Calcium currents and their modulation by 5-HT were studied using both whole-cell and single-channel patch-clamp techniques in acutely isolated adult rat dorsal raphe neurons. Evidence for three types of Ca channels (T, N, L) was obtained in both whole-cell and single-channel experiments. Approximately 4% of the total high-threshold Ca current (L-type) was sensitive to dihydropyridines (DHPs) while approximately 40% of the Ca current (N-type) was sensitive to omega-conotoxin (omega-CgTx). About 56% of the whole-cell current was insensitive to either DHPs or omega-CgTx and may thus represent a different kind of Ca current. 5-HT reduced raphe neuron Ca currents by approximately 50%, while slowing activation. 5-HT inhibited both omega-CgTx-sensitive and -insensitive Ca current. Inhibition by 5-HT was voltage dependent; prepulses to +80 mV lasting for 20 msec almost completely abolished the 5-HT-mediated inhibition. The voltage dependence of the response to 5-HT suggested that trains of action potentials might overcome the inhibition due to 5-HT. Trains of brief depolarizations were used to simulate action potentials; only about 5% of the 5-HT-induced inhibition was relieved by the trains. These results suggest that while large depolarizations could restore the Ca current inhibited by 5-HT, physiological stimuli, such as trains of action potentials, could not. The action of 5-HT was made irreversible by inclusion of GTP-gamma-S in the patch pipette, suggesting a G-protein mediation of the response to 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARY1. An inwardly rectifying K+ current activated by serotonin (5-HT) was recorded from acutely isolated adult dorsal raphe (DR) neurones using the whole-cell recording mode of the patch clamp technique.

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Previous studies have indicated that stimulation of neuronal inhibitory receptors, such as the serotonin 1A receptor (5-HT 1A -R), could cause attenuation of the activity of both N-type Ca 2ϩ channels and Nmethyl-D-aspartic acid receptors, thus resulting in protection of neurons against excitotoxicity. The purpose of this study was to investigate if the 5-HT 1A -R is also coupled to an alternative pathway that culminates in suppression of apoptosis even in cells that are deficient in Ca 2ϩ channels. Using a hippocampal neuron-derived cell line (HN2-5) that is Ca 2ϩ channel-deficient, we demonstrate here that an alternative pathway is responsible for 5-HT 1A -R-mediated protection of these cells from anoxia-triggered apoptosis, assessed by deoxynucleotidyltransferase-mediated dUTP nick end-labeling ( TUNEL). The 5-HT 1A -R agonist-evoked protection was eliminated in the presence of pertussis toxin and also required phosphorylation-mediated activation of mitogen-activated protein kinase (MAPK), as evidenced by the elimination of the agonist-elicited rescue of neuronal cells by the MAPK kinase inhibitor PD98059 but not by the phosphatidylinositol 3-kinase (PI-3K) inhibitor wortmannin. Furthermore, agonist stimulation of the 5-HT 1A -R caused a 60% inhibition of anoxia-stimulated caspase 3-like activity in the HN2-5 cells, and this inhibition was abrogated by PD98059 but not by wortmannin. Although agonist stimulation of the 5-HT 1A -R caused an activation of PI-3K␥ in HN2-5 cells, our results showed that this PI-3K␥ activity was not linked to the 5-HT 1A -R-promoted regulation of caspase activity and suppression of apoptosis. Thus, in the neuronal HN2-5 cells, agonist binding to the 5-HT 1A -R results in MAPK-mediated inhibition of a caspase 3-like enzyme and a 60 -70% suppression of anoxia-induced apoptosis through a Ca 2ϩ channel-independent pathway. Key Words: Serotonin 5-HT 1A receptor-Neuroprotection-Mitogen-activated protein kinaseAnoxia-Apoptosis.