All forms of locomotion are repetitive motor activities that require coordinated bilateral activation of muscles. The executive elements of locomotor control are networks of spinal neurons that determine gait pattern through the sequential activation of motor-neuron pools on either side of the body axis. However, little is known about the constraints that link left-right coordination to locomotor speed. Recent advances have indicated that both excitatory and inhibitory commissural neurons may be involved in left-right coordination. But the neural underpinnings of this, and a possible causal link between these different groups of commissural neurons and left-right alternation, are lacking. Here we show, using intersectional mouse genetics, that ablation of a group of transcriptionally defined commissural neurons--the V0 population--leads to a quadrupedal hopping at all frequencies of locomotion. The selective ablation of inhibitory V0 neurons leads to a lack of left-right pattern at low frequencies, mixed coordination at medium frequencies, and alternation at high locomotor frequencies. When ablation is targeted to excitatory V0 neurons, left-right alternation is present at low frequencies, and hopping is restricted to medium and high locomotor frequencies. Therefore, the intrinsic logic of the central control of locomotion incorporates a modular organization, with two subgroups of V0 neurons required for the existence of left-right alternating modes at different speeds of locomotion. The two molecularly distinct sets of commissural neurons may constrain species-related naturally occurring frequency-dependent coordination and be involved in the evolution of different gaits.

The dorsal horn of the spinal cord is critical to processing distinct modalities of noxious and innocuous sensation, but little is known of the neuronal subtypes involved, hampering efforts to deduce principles governing somatic sensation. Here we used single-cell RNA sequencing to classify sensory neurons in the mouse dorsal horn. We identified 15 inhibitory and 15 excitatory molecular subtypes of neurons, equaling the complexity in cerebral cortex. Validating our classification scheme in vivo and matching cell types to anatomy of the dorsal horn by spatial transcriptomics reveals laminar enrichment for each of the cell types. Neuron types, when combined, define a multilayered organization with like neurons layered together. Employing our scheme, we find that heat and cold stimuli activate discrete sets of both excitatory and inhibitory neuron types. This work provides a systematic and comprehensive molecular classification of spinal cord sensory neurons, enabling functional interrogation of sensory processing.

Neural networks in the spinal cord known as central pattern generators produce the sequential activation of muscles needed for locomotion. The overall locomotor network architectures in limbed vertebrates have been much debated, and no consensus exists as to how they are structured. Here, we use optogenetics to dissect the excitatory and inhibitory neuronal populations and probe the organization of the mammalian central pattern generator. We find that locomotor-like rhythmic bursting can be induced unilaterally or independently in flexor or extensor networks. Furthermore, we show that individual flexor motor neuron pools can be recruited into bursting without any activity in other nearby flexor motor neuron pools. Our experiments differentiate among several proposed models for rhythm generation in the vertebrates and show that the basic structure underlying the locomotor network has a distributed organization with many intrinsically rhythmogenic modules.channelrhodopsin-2 | halorhodopsin | motor neurons | interneurons S pinal cord networks that drive and coordinate walking are, to a large extent, innate. Even in species that do not walk at birth, such as rodents and humans, the networks that generate walking are already present (1, 2). The early development of functional locomotor networks has been exploited and studied in the neonatal rodent spinal cord in vitro preparation in which locomotor-like activity can be induced pharmacologically or by electrical activation of descending or afferent fibers (3-6). The mammalian locomotor networks have also been studied by using the adult cat where locomotor-like activity similarly can be induced pharmacologically or electrically (7-9). A general notion from these studies is that forelimb and hindlimb locomotion are controlled by independent limb-controlling circuits (10) and that rhythm-generating excitatory neurons, and pattern-generating neurons, interact to produce the coordinated motor output (11)(12)(13)(14). The network layout within these limb-controlling circuits has, however, been debated. A number of conceptual models have been advanced. The classical half-center model asserts that flexor and extensor bursting is generated by two reciprocally coupled half-centers driving all flexors and extensors (8, 15). The flexor burst generator model is asymmetric and consists of a flexor burst generator that provides active excitation of flexor motor neurons and inhibition of extensor motor neurons that are otherwise tonically active (14,16,17). In response to evidence that the central pattern generator (CPG) could produce a more complex motor output than just a mere flexor-extensor alternation (9), the unit burst generator (UBG) model was proposed (18). According to this theory, separate modules can generate a rhythm in close muscle synergies and are distributed around each joint (18,19), or in the swimming network in each hemisegment (20). The UBGs therefore generate a local rhythmic activity that during locomotion will be recruited so that they form an interconnected n...

Central pattern generators (CPGs) are spinal neuronal networks required for locomotion. Glutamatergic neurons have been implicated as being important for intrinsic rhythm generation in the CPG and for the command signal for initiating locomotion, although this has not been demonstrated directly. We used a newly generated vesicular glutamate transporter 2-channelrhodopsin2-yellow fluorescent protein (Vglut2-ChR2-YFP) mouse to directly examine the functional role of glutamatergic neurons in rhythm generation and initiation of locomotion. This mouse line expressed ChR2-YFP in the spinal cord and hindbrain. ChR2-YFP was reliably expressed in Vglut2-positive cells and YFP-expressing cells could be activated by light. Photo-stimulation of either the lumbar spinal cord or the caudal hindbrain was sufficient to both initiate and maintain locomotor-like activity. Our results indicate that glutamatergic neurons in the spinal cord are critical for initiating or maintaining the rhythm and that activation of hindbrain areas containing the locomotor command regions is sufficient to directly activate the spinal locomotor network.

SHP (NROB2) is an atypical orphan nuclear receptor that lacks a DNA-binding domain but contains a putative ligand-binding domain. Previous studies have revealed that SHP interacts with a variety of nuclear receptors and inhibits their transcriptional activity, thereby acting as a corepressor. In this report we identify the glucocorticoid receptor (GR) as a novel downstream target receptor for SHP inhibition. SHP potently inhibits dexamethasone-induced transcriptional GR activity in mammalian cells, and the inhibition involves a functional second NR-box within SHP. Interestingly, this motif shows a high homology with the NR-box in the glucocorticoid and cAMP-inducible GR coactivator PGC-1, indicating similar binding specificity and shared target receptors. We show that SHP antagonizes PGC-1 coactivation and, in addition, we identify the PGC- 1-regulated phospho(enol)pyruvate carboxykinase (PEPCK) promoter as a novel target promoter for SHP inhibition. This implies a physiologically relevant role for SHP in modulating hepatic glucocorticoid action. Furthermore, when coexpressing green fluorescent protein-tagged GR together with SHP, an intranuclear redistribution of GR was observed. As inhibition-deficient SHP mutants were unable to induce this redistribution, intranuclear tethering of target receptors may represent yet another, previously uncovered, aspect of SHP inhibition.