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ACTIVITIES OF ACETYLCHOLINE-METABOLIZING ENZYMES IN THE MASTICATORY MOTONEURONS OF THE RAT

大鼠咀嚼运动神经元乙酰胆碱代谢酶的活性

基本信息

批准号：
07838032
负责人：
KAWAGISHI Shigenori
金额：
$ 1.15万
依托单位：
KYUSHU DENTAL COLLEGE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

Masticatory alpha motoneurons in the rat were examined quantitatively for their activity of the acetylcholine-degrading enzyme, acetylcholinesterase (AChE). After the anterior digastric (jaw-opening) and masseter (law-closing) motoneurons were retrogradely labeled by the fluorescent tracers nuclear yellow and bisbenzimide respectively, the rats were administered with the ir-reversible AChE inhibitor diisopropyl fluorophosphate (DFP) and then allowed to survive for 3 hr for the synthesis of new enzymes. Serial frozen 20 mum sections of the brainstems were prepared and processed for AChE histochemistry using acetylthiocholine iodide as the substrate. The AChE activity of motoneurons labeled by the fluorescence was determined by measuring the absorbance at 470 nm in the cytoplasm using a microspectrophotometer. In the suckling and young adult animals, the anterior digastric motoneurons showed significantly higher AChE activity than the masseter motoneurons (p<0.001), but in the old rats such differences were not detected. In the young adult rats, there was no relationship between the AChE activities and soma areas of alpha motoneurons which were measured using an image analyzer. The activity of transmitter-synthesizing enzyme, choline acetyltransferase (ChAT), in the young rats was also examined. Thirty mum sections of the brainstems were prepared from the DFP-untreated rats and processed for ChAT immunohistochemisry using the peroxidase -antiperoxidase complex. The ChAT activity of motoneurons was measured as the absorbance at 450 nm of the peroxidase-reaction-products ; the ChAT activity was also significantly higher in the anterior digastric than in the masseter motoneurons (p<0.001).

定量测定了大鼠咀嚼α运动神经元乙酰胆碱降解酶乙酰胆碱酯酶（AChE）的活性。分别用荧光示踪剂核黄和双苯并胺逆行标记大鼠的前二腹肌（开颌）和咬肌（闭颌）运动神经元后，给予不可逆乙酰胆碱酯酶抑制剂氟磷酸二异丙基（diisopropyl fluorophosphate， DFP），然后让大鼠存活3小时以合成新的酶。以乙酰硫代胆碱碘化为底物，制备连续冷冻的20块脑干切片，进行乙酰胆碱胆碱酯酶组织化学处理。荧光标记的运动神经元乙酰胆碱酯酶活性用微分光光度计测定细胞质在470 nm处的吸光度。在哺乳期和幼龄动物中，二腹肌前运动神经元的AChE活性明显高于咬肌运动神经元（p<0.001），但在老年大鼠中没有发现这种差异。在年轻的成年大鼠中，使用图像分析仪测量的AChE活性与α运动神经元的体面积之间没有关系。测定了幼龄大鼠递质合成酶胆碱乙酰转移酶（ChAT）的活性。使用过氧化物酶-抗过氧化物酶复合物制备30块经dfp处理的大鼠脑干切片，进行ChAT免疫组织化学处理。以过氧化物酶反应产物450 nm处的吸光度测定运动神经元的ChAT活性；二腹肌前部的ChAT活性也显著高于咬肌运动神经元（p<0.001）。