Optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus controls locomotion in a mouse model of Parkinson's disease.

In Parkinson's disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.

Locomotor pattern generation and descending control: a historical perspective.

The ability to generate and control locomotor movements depends on complex interactions between many areas of the nervous system, the musculoskeletal system, and the environment. How the nervous system manages to accomplish this task has been the subject of investigation for more than a century. In vertebrates, locomotion is generated by neural networks located in the spinal cord referred to as central pattern generators. Descending inputs from the brain stem initiate, maintain, and stop locomotion as well as control speed and direction. Sensory inputs adapt locomotor programs to the environmental conditions. This review presents a comparative and historical overview of some of the neural mechanisms underlying the control of locomotion in vertebrates. We have put an emphasis on spinal mechanisms and descending control.

Descending Dopaminergic Inputs to Reticulospinal Neurons Promote Locomotor Movements.

Meso-diencephalic dopaminergic neurons are known to modulate locomotor behaviors through their ascending projections to the basal ganglia, which in turn project to the mesencephalic locomotor region, known to control locomotion in vertebrates. In addition to their ascending projections, dopaminergic neurons were found to increase locomotor movements through direct descending projections to the mesencephalic locomotor region and spinal cord. Intriguingly, fibers expressing tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis, were also observed around reticulospinal neurons of lampreys. We now examined the origin and the role of this innervation. Using immunofluorescence and tracing experiments, we found that fibers positive for dopamine innervate reticulospinal neurons in the four reticular nuclei of lampreys. We identified the dopaminergic source using tracer injections in reticular nuclei, which retrogradely labeled dopaminergic neurons in a caudal diencephalic nucleus (posterior tuberculum [PT]). Using voltammetry in brain preparations isolated in vitro, we found that PT stimulation evoked dopamine release in all four reticular nuclei, but not in the spinal cord. In semi-intact preparations where the brain is accessible and the body moves, PT stimulation evoked swimming, and injection of a D1 receptor antagonist within the middle rhombencephalic reticular nucleus was sufficient to decrease reticulospinal activity and PT-evoked swimming. Our study reveals that dopaminergic neurons have access to command neurons that integrate sensory and descending inputs to activate spinal locomotor neurons. As such, our findings strengthen the idea that dopamine can modulate locomotor behavior both via ascending projections to the basal ganglia and through descending projections to brainstem motor circuits.SIGNIFICANCE STATEMENT Meso-diencephalic dopaminergic neurons play a key role in modulating locomotion by releasing dopamine in the basal ganglia, spinal networks, and the mesencephalic locomotor region, a brainstem region that controls locomotion in a graded fashion. Here, we report in lampreys that dopaminergic neurons release dopamine in the four reticular nuclei where reticulospinal neurons are located. Reticulospinal neurons integrate sensory and descending suprareticular inputs to control spinal interneurons and motoneurons. By directly modulating the activity of reticulospinal neurons, meso-diencephalic dopaminergic neurons control the very last instructions sent by the brain to spinal locomotor circuits. Our study reports on a new direct descending dopaminergic projection to reticulospinal neurons that modulates locomotor behavior.

S100ß-mediated astroglial control of firing and input processing in layer 5 pyramidal neurons of the mouse visual cortex.

