Harry S. Truman Memorial Veterans Hospital and Department of Neurology, University of Missouri, Columbia, USA.
Alcohol Clin Exp Res. 2010 May;34(5):813-8. doi: 10.1111/j.1530-0277.2010.01153.x. Epub 2010 Feb 24.
Adenosine is implicated to play a pivotal role in mediating many neuronal responses to ethanol. While in vitro studies performed in cell culture have demonstrated that acute ethanol exposure increases extracellular adenosine levels, this effect has not been demonstrated, in vivo, in the brain. We performed an in vivo microdialysis study to examine the effects of local ethanol perfusion on extracellular levels of adenosine in the basal forebrain (BF).
Under sterile conditions and using a standard surgical protocol, adult male Sprague-Dawley rats were implanted with unilateral microdialysis guide cannula targeted toward the BF. Following postoperative recovery, the microdialysis probe was inserted. After allowing at least 12 to 16 hours for probe insertion recovery, the experiment was begun. Artificial cerebrospinal fluid (aCSF) was perfused (0.7 microl/min) for 80 minutes, and 4 x 20-minute pre-ethanol baseline samples were collected. Subsequently, 30, 100, and 300 mM doses of ethanol were perfused. Each ethanol dose was perfused for 80 minutes, and 4 x 20-minute samples were collected. Finally, aCSF was perfused, and 4 x 20 postethanol samples were collected. Adenosine in the microdialysate was separated and measured with HPLC coupled with an UV detector. On completion, the animals were euthanized, brain removed and processed for histology.
Local ethanol perfusion in the BF produced a significant increase in extracellular adenosine with the highest dose of 300 mM ethanol producing a 4-fold increase. Cresyl violet (Nissl) staining did not indicate any toxic damage in the area surrounding the probe tip. Choline acetyltransferase immunohistochemistry revealed that all microdialysis probe sites were localized in the BF.
Our study is the first to demonstrate that ethanol acts directly in the brain to increase extracellular adenosine.
腺苷被认为在介导许多神经元对乙醇的反应中起着关键作用。虽然在细胞培养中进行的体外研究表明，急性乙醇暴露会增加细胞外腺苷水平，但在体内，在大脑中尚未证明这一效应。我们进行了一项体内微透析研究，以检查局部乙醇灌注对基底前脑（BF）细胞外腺苷水平的影响。
在无菌条件下，使用标准手术方案，将成年雄性 Sprague-Dawley 大鼠植入单侧微透析导向管，靶向 BF。术后恢复后，插入微透析探针。在允许探针插入恢复至少 12-16 小时后，开始实验。人工脑脊液（aCSF）以 0.7 微升/分钟的速度灌注（80 分钟），并收集 4 x 20 分钟的乙醇前基线样本。随后，灌注 30、100 和 300mM 剂量的乙醇。每个乙醇剂量灌注 80 分钟，并收集 4 x 20 分钟的样本。最后，灌注 aCSF，并收集 4 x 20 分钟的乙醇后样本。用 HPLC 与紫外检测器分离和测量微透析液中的腺苷。完成后，处死动物，取出大脑并进行组织学处理。
BF 中的局部乙醇灌注导致细胞外腺苷显著增加，最高剂量的 300mM 乙醇产生 4 倍的增加。亚甲蓝（Nissl）染色未显示探针尖端周围区域有任何毒性损伤。胆碱乙酰转移酶免疫组织化学显示，所有微透析探针部位均位于 BF 中。
我们的研究首次表明，乙醇直接作用于大脑以增加细胞外腺苷。
