Extended Data Fig. 10: Controls and cannulae placements for chemogenetic inactivation experiments.

a, Summary in vitro data for hM4D-neurexin/ChR2-expressing VGluT2⁺ dmSC neurons before (baseline) and after CNO application (CNO), showing no effect of CNO on action potential firing in response to current injection (left, n = 6 cells, P = 0.8738 for main effect of CNO, two-way repeated measured ANOVA; inset shows example traces to two current steps) or to 473-nm light-evoked ChR2 activation (right, n = 9 cells, P = 0.7006 for main effect of CNO, two-way repeated measured ANOVA). Error bars are s.e.m. b, Application of CNO reduces dmSC–dPAG excitatory synaptic transmission by 71 ± 7% (n = 10 cells, P = 6.19 × 10⁻⁶, two-tailed t-test between baseline and CNO). c, Disrupting mSC–dPAG synapses with CNO microinfusion in behaving mice blocks visually evoked escape behaviour (n = 3 mice, P = 0.036, U-test). d, Doubling the intensity or frequency of mSC stimulation while locally blocking mSC-dPAG synapses is not sufficient to rescue escape behaviour (n = 5 mice, P = 0.11 for intensity, U-test; P = 0.42 for frequency, U-test; both comparisons against escape probability after local block in baseline conditions shown in Fig. 4l). e, Cannula placements for local inactivation experiments with CNO at the SC–PAG synapse (left) and at the SC–LP synapse (right). The tip of the internal cannulae is indicated by yellow circles (for experiments with optogenetic stimulation of dmSC VGluT2⁺ cells) and brown circles (for experiments with visual stimulation). Coordinates are in mm and from bregma. Mouse brain images adapted from Franklin and Paxinos⁴⁶ and reproduced with permission from Elsevier. Box-and-whisker plots show median, IQR and range.