Molecular mechanisms of exocytotic events in single ventral midbrain dopaminergic neurons : amprometric detection and characterization

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Dopaminergic neurons were enzymatically dissociated from slices of ventral midbrain from postnatal rat pups (first 2 days after birth). The neurons were plated onto glial monolayers and maintained in culture for several weeks. Dopaminergic phenotype was confirmed by the immunocytochemistry of tyrosine hydroxylase. After 2-6 weeks in culture, dopaminergic neurons had cell bodies 20-40μm in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 3-5 pm in diameter. Amperometric detection of single exocytotic events was monitored with a carbon fiber microelectrode (5μm diameter). The applied voltage was +700mV versus the reference electrode. To characterize a single exocytotic event that is detected as a single current spike, the spike parameters (rise time, middle width and area) were analyzed. Exposure of the cells to 90 mM KC1 (1000 hps, 30 sec) evoked exocytotic events with distinctive kinetics. The values of rise time and middle width in dopaminergic neurons were approx. 10-and 20-fold less than those observed in cultured adrenal chromaffin cells, suggesting much faster exocytotic events in the neurons than adrenal chromaffin cells.