Membrane properties of identified mesencephalic dopamine neurons in primary dissociated cell culture.

Abstract

Dopamine (DA)-containing neurons in primary dissociated cell cultures derived from the embryonic mouse mesencephalon (day E13) were studied by histochemical and electrophysiological techniques. DA neurons exhibited two distinct morphologies, fusiform and multipolar, tended to reside in groups and organize dendrites into common fascicles. While these neurons expressed the cell-surface marker acetylcholinesterase, the presence of this enzyme could not be used to identify DA neurons unequivocally, since it was also observed in nondopaminergic cells. Neurons were therefore identified as DA by their distinct morphology, and this identification was validated with a double-labeling procedure that entailed the intracellular deposition of a fluorescent dye (Lucifer yellow or ethidium bromide), followed by processing for tyrosine hydroxylase immunocytochemistry. DA neurons identified in this manner were observed to have resting membrane potentials between -50 and -75 mV, input resistances of 50-360 M omega, and membrane time constants of 4.1-14.1 msec. Forty-seven percent of these cells displayed spontaneous activity that was irregular in nature and often contained bursts (burst length was between two and six action potentials). The DA neurons displayed a variety of ionic conductances, including (1) a Na+ conductance (gNa) that underlies the action potential, (2) Ca2+ conductances (gCa) that mediate the nonsomatic low- and high-threshold spikes observed, and (3) at least three K+ conductances (gK). Voltage-clamp analysis revealed several distinct transmembrane ionic currents, including (1) a large, rapidly inactivating tetrodotoxin-sensitive inward Na+ current (INa), (2) a 4-aminopyridine-sensitive, transient early outward K+ current that required a conditioning hyperpolarization of the membrane to be activated by a subsequent depolarization (A-current, IA), (3) a slowly developing inward current that was seen only after a conditioning hyperpolarization of the membrane and that was dependent on the presence of external Ca2+ ions (ICa), and (4) a late-onset, noninactivating K+ current. Between 25% and 54% of the late-onset K+ current was Ca(2+)-dependent and was not affected by tetraethylammonium ions. This current was termed IAHP. The remaining current was not sensitive to changes in the extracellular Ca2+ concentration but was blocked by external tetraethylammonium. This current was termed IK. The direct pressure application of DA (1-200 microM) onto the soma dose-dependently hyperpolarized these neurons; this effect was potentiated by the presence of the catecholamine reuptake blocker cocaine hydrochloride (10-200 microM). Under voltage-clamp conditions, DA was observed to increase IK significantly and had little effect on IAHP.(ABSTRACT TRUNCATED AT 400 WORDS)