Neural circuitry in the lumbar spinal cord governs two principal features of locomotion, rhythm and pattern, which reflect intra- and interlimb movement. These features are functionally organized into a hierarchy that precisely controls stepping in a stereotypic, speed-dependent fashion. Here, we show that a specific component of the locomotor pattern can be independently manipulated. Silencing spinal L2 interneurons that project to L5 selectively disrupts hindlimb alternation allowing a continuum of walking to hopping to emerge from the otherwise intact network. This perturbation, which is independent of speed and occurs spontaneously with each step, does not disrupt multi-joint movements or forelimb alternation, nor does it translate to a non-weight-bearing locomotor activity. Both the underlying rhythm and the usual relationship between speed and spatiotemporal characteristics of stepping persist. These data illustrate that hindlimb alternation can be manipulated independently from other core features of stepping, revealing a striking freedom in an otherwise precisely controlled system.

Within the cervical and lumbar spinal enlargements, central pattern generating (CPG) circuitry produces the rhythmic output necessary for limb coordination during locomotion. Long propriospinal neurons that inter-connect these CPGs are thought to secure hindlimb-forelimb coordination, ensuring that diagonal limb pairs move synchronously while the ipsilateral limb pairs move out-of-phase during stepping. Here, we show that silencing long ascending propriospinal neurons (LAPNs) that interconnect the lumbar and cervical CPGs disrupts left-right limb coupling of each limb pair in the adult rat during overground locomotion on a high-friction surface. These perturbations occurred independent of the locomotor rhythm, intralimb coordination, and speed-dependent (or any other) principal features of locomotion. Strikingly, the functional consequences of silencing LAPNs are highly context-dependent; the phenotype was not expressed during swimming, treadmill stepping, exploratory locomotion, or walking on an uncoated, slick surface. These data reveal surprising flexibility and context-dependence in the control of interlimb coordination during locomotion.

Long ascending propriospinal neurons (LAPNs) are a subpopulation of spinal cord interneurons that directly connect the lumbar and cervical enlargements. Previously we showed, in uninjured animals, that conditionally silencing LAPNs disrupted left-right coordination of the hindlimbs and forelimbs in a context-dependent manner, demonstrating that LAPNs secure alternation of the fore- and hindlimb pairs during overground stepping. Given the ventrolateral location of LAPN axons in the spinal cord white matter, many likely remain intact following incomplete, contusive, thoracic spinal cord injury (SCI), suggesting a potential role in the recovery of stepping. Thus, we hypothesized that silencing LAPNs after SCI would disrupt recovered locomotion. Instead, we found that silencing spared LAPNs post-SCI improved locomotor function, including paw placement order and timing, and a decrease in the number of dorsal steps. Silencing also restored left-right hindlimb coordination and normalized spatiotemporal features of gait such as stance and swing time. However, hindlimb-forelimb coordination was not restored. These data indicate that the temporal information carried between the spinal enlargements by the spared LAPNs post-SCI is detrimental to recovered hindlimb locomotor function. These findings are an illustration of a post-SCI neuroanatomical-functional paradox and have implications for the development of neuronal- and axonal-protective therapeutic strategies and the clinical study/implementation of neuromodulation strategies.

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Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.