KEY POINTS: Inputs impinging on layer 5 pyramidal neurons perform essential operations as these cells represent one of the most important output carriers of the cerebral cortex. However, the contribution of astrocytes, a type of glial cell, to these operations is poorly documented. Here we found that optogenetic activation of astrocytes in the vicinity of layer 5 in the mouse primary visual cortex induces spiking in local pyramidal neurons through Nav1.6 ion channels and prolongs the responses elicited in these neurons by stimulation of their distal inputs in cortical layer 1. This effect partially involved glutamatergic signalling but relied mostly on the astrocytic calcium-binding protein S100ß, which regulates the concentration of calcium in the extracellular space around neurons. These findings show that astrocytes contribute to the fundamental computational operations of the cortex by acting on the ionic environment of neurons. ABSTRACT: The most complex cerebral functions are performed by the cortex, whose most important output is carried out by its layer 5 pyramidal neurons. Their firing reflects integration of the sensory and contextual information that they receive. There is evidence that astrocytes influence cortical neuron firing through the release of gliotransmitters such as ATP, glutamate or GABA. These effects have been described at the network and at the synaptic levels, but it is still unclear how astrocytes influence neuron input-output transfer function at the cellular level. Here, we used optogenetic tools coupled with electrophysiological, imaging and anatomical approaches to test whether and how astrocytic activation affected processing of distal inputs to layer 5 pyramidal neurons (L5PNs). We show that optogenetic activation of astrocytes near L5PN cell body prolonged firing induced by distal inputs to L5PNs and potentiated their ability to trigger spikes. The observed astrocytic effects on L5PN firing involved glutamatergic transmission to some extent but relied mostly on release of S100ß, an astrocytic Ca2+ -binding protein that decreases extracellular Ca2+ once released. This astrocyte-evoked decrease in extracellular Ca2+ elicited firing mediated by activation of Nav1.6 channels. Our findings suggest that astrocytes contribute to the cortical fundamental computational operations by controlling the extracellular ionic environment.

The serotonin reuptake blocker citalopram destabilizes fictive locomotor activity in salamander axial circuits through 5-HT1A receptors.

Serotoninergic (5-HT) neurons are powerful modulators of spinal locomotor circuits. Most studies on 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. In mammals, such molecules were shown to destabilize the fictive 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.NEW & NOTEWORTHY Little is known about the modulation exerted by endogenous serotonin on axial locomotor circuits in tetrapods. Using axial ventral root recordings in salamanders, we found that a serotonin reuptake blocker destabilized fictive locomotor activity through 5-HT1A receptors. Our anatomical results suggest that serotonin is released on motoneurons and glutamatergic interneurons possibly involved in rhythmogenesis. Our study suggests that common serotoninergic mechanisms modulate axial motor circuits in amphibians and limb motor circuits in reptiles and mammals.

A descending dopamine pathway conserved from basal vertebrates to mammals.

Dopamine neurons are classically known to modulate locomotion indirectly through ascending projections to the basal ganglia that project down to brainstem locomotor networks. Their loss in Parkinson's disease is devastating. In lampreys, we recently showed that brainstem networks also receive direct descending dopaminergic inputs that potentiate locomotor output. Here, we provide evidence that this descending dopaminergic pathway is conserved to higher vertebrates, including mammals. In salamanders, dopamine neurons projecting to the striatum or brainstem locomotor networks were partly intermingled. Stimulation of the dopaminergic region evoked dopamine release in brainstem locomotor networks and concurrent reticulospinal activity. In rats, some dopamine neurons projecting to the striatum also innervated the pedunculopontine nucleus, a known locomotor center, and stimulation of the dopaminergic region evoked pedunculopontine dopamine release in vivo. Finally, we found dopaminergic fibers in the human pedunculopontine nucleus. The conservation of a descending dopaminergic pathway across vertebrates warrants re-evaluating dopamine's role in locomotion.

Nigral Glutamatergic Neurons Control the Speed of Locomotion.

The mesencephalic locomotor region (MLR) plays a crucial role in locomotor control. In vertebrates, stimulation of the MLR at increasing intensities elicits locomotion of growing speed. This effect has been presumed to result from higher brain inputs activating the MLR like a dimmer switch. Here, we show in lampreys (Petromyzon marinus) of either sex that incremental stimulation of a region homologous to the mammalian substantia nigra pars compacta (SNc) evokes increasing activation of MLR cells with a graded increase in the frequency of locomotor movements. Neurons co-storing glutamate and dopamine were found to project from the primal SNc to the MLR. Blockade of glutamatergic transmission largely diminished MLR cell responses and locomotion. Local blockade of D1 receptors in the MLR decreased locomotor frequency, but did not disrupt the SNc-evoked graded control of locomotion. Our findings revealed the presence of a glutamatergic input to the MLR originating from the primal SNc that evokes graded locomotor movements.SIGNIFICANCE STATEMENT The mesencephalic locomotor region (MLR) plays a crucial role in the control of locomotion. It projects downward to reticulospinal neurons that in turn activate the spinal locomotor networks. Increasing the intensity of MLR stimulation produces a growing activation of reticulospinal cells and a progressive increase in the speed of locomotor movements. Since the discovery of the MLR some 50 years ago, it has been presumed that higher brain regions activate the MLR in a graded fashion, but this has not been confirmed yet. Here, using a combination of techniques from cell to behavior, we provide evidence of a new glutamatergic pathway activating the MLR in a graded fashion, and consequently evoking a progressive increase in locomotor output.

