Effects of propofol on the potassium channel on the rat midbrain dopaminergic neurons.

背景：异丙酚具有成瘾性。成瘾的神经元机制被认为与起源于腹侧被盖区（VTA）的中脑多巴胺系统的激活有关。在这项研究中，我们计划使用膜片钳技术来表征异丙酚对VTA多巴胺能神经元兴奋性的影响。方法：取幼龄大鼠含VTA的脑切片。采用全细胞膜片钳技术记录VTA神经元的膜电位和膜电流。我们获得了中脑多巴胺能神经元在负电流注入下表现出电压凹陷的数据，以及在电压箝位模式下对超极化步骤的响应中表现出时间依赖的内向电流。异丙酚被稀释到人工脑脊液（ACSF）中，最终浓度为0.5 ~ 50μM，浴敷6 ~ 16min，直至反应稳定。在电流箝位模式下记录了更多的离子电位，在电压箝位模式下记录了不同设置下保持电位为-60mV时的膜电流。采用选择性GAGAA激动剂THIP（4,5,6,7-四氢异恶唑[5,4-c]吡啶-3-醇，3和30μM）进行了相同的实验。在电压箝位模式的部分实验中，ACSF中添加100□M的微毒素阻断GABAA受体。结果：在电流箝位下，8 / 18个神经元在0.5μ m和5μM丙泊酚灌注下动作电位升高（对照组：9.3±4.2 spike /min, 0.5μ m:27.5±0.1 spike /min, 5μM:19.0±8.2 spike /min，洗脱组：1.8±0.8 spike /min, p<0.05）。另外10个神经元的动作电位保持不变或受到抑制（对照组：0.75±0.71 spike /min, 0.5μM:0.43±0.37 spike /min, 5μM:0.06±0.04 spike /min，洗脱期：几乎没有峰值，p>0.05）。低浓度丙泊酚激活神经元的膜电位较低（丙泊酚激活神经元的膜电位为-53.4±4.7 mV，丙泊酚不敏感神经元的膜电位为-55.5±4.0mV, p<0.05），其固有兴奋性较丙泊酚不敏感或抑制神经元高。THIP灌注后，4个神经元中有2个在3μM时动作电位升高，而在30μM时动作电位完全抑制。在电压箝位实验中，0.5μ m和5μ m异丙酚诱导11个神经元中的6个神经元产生阳离子内流或阴离子外流（内向电流组：对照组：0.35±0.18pA/pF, 0.5μ m:-0.76±0.20 pA/pF, 5μ m:-0.93±0.20 pA/pF，冲刷：0.48±0.51pA/pF, n=6；外向电流组：对照组：-0.36±0.11 pA/pF, 0.5μ m: -0.31±0.14pA/pF, 5pM:0.15±0.24pA/pF，冲刷：0.73±0.46pA/pF, n=5）。THIP 3μM对7个神经元不能产生明显的向内电流，而30μM诱导明显的向外电流。在ACSF中添加100□M的微毒素后，低糖异丙酚诱导的内向电流被完全抑制。结论：低浓度异丙酚能激活部分VTA域能神经元，与异丙酚不敏感或抑制的神经元相比，VTA域能神经元具有更浅的膜电位和更强的内在兴奋性。选择性GABAA激动剂THIP部分模拟了异丙酚的作用，微毒素阻断了其作用，这可能与异丙酚对VTA神经元GABAA受体的激活有关。这一结果可以解释先前的体内研究^1.1。Anesth Analg 2002；95: 915.9少

Background : Propofol has an aspect of addictive agent. Neuronal mechanisms of addiction are thought to be related with the activation of midbrain dopamine system originated from ventral tegmental area (VTA). In this study, we planed to characterize propofol effects for the excitability of VTA dopaminergic neurons using patch clamp technique.Method : Brain slices containing VTA were obtained from young rats. Membrane potentials and membrane currents of the VTA neurons were recorded using whole-cell patch clamp technique. We obtained data from the neurons exhibiting voltage sag under current clamp mode in response to negative current injection and time-ependent inward current under voltage clamp mode in response to hyperpolarizing steps, which is characteristic to midbrain dopaminergic neurons. Propofol was diluted into artificial cerebrospinal fluid (ACSF) to a final concentration of 0.5 to 50μM and bath-applied for 6 to 16min until responses were stabilized. Membrane potential and act … More ion potentials were recorded under current clamp mode and membrane currents at a holding potential of -60mV were recorded under voltage clamp mode in the different setting. Same experiments were done using THIP (4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridine-3-ol, 3 and 30μM), a selective GAGAA agonist. In some experiments of voltage clamp mode, ACSF was supplemented with 100□M of picrotoxin for blocking GABAA receptor.Result : Action potentials were increased with 0.5 and 5μM of propofol perfusion in 8 of 18 neurons under current clamp configuration (control : 9.3±4.2 spikes/min, 0.5μM:27.5±0.1 spikes/min, 5μM:19.0±8.2 spikes/min, washout : 1.8±0.8 spikes/min, p<0.05). In other ten neurons, action potentials were unchanged or suppressed (control: 0.75±0.71 spikes/min, 0.5μM:0.43±0.37 spikes/min, 5μM:0.06±0.04 spikes/min, washout : almost no spikes, p>0.05). Neurons activated low-ose propofol showed shallower membrane potential (propofol activated neurons: -53.4±4.7 mV, propofol insensitive neurons : -55.5±4.0mV, p<0.05), and more intrinsic excitability compared to propofol insensitive or depressed neurons. With THIP perfusion, action potentials were increased in two of four neurons at 3μM although action potential was completely suppressed at 30μM. In the voltage clamp experiment, 0.5 and 5μM of propofol induced inward currents, which represents cation influx or anion exflux, in 6 of 11 neurons (inward current group : control : 0.35±0.18pA/pF, 0.5μM:-0.76±0.20 pA/pF, 5μM:-0.93±0.20 pA/pF, washout : 0.48±0.51pA/pF, n=6, outward current group : control : -0.36±0.11 pA/pF, 0.5μM: -0.31±0.14pA/pF, 5pM:0.15±0.24pA/pF, washout : 0.73±0.46pA/pF, n=5). THIP 3μM could not induce significant inward current for seven neurons and 30μM induced marked outward current. Inward currents induced by low-ose propofol completely suppressed when 100□M of picrotoxin was added in ACSF.Conclusion : Low-ose propofol can activate some population of VTA domaminergic neurons, which has shallower membrane potential and more intrinsic excitability than the propofol-insensitive or suppressed neurons. THIP, selective GABAA agonist, partially mimicked the propofol effect and picrotoxin blocked it, this phenomenon would be related to GABAA receptor activation of propofol on VTA neurons. This result can account for previous in vivo study^1.1. Anesth Analg 2002 ; 95 : 915.9 Less