a, b, Upper panels: Experimental set-ups. Middle panels: Colour plot of individual trails following stimulation of CnF- or PPN-Vglut2/ChR2-Ns (). The x-axis represents time and the y-axis represents different trials obtained with different frequencies of stimulation. Data are aligned to onset of stimulation. The colour gradient illustrates speed, with dark blue representing no movement and colours towards yellow representing increase of speed (up to 120 cm/s) of the animal in the linear corridor. Lower panels: Speed profiles obtained as average of the movements at each stimulation frequency.
c, Latencies to onset of locomotion from stimulation of PPN- (red) and CnF-Vglut2/ChR2-Ns (blue) as a function of the stimulation frequency. Error bars indicate the 25^th and 75^th percentile of the distribution.
d, Post-stimulus locomotor speed plotted against pre-stimulus locomotor speed in Vglut2^Cre mice injected with AAV-DIO-ChR2-mCherry in PPN (n = 50 from 4 mice).
e, Step frequency plotted against speed of locomotion for stimulation of PPN-Vglut2/ChR2-Ns (red circles, n = 84 from 5 mice) or CnF-Vglut2/ChR2-Ns (blue circles, n = 173 from 9 mice).

a, Expression of ChR2 and fibre tip positions in CnF (left) and PPN (right) for data in and . Coronal brain sections with viral expression from injected Vglut2^Cre mice where superimposed on sections redrawn from a mouse brain atlas . The dark contour colour indicates centre of expression while the lighter contour colour indicates the border of the most extended expression. Round dot indicates tip of the fibre.
b, Expression of ChR2 and fibre tip positions in PPN data in .
The mouse brain schematics in this figure have been reproduced with permission from Elsevier .

a–c, Top panels: Schematics showing the implantation of the optical fibre to stimulate inhibitory cells in CnF (a) and PPN (b), and cholinergic cells in PPN (c). AAV-DIO-ChR2 virus was injected in Vgat^Cre mice to target inhibitory cells while cholinergic neurons expressed ChR2 transgenetically by crossing ChAT^Cre with RC26-ChR2^flx/flx mice. Experiments where performed 3 – 4 weeks after injection of virus with animals was locomoting spontaneously in a linear corridor. Middle and lower panels show colour plots where the x-axis represents time and the y-axis represents different trials of stimulation, when the animals were not locomoting (middle panels, “Still”) or when they were locomoting (lower panels, “Moving”) before the stimulation. Data are aligned to the onset of stimulation (dotted lines). The colour gradient illustrates speed, with dark blue representing no movement and colours towards yellow representing increase of speed (up to 60 – 80 cm/s) of the animal in the linear corridor.
Speed before vs after stimulation. CnF-Vgat-INs: from still, p > 0.05, Sign-rank test (two sided) (n = 18, N =2); when moving, from 27.9 cm/s to 4.2 cm/s p<0.05, Sign-rank test (n = 22, N = 2). PPN-Vgat-INs: from still, p > 0.05 (n = 5, N = 2); when moving from 27.6 cm/s vs. to 8.6 cm/s, p < 0.05 Sign-rank test (two sided) (n = 34, N = 2). Stimulation of long-projecting cholinergic cells in PPN: from still, p > 0.05, Sign-rank test (n = 102, N = 5); when moving: before 47.3 cm/s vs. after 22.9 cm/s, p < 0.05, Sign-rank test (two-sided) (n = 88, N = 5). Number of trials, n and animals, N.
d, Summary diagram of viral injection sites and fibre positions in Vgat^Cre mice in CnF (left) and PPN (right).
e, Summary diagram for fibre positions in ChAT^Cre mice.
The mouse brain schematics in this figure have been reproduced with permission from Elsevier .

a, Expression of iDREADD in CnF (left – N = 9) or PPN-Vglut2-Ns (right, N = 9) for animals used in .
b, Example of Coronal section showing expression pattern of iDREADD in CnF-Vglut2-Ns. Scale bar: 500 μm.
c, Coronal section showing expression pattern of iDREADD in PPN-Vglut2-Ns.
Scale bar: 500 μm.
The mouse brain schematics in this figure have been reproduced with permission from Elsevier .

a, Treadmill experiments with saline (black bar) and Clozapine-N-oxide (CNO) (orange bar) (1mg/kg) (right) injected i.p. in wild-type animals (N = 7). Average speeds to the left and peak speeds to the right. There was no significant difference in these parameters between saline and CNO sign-rank, two-sided (p > 0.45).
b–d, Diagrams of injections of AAV-DIO-hM4D(Gi)-mCherry in Vglut2^Cre mice in CnF (b), PPN (c) or CnF+PPN (d). Clozapine-N-oxide (CNO) was injected i.p. and locomotor performance was tested on a treadmill.
e–g, Graphs show the development of the effect of the inhibition of glutamatergic cells in CnF (e, N=3), PPN (f, N=3) or CnF+PPN (g, N=5) on maximal locomotor speed over time. Grey bars, baseline. Orange bars, different time points after CNO administration. Points shows individual trials.

a, Latencies for light-activation of all cells included in the analysis.
b, Distribution of CnF-Vglut2/ChR2-Ns (blue bars, n=79/169) and PPN-Vglut2/ChR2-Ns (red bars, n=105/493) showing correlation of firing activity with locomotor speed of the animal. Grey bars show, in both panels, neurons with no significant correlation with the speed (Spearman correlation test p > 0.05).

a, Expression of ChR2 and fibre tip positions in CnF (left) or PPN (right) for animals used in . The mouse brain schematics in this figure have been reproduced with permission from Elsevier .