Forebrain dopamine neurons project down to a brainstem region controlling locomotion.

The contribution of dopamine (DA) to locomotor control is traditionally attributed to ascending dopaminergic projections from the substantia nigra pars compacta and the ventral tegmental area to the basal ganglia, which in turn project down to the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion in vertebrates. However, a dopaminergic innervation of the pedunculopontine nucleus, considered part of the MLR, was recently identified in the monkey. The origin and role of this dopaminergic input are unknown. We addressed these questions in a basal vertebrate, the lamprey. Here we report a functional descending dopaminergic pathway from the posterior tuberculum (PT; homologous to the substantia nigra pars compacta and/or ventral tegmental area of mammals) to the MLR. By using triple labeling, we found that dopaminergic cells from the PT not only project an ascending pathway to the striatum, but send a descending projection to the MLR. In an isolated brain preparation, PT stimulation elicited excitatory synaptic inputs into patch-clamped MLR cells, accompanied by activity in reticulospinal cells. By using voltammetry coupled with electrophysiological recordings, we demonstrate that PT stimulation evoked DA release in the MLR, together with the activation of reticulospinal cells. In a semi-intact preparation, stimulation of the PT elicited reticulospinal activity together with locomotor movements. Microinjections of a D1 antagonist in the MLR decreased the locomotor output elicited by PT stimulation, whereas injection of DA had an opposite effect. It appears that this descending dopaminergic pathway has a modulatory role on MLR cells that are known to receive glutamatergic projections and promotes locomotor output.

Dopamine-sensitive neurons in the mesencephalic locomotor region control locomotion initiation, stop, and turns.

The locomotor role of dopaminergic neurons is traditionally attributed to their ascending projections to the basal ganglia, which project to the mesencephalic locomotor region (MLR). In addition, descending dopaminergic projections to the MLR are present from basal vertebrates to mammals. However, the neurons targeted in the MLR and their behavioral role are unknown in mammals. Here, we identify genetically defined MLR cells that express D1 or D2 receptors and control different motor behaviors in mice. In the cuneiform nucleus, D1-expressing neurons promote locomotion, while D2-expressing neurons stop locomotion. In the pedunculopontine nucleus, D1-expressing neurons promote locomotion, while D2-expressing neurons evoke ipsilateral turns. Using RNAscope, we show that MLR dopamine-sensitive neurons comprise a combination of glutamatergic, GABAergic, and cholinergic neurons, suggesting that different neurotransmitter-based cell types work together to control distinct behavioral modules. Altogether, our study uncovers behaviorally relevant cell types in the mammalian MLR based on the expression of dopaminergic receptors.

From lamprey to salamander: an exploratory modeling study on the architecture of the spinal locomotor networks in the salamander.

The evolutionary transition from water to land required new locomotor modes and corresponding adjustments of the spinal "central pattern generators" for locomotion. Salamanders resemble the first terrestrial tetrapods and represent a key animal for the study of these changes. Based on recent physiological data from salamanders, and previous work on the swimming, limbless lamprey, we present a model of the basic oscillatory network in the salamander spinal cord, the spinal segment. Model neurons are of the Hodgkin-Huxley type. Spinal hemisegments contain sparsely connected excitatory and inhibitory neuron populations, and are coupled to a contralateral hemisegment. The model yields a large range of experimental findings, especially the NMDA-induced oscillations observed in isolated axial hemisegments and segments of the salamander Pleurodeles waltlii. The model reproduces most of the effects of the blockade of AMPA synapses, glycinergic synapses, calcium-activated potassium current, persistent sodium current, and [Formula: see text]-current. Driving segments with a population of brainstem neurons yields fast oscillations in the in vivo swimming frequency range. A minimal modification to the conductances involved in burst-termination yields the slower stepping frequency range. Slow oscillators can impose their frequency on fast oscillators, as is likely the case during gait transitions from swimming to stepping. Our study shows that a lamprey-like network can potentially serve as a building block of axial and limb oscillators for swimming and stepping in salamanders.

