Buckley S T, Eckert A L, Dodd P R
Department of Biochemistry, University of Queensland, Brisbane, Australia.
Ann N Y Acad Sci. 2000 Sep;914:58-64. doi: 10.1111/j.1749-6632.2000.tb05183.x.
Long-term alcohol abuse is known to target specific areas of the brain such as the superior frontal cortex (SFC), resulting in neuronal cell loss. Abnormal transmission of the inhibitory neurotransmitter GABA may contribute to this damage. Previous work in our laboratory has found differential expression and distribution of certain a subunit genes of the GABAA receptor in the SFC of human alcoholic brain, suggesting that differences in GABAA receptor subunit expression could give rise to the locally altered GABAA pharmacology which is associated with alcohol abuse. A competitive RT-PCR assay has been developed to study the expression of the GABAA receptor beta-subunit genes beta1, beta2, and beta3. A single set of primers homologous to all three beta isoform sequences has been shown to amplify each of the beta isoforms from mRNA isolated from human brain tissue obtained at autopsy. An internal standard has been designed which is identical to the target except for a 61-bp deletion and a unique restriction enzyme (RE) site. This is co-amplified with the target sequences to allow amplification efficiency to be assessed and thus enable the quantitation of gene expression. A range of GABAA receptor ligands were used to look at differential distribution of receptor subtypes in the cortical laminae by autoradiography. Differences in distribution of the ligands were demonstrated, consistent with a hypothesis of alcohol-induced variations in the expression of receptor subunits.
长期酗酒已知会靶向大脑的特定区域，如额上皮质（SFC），导致神经元细胞丢失。抑制性神经递质γ-氨基丁酸（GABA）的异常传递可能导致这种损伤。我们实验室之前的研究发现，人类酒精性脑的SFC中，GABAA受体某些α亚基基因存在差异表达和分布，这表明GABAA受体亚基表达的差异可能导致局部GABAA药理学改变，而这与酒精滥用有关。已开发出一种竞争性逆转录聚合酶链反应（RT-PCR）检测方法来研究GABAA受体β亚基基因β1、β2和β3的表达。已证明一组与所有三种β异构体序列同源的引物可从尸检获得的人类脑组织分离的mRNA中扩增出每种β异构体。已设计了一种内标，除了有一个61碱基对的缺失和一个独特的限制性内切酶（RE）位点外，它与靶标相同。它与靶标序列共同扩增，以评估扩增效率，从而实现基因表达的定量。使用一系列GABAA受体配体，通过放射自显影观察受体亚型在皮质层中的差异分布。已证明配体分布存在差异，这与酒精诱导受体亚基表达变化的假设一致。