a, b, AAV-EF1a-FLEX-GTB helper virus followed by EnvA G-deleted-Rabies-mCherry virus were unilaterally injected in the PPN (left, red) or the CnF (right, blue) in Vglut2^Cre mice to trace inputs to glutamatergic neurons. a, Schematics summarising the inputs to PPN- Vglut2-Ns (red) and CnF-Vglut2-Ns (blue) neurons. The thickness of the arrows indicates the amount of connectivity based on the counts of the normalized number of neurons (b). Dashed arrows indicate sparse connectivity. CnF, cuneiform nucleus; IC, inferior colliculus; PAG, periaqueductal grey; PPN, pedunculopontine nucleus.

a, Simultaneous unilateral injection (left) of AAV-DIO-ChR2 virus in the CnF (mCherry, red) and the PPN (eYFP, green) in Vglut2^Cre animals (N = 3). Sagittal view of the brain (right) displaying the location in the brainstem (1 – 4) and spinal cord (5) of the coronal sections shown in c.
b, Coronal section showing ipsilateral (left side) and contralateral projection areas from glutamatergic CnF and PPN neurons.
c1–5, Schematics and coronal sections showing projection areas from glutamatergic PPN (left, green) and CnF (right, red) neurons onto nuclei in the pons, medulla and spinal cord. In the schematics, the darker shades delineate the areas with the highest density of projections. In coronal sections labelled processes are seen in black. Anatomical landmarks are indicated in the schematics. 4V, fourth ventricle; 7N, facial motor nucleus; IOM, inferior olive, medial nucleus; IRt, intermediate reticular nucleus; Gi, gigantocellular nucleus; GiA, gigantocellular reticular nucleus, alpha part; GiV, gigantocellular reticular nucleus, ventral part; IRt, intermediate reticular nucleus; LC, locus coeruleus; LPGi, lateral paragigantocellular nucleus; LRt, lateral reticular nucleus; MdV, medullary reticular nucleus, ventral part; PnC, pontine reticular nucleus, caudal part; PnV, pontine reticular nucleus, ventral part; py, pyramidal tract; pyx, pyramidal decussation; RMg, raphe magnus; RPa, raphe pallidus; ROb, raphe obscurus. The mouse brain schematics in this figure have been reproduced with permission from Elsevier . Scale bars, 200 μm.

a,b,c, Neurotransmitter identity and localization.
d,e,f,g, Locomotor examples induced by optical stimulation of CnF (d,e) and PPN (f,g).
h, Maximum speed evoked by different stimulations of PPN (red; N=5, n=67) and CnF (blue; N 9, n=148). Error bars indicate the 25^th and 75^th percentile of the distribution.
i, Fraction of trials at a given maximum speed (inset; ***, p<0.001, two-tailed U-test).
j, Probability of obtaining different gaits (CnF-upper or PPN-lower panel).

a,b, Bilateral inhibition with iDREADDs in Vglut2^Cre mice in CnF or/and PPN. Average (a) and maximum (b) speed of the animal before vs. after CNO administration (*, p<0.05, two-tailed sign-rank test).

a,b, Injection of DREADDs bilaterally in CnF of Vglut2^Cre and probability of evoking gallop/bound before and after inactivation of CnF with CNO during air-puff induced escaping behaviour (p=0.0312, two-tailed sign-rank test, N=6).
c–d, Maximum speeds of locomotion (c) combining inhibitory DREADDs in the PPN, and optogenetic activation of CnF at 50 Hz before (grey, n=13 repetitions, N=4 mice) and after CNO (orange bars, n=12 repetitions, N=4 mice, p<0.05, two-tailed U-test) and their relationship with gaits (d).
Drawing in Fig. 3a reproduced with permission from Mattias Karlen.

a,b, Examples of neuronal firing at different speed of treadmill induced locomotion in CnF (a) and PPN (b).
c, Average responses at rest (left) in CnF (n=79) and PPN (n=105, p<0.001, two-tailed U-test) and during movements (right, p<0.001, two-tailed U-test).
d, Speed Selectivity Index (see text and Methods). PPN-Vglut2 neurons were more selective at lower speed while CnF-Vglut2 were more selective at higher speed (*, p<0.05, two-tailed U-test with post-hoc Bonferroni correction). Data are mean ± s.e.m.

a,b,c, During exploratory hole-board experiments (a), bilateral inactivation of CnF (b, N = 6) did not reduce either the frequency (left, p > 0.05) or the fraction of time (right, p > 0.05) of head-dips. Bilateral inactivation of PPN (c, N=6) reduced both parameters (left, p=0.031, right, p=0.031, both two-tailed sign-rank tests).
d,e, Optogenetic stimulation of CnF (N=2) induced a decrease in number (d–left, p=0.0023) but not in the fraction of time of head-dipping (d–right, p>0.05) while stimulation of PPN (N=4) increased (e) them (left p<0.001, right p=0.0218, all two-tailed sing-rank tests). Drawing in Fig. 5a reproduced with permission from Mattias Karlen.

a,b, Reconstruction of projections to CnF (b, N=3, upper row) or PPN (b, N=3, lower row) as revealed by mono-synaptically restricted trans-synaptic retrograde labelling.
c–e, Regional distribution (median, N=3) of neurons projecting to CnF (blue) and to PPN (red) normalized to the number of primary infected neurons in either structure.
f, Examples of labelled neurons (black) in the substantia nigra projecting onto glutamatergic CnF (left) or PPN (right) neurons. a, Scale bar, 20 μm. f, Scale bars, 500 μm. IC, inferior colliculus; SNc, substantia nigra pars compacta; SNl, substantia nigra pars lateralis; SNr, substantia nigra pars reticulata; PAG, periaqueductal grey; PSTh, parasubthalamic nucleus; STh, subthalamic nucleus; ZI, zona incerta. The mouse brain schematics in this figure have been reproduced with permission from Elsevier .