Effects of Serotonin on Caudal Raphe Neurons: Inhibition of N- and P/Q-Type Calcium Channels and the Afterhyperpolarization

Abstract

Bayliss, Douglas A., Yu-Wen Li, and Edmund M. Talley. Effects of serotonin on caudal raphe neurons: inhibition of N- and P/Q-type calcium channels and the afterhyperpolarization. J. Neurophysiol. 77: 1362–1374, 1997. We characterized whole cell barium currents through calcium channels and investigated the effects of serotonin (5-HT) on calcium channel currents and firing behavior in visualized caudal raphe neurons of the neonatal rat in brain stem slices (n = 201). A subpopulation of recorded neurons was recovered after staining for tryptophan hydroxylase (TPH), the 5-HT synthesizing enzyme (n = 21); of those cells, 86% were TPH immunoreactive, suggesting that the majority of recorded neurons was serotonergic. Calcium channel currents began to activate at about −40 mV in caudal raphe neurons and showed a peak amplitude of 952.2 ± 144.2 (SE) pA at −10 mV. A small low-voltage-activated current was also observed (∼22 pA). Calcium channel currents were potently inhibited by bath-applied 5-HT in most cells tested (∼90%). The EC₅₀ for inhibition of calcium current by 5-HT was 0.1 μM; a saturating concentration (1.0 μM) blocked ∼49% of the current evoked at 0 mV from a holding potential of −70 mV (n = 101). Current inhibition was associated with a slowing of activation kinetics and a shift in the peak of the current-voltage relationship, and was partially relieved by strong depolarizations. Current inhibition by 5-HT was mimicked by 8-OH-DPAT, a specific 5-HT_1A agonist, and blocked by the 5-HT_1A antagonists NAN 190 and (+)WAY 100135, but was unaffected by ketanserin, a 5-HT_2A/C antagonist. ω-Conotoxin GVIA (ω-CgTx)-sensitive N-type channels and ω-agatoxin IVA (ω-AgaIVA)-sensitive P/Q-type channels together accounted for most of the calcium current (36 and 37%, respectively). Nimodipine had no effect on calcium current, indicating that caudal raphe neurons do not express dihydropyridine-sensitive L-type currents. A substantial residual current (27%) remained after application of ω-CgTx, ω-AgaIVA, and nimodipine. Most of the 5-HT-sensitive calcium current was blocked by ω-CgTx and ω-AgaIVA; 5-HT had little effect on the residual current. Inhibition of calcium current by 5-HT was irreversible when GTPγS, a nonhydrolyzable guanosine 5′-triphosphate (GTP) analogue, was substituted for GTP in the pipette. In addition, the effects of 5-HT were blocked by pretreating slices with pertussis toxin (PTX). Together these data indicate that inhibition of N- and P/Q-type calcium current in serotonergic caudal raphe neurons is mediated by a 5-HT_1A receptor via PTX-sensitive G proteins. Under current clamp, calcium channel toxins (ω-CgTx and ω-AgaIVA) and 5-HT each caused a decrease in the spike afterhyperpolarization and enhanced the repetitive firing response to injected current. The similar effects of 5-HT and the calcium channel toxins on firing behavior suggest that those effects of 5-HT were secondary to inhibition of N- and P/Q-type calcium channels.