Analysis of ionic channels of medullary respiratory neurons by whole-cell patch-clamp recordings

本研究采用全细胞膜片钳法对哺乳动物髓质呼吸神经元的膜离子通道特性进行了分析。本研究重点分析了新生大鼠脑干-脊髓分离制剂中吸气前（Pre-I）、吸气前（Insp）和呼气强直（Exp）神经元的主要Ca通道。这些呼吸神经元记录在延髓吻侧腹外侧。TTX阻断na通道、Cs +和TEA^+阻断k通道后，至少出现4种钙依赖性膜电位反应：1)高阈值Ca^<2+>峰（HTS），阈值为-40 ~ -20mV。在所有类型的呼吸神经元中均观察到HTS。2)包括低阈值Ca^<2+>尖峰（LTS）在内的反弹响应；超极化电流脉冲终止时的去极化响应。低阈值Ca^<2+>后去极化（L-ADP），在保持电位低于-50mV时诱导的LTS，在Pre-I、Insp或Exp神经元中经常观察到，这些神经元倾向于…这是在保持电位大于-45至-50 mV时观察到的，并且振幅随着膜超极化的进展而增加。在部分Insp神经元中观察到L-ADP。4)中阈值Ca^<2+>平台电位（ITP），阈值范围为-50 ~ -40mV。该响应的特征是由静息电位水平（约-50mV）的短暂去极化引起的缓慢再生去极化，以及在去极化电流脉冲注入结束时的后去极化。振幅随膜超极化而减小。在大约一半的Pre-I神经元和III型（从Pre-I接收抑制性突触输入）Insp神经元中检测到ITP。药理研究表明，p型和n型钙通道参与ITP反应。本研究表明，新生大鼠骨髓呼吸神经元具有一套复杂的钙依赖膜电导，可能与呼吸爆发的产生和维持有关。少

The purpose of the present study was to analyze properties of membrane ionic channels of mammalian medullary respiratory neurons by whole-cell patch-clamp recordings. I focused on analysis of mainly Ca channels of pre-inspiratory (Pre-I), inspiratory (Insp) and tonic-expiratory (Exp) neurons in brainstem-spinal cord preparations isolated from newborn rats. These respiratory neurons were recorded in the rostral ventrolateral medulla. After Na-channel blockade by TTX and K-channel blockade by Cs^+ and TEA^+, at least 4types of Ca-dependent membrane potential responses were revealed : 1) High-threshold Ca^<2+> spike (HTS) which has the threshold of -40 to -20mV.The HTS was observed in all types of respiratory neurons analyzed. 2) Rebound responses including low-threshold Ca^<2+> spike (LTS) ; depolarizing responses to termination of hyperpolarizing current pulses. The LTS that was induced at holding potential below -50mV,was observed frequently in Pre-I,Insp or Exp neurons that tended to … More show continuous firings (i.e.tonic pacemaker activity) in the perfusate with or without TTX.3) Low-threshold Ca^<2+> after-depolarization (L-ADP). This was observed at holding potential more negative than -45 to -50 mV and the amplitude increased with progressive membrane hyperpolarization. The L-ADP was observed in some Insp neurons. 4) Intermediate-threshold Ca^<2+> plateau potential (ITP) of which threshold ranged from -50 to -40mV.This response was characterized with slow regenerative depolarization induced by brief depolarization from resting potential level (about -50mV) and with afterdepolarization at termination of depolarizing current pulse injection. The amplitude decreased with membrane hyperpolarization. The ITP was detected in about half of Pre-I neurons and type III (receiving inhibitory synaptic input from Pre-I) Insp neurons. Pharmacological study suggested that P-and also N-type Ca channels are involved in ITP responses. The present study shows that medullary respiratory neurons in newborn rat preparations have a complex set of Ca-dependent membrane conductances, which may be relevant for the respiratory burst generation and maintenance. Less