A key function of the mesencephalic locomotor region (MLR) is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Here, we investigated whether freely behaving mice could brake or turn, based on environmental cues during MLR stimulation. We photostimulated the cuneiform nucleus (part of the MLR) in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. In the linear corridor, gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. In the open-field arena, optogenetic stimulation of the MLR evoked locomotion, and increasing laser power increased locomotor speed. Mice could brake and make sharp turns (~90°) when approaching a corner during MLR stimulation in the open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turn angle. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short-latency spiking in MLR neurons. Our results strengthen the idea that different brainstem neurons convey braking/turning and MLR speed commands in mammals. Our study also shows that Vglut2-positive neurons of the cuneiform nucleus are a relevant target to increase locomotor activity without impeding the ability to brake and turn when approaching obstacles, thus ensuring smooth and adaptable navigation. Our observations may have clinical relevance since cuneiform nucleus stimulation is increasingly considered to improve locomotion function in pathological states such as Parkinson’s disease, spinal cord injury, or stroke.

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Serotoninergic (5-HT) neurons are powerful modulators of spinal locomotor circuits. Most studies about 5-HT modulation focused on the effect of exogenous 5-HT and these studies provided key information about the cellular mechanisms involved. Less is known about the effects of increased release of endogenous 5-HT with selective serotonin reuptake inhibitors. Such molecules were shown to destabilize the locomotor output of spinal limb networks through 5-HT1A receptors. However, in tetrapods little is known about the effects of increased 5-HT release on the locomotor output of axial networks, which are coordinated with limb circuits during locomotion from basal vertebrates to mammals. Here, we examined the effect of citalopram on fictive locomotion generated in axial segments of isolated spinal cords in salamanders, a tetrapod where raphe 5-HT reticulospinal neurons and intraspinal 5-HT neurons are present as in other vertebrates. Using electrophysiological recordings of ventral roots, we show that fictive locomotion generated by bath-applied glutamatergic agonists is destabilized by citalopram. Citalopram-induced destabilization was prevented by a 5-HT1A receptor antagonist, whereas a 5-HT1A receptor agonist destabilized fictive locomotion. Using immunofluorescence experiments, we found 5-HT-positive fibers and varicosities in proximity with motoneurons and glutamatergic interneurons that are likely involved in rhythmogenesis. Our results show that increasing 5-HT release has a deleterious effect on axial locomotor activity through 5-HT1A receptors. This is consistent with studies in limb networks of turtle and mouse, suggesting that this part of the complex 5-HT modulation of spinal locomotor circuits is common to limb and axial networks in limbed vertebrates.

Background: Stimulation of the Mesencephalic Locomotor Region (MLR) is increasingly considered as a target to improve locomotor function in Parkinson's disease and spinal cord injury. A key function of the MLR is to control the speed of forward symmetrical locomotor movements. However, the ability of freely moving mammals to integrate environmental cues to brake and turn during MLR stimulation is poorly documented. Objective/hypothesis: We investigated whether freely behaving mice could brake or turn based on environmental cues during MLR stimulation. Methods: We stimulated the cuneiform nucleus in mice expressing channelrhodopsin in Vglut2-positive neurons in a Cre-dependent manner (Vglut2-ChR2-EYFP) using optogenetics. We detected locomotor movements using deep learning. We used patch-clamp recordings to validate the functional expression of channelrhodopsin and neuroanatomy to visualize the stimulation sites. Results: Optogenetic stimulation of the MLR evoked locomotion and increasing laser power increased locomotor speed. Gait diagram and limb kinematics were similar during spontaneous and optogenetic-evoked locomotion. Mice could brake and make sharp turns (~90⁰) when approaching a corner during MLR stimulation in an open-field arena. The speed during the turn was scaled with the speed before the turn, and with the turning angle. In a reporter mouse, many Vglut2-ZsGreen neurons were immunopositive for glutamate in the MLR. Patch-clamp recordings in Vglut2-ChR2-EYFP mice show that blue light evoked short latency spiking in MLR neurons. Conclusion: MLR glutamatergic neurons are a relevant target to improve locomotor activity without impeding the ability to brake and turn when approaching an obstacle, thus ensuring smooth and adaptable navigation.

During the last 50 years, the serotonergic (5-HT) system was reported to exert a complex modulation of locomotor activity. Here, we focus on two key factors that likely contribute to such complexity. First, locomotion is modulated directly and indirectly by 5-HT neurons. The locomotor circuitry is directly innervated by 5-HT neurons in the caudal brainstem and spinal cord. Also, indirect control of locomotor activity results from ascending projections of 5-HT cells in the rostral brainstem that innervate multiple brain centers involved in motor action planning. Second, each approach used to manipulate the 5-HT system likely engages different 5-HT-dependent mechanisms. This includes the recruitment of different 5-HT receptors, which can have excitatory or inhibitory effects on cell activity. These receptors can be located far or close to the 5-HT release sites, making their activation dependent on the level of 5-HT released. Here we review the activity of different 5-HT nuclei during locomotor activity, and the locomotor effects of 5-HT precursors, exogenous 5-HT, selective 5-HT reuptake inhibitors (SSRI), electrical or chemical stimulation of 5-HT neurons, genetic deletions, optogenetic and chemogenetic manipulations. We highlight both the coherent and controversial aspects of 5-HT modulation of locomotor activity from basal vertebrates to mammals. This mini review may hopefully inspire future studies aiming at dissecting the complex effects of 5-HT on locomotor function.