The overall goal of this proposal is to increase our understanding of the molecular pathophysiology of nerve injury. The proposed studies are designed to reveal a possible molecular mechanism for ectopic impulse generation on the basis of altered Na+ channel expression in the somatic membrane of chronically injured DRG neurons. A modified patch-clamp method for recording macroscopic currents from mammalian neurons will be used. Na+ currents will be characterized in embryonic, neonatal, adult and axotomized adult DRG neurons. The potential library of Na+ channels which can be expressed on DRG somatic membrane will be determined. Specific objectives: (1) To survey Na+ channels in small, medium, and large DRG neurons in adult rats, to differentiate channel subpopulations as they occur in cells of different sensory modality. (2) To reveal changes in Na+ channel populations in somatic membrane of DRG neurons with age. (3) To reveal the regional distribution of Na+ channel subpopulations in a single DRG neuron. (4) To determine changes in somatic membrane Na+ channel properties in response to chronic injury in adult DRG neurons. Methods: Cultured DRG neurons exised from adult, neonatal, and embryonic rat will be used. In experiments on injured neurons, the sciatic nerve will be ligated 19 to 21 days prior to excision of the ipsilateral lumbar DRGs. Macroscopic Na+ currents will be recorded via patch-clamp of whole cells and of fragments of membrane ('blebs') placed in a customized perfusion/dialysis chamber. Pulse protocols, data acquisition, and analysis will be computer controlled. The 'P/4' method will be applied to minimized leak and capacity currents. Na+ channels will be characterized according to sensitivity to block by tetrodotoxin, steady-state activation and inactivation curves, and kinetic properties. Comparisons of Na+ channel properties will be done among cells of different size, age, and among different portions of membrane (somatic, neuritic, axonal). Properties of Na+ channels obtained from somatic membrane of injured adult neurons will be compared with those of healthy adults and neonates to reveal the subpopulation of channels accessed in response to injury.
| Oyelese, A A; Rizzo, M A; Waxman, S G et al. (1997) Differential effects of NGF and BDNF on axotomy-induced changes in GABA(A)-receptor-mediated conductance and sodium currents in cutaneous afferent neurons. J Neurophysiol 78:31-42 |
| Honmou, O; Utzschneider, D A; Rizzo, M A et al. (1994) Delayed depolarization and slow sodium currents in cutaneous afferents. J Neurophysiol 71:1627-37 |
