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. 2018 Jan 25;553(7689):455-460.
doi: 10.1038/nature25448. Epub 2018 Jan 17.

Midbrain Circuits That Set Locomotor Speed and Gait Selection

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Free PMC article

Midbrain Circuits That Set Locomotor Speed and Gait Selection

V Caggiano et al. Nature. .
Free PMC article

Abstract

Locomotion is a fundamental motor function common to the animal kingdom. It is implemented episodically and adapted to behavioural needs, including exploration, which requires slow locomotion, and escape behaviour, which necessitates faster speeds. The control of these functions originates in brainstem structures, although the neuronal substrate(s) that support them have not yet been elucidated. Here we show in mice that speed and gait selection are controlled by glutamatergic excitatory neurons (GlutNs) segregated in two distinct midbrain nuclei: the cuneiform nucleus (CnF) and the pedunculopontine nucleus (PPN). GlutNs in both of these regions contribute to the control of slower, alternating-gait locomotion, whereas only GlutNs in the CnF are able to elicit high-speed, synchronous-gait locomotion. Additionally, both the activation dynamics and the input and output connectivity matrices of GlutNs in the PPN and the CnF support explorative and escape locomotion, respectively. Our results identify two regions in the midbrain that act in conjunction to select context-dependent locomotor behaviours.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Speed control of locomotion from glutamatergic neurons in cuneiform and pedunculopontine nuclei
a, b, Upper panels: Experimental set-ups. Middle panels: Colour plot of individual trails following stimulation of CnF- or PPN-Vglut2/ChR2-Ns (Figure 1). 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 25th and 75th percentile of the distribution. d, Post-stimulus locomotor speed plotted against pre-stimulus locomotor speed in Vglut2Cre 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).
Extended Data Figure 2
Extended Data Figure 2. Summary of ChR2 expression in CnF and PPN for behavioural data in Figure 1 and Extended Data Figure 1ab
a, Expression of ChR2 and fibre tip positions in CnF (left) and PPN (right) for data in Figure 1 and Extended Figure 1a,b,c,e. Coronal brain sections with viral expression from injected Vglut2Cre 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 Extended Figure 1d. The mouse brain schematics in this figure have been reproduced with permission from Elsevier .
Extended Data Figure 3
Extended Data Figure 3. Activation of inhibitory neurons in CnF or PPN and cholinergic neurons in PPN does not initiate locomotion but may modulate on-going locomotion
ac, 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 VgatCre mice to target inhibitory cells while cholinergic neurons expressed ChR2 transgenetically by crossing ChATCre with RC26-ChR2flx/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 VgatCre mice in CnF (left) and PPN (right). e, Summary diagram for fibre positions in ChATCre mice. The mouse brain schematics in this figure have been reproduced with permission from Elsevier .
Extended Data Figure 4
Extended Data Figure 4. Summary diagram of iDREADD injection sites in CnF and PPN
a, Expression of iDREADD in CnF (left – N = 9) or PPN-Vglut2-Ns (right, N = 9) for animals used in Figure 2. 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 .
Extended Data Figure 5
Extended Data Figure 5. Control for CNO injection and time course of silencing effect of glutamatergic neurons in the cuneiform and pedunculopontine nuclei
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). bd, Diagrams of injections of AAV-DIO-hM4D(Gi)-mCherry in Vglut2Cre 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.
Extended Data Figure 6
Extended Data Figure 6. Latencies for light-activation of PPN and CnF neurons and fractions of CnF and PPN-Vglut2/ChR2-Ns with speed related activity
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).
Extended Data Figure 7
Extended Data Figure 7. Summary of injection in PPN and CnF for hole-board stimulation experiments
a, Expression of ChR2 and fibre tip positions in CnF (left) or PPN (right) for animals used in figure 5 d,e. The mouse brain schematics in this figure have been reproduced with permission from Elsevier .
Extended Data Figure 8
Extended Data Figure 8. Connectivity between PPN and CnF
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 Vglut2Cre 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.
Extended Data Figure 9
Extended Data Figure 9. The cuneiform and pedunculopontine nuclei have different descending output matrices
a, Simultaneous unilateral injection (left) of AAV-DIO-ChR2 virus in the CnF (mCherry, red) and the PPN (eYFP, green) in Vglut2Cre 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.
Figure 1
Figure 1. Speed-gait control of locomotion from glutamatergic neurons in cuneiform and pedunculopontine nuclei
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 25th and 75th 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).
Figure 2
Figure 2. PPN and CnF provide dual control of slower locomotion
a,b, Bilateral inhibition with iDREADDs in Vglut2Cre 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).
Figure 3
Figure 3. Glutamatergic neurons in the cuneiform nucleus are needed and sufficient for fast synchronous locomotion
a,b, Injection of DREADDs bilaterally in CnF of Vglut2Cre 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.
Figure 4
Figure 4. Coding of speed in glutamatergic neurons in cuneiform and pedunculopontine nuclei
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.
Figure 5
Figure 5. Selection of exploration from PPN
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.
Figure 6
Figure 6. Neurons in cuneiform and pedunculopontine nuclei have differential input matrices
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. ce, 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 .

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