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

研究背景：异丙酚具有成瘾性。成瘾的神经元机制被认为与中脑腹侧被盖区（VTA）多巴胺系统的激活有关。本研究采用膜片钳技术观察异丙酚对大鼠腹侧被盖区多巴胺能神经元兴奋性的影响。采用全细胞膜片钳技术记录腹侧被盖区神经元的膜电位和膜电流。我们获得的数据显示，在电流钳模式下的负电流注入和电压钳模式下的时间依赖性内向电流响应于超极化步骤，这是典型的中脑多巴胺能神经元的电压凹陷。将丙泊酚稀释到人工脑脊液（ACSF）中至终浓度为0.5 - 50μM，并浴用6 - 16 min，直至反应稳定。膜电位和ACT ...更多信息 在不同的设置下，在电流钳模式下记录离子电位，在电压钳模式下记录保持电位为-60mV的膜电流。使用THIP（4，5，6，7-四氢异恶唑并[5，4-c]吡啶-3-醇，3和30μM）（一种选择性GAGAA激动剂）进行了相同的实验。结果：0.5 μ M和5μM异丙酚灌流可使电流钳下18个神经元中的8个动作电位增加（对照组：9.3± 4.2spikes/min，0.5μM：27.5± 0.1spikes/min，5μ M：19.0± 8.2spikes/min，洗脱组：1.8± 0.8spikes/min，p<0.05）。在其他10个神经元中，动作电位不变或被抑制（对照：0.75±0.71个峰/min，0.5μM：0.43±0.37个峰/min，5μM：0.06±0.04个峰/min，洗脱：几乎没有峰，p>0.05）。与丙泊酚不敏感或抑制的神经元相比，低浓度丙泊酚激活的神经元显示出更浅的膜电位（丙泊酚激活的神经元：-53.4 ~4.7 mV，丙泊酚不敏感的神经元：-55.5 ~4.0 mV，p<0.05）和更多的内在兴奋性。在THIP灌注下，3μM时4个神经元中有2个的动作电位增加，但在30μM时动作电位完全受到抑制。在电压钳实验中，0.5和5μM异丙酚在11个神经元中的6个诱发了代表阳离子内流或阴离子外流的内向电流（内向电流组：对照：0.35±0.18pA/pF，0.5μM：-0.76 ~0.20 pA/pF，5μM：-0.93 ~0.20 pA/pF，洗脱：0.48±0.51pA/pF，n=6，外向电流组：对照：-0.36 ~0.11 pA/pF，0.5μM：-0.31 ~0.14pA/pF，5pM：0.15±0.24pA/pF，洗脱：0.73±0.46pA/pF，n=5）。THIP 3μM不能诱导7个神经元产生明显的内向电流，30μM诱导明显的外向电流。结论：低浓度异丙酚可激活部分腹侧被盖区多巴胺能神经元，这些神经元的膜电位比异丙酚不敏感或抑制的神经元更浅，具有更强的内在兴奋性。选择性GABAA受体激动剂THIP可部分模拟异丙酚的效应，而印防己毒素可阻断THIP的这种效应，这可能与异丙酚激活腹侧被盖区神经元上的GABAA受体有关。这一结果可以解释先前的体内研究^[1.1]。Anesth Analg 2002 ; 95：915.9减

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