Decoding the mechanisms of gait generation in salamanders by combining neurobiology, modeling and robotics.

Vertebrate animals exhibit impressive locomotor skills. These locomotor skills are due to the complex interactions between the environment, the musculo-skeletal system and the central nervous system, in particular the spinal locomotor circuits. We are interested in decoding these interactions in the salamander, a key animal from an evolutionary point of view. It exhibits both swimming and stepping gaits and is faced with the problem of producing efficient propulsive forces using the same musculo-skeletal system in two environments with significant physical differences in density, viscosity and gravitational load. Yet its nervous system remains comparatively simple. Our approach is based on a combination of neurophysiological experiments, numerical modeling at different levels of abstraction, and robotic validation using an amphibious salamander-like robot. This article reviews the current state of our knowledge on salamander locomotion control, and presents how our approach has allowed us to obtain a first conceptual model of the salamander spinal locomotor networks. The model suggests that the salamander locomotor circuit can be seen as a lamprey-like circuit controlling axial movements of the trunk and tail, extended by specialized oscillatory centers controlling limb movements. The interplay between the two types of circuits determines the mode of locomotion under the influence of sensory feedback and descending drive, with stepping gaits at low drive, and swimming at high drive.

Dopamine control of downstream motor centers.

The role of dopamine in the control of movement is traditionally associated with ascending projections to the basal ganglia. However, more recently descending dopaminergic pathways projecting to downstream brainstem motor circuits were discovered. In lampreys, salamanders, and rodents, these include projections to the downstream Mesencephalic Locomotor Region (MLR), a brainstem region controlling locomotion. Such descending dopaminergic projections could prime brainstem networks controlling movement. Other descending dopaminergic projections have been shown to reach reticulospinal cells involved in the control of locomotion. In addition, dopamine directly modulates the activity of interneurons and motoneurons. Beyond locomotion, dopaminergic inputs modulate visuomotor transformations within the optic tectum (mammalian superior colliculus). Loss of descending dopaminergic inputs will likely contribute to pathological conditions such as in Parkinson's disease.

The Mesencephalic Locomotor Region: Multiple Cell Types, Multiple Behavioral Roles, and Multiple Implications for Disease.

The mesencephalic locomotor region (MLR) controls locomotion in vertebrates. In humans with Parkinson disease, locomotor deficits are increasingly associated with decreased activity in the MLR. This brainstem region, commonly considered to include the cuneiform and pedunculopontine nuclei, has been explored as a target for deep brain stimulation to improve locomotor function, but the results are variable, from modest to promising. However, the MLR is a heterogeneous structure, and identification of the best cell type to target is only beginning. Here, I review the studies that uncovered the role of genetically defined MLR cell types, and I highlight the cells whose activation improves locomotor function in animal models of Parkinson disease. The promising cell types to activate comprise some glutamatergic neurons in the cuneiform and caudal pedunculopontine nuclei, as well as some cholinergic neurons of the pedunculopontine nucleus. Activation of MLR GABAergic neurons should be avoided, since they stop locomotion or evoke bouts flanked with numerous stops. The MLR is also considered a potential target in spinal cord injury, supranuclear palsy, primary progressive freezing of gait, or stroke. Better targeting of the MLR cell types should be achieved through optimized deep brain stimulation protocols, pharmacotherapy, or the development of optogenetics for human use.

From retina to motoneurons: A substrate for visuomotor transformation in salamanders.

