Using an upright preparation to identify and characterize locomotor related neurons across the transverse plane of the neonatal mouse spinal cord

Abstract

Background: The basic rhythmicity underlying stepping in mammals is generated by a neural network, situated in the spinal cord, known as the locomotor central pattern generator (CPG). While a molecular approach has provided information regarding neuronal populations that participate in locomotor activity and their specific function, the distributed nature of the locomotor CPG has made it difficult to identify and characterize the specific neurons belonging to each population that are rhythmically-active during stepping.

New method: We describe a preparation in which we isolate the spinal cord from a neonatal mouse, section it at a lumbar segment, situate it in an upright orientation under the objective lens of a 2- photon microscope, and evoke fictive locomotion.

Results: This preparation allows us to image rhythmic Ca²⁺ oscillations in spinal neurons, and visually identify those that are involved in fictive locomotor activity. We can then characterize unique features of these neurons.

Comparison with existing methods: This builds on existing fictive locomotor preparations and is the first which allows for the visual identification of locomotor related neurons spanning the transverse plane of the spinal cord, facilitating their electrophysiological and anatomical characterization CONCLUSIONS: This approach promises to provide new information regarding the distribution of the locomotor CPG in the transverse plane, the characteristics of its component interneurons, as well as the cellular mechanisms and network properties which underlie rhythm generation. By altering the location of Ca²⁺ indicator application it can also be used to identify and characterize neurons involved in other facets of sensorimotor processing.

Keywords: CPG; Ca2+ imaging; Locomotion; Morphology; Whole Cell recording.