EXCITOTOXICITY AND SODIUM PUMP IN DOPAMINE NEURONS

DESCRIPTION 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.