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

背景：异丙酚具有一定的成瘾性。成瘾的神经机制被认为与腹侧被盖区(VTA)起源的中脑多巴胺系统的激活有关。本研究拟用膜片钳技术研究异丙酚对VTA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VTA神经元的膜电位和膜电流。我们从中脑多巴胺能神经元在电流钳模式下的电压下降和电压钳模式下的时间依赖性内向电流对超极化步骤的反应中获得了数据。将异丙酚稀释到人工脑脊液中，最终浓度为0.5~50μM，浸浴6~16min，直至反应稳定。膜电位与ACT…在不同设置下，在电流钳模式下记录到更多的离子电位，在电压钳模式下记录到-60 mV的膜电流。用选择性GAGAAA激动剂THIP(4，5，6，7-四氢异恶唑[5，4-c]吡啶-3-醇，3和30μM)进行相同的实验。结果：在电压钳模式下，灌流0.5和5μM的异丙酚可使18个神经元中的8个神经元的动作电位增加(对照组：9.3±4.2次/分，0.5μM：2 7.5±0.1次/分，5μM：19.0±8.2次/分，冲洗：1.8±0.8次/分，p&lt；0.0 5)。在其余10个神经元中，动作电位不变或被抑制(对照组：0.75±0.71个峰/分，0.5μM：0.43±0.37个峰/分，5μM：0.06±0.04个峰/分，冲洗：几乎没有峰电位，p&gt；0.05)。与异丙酚不敏感或抑制神经元相比，低剂量异丙酚激活的神经元表现出较浅的膜电位(异丙酚激活的神经元：-53.4±4.7 mV，异丙酚不敏感的神经元：-55.5±4.0 mV，p&lt；0.05)，而内源性兴奋性更强。在电压钳实验中，0.5和5μM的异丙酚在11个神经元中的6个(内向电流组：对照组：0.35±0.18pA/pF，0.5μM：-0.76±0.20pA/pF，5 mBM：-0.93±0.20pA/pF，洗脱：0.48±0.51pA/pF，n=6，外向电流组：-0.36±0.11pA/pF，0.5μ男：-0.31±0.14pA/pF，5 PM：0.15±0.24pA/pF，洗脱：0.73±0.46pA/pF，n=5)。THIP-3μ-M对7个神经元无明显内向电流，30μ-M可引起明显的外向电流。在ACSF中加入100M的印防己毒素，可完全抑制低剂量异丙酚诱发的内向电流。结论：低剂量异丙酚可激活部分VTA区神经元，其膜电位比异丙酚不敏感或抑制的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部分模拟异丙酚的作用并被印防己毒素阻断，这一现象可能与异丙酚激活VTA神经元的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：减少915.9

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