Research reportGenesis of spontaneous rhythmic motor patterns in the lumbosacral spinal cord of neonate mouse
Introduction
Preparations of vertebrate spinal cord isolated in vitro have been developed to investigate both the organisation and localisation of the central pattern generators (CPGs) responsible for locomotion. With such preparations, rhythmic motor patterns that can be considered as `locomotor-like' according to their frequency (0.1–1 Hz) and bilateral alternation were generally obtained by pharmacological activation. Specifically, although spontaneous motor rhythms can be obtained from isolated spinal cord of lower vertebrates 6, 35, CPGs for locomotion have been studied mainly in in vitro spinal cord of lamprey [7]and Xenopus embryo [11]after activation by excitatory amino acids (EAA) agonists. In the same way, for higher vertebrates, locomotor activity has been routinely induced in an in vitro model of newborn rat by application of drugs such as 5HT and EAA agonists 9, 19, 22, 32, dopamine 19, 32or acetylcholine [32]. Locomotor activity with similar characteristics was also evoked in newborn rat preparations by electrical stimulation of brainstem areas [2], somatosensory afferent pathways [32], or the lumbosacral enlargement [18]. To date, the ability of isolated spinal cord from higher vertebrates to elaborate spontaneous activity in vitro has been almost exclusively examined using the chick embryo for developmental studies of motor activity 3, 17, 28. Only episodic spontaneous activity has been reported in some newborn rat in vitro preparations [31]and more recently, mouse lumbosacral explants have also been shown to be capable of generating spontaneous episodes of motor activity that were recorded from hindlimb muscles 16, 34.
The major role of EAA receptors in the genesis of fictive locomotion in higher vertebrates has clearly derived from the studies mentioned above and was later confirmed by using EAA antagonists both in vitro 3, 9and in vivo 13, 14. In parallel, decreasing the spinal extracellular concentration of Mg2+ was shown to be able to enhance the activation of locomotion by EAA in vertebrates [25].
In the present study, the characterisation of both spontaneous and Mg2+-free-induced activities is proposed to reflect the spinal capabilities of motor rhythm generation in the mouse. A major advantage of the mouse is that future studies can exploit transgenic animals to study the operation of locomotor networks. In addition, this study constitutes a first step in the pursuit of a simpler mammalian preparation that is still capable of generating motor rhythms in vitro (see Ref. [4]).
Section snippets
Reduced spinal cord preparations
Experiments were performed on neonatal mice (0 to 5-day-old). Timed pregnant mice were obtained from our laboratory's breeding centre. All animals were deeply anaesthetised with ether. In some experiments, it was first verified that rhythmic motor patterns persisted at the lumbosacral level after thoracic transection removing the cervical enlargement. The posterior vertebral column was then systematically excised from T5–T7. The tissue was placed in a 25-ml recording chamber where the caudal
Results
In 32 preparations of neonatal mouse, continuous rhythmic motor patterns were obtained on contralateral ventral roots either spontaneously under normal superfusion medium or by using Mg2+-free medium.
Once motor activity was recorded on lumbar ventral roots, the rostrocaudal extension of the outputs of this rhythmic activity was systematically monitored on some ventral roots from T7. This revealed that rhythmic motor patterns could occur in lumbar and sacral roots (L1 to S3) but was never
Discussion
In the present study performed on preparations of neonatal mouse spinal cord isolated in vitro, the monitoring of rostrocaudal extension of both spontaneous and Mg2+-free-induced motor outputs enabled us to characterise two different rhythmic motor patterns at two developmental periods, P0–2 and P3–5. The effect of Mg2+-free medium, which is known to be mediated via NMDA receptors, and the effect of antagonists of both NMDA and non-NMDA receptors provide strong evidence for the involvement of
Acknowledgements
We would like to thank Michel Chaigniau, Jean-Louis Salat et Coralie Reclus for their thorough technical assistance, and Novo Nordisk Laboratory (Denmark) for providing NBQX. We are also grateful to Drs. Michael O'Donovan, John Simmers and Thomas Durkin for valuable comments on the manuscript and for English revision. This study was supported by the Université Bordeaux I and the CNRS, and it has also benefited of financial support from the `Institut pour la Recherche sur la Moelle Epinière'
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