Fig. 4: GRP⁺ interneurons are the direct downstream targets of NPY⁺ interneurons with monosynaptic connections.

A–Fc-Fos expression in the SDH after intrathecal injected saline (15 spinal cord sections from 5 mice were analyzed) or morphine (0.3 nmol, 23 spinal cord sections from 8 mice were analyzed) or morphine + NPY (0.3 nmol + 10 μg, 18 spinal cord sections from 6 mice were analyzed) in WT mice, one way ANOVA, Dunnett’s multiple comparisons (B–E). A Co-expression of Grp (red) and c-Fos (white) mRNA in the SDH after intrathecal injection of morphine. Yellow arrows indicate Grp double-labeled with c-Fos. B The cell numbers of Grp and c-Fos double-positive neurons in the SDH. C The ratio of c-Fos⁺ neurons in Grp⁺ neurons. D Co-expression of Grpr (Green) and c-Fos (White) mRNA in the SDH after intrathecal injected morphine. Yellow arrows indicate Grpr double-labeled with c-Fos. E The cell numbers of Grpr and c-Fos double-positive neurons in the SDH. F The ratio of c-Fos⁺ neurons in Grpr⁺ neurons. G Spinal dorsal horn injection of rAAV-CAG-DIO-mWGA-mCherry virus in Npy^Cre mice. H mWGA-mCherry signals were detected in both Npy⁺ (Green) and Grp⁺ (White) interneurons, yellow and white arrows indicate mWGA-mCherry double-labeled with Npy and Grp, respectively. I mWGA-mCherry signals were detected in Npy⁺ (White), but only a few in Grpr⁺ (Green) interneurons, yellow arrows indicate mWGA-mCherry double-labeled with Npy. J Quantification of mWGA-mCherry double-labeled Grp and Grpr. 9 and 6 spinal cord sections from 3 mice were analyzed, respectively, student’s unpaired two-tailed t test. K Quantification of Grp⁺ labeled HSV-mCherry in the SDH. 10 spinal cord sections from five mice were analyzed. L HSV-mCherry signals were detected in Grp⁺ (White) interneurons, yellow and gray arrows indicate HSV-mCherry double-labeled with Grp and AAV helper, respectively. M Quantification of Npy⁺ labeled RV-CVS-EnvA-ΔG-tdTomato in the SDH. 12 spinal cord sections from five mice were analyzed. N RV-CVS-EnvA-ΔG-tdTomato signals were detected in Npy⁺ (White) interneurons, yellow and purple arrows indicate RV-CVS-EnvA-ΔG-tdTomato double-labeled with Npy and AAV helper, respectively. Cells containing ≥ 5 RNAscope puncta were considered positive expressions. Data are shown as means ± SEM. Scale bars and P values are indicated in the figures. Source data are provided as a Source Data file. Created in BioRender. Wang, Z. (2025) https://BioRender.com/8lt9u0c.