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

Abstract

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.

In vitro longitudinal lumbar spinal cord preparations to study sensory and recurrent motor microcircuits of juvenile mice

“…However it is not suitable for recording recurrent circuits, that are mostly located in the ventral horn. A dorsal horn ablated spinal cord preparation has already been described, and used in neonatal cords to record from V2a, Shox2, and Chx10 interneurons, as well as for fictive locomotion (Bhumbra & Beato, 2018; Dougherty & Kiehn, 2010; Li et al, 2019). Here we report that the dorsal horn-ablated preparation (Fig.…”

Candidate Interneurons Mediating the Resetting of the Locomotor Rhythm by Extensor Group I Afferents in the Cat

“…There are a more limited number of research publications of more direct significance to our present work, and that include the cholinergic V0C interneurons (Zagoraiou et al, 2009), the Hb9 interneurons (Wilson et al 2005 andCaldeira et al 2017), and not least the Shox2 excitatory interneurons described by Dougherty et al (2013). Li et al (2019) have recently investigated whether a group of Shox2-nonV2a neurons (presumed rhythm-generating neurons; see Dougherty et al, 2013) also received short latency excitation from extensor gr I afferents in the neonatal mouse. The work by Li et al (2019) is still not conclusive with regard to the specific short-latency excitation from extensor gr I afferents, but certainly opens for the possibility of a successful analysis at the neonatal stage in the mouse.…”

“…Li et al (2019) have recently investigated whether a group of Shox2-nonV2a neurons (presumed rhythm-generating neurons; see Dougherty et al, 2013) also received short latency excitation from extensor gr I afferents in the neonatal mouse. The work by Li et al (2019) is still not conclusive with regard to the specific short-latency excitation from extensor gr I afferents, but certainly opens for the possibility of a successful analysis at the neonatal stage in the mouse. It also provides a link between a large body of studies in the adult cat and the modern approaches investigating electrophysiology and anatomy of neurons expressing selective transcriptional factors identified and manipulated by genetic tools.…”

Covert actions of epidural stimulation on spinal locomotor circuits

“…ES uses sensory afferent fibers to access spinal locomotor circuitry in an attempt to facilitate motor function after SCI (10,16,32,33,(51)(52)(53)(54)(55). We previously showed that sensory afferent transmission to Shox2 interneurons is both excitatory and inhibitory in uninjured mice (25,56) but changes to mainly excitatory after chronic (>7 weeks) SCI (25).…”