The long-term goal of this research is to understand the developmental expression of the neuronal genome. The proposal focuses on the development of ion channels in motoneurons. motoneurons were selected for examination because (1) They represent a relatively homogeneous population which, with recent technical advances, can be experimentally identified after enzymatic dissociation, (2) The timing of important events in their in vivo developmental history has been established, and (3) Mutations have been identified that profoundly alter neuromuscular development. One project aims to characterize ionic currents in motoneurons from animals at important in vivo developmental stages: prior to the period of motoneuron death, immediately after this period during which half to two-thirds of these cells die, before and after the period of synapse elimination when about half the neuromuscular contacts made by a motoneuron are withdrawn, and after the postnatal maturation of the neuromuscular system.. after retrograde labeling by injection of dye into hindlimbs, identified motoneurons will be isolated by ezymatic dissociation of the spinal cords of both chicks and mice. ionic currents will be measured with the whole-cell patch clamp technique. The developmental expression of motoneuronal ion channels will be further dissected using mutants: crooked neck dwarf chicks and muscular dysgenic mice both suffer a fatal mutation that abolishes skeletal muscle contraction and largely eliminates motoneuronal death. The experiments will determine whether motoneurons in these animals retain the electrophysiological features displayed by motoneurons before cell death in normal animals. A pharmacological approach, that complements this genetic approach, will be to characterize ion channel development in chicks paralyzed by in ovo injections of curare, which others have shown largely abolishes motoneuron death. Another mutation that will be experimentally exploited is motor endplate disease (med) in mice, a fatal mutation that causes the failure of evoked transmitter release at the motor endplate. The experiments will test the hypothesis that a defect in a motoneuronal calcium current is responsible for the pathophysiology of this disease. The in vitro development of motoneuron ion channels will be determined by measuring ionic currents at varying times after plating motoneurons in culture. The effects of motoneuron growth factors on ionic currents will be characterized since this will help provide a more mechanistic understanding for the action of these growth factors.
