Fig. 3: Commissural Vglut2⁺ neurons in PnO link SNr to Gi.

a, Viral screening strategy for identifying link between SNr and Gi. AAV_retro-fDIO-tdTom was injected in Gi_left, followed by injection of AAV1-FlpO in SNr_right. This screen uncovered only one neuronal population which links SNr to Gi located in the PnO (PnO_right, ipsilateral to SNr and contralateral to Gi). b, Anatomy of SNr recipient, PnO → Gi projection neurons. Neurons exhibit caudally projecting axons which decussate at −5.0 mm AP and terminate in the contralateral Gi. Caudal brainstem sections exhibit only sparse axonal projections. Images are representative of n = 6 mice from 2 independent experiments. c, Triple in situ hybridization for tdTom, Vglut2 and Vgat indicates 93.4 ± 1.2% of tdTom⁺ (SNr recipient, PnO → Gi projection) neurons co-expressed Vglut2 whereas 6.6 ± 1.2% of tdTom⁺ neurons co-expressed Vgat. ***P = 4.2 × 10⁻⁵; two-tailed paired t-test; n = 4 mice from 1 experiment. See also Supplementary Table 1. d, tdTom labeling of SNr recipient, PnO → Gi projection neurons in GlyT2^GFP or Gad67^GFP mice. SNr recipient, PnO → Gi projection (tdTom⁺) neurons are predominantly GFP⁻, indicating they are not glycinergic or GABAergic. GlyT2^GFP, ***P = 3.0 × 10⁻⁵; Gad67^GFP, ***P = 4.0 × 10⁻⁷; two-tailed paired t-test; n = 3 mice from 1 experiment for GlyT2^GFP and n = 4 mice from 2 independent experiments for Gad67^GFP. Box-and-whisker plots in c and d give the median, the 25th and 75th percentiles and the range. e, Density plots representing the location of soma in the coronal, horizontal and sagittal planes. Population data are superimposed on plates redrawn from Paxinos and Franklin’s reference atlas³⁷. Plates were selected based on the mean AP, DV or ML value of all PnO-Vglut2_contra neurons registered. f, Quantification of PnO-Vglut2_contra neurons in the AP, DV and ML axis relative to bregma, n = 3 mice. Error bars in f give the s.e.m.