Identification of adult spinal Shox2 neuronal subpopulations based on unbiased computational clustering of electrophysiological properties.

Spinal cord neurons integrate sensory and descending information to produce motor output. The expression of transcription factors has been used to dissect out the neuronal components of circuits underlying behaviors. However, most of the canonical populations of interneurons are heterogeneous and require additional criteria to determine functional subpopulations. Neurons expressing the transcription factor Shox2 can be subclassified based on the co-expression of the transcription factor Chx10 and each subpopulation is proposed to have a distinct connectivity and different role in locomotion. Adult Shox2 neurons have recently been shown to be diverse based on their firing properties. Here, in order to subclassify adult mouse Shox2 neurons, we performed multiple analyses of data collected from whole-cell patch clamp recordings of visually-identified Shox2 neurons from lumbar spinal slices. A smaller set of Chx10 neurons was included in the analyses for validation. We performed k-means and hierarchical unbiased clustering approaches, considering electrophysiological variables. Unlike the categorizations by firing type, the clusters displayed electrophysiological properties that could differentiate between clusters of Shox2 neurons. The presence of clusters consisting exclusively of Shox2 neurons in both clustering techniques suggests that it is possible to distinguish Shox2+Chx10- neurons from Shox2+Chx10+ neurons by electrophysiological properties alone. Computational clusters were further validated by immunohistochemistry with accuracy in a small subset of neurons. Thus, unbiased cluster analysis using electrophysiological properties is a tool that can enhance current interneuronal subclassifications and can complement groupings based on transcription factor and molecular expression.

Spinal cord injury alters spinal Shox2 interneurons by enhancing excitatory synaptic input and serotonergic modulation while maintaining intrinsic properties in mouse.

Neural circuitry generating locomotor rhythm and pattern is located in the spinal cord. Most spinal cord injuries (SCI) occur above the level of spinal locomotor neurons; therefore, these circuits are a target for improving motor function after SCI. Despite being relatively intact below the injury, locomotor circuitry undergoes substantial plasticity with the loss of descending control. Information regarding cell-type specific plasticity within locomotor circuits is limited. Shox2 interneurons (INs) have been linked to locomotor rhythm generation and patterning, making them a potential therapeutic target for the restoration of locomotion after SCI. The goal of the present study was to identify SCI-induced plasticity at the level of Shox2 INs in a complete thoracic transection model in adult male and female mice. Whole cell patch clamp recordings of Shox2 INs revealed minimal changes in intrinsic excitability properties after SCI. However, afferent stimulation resulted in mixed excitatory and inhibitory input to Shox2 INs in uninjured mice which became predominantly excitatory after SCI. Shox2 INs were differentially modulated by serotonin (5-HT) in a concentration-dependent manner in uninjured conditions but following SCI, 5-HT predominantly depolarized Shox2 INs. 5-HT7 receptors mediated excitatory effects on Shox2 INs from both uninjured and SCI mice, but activation of 5-HT2B/2C receptors enhanced excitability of Shox2 INs only after SCI. Overall, SCI alters sensory afferent input pathways to Shox2 INs and 5-HT modulation of Shox2 INs to enhance excitatory responses. Our findings provide relevant information regarding the locomotor circuitry response to SCI that could benefit strategies to improve locomotion after SCI.SIGNIFICANCE STATEMENTCurrent therapies to gain locomotor control after SCI target spinal locomotor circuitry. Improvements in therapeutic strategies will require a better understanding of the SCI-induced plasticity within specific locomotor elements and their controllers, including sensory afferents and serotonergic modulation. Here, we demonstrate that excitability and intrinsic properties of Shox2 interneurons, which contribute to the generation of the locomotor rhythm and pattering, remain intact after SCI. However, SCI induces plasticity in both sensory afferent pathways and serotonergic modulation, enhancing the activation and excitation of Shox2 interneurons. Our findings will impact future strategies looking to harness these changes with the ultimate goal of restoring functional locomotion after SCI.

Flexor and Extensor Ankle Afferents Broadly Innervate Locomotor Spinal Shox2 Neurons and Induce Similar Effects in Neonatal Mice.

Central pattern generators (CPGs) in the thoracolumbar spinal cord generate the basic hindlimb locomotor pattern. The locomotor CPG integrates descending commands and sensory information from the periphery to activate, modulate and halt the rhythmic program. General CPG function and response to sensory perturbations are well described in cat and rat models. In mouse, roles for many genetically identified spinal interneurons have been inferred from locomotor alterations following population deletion or modulation. However, the organization of afferent input to specific genetically identified populations of spinal CPG interneurons in mouse remains comparatively less resolved. Here, we focused on a population of CPG neurons marked by the transcription factor Shox2. To directly test integration of afferent signaling by Shox2 neurons, sensory afferents were stimulated during patch clamp recordings of Shox2 neurons in isolated spinal cord preparations from neonatal mice. Shox2 neurons broadly displayed afferent-evoked currents at multiple segmental levels, particularly from caudal dorsal roots innervating distal hindlimb joints. As dorsal root stimulation may activate both flexor- and extensor-related afferents, preparations preserving peripheral nerves were used to provide more specific activation of ankle afferents. We found that both flexor- and extensor-related afferent stimulation were likely to evoke similar currents in a given Shox2 neuron, as assessed by response polarity, latency, duration and amplitude. It has been proposed that Shox2 neurons can be divided into neurons which contribute to rhythm generation and neurons that are premotor by the absence and presence of the V2a marker Chx10, respectively. Response to afferent stimulation did not differ based on Chx10 expression. Although currents evoked in response to flexor and extensor afferent activation did not follow expected functional antagonism, they were consistent with the observation that stimulation of flexor- and extensor-related afferents both reset the phase of ongoing fictive locomotion to flexion in neonatal mice. Together, the data suggest that Shox2 neurons are interposed in multiple sensory pathways and low threshold proprioceptive input reinforces sensory perturbation of ongoing locomotion by similarly activating or inhibiting both the rhythm and patterning layers of the CPG.