| Abstract: | Reliable methods for coculturing dissociated rat brainstem cells together with dorsal root ganglion (DRG) cells have been established. The cells were characterized using autoradiographic, morphological, immunocytochemical and electrophysiological techniques. Light-level microscopy showed the cocultures to be extensively invaded by serotonin (5-HT)-containing neuronal processes and intense clustering of 5-HT-containing varicosities to occur in the vicinity of large round phase-bright neurones thought to be DRGs. Rather extensive fine ramification of the neuronal processes throughout the culture dish were visualized using scanning electron microscopy. Intracellular recording showed the brainstem neurones to be spontaneously active, electrically excitable and sensitive to a variety of transmitter candidates including serotonin (5-HT) and gamma-aminobutyric acid. Several different responses to 5-HT have been observed. These include a depolarization accompanied by an increase in membrane resistance, a depolarization accompanied by a decrease in membrane resistance and a hyperpolarization accompanied by an increase in membrane resistance. As established by others, DRG cells grown in isolation were always quiescent. The application of 5-HT produced no effect on membrane potential, resistance or excitability. In the brainstem-DRG cocultures 52% of DRG cells exhibited synaptic activity and occasional spontaneous spiking, both of which were abolished in the presence of tetrodotoxin or low-Ca2+/high-Mg2+ solution. Transmission electron microscopy confirmed the presence of synapses on the DRG cells. The spontaneously active DRG cells were also found to be sensitive to the application of 5-HT. Thus it appears that a source of 5-HT nerve terminals may regulate the development and pharmacological sensitivity of primary afferent neurones in culture. |