The transformation of visual input into motor output is essential to approach a target or avoid a predator. In salamanders, visually guided orientation behaviors have been extensively studied during prey capture. However, the neural circuitry involved is not resolved. Using salamander brain preparations, calcium imaging and tracing experiments, we describe a neural substrate through which retinal input is transformed into spinal motor output. We found that retina stimulation evoked responses in reticulospinal neurons of the middle reticular nucleus, known to control steering movements in salamanders. Microinjection of glutamatergic antagonists in the optic tectum (superior colliculus in mammals) decreased the reticulospinal responses. Using tracing, we found that retina projected to the dorsal layers of the contralateral tectum, where the dendrites of neurons projecting to the middle reticular nucleus were located. In slices, stimulation of the tectal dorsal layers evoked glutamatergic responses in deep tectal neurons retrogradely labeled from the middle reticular nucleus. We then examined how tectum activation translated into spinal motor output. Tectum stimulation evoked motoneuronal responses, which were decreased by microinjections of glutamatergic antagonists in the contralateral middle reticular nucleus. Reticulospinal fibers anterogradely labeled from tracer injection in the middle reticular nucleus were preferentially distributed in proximity with the dendrites of ipsilateral motoneurons. Our work establishes a neural substrate linking visual and motor centers in salamanders. This retino-tecto-reticulo-spinal circuitry is well positioned to control orienting behaviors. Our study bridges the gap between the behavioral studies and the neural mechanisms involved in the transformation of visual input into motor output in salamanders.

The transformation of a unilateral locomotor command into a symmetrical bilateral activation in the brainstem.

A unilateral activation of the mesencephalic locomotor region (MLR) produces symmetrical bilateral locomotion in all vertebrate species tested to date. How this occurs remains unresolved. This study examined the possibility that the symmetry occurred at the level of the inputs from the MLR to reticulospinal (RS) cells. In lamprey semi-intact preparations, we recorded intracellular responses of pairs of large, homologous RS cells on both sides to stimulation of the MLR on one side. The synaptic responses on both sides were very similar in shape, amplitude, and threshold intensity. Increasing MLR stimulation intensity produced a symmetrical increase in the magnitude of the responses on both sides. Ca(2+) imaging confirmed the bilateral activation of smaller-sized RS cells as well. In a high-divalent cation solution, the synaptic responses of homologous RS cells persisted and exhibited a constant latency during high-frequency stimulation. Moreover, during gradual replacement of normal Ringer's solution with a Ca(2+)-free solution, the magnitude of responses showed a gradual reduction with a similar time course in the homologous RS cells. These results support the idea that the MLR projects monosynaptically to RS cells on both sides with symmetrical inputs. During locomotion of the semi-intact preparation, the discharge pattern was also very similar in homologous bilateral RS cells. Anatomical experiments confirmed the presence of MLR neurons projecting ipsilaterally to the reticular formation intermingled with neurons projecting contralaterally. We conclude that the bilaterally symmetrical MLR inputs to RS cells are likely contributors to generating symmetrical locomotor activity.

Heterogeneous expression of dopaminergic markers and Vglut2 in mouse mesodiencephalic dopaminergic nuclei A8-A13.

Co-transmission of glutamate by brain dopaminergic (DA) neurons was recently proposed as a potential factor influencing cell survival in models of Parkinson's disease. Intriguingly, brain DA nuclei are differentially affected in Parkinson's disease. Whether this is associated with different patterns of co-expression of the glutamatergic phenotype along the rostrocaudal brain axis is unknown in mammals. We hypothesized that, as in zebrafish, the glutamatergic phenotype is present preferentially in the caudal mesodiencephalic DA nuclei. Here, we used in mice a cell fate mapping strategy based on reporter protein expression (ZsGreen) consecutive to previous expression of the vesicular glutamate transporter 2 (Vglut2) gene, coupled with immunofluorescence experiments against tyrosine hydroxylase (TH) or dopamine transporter (DAT). We found three expression patterns in DA cells, organized along the rostrocaudal brain axis. The first pattern (TH-positive, DAT-positive, ZsGreen-positive) was found in A8-A10. The second pattern (TH-positive, DAT-negative, ZsGreen-positive) was found in A11. The third pattern (TH-positive, DAT-negative, ZsGreen-negative) was found in A12-A13. These patterns should help to refine the establishment of the homology of DA nuclei between vertebrate species. Our results also uncover that Vglut2 is expressed at some point during cell lifetime in DA nuclei known to degenerate in Parkinson's disease and largely absent from those that are preserved, suggesting that co-expression of the glutamatergic phenotype in DA cells influences their survival in Parkinson's disease.

