依托咪酯降低新生大鼠离体脊髓腹角神经元的兴奋性及抑制nAChR的功能

Etomidate reduces excitability of the neurons and suppresses the function of nAChR ventral horn in the spinal cord of neonatal rats

目的研究依托咪酯(ET)对脊髓腹角神经元电生理特性及烟碱型乙酰胆碱受体(nAChR)的影响。方法选用19只7~12 d新生SD大鼠,麻醉后,将含有腰骶膨大的脊髓分离并切片,用木瓜蛋白酶(0.18 g/30 mL人工脑脊液)消化切片并孵育40 min,显微镜下选取腹角,用抛光的巴斯德吸管进行急性机械分离神经元,对贴壁的健康神经元结合药理学方法进行穿孔膜片钳记录实验。在电流钳模式下,先记录脊髓腹角神经元自发动作电位(AP),然后结合预处理给药方式,分别记录不同浓度的ET对脊髓腹角神经元自发AP影响。在电压钳模式下,先应用尼古丁在脊髓腹角神经元诱导出内向电流,然后结合预处理给药方式,记录在不同浓度的ET、不同钳制电位以及不同使用时间的情况下,ET对尼古丁在脊髓腹角神经元诱导的内向电流的影响。结果急性分离的脊髓腹角神经元状态良好,具有形状多样的胞体和完整的突起;共记录到21例脊髓腹角神经元有自发AP,经0.3、3.0、30.0μmol/L(3.0μmol/L相当于临床浓度)不同浓度的ET持续灌流2 min后,与给药前比较,结果12例神经元的AP幅度、锋电位幅度及超射分别被浓度依赖性抑制(P<0.01);自发放电频率降低(P<0.01)。另外9例神经元的AP被3.0或30.0μmol/L的ET完全取消;在相同钳制电位下(VH=-70 mV),分别经0.3、3.0和30.0μmol/L的ET预处理2 min后,对0.4 mmol/L尼古丁诱导的电流幅度显示出浓度依赖性压抑作用(P<0.01,n=7)。将钳制电位分别设定为-30、-50、-70 mV,应用30.0μmol/L的ET预处理2 min后,对0.4 mmol/L尼古丁诱导的电流幅度呈电压依赖性压抑作用(P<0.01,每个钳制电位下n=6)。在30.0μmol/L的ET预处理6 min过程中,分别于0、2、4、6 min时先后4次暴露于0.4 mmol/L的尼古丁(每次暴露时间为2 s),随着暴露次数增多,尼古丁电流幅度逐渐减小;但若在6 min的预处理过程中,仅在开始(0 min)和结束(6 min)时两次暴露于相同浓度尼古丁,则6 min时,电流幅度抑制率较4次暴露尼古丁时的抑制率明显降低(P<0.01,n=6)。结论ET以浓度依赖的方式降低脊髓腹角神经元的兴奋性,并且以浓度依赖、电压依赖和使用依赖的方式压抑nAChR功能。

Objective To investigate the effects of etomidate on electrophysiological properties and nicotinic acetylcholine receptors(nAChRs)of ventral horn neurons in the spinal cord.Methods The spinal cord containing lumbosacral enlargement was isolated from 19 neonatal SD rats aged 7-12 days.The spinal cord were sliced and digested with papain(0.18 g/30 mL artificial cerebrospinal fluid)and incubated for 40 min.At the ventral horn,acute mechanical separation of neurons was performed with fire-polished Pasteur pipettes,and perforated patch-clamp recordings combined with pharmacological methods were employed on the adherent healthy neurons.In current-clamp mode,the spontaneous action potential(AP)of the ventral horn neurons in the spinal cord was recorded.The effects of pretreatment with different concentrations of etomidate on AP recorded in the ventral horn neurons were examined.In the voltage-clamp mode,nicotine was applied to induce inward currents in the ventral horn neurons,and the effect of pretreatment with etomidate on the inward currents induced by nicotine were examined with different etomidate concentrations,different holding potentials and different use time.Results The isolated ventral horn neurons were in good condition with large diverse somata and intact processes.The isolated spinal ventral horn neurons(n=21)had spontaneous action potentials,and were continuously perfused for 2 min with 0.3,3.0 and 30.0μmol/L etomidate.Compared with those before administration,the AP amplitude,spike potential amplitude and overshoot were concentration-dependently suppressed(P<0.01),and spontaneous discharge frequency was obviously reduced(P<0.01,n=12).The APs of the other 9 neurons were completely abolished by etomidate at 3.0 or 30μmol/L.At the same holding potential(VH=-70 mV),pretreatment with 0.3,3.0 or 30.0μmol/L etomidate for 2 min concentration-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine(P<0.01,n=7).At the holding potentials of-30,-50,and-70 mV,pretreatment with 30.0μmol/L etomidate for 2 min voltage-dependently suppressed the current amplitude induced by 0.4 mmol/L nicotine(P<0.01,n=6 for each holding potential).During the 6 min of 30.0μmol/L etomidate pretreatment,the clamped cells were exposed to 0.4 mmol/L nicotine for 4 times at 0,2,4,and 6 min(each exposure time was 2 s),and the nicotinic current amplitude decreased gradually as the number of exposures increased.But at the same concentration,two nicotine exposures(one at the beginning and the other at the end of the 6 min pretreatment)resulted in a significantly lower inhibition rate compared with 4 nicotine exposures(P<0.01,n=6).Conclusion etomidate reduces the excitability of the spinal ventral neurons in a concentration-dependent manner and suppresses the function of nAChR in a concentration-,voltage-,and use-dependent manner.