An In Vitro mammalian model for investigating locomotor pattern generation

The goal of our research was to establish an in vitro mammalian spinal preparation suitable for investigating the mechanisms underlying motor pattern generation. The objective of the study was to compare the step cycle timing of intact, freely moving neonatal and adult Balb/C mice with the pattern generating capability of spinal cord-hindlimb explants taken from animals aged from birth to 4 days. The analysis was based on EMG recordings from the gastrocnemius (G) and tibialis anterior (TA) muscles, an antagonist set of muscles used in stepping. In intact animals, electrodes were placed in the G and TA muscles to ascertain the step cycle pattern and timing. Adult mice displayed reciprocal activation of the G and TA muscles and a faster cycle periods than the neonates. The neonates also exhibited reciprocal activation of the muscles, as well as asymmetrical activation and synchronous bursting.

For the in vitro preparation, neonatal mice were anesthetized, electrodes were positioned into the G and TA muscles, and the spinal cord with the hindlimbs attached were surgically removed and perfused with ACSF. The spinal cord-hindlimb explant was investigated under 3 types of conditions: (1) nonhemisected spinal cord with Mg$\backslash sp++$ present in the ACSF; (2) nonhemisected spinal cord without Mg$\backslash sp++$ present in the ACSF; (3) hemisected spinal cord without Mg$\backslash sp++$ in the ACSF. The pattern generating capability was first investigated spontaneously, and afterwards upon pharmacological stimulation with NMDA (an excitatory amino acid which is inhibited by Mg$\backslash sp++$). Spontaneous bursting activity rarely occurred. In contrast, NMDA increased the occurrence of rhythmicity, especially in the absence of Mg$\backslash sp++$. When rhythmic activity occurred spontaneously or was evoked by NMDA, rhythm was usually present in one muscle and faster step cycles were exhibited than in the intact neonate. There was a higher incidence of rhythmicity in the day 2 nonhemisected Mg$\backslash sp++$ free explant. This preparation, with pharmacological stimulation, is useful for further investigation of motor pattern generation.