The objectives of this proposal are to characterize the changes in electrical and pharmacological properties of spinal motoneurons during embryonic development and after injury. We will determine: 1) the influence of newly formed central and peripheral synapses on motoneuron properties, and 2) the effects of sensory deprivation and axotomy on motoneuron differentiation. These studies will be carried out using two preparations: 1) isolated lumbar segments of rat spinal cord, in which functional sensorimotor pathways are preserved for at least 9 hours, and 2) spinal cord-muscle explants in which motoneurons are viable for 4-6 days. The isolated spinal cord of rat embryos is particularly suitable for studying neural and synaptic responses to known concentrations of ions and drugs. We have been using this preparation to study the pattern and time course of formation of excitatory and inhibitory synapses in spinal cord, and to examine the effects of the new synapses on motoneuron properties. The results of the proposed studies will establish the pattern of motoneuron differentiation in vivo, and will guide our future studies on motoneuron development in organ culture. Isolated segments of thoracic spinal cord with their associated intercostal muscles will be maintained in vitro for a period of several days. The advantage of using spinal cord-intercostal muscle explants are: 1) motoneurons are cultured in continuity with the muscles they normally innervate in vivo, and 2) we have previously described the chronology of changes that occur during development of intercostal neuromuscular junctions in vivo and in organ culture. We will thus be able to correlate these changes with differentiation of motoneurons that innervate these muscles. Maintaining these explants in a controlled environment provides valuable means for determining the extracellular factors that influence motoneuron properties and the formation of specific central neural pathways. Acquisition of motoneuron specific characteristics arises from interactions between intrinsic instructions and extracellular factors, but little is known about their relative contributions to differentiation of mammalian neurons. The proposed studies will extend our understanding about the way functional neural circuits develop, and the effects of newly formed pathways on neuron differentiation. This understanding should provide insight into mechanisms underlying long-term interactions between neurons in the central nervous system.
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