PICK1在瑞芬太尼诱发幼鼠脊髓背角神经元AMPA受体微小兴奋性突触后膜电流及AMPA受体表达中的作用

Role of PICK1 in remifentanil-induced miniature excitatory postsynaptic currents mediated by AMPA receptors in spinal dorsal horn neurons and in expression of AMPA receptors in juvenile rats

目的评价蛋白激酶Cα相互作用蛋白1（PICK1）在瑞芬太尼诱发幼鼠脊髓背角神经元使君子酸（AMPA）受体微小兴奋性突触后膜电流（mEPSCs）及AMPA受体表达中的作用。方法出生14～18 d雄性SD幼鼠36只，体重50～60 g，麻醉后处死取腰段脊髓，随机取18只幼鼠腰段脊髓，制备400 μm厚脊髓切片108张（用于全细胞膜片钳检测），余腰段脊髓，制备5 μm厚脊髓切片108张（用于免疫荧光检测），分别采用随机数字表法分为3组（n＝36）：空白对照组（C组）在人工脑脊液中孵育90 min；瑞芬太尼组（R组）在含终浓度4 nmol/L瑞芬太尼的人工脑脊液中孵育90 min；瑞芬太尼＋PICK抑制剂组（R＋PICKi组）在含终浓度4 nmol/L瑞芬太尼及50 μmol/L PICK抑制剂FSC231的人工脑脊液中孵育90 min。采用全细胞膜片钳检测AMPA受体介导的mEPSCs的振幅与时间间隔，采用免疫荧光法检测AMPA受体的表达。结果与C组比较，R组和R＋PICKi组mEPSCs的振幅增大、时间间隔缩短，GluR1和GluR3表达上调，GluR2表达下调（P〈0.05）；与R组比较，R＋PICKi组mEPSCs的振幅减小、时间间隔延长，GluR3表达下调，GluR2表达上调（P〈0.05），GluR1表达差异无统计学意义（P〉0.05）。结论PICK1参与了瑞芬太尼诱发幼鼠脊髓背角神经元AMPA受体mEPSCs及AMPA受体表达的过程，可能是瑞芬太尼诱发痛觉过敏形成的机制。

Objective To evaluate the role of PICK1 in remifentanil-induced miniature excitatory postsynaptic currents（mEPSCs）mediated by AMPA receptors in spinal dorsal horn neurons and in the expression of AMPA receptors in juvenile rats.Methods Thirty-six male Sprague-Dawley rats, aged 14-18 days, weighing 50-60 g, were used in the study.Eighteen rats were randomly selected, their lumbar segments of the spinal cord were immediately removed, and 108 spinal cord slices（400 μm thick, for whole-cell patch-clamp recording）and 108 spinal cord slices（5 μm thick, for immunofluorescence detection）were prepared.The 108 slices with two kinds of thickness were divided into 3 groups（n=36 each）using a random number table： blank control group（group C）, remifentanil group（group R）and remifentanil plus PICK inhibitor group（group R＋ PICKi）. Spinal cord slices were incubated in artificial cerebrospinal fluid（ACSF）for 90 min in group C. Spinal cord slices were incubated for 90 min in ACSF containing remifentanil at the final concentration of 4 mnol/L in group R. Spinal cord slices were incubated for 90 min in ACSF containing remifentanil at the final concentration of 4 nmol/L and 50 μmol PICK inhibitor in group R＋ PICKi.The whole-cell patch-clamp technique was used to record the amplitude and time interval of AMPA receptors-mediated mEPSCs.The method of immunofluorescence was used to detect the expression of AMPA receptors.Results Compared with group C, the amplitude of mEPSCs was significantly increased, and the time interval of mEPSCs was shortened, the expression of GluR1 and GluR3 was up-regulated, and the expression of GluR2 was down-regulated in R and R＋ PICKi groups（P〈0.05）. Compared with group R, the amplitude of mEPSCs was significantly decreased, and the time interval was prolonged, the expression of GluR3 was down-regulated, the expression of GluR2 was up-regulated（P〈0.05）, and no significant change was found in GluR1 expression in group R＋ PICKi（P〉0.05）.Conclusion PICK1 is involved in the process of remifentanil-induced mEPSCs mediated by AMPA receptors in spinal dorsal horn neurons and of expression of AMPA receptors in juvenile rats, which may be the mechanism underlying remifentanil-induced hyperalgesia.