Fig. 1

Sound-evoked responses in CnF and PPN neurons. (A) Vglut2-cre, VGAT-cre or ChAT-cre mice were injected in the cuneiform nucleus (CnF) or pedunculopontine nucleus (PPN) with an adeno-associated virus encoding for the genetically encoded calcium (Ca²⁺) indicator jGCaMP7f in a cre-dependent manner (see Methods) and implanted with an optic fiber ∼150 μm above the injection site (Figure S1). Fiber photometry recordings were done using two LEDs for isosbestic and jGCaMP7f signals (405 and 465 nm), a filter cube (FC) and a photodetector (PD). A clinking metallic sound (86.9 ± 0.5 dB, spectrogram in Figures S2A–C) was applied every 60.1 s ± 0.3 s, ∼1 m away from an open-field arena (see Methods). (B–F) Top, Ca²⁺ responses in the CnF or PPN evoked by sound stimulation. Each line illustrates a trial. Sound stimulus onset is indicated by a black vertical dotted line. Ca²⁺ responses are expressed in Z-score (see Methods), with warmer colors indicating stronger responses. Bottom, mean Z-scores ± SEM are illustrated. (G–L) Sound-evoked mean Ca²⁺ response ± SEM (top) and locomotor speed ± SEM (bottom) obtained in each mouse (M) (1–4 series of 4–10 trials per animal). The number of mice (N) used is indicated. The colored rings illustrate the mean proportion of trials in which mice displayed a locomotor response (speed > 3 cm/s for at least 0.5 s^8,9) during the 5 s after sound stimulation. (M–R) mean Ca²⁺ signal before (− 5 to 0 s), during (0 to 5 s) and after sound stimulation (5 to 10 s) (9–36 trials per animal; *P < 0.05, **P < 0.01, ***P < 0.001, Student-Newman-Keuls test after a significant one-way repeated measures ANOVA; ⁺P < 0.05, Student-Newman-Keuls test after a significant Friedman ANOVA on ranks). (S–X) Comparison of mean Ca²⁺ signals in trials with (blue) or without (grey) a locomotor response during the 5 s following sound stimulation (ns, not significant, ***P < 0.001 paired t-test; NS, not significant, Wilcoxon test; mice without locomotor trials or without non-locomotor trials were excluded from the analysis).