Extended Data Fig. 7: dPAG neurons receive input from mainly excitatory cells in the SC and do not project back to the SC.

a, Image showing starter dPAG VGlut2⁺ cells expressing both TVA–GFP and RV–mCherry and presynaptic cells expressing RV–mCherry only (left), and corresponding schematic (right) illustrating the position of starter dPAG (blue) and presynaptic SC cells (pink) across deep, intermediate and superficial SC layers (same as shown in Fig. 4a). b, Kernel density estimation curves for the axial position of presynaptic SC cells for each layer (82.9 ± 2.6% of 1,770 cells are located within the medial bisection of ipsilateral SC, n = 3 mice). c, Image showing presynaptic cells in the mSC infected with rabies virus (red) from starter neurons in the dPAG of a VGluT2::eYFP mouse (left). Box indicates area magnified shown on the right. Yellow cells are VGluT2⁺ mSC presynaptic neurons. d, Summary quantification of the percentage of presynaptic cells in the mSC that express VGluT2⁺ (mean = 87.9 ± 1.0%, n = 4 mice). e, Image showing injection of rAAV2-retro in the mSC (left) and no retrogradely labelled cells in the dPAG (bottom, left), while retrograde labelling is present in the auditory cortex for comparison (bottom, right). Similarly, rabies virus injected in the mSC shows a lack of presynaptic cells in the dPAG (right), suggesting a predominantly feed-forward connectivity arrangement between the mSC and dPAG (note, however, that it cannot be excluded that both rAAV2-retro and rabies display selective tropism that prevents labelling of dPAG neurons). f, Summary quantification for retrogradely labelled cells in the dPAG and auditory cortex after mSC rAAV2-retro (n = 3 mice) or rabies infection (n = 3 mice). Box-and-whisker plots show median, IQR and range.