N-methyl-D-aspartate (NMDA) produces burst firing in dopamine neurons in vitro, and the hyperpolarization that occurs between bursts requires activation of an electrogenic sodium pump. Furthermore, pump current may help protect dopamine neurons against toxic effects of excitatory amino acids. Whole-cell pipettes and microelectodes will be used to study pump current in dopamine neurons in the brain slice. Outward pump current will be evoked by dialyzing the contents of whole-cell pipettes with solution containing raised concentrations of sodium (20-80 mM). the magnitude of current capable of being generated by the sodium pump will be established, and it's dependence upon voltage, intracellular sodium, and extracellular potassium will be characterized. Strophanthidin (1 mM), a cardiac glycoside which inhibits Na+/K+ ATPase, will be superfused to confirm that the outward current is produced by a pump.Dopamine, which converts burst-firing to single-spike firing, will be investigated for a possible inhibitory effect on pump current. Isoproterenol, carbachol, and substance P will also be studied, because other investigators have suggested that substances which alter adenylate cyclase activity (isoproterenol) or phosphoinositide turnover (carbachol and substance P) may regulate activity of Na+/K+ ATPase. In other experiments, microelectrodes or whole-cell pipettes will be used to record pump current which will be evoked by superfusing the slice with NMDA (20 uM).Toxic effects of NMDA, which occur at 50-100 uM, will be quantified by measuring an inward current which does not recover upon washing NMDA. It is predicted that dialyzing the cell contents with zero sodium or calcium will protect cells from this persistent inward current. Furthermore, strophanthidin (1 uM) may shift the NMDA concentration- toxicity curve to the left, and this may be prevented by removing Ca2+ from the superfusate. Finally, digoxin will be injected systemically into mice to test its ability to potentiate depletion of striatal dopamine by 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP). This is the expected result because the neurotoxic effect of MPTP may be mediated by a reduction in the ability of dopamine cells to generate ATP needed for fueling the sodium pump. Results of this study may have important implications for the treatment of patients with Parkinson's disease.
