells/7–8 mice in each group; χ2 test). l The quantitation of responsive IPSC amplitudes from SST-INs onto FS PV-INs (n = 25–29 cells/7 mice in each group; Mann–Whitney U test). m Schematic of the recording strategy in pyramidal neurons in the same slice. n Representative traces of light-evoked response from SST-INs onto a pyramidal neuron. o Quantification of the responsive probability from SST-INs onto a pyramidal neuron in slice (n = 27–38 cells/7–8 mice in each group; χ2 test). p Quantitation of responsive IPSC amplitudes from SST-INs onto pyramidal neurons (n = 26–28 cells/7–8 mice in each group; Mann–Whitney U test). q, r Representative recording of AP traces (q) and induced spikes (r) in pyramidal neurons nearby the SST-INs expressing mCherry or Rac1-DN after saline or morphine exposure (n = 26 cells/6 mice in Saline mCherry group, n = 32 cells/7 mice in Morphine mCherry group, 33 cells/7 mice in Morphine Rac1-DN group; two-way RM ANOVA by the Bonferroni’s post-hoc test. Saline mCherry vs. Morphine mCherry, current: F(25,1400) = 503.1, P < 0.0001, treatment: F(1,56) = 8.02, P = 0.0064, interaction: F(25,1400) = 4.988, P < 0.0001; Morphine mCherry vs. Morphine Rac1-DN, current: F(25,1575) = 452.3, P < 0.0001; virus: F(1,63) = 5.106, P = 0.0273, interaction: F(25,1575) = 3.244, P < 0.0001). Data are presented as mean ± SEM; n.s. not significant; *P < 0.05 and ****P < 0.0001