Schneider S P, Eckert W A, Light A R
Department of Anatomy and Neuroscience Program, Michigan State University, East Lansing, Michigan 48824, USA.
J Neurophysiol. 1998 Dec;80(6):2954-62. doi: 10.1152/jn.1998.80.6.2954.
Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954-2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the mu-opioid agonist (-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1-5 microM DAMGO caused a robust and repeatable hyperpolarization in membrane potential (Vm) and decrease in neuronal input resistance (RN) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5'-triphosphate (GTP; 100 microM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S; 100 microM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S; 500 microM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate mu-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.
阿片类物质激活的脊髓背角胶状质神经元全细胞记录中的突触后内向整流钾电流。《神经生理学杂志》80: 2954 - 2962, 1998年。利用来自仓鼠和大鼠脊髓分离横切片的紧密封全细胞记录，我们研究了μ - 阿片受体激动剂（-丙氨酸2，N - 甲基苯丙氨酸4，甘氨酸5 - 醇） - 脑啡肽（DAMGO）对脊髓背角胶状质（SG）神经元膜电位和电导的影响。我们观察到，在浴槽中加入1 - 5微摩尔DAMGO可使60%（27/45）的仓鼠神经元和39%（9/23）的大鼠神经元的膜电位（Vm）出现强烈且可重复的超极化，神经元输入电阻（RN）降低，但仅当膜片钳微管内液中含有ATP（2毫摩尔）和鸟苷5'-三磷酸（GTP；100微摩尔）时才显著。在大鼠SG神经元中观察到超极化的半数有效剂量（ED50）为50纳摩尔。由于在其他系统中已显示阿片类物质的作用是由G蛋白介导的，我们测试了SG神经元中阿片类物质超极化对核苷酸的需求是否是由于G蛋白激活所致。用不可水解的GTP类似物鸟苷 - 5'-O - （3 - 硫代三磷酸）（GTP - γ - S；100微摩尔）替代GTP，这使得DAMGO能够激活不可逆的膜超极化。此外，细胞内应用鸟苷 - 5'-O - （2 - 硫代二磷酸）（GDP - β - S；
