Bayliss D A, Li Y W, Talley E M
Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.
J Neurophysiol. 1997 Mar;77(3):1362-74. doi: 10.1152/jn.1997.77.3.1362.
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 (approximately 22 pA). Calcium channel currents were potently inhibited by bath-applied 5-HT in most cells tested (approximately 90%). The EC50 for inhibition of calcium current by 5-HT was 0.1 microM; a saturating concentration (1.0 microM) blocked approximately 40% 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-HT1A agonist, and blocked by the 5-HT1a antagonists NAN 190 and (+) WAY 100135, but was unaffected by ketanserin, a 5-HT2A/C antagonist. omega-Conotoxin GVIA (omega-CgTx)-sensitive N-type channels and omega-agatoxin IVA (omega-AgaIVA)-sensitive P/Q-type channels together accounted for most of the calcium current (36 and 37%, respectively). Nimodipine had no effect on the calcium current, indicating that caudal raphe neurons do not express dihydropyridine-sensitive L-type currents. A substantial residual current (27%) remained after application of omega-CgTx, omega-AgaIVA, and nimodipine. Most of the 5-HT-sensitive calcium current was blocked by omega-CgTx and omega-AgaIVA; 5-HT had little effect on the residual current. Inhibition of calcium current by 5-HT was irreversible when GTP gamma S, 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-HT1A receptor via PTX-sensitive G proteins. Under current clamp, calcium channel toxins (omega-CgTx and omega-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.
我们对通过钙通道的全细胞钡电流进行了特性分析，并研究了血清素（5-羟色胺，5-HT）对新生大鼠脑干切片中可视化的中缝尾部神经元的钙通道电流及放电行为的影响（n = 201）。对记录的神经元亚群进行色氨酸羟化酶（TPH）染色后进行回收，TPH是5-HT合成酶（n = 21）；在这些细胞中，86%呈TPH免疫反应性，这表明大多数记录的神经元是血清素能神经元。中缝尾部神经元的钙通道电流在约-40 mV时开始激活，在-10 mV时显示出952.2±144.2（SE）pA的峰值幅度。还观察到一个小的低电压激活电流（约22 pA）。在大多数测试细胞中（约90%），通过浴加5-HT可有效抑制钙通道电流。5-HT抑制钙电流的EC50为0.1 microM；饱和浓度（1.0 microM）可阻断从-70 mV的钳制电位在0 mV诱发电流的约40%（n = 101）。电流抑制与激活动力学减慢以及电流-电压关系峰值的偏移有关，并且通过强去极化可部分缓解。5-HT对电流的抑制作用可被特异性5-HT1A激动剂8-OH-DPAT模拟，并被5-HT1a拮抗剂NAN 190和（+）WAY 100135阻断，但不受5-HT2A/C拮抗剂酮色林的影响。ω-芋螺毒素GVIA（ω-CgTx）敏感的N型通道和ω-阿加毒素IVA（ω-AgaIVA）敏感的P/Q型通道共同构成了大部分钙电流（分别为36%和37%）。尼莫地平对钙电流无影响，表明中缝尾部神经元不表达对二氢吡啶敏感的L型电流。在应用ω-CgTx、ω-AgaIVA和尼莫地平后，仍存在大量残余电流（27%）。大部分对5-HT敏感的钙电流被ω-CgTx和ω-AgaIVA阻断；5-HT对残余电流影响很小。当在移液管中用不可水解的鸟苷5'-三磷酸（GTP）类似物GTPγS替代GTP时，5-HT对钙电流的抑制作用不可逆。此外，用百日咳毒素（PTX）预处理切片可阻断5-HT的作用。这些数据共同表明，血清素能中缝尾部神经元中N型和P/Q型钙电流的抑制是由5-HT1A受体通过对PTX敏感的G蛋白介导的。在电流钳制下，钙通道毒素（ω-CgTx和ω-AgaIVA）和5-HT均导致动作电位后超极化减小，并增强了对注入电流的重复放电反应。5-HT和钙通道毒素对放电行为的类似作用表明，5-HT的这些作用继发于对N型和P/Q型钙通道的抑制。
