Morphological and physiological properties of serotonergic neurons in dissociated cultures from the postnatal rat dorsal raphe nucleus

We have developed dissociated primary cultures of the dorsal raphe nucleus from postnatal 9–12-day-old rats. The nucleus was dissected out from brain slices, dissociated, and cultured over a glial feeder layer. Serotonin immunocytochemistry revealed that 62% of cultured neurons were serotonergic. There was no significant difference in diameters between serotonergic and non-serotonergic neurons. With the whole-cell patch-clamp method, cultured neurons were tested for responses to 8-hydroxydipropylaminotetraline (8-OH-DPAT, a selective agonist for 5-HT1A), and then treated with serotonin immunocytochemistry. Ninety-two percent of neurons responding to 8-OH-DPAT were serotonergic. These results were used to identify serotonergic neurons. In most cases, serotonergic neurons did not show spontaneous firings of action potentials. Constant current depolarizations elicited trains of action potentials that usually did not show marked adaptation. Application of 8-OH-DPAT inhibited action potential firing. The current–voltage relation of the 8-OH-DPAT-induced current indicated an inward rectification with its reversal potential near EK. Serotonergic neurons were depolarized by phenylephrine, bombesin, and gastrin-releasing peptide. This culture system will serve as a useful tool for elucidating the cellular, physiological, and molecular properties of brain serotonergic neurons.