Thompson C L, Pollard S, Stephenson F A
Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, U.K.
Neuropharmacology. 1996;35(9-10):1337-46. doi: 10.1016/s0028-3908(96)00114-1.
We have studied the postnatal development of GABAA receptor alpha 1 and alpha 6 subunits expressed by primary cultures of cerebellar granule cells originating from 2-day-old (postnatal day 2, P2) and 10-day-old (P10) rat neonates. At these ages, the granule cells are at distinct stages of cerebellar development. In both cases, GABAA receptor alpha 1 and alpha 6 subunit-like immunoreactivities were detected, and displayed temporal expression profiles that were correlated with the maturity of the cerebella from which the cultured granule cells were derived. Using two different specificity anti-alpha 1 subunit-specific antibodies, immunoreactive species with M(r) 53,000 Da and 54,000 Da were detected by immunoblotting. The lower 53,000-Da band co-migrated with the alpha 1 subunit-like immunoreactivity detected in GABAA receptors purified from adult rat forebrain by benzodiazepine affinity chromatography. This 53,000-Da alpha 1 subunit-like immunoreactive species was detected at day 1 in vitro (1 DIV) in P10 cultures and 3-5 DIV in P2 cultures. The GABAA receptor alpha 6 subunit-like immunoreactivity (58,000 Da) was not detected until 5-7 DIV in P10 and 9-11 DIV in P2-derived cultures. The appearance of alpha 6 subunit-like immunoreactivity was paralleled by an up-regulation of alpha 1 subunit expression and a concomitant increase in diazepam-insensitive (DZ-IS) [3H]Ro 15-4513 binding activity, a pharmacological characteristic of alpha 6 and alpha 1 alpha 6-subunit-containing GABAA receptors (Pollard et al. J. Biol. Chem., 270, 21,285-21,290, (1995)). Antagonism of both non-NMDA and NMDA subtypes of ionotropic glutamate receptors did not significantly affect the developmental profile, the level of GABAA receptor alpha 6 subunit or the total DZ-IS or DZ-S [3H]Ro 15-4513 binding activities expressed by these neurons. These results provide further evidence that the expression of specific GABAA receptor subunit genes is subject to differential regulation. Furthermore, developmental expression of the GABAA receptor alpha 6 subunit gene by these neurons is either a preprogrammed event or is initiated by an environmental cue that is received early in granule cell development, and it is not a result of afferent activation of ionotropic glutamate receptors.
我们研究了源自2日龄（出生后第2天，P2）和10日龄（P10）新生大鼠的小脑颗粒细胞原代培养物中GABAA受体α1和α6亚基的出生后发育情况。在这些年龄段，颗粒细胞处于小脑发育的不同阶段。在这两种情况下，均检测到了GABAA受体α1和α6亚基样免疫反应性，并且其呈现出与所培养颗粒细胞来源的小脑成熟度相关的时间表达谱。使用两种不同特异性的抗α1亚基特异性抗体，通过免疫印迹检测到分子量为53,000 Da和54,000 Da的免疫反应性条带。较低的53,000 Da条带与通过苯二氮䓬亲和色谱从成年大鼠前脑纯化的GABAA受体中检测到的α1亚基样免疫反应性共同迁移。在P10培养物的体外第1天（1 DIV）和P2培养物的3 - 5 DIV检测到这种53,000 Da的α1亚基样免疫反应性条带。直到P10培养物的5 - 7 DIV和P2来源培养物的9 - 11 DIV才检测到GABAA受体α6亚基样免疫反应性（58,000 Da）。α6亚基样免疫反应性的出现伴随着α1亚基表达的上调以及地西泮不敏感（DZ - IS）[3H]Ro 15 - 4513结合活性的相应增加，这是含α6和α1α6亚基的GABAA受体的药理学特征（Pollard等人，《生物化学杂志》，270，21,285 - 21,290，（1995））。离子型谷氨酸受体的非NMDA和NMDA亚型的拮抗剂均未显著影响这些神经元的发育谱、GABAA受体α6亚基水平或总的DZ - IS或DZ - S [3H]Ro 15 - 4513结合活性。这些结果进一步证明了特定GABAA受体亚基基因的表达受到差异调节。此外，这些神经元中GABAA受体α6亚基基因的发育表达要么是一个预先编程的事件，要么是由颗粒细胞发育早期接收到的环境信号引发的，而不是离子型谷氨酸受体传入激活的结果。
