Fig. 5: The CN sends monosynaptic glutamatergic projections to RtTg glutamatergic neurons.

a Schematic for the anterograde tracing from CN to RtTg. b Left: representative images showing RtTg tdT⁺ neurons co-labelled with glutamate (white arrowheads). Scale bar, 300 μm. Right: pie chart indicating the percentage of RtTg tdT⁺ neurons co-labelled with or without glutamate. c Schematic for the optogenetic activation of CN neuronal terminals and the simultaneous whole-cell patch clamp recording of RtTg neurons. d Sample traces (left) and quantitative analysis (right) for EPSCs of RtTg neurons evoked by photostimulation (blue bar, 1 ms) before and after bath application of KYNA. TTX and 4-AP were added into aCSF (n = 6 cells from 4 mice; P = 0.0003). The doughnut chart shows the percentage of RtTg neurons responsive or not responsive to the photostimulation. e Left: schematic for the optogenetic activation of CN glutamatergic neuron terminals in the RtTg. Right: a representative image showing EGFP⁺ terminals in the RtTg. Scale bar, 300 μm. f Startle amplitudes evoked by photostimulation of ChR2⁺ terminals or EGFP⁺ terminals in the RtTg of Vglut2-Cre mice (n = 15 stimuli from 5 mice per group; P = 7.76 × 10⁻⁷). Error bar represents mean ± s.e.m. Significance was assessed using two-sided paired t-tests in d, and two-sided unpaired t-tests in f. *** P < 0.001; ns, not significant.