Midbrain dopaminergic neurons from postnatal rat in long-term primary culture

Midbrain dopamine neurons project extensively throughout the vertebrate forebrain and influence a wide variety of brain functions. These neurons, which are believed to form a major brain reward system, are involved in initiation and control of motor programs, addictive behaviors, and determination of mood. Given their critical role in behavioral function, relatively little is known about their fundamental cellular physiological and pharmacological properties. A long-term dissociated culture system for postnatal rat dopamine neurons was developed to permit both acute and chronic studies of these cells. Dopamine neurons were dissociated from slices of ventral midbrain from neonatal rat pups and maintained in cell culture for several months. The dopaminergic phenotype was confirmed by catecholamine fluorescence and by tyrosine hydroxylase immunocytochemistry. After four weeks in culture, dopamine neurons had cell bodies 10-40 microns in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 0.5-2 microns in diameter. Electrophysiological recordings were made from 71 dopamine neurons identified by 5,7-dihydroxytryptamine fluorescence after six to 67 days in culture. The neurons had resting potentials of -51 +/- 5 mV, broad action potentials with durations of 2.9 +/- 1.3 ms, and the majority of the neurons (65%) displayed anomalous rectification. Most dopamine neurons in culture fired spontaneously in a pacemaker-like manner with a frequency of 2.3 +/- 1.3 Hz, or in a bursting pattern, typically having two to seven action potentials per burst. All neurons tested had glutamate and gamma-aminobutyric acid receptors, and 90% of neurons responded to dopamine or quinpirole with inhibition of firing, suggesting the presence of dopamine autoreceptors. Some neurons were inhibited by concentrations of quinpirole as low as 10 nM. The results show that midbrain dopamine neurons can be maintained in dissociated cell culture for periods of several months. These neurons can be identified prior to electrophysiological recording, and they express many of the physiological characteristics that have been reported for midbrain dopamine neurons in vivo.