Emergence of robust self-organized undulatory swimming based on local hydrodynamic force sensing.

Undulatory swimming represents an ideal behavior to investigate locomotion control and the role of the underlying central and peripheral components in the spinal cord. Many vertebrate swimmers have central pattern generators and local pressure-sensitive receptors that provide information about the surrounding fluid. However, it remains difficult to study experimentally how these sensors influence motor commands in these animals. Here, using a specifically designed robot that captures the essential components of the animal neuromechanical system and using simulations, we tested the hypothesis that sensed hydrodynamic pressure forces can entrain body actuation through local feedback loops. We found evidence that this peripheral mechanism leads to self-organized undulatory swimming by providing intersegmental coordination and body oscillations. Swimming can be redundantly induced by central mechanisms, and we show that, therefore, a combination of both central and peripheral mechanisms offers a higher robustness against neural disruptions than any of them alone, which potentially explains how some vertebrates retain locomotor capabilities after spinal cord lesions. These results broaden our understanding of animal locomotion and expand our knowledge for the design of robust and modular robots that physically interact with the environment.

Freely Behaving Mice Can Brake and Turn During Optogenetic Stimulation of the Mesencephalic Locomotor Region.

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.

Segmental oscillators in axial motor circuits of the salamander: distribution and bursting mechanisms.

The rhythmic and coordinated activation of axial muscles that underlie trunk movements during locomotion are generated by specialized networks in the spinal cord. The operation of these networks has been extensively investigated in limbless swimming vertebrates. But little is known about the architecture and functioning of the axial locomotor networks in limbed vertebrates. We investigated the rhythm-generating capacity of the axial segmental networks in the salamander (Pleurodeles waltlii). We recorded ventral root activity from hemisegments and segments that were surgically isolated from the mid-trunk cord and chemically activated with bath-applied N-methyl-d-aspartate (NMDA). We provide evidence that the rhythmogenic capacity of the axial network is distributed along the mid-trunk spinal cord without an excitability gradient. We demonstrate that the burst generation in a hemisegment depends on glutamatergic excitatory interactions. Reciprocal glycinergic inhibition between opposite hemisegments ensures left-right alternation and lowers the rhythm frequency in segments. Our results further suggest that persistent sodium current contributes to the rhythmic regenerating process both in hemisegments and segments. Burst termination in hemisegments is not achieved through the activation of apamine-sensitive Ca(2+)-activated K(+) channels and burst termination in segments relies on crossed glycinergic inhibition. Together our results indicate that the basic design of the salamander axial network is similar to most of axial networks investigated in other vertebrates, albeit with some significant differences in the cellular mechanism that underlies segmental bursting. This finding supports the view of a phylogenetic conservation of basic building blocks of the axial locomotor network among the vertebrates.

Walking with Salamanders: From Molecules to Biorobotics.

How do four-legged animals adapt their locomotion to the environment? How do central and peripheral mechanisms interact within the spinal cord to produce adaptive locomotion and how is locomotion recovered when spinal circuits are perturbed? Salamanders are the only tetrapods that regenerate voluntary locomotion after full spinal transection. Given their evolutionary position, they provide a unique opportunity to bridge discoveries made in fish and mammalian models. Genetic dissection of salamander neural circuits is becoming feasible with new methods for precise manipulation, elimination, and visualisation of cells. These approaches can be combined with classical tools in neuroscience and with modelling and a robotic environment. We propose that salamanders provide a blueprint of the function, evolution, and regeneration of tetrapod locomotor circuits.