Altered gabaa receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol.
Altered gabaa receptor subunit and splice variant expression in rats treated with chronic intermittent ethanol.
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DOI：
10.1111/j.1530-0277.2001.tb02285.x
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发表时间：
2001-06
期刊：
影响因子：
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通讯作者：
J. Petrie;D. W. Sapp;R. Tyndale;M. Park;M. Fanselow;R. Olsen
中科院分区：
文献类型：
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作者：
J. Petrie;D. W. Sapp;R. Tyndale;M. Park;M. Fanselow;R. Olsen
背景：先前已经证明，对大鼠间歇性长期给予乙醇会产生过度兴奋的点燃样状态，伴随着海马中抑制性突触传递的减少和γ-氨基丁酸A型（GABAA）受体的变化。需要更多关于GABAA受体的详细变化及其时程和持续性的信息，以及与慢性连续乙醇后变化的比较。方法采用放射性配体结合、多肽光亲和标记、逆转录酶-聚合酶链反应（RT-PCR）和原位杂交技术估计受体亚基信使RNA（mRNA）水平等方法，分析慢性间歇性乙醇（CIE）后大鼠脑中GABAA受体的变化。结果通过光亲和标记证实CIE大鼠具有苯二氮卓类部分反向激动剂和乙醇解毒剂配体Ro 15 -4513的增加的GABAA受体结合，这是由于小脑中α 6亚基多肽的表达增加。通过使用RT-PCR的mRNA水平的估计没有发现任何显着增加α 6或在几个其他受体亚基在几个脑区，但在γ 2亚基的长和短剪接变体（L/S）的比例下降检测到在海马，特别是CA 1区。结论：CIE可引起大鼠GABAA受体的改变。通过使用[3 H] Ro 15 -4513与地西泮不敏感位点的结合和使用该配体进行光亲和标记后57-kDa α 6多肽水平的增加，证明小脑中α 6亚基的增加。这种增加出现在30次乙醇剂量后，并在停止乙醇后1周恢复正常。小脑α 6亚基受体的瞬时变化，也据报道，慢性连续乙醇后，因此不太可能解释CIE中观察到的持续过度兴奋，点燃，癫痫易感状态。然而，海马中γ 2亚基L/S剪接变体比率的显著降低意味着GABAA受体功能的变化，可能涉及蛋白激酶C引起的蛋白磷酸化。与突触可塑性相关的受体运输和周转的改变可能有助于观察到的海马抑制减少和CIE产生的酒精依赖的其他迹象。
BACKGROUND Intermittent chronic administration of ethanol to rats has been shown previously to produce a hyperexcitable, kindling-like state, accompanied by reduced inhibitory synaptic transmission in the hippocampus and changes in gamma-aminobutyric acid type A (GABAA) receptors. Further information is needed on the detailed changes in GABAA receptors and their time course and persistence, as is comparison to changes after chronic, continuous ethanol. METHODS GABAA receptors were analyzed in the rat brain after chronic intermittent ethanol (CIE) by using radioligand binding, photoaffinity labeling of polypeptides, and estimates of messenger RNA (mRNA) levels of receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS CIE rats were confirmed to have increased GABAA receptor binding of the benzodiazepine partial inverse agonist and ethanol antidote ligand Ro15-4513, due to increased expression of the alpha6 subunit polypeptide in the cerebellum, shown by photoaffinity labeling. Estimates of mRNA levels by use of RT-PCR did not reveal any significant increase in alpha6 or in several other receptor subunits in several brain regions, but a decrease in the ratio of the long and short splice variants (L/S) of the gamma2 subunit was detected in the hippocampus, especially the CA1 region. CONCLUSIONS Changes in GABAA receptors were found in rats given CIE. Increased alpha6 subunit in the cerebellum was demonstrated by using both the binding to diazepam-insensitive sites for [3H]Ro15-4513 and increased levels of the 57-kDa alpha6 polypeptide after photoaffinity labeling with this ligand. This increase appeared after 30 doses of ethanol and decayed to normal 1 week after ethanol was discontinued. The transient change in cerebellar alpha6 subunit-containing receptors, also reportedly seen after chronic continuous ethanol, is thus unlikely to account for the persistently hyperexcitable, kindled, seizure-susceptible state seen in CIE. However, the significant decrease in gamma2 subunit L/S splice variant ratio in the hippocampus implies changes in GABAA receptor function, possibly involving protein phosphorylation by protein kinase C. Altered receptor trafficking and turnover associated with synaptic plasticity may contribute to the observed reduced inhibition in the hippocampus and other signs of alcohol dependence produced by CIE.