Effects of melatonin on learning abilities, cholinergic fibers and nitric oxide synthase expression in rat cerebral cortex

Bin Xu; Junpao Chen; Hailing Zhao

摘要：BACKGROUND: Melatonin is a kind of hormones derived from pineal gland. Recent researches demonstrate that melatonin is characterized by anti-oxidation, anti-senility and destroying free radicals. While, effect and pathogenesis of pineal gland on learning ability should be further studied.OBJ ECTIVE: To investigate the effects of pinealectomy on learning abiliy, distribution of cholinesterase and expression of neuronal nitric oxide synthase (nNOS) in cerebral cortex of rats and probe into the effect of melatonin on learning ability, central cholinergic system and nNOS expression.DESIGN: Randomized grouping design and animal study.SETTING: Department of Neurology, the 187 Hospital of Chinese PLA.MATERIALS: A total of 12 male SD rats, of normal learning ability testing with Y-tape maze, of clean grade,weighing 190-210 g, aged 6 weeks, were selected in this study.METHODS: The experiment was carried out in the Department of Neurology, Zhujiang Hospital from July 1997to June 2000. All SD rats were divided into experimental group (n =6,pinealectomy) and control group (n =6, sham operation). Seven days later, rats in both two groups were continuously fed for 33 days. ①Learning ability test: The learning ability of rats was tested by trisection Y-type maze and figured as attempting times. ②Expression of acetylcholinesterase (AchE) was detected by enzyme histochemistry and nNOS was measured by SABC method. ③ Quantitative analysis of AchE fibers: AchE fibers density in unit area (surface density)was surveyed with Leica Diaplan microscope and Leica Quantimet 500+ image analytic apparatus and quantitative parameter was set up for AchE fibers covering density (μm2) per 374 693.656 μm2, moreover, the AchE fibers density was measured in Ⅱ -Ⅳ layers of motor and somatosensory cortex (showing three layers per field of vision at one time), in radiative, lacunaria and molecular layers of CA1, CA2 and CA3 areas, and in lamina multiforms of dentate gyrus. Three tissue slices were picked up randomly in the same part of each rat,together six tissue slices for nNOS expression and four near view (× 400) were selected in the parts of right neocortex, medial septal nucleus-diagonal band nucleus (SM-DB), corpus striatus and hippocampus to count nNOS-positive cells.MAIN OUTCOME MEASURES: Learning ability; distribution and quantitative analysis of AchE fibers; expression of nNOS in various cerebral areas.before operation in the experimental group [(14.67±4.97) times] were consistent with those in the control group[(14.33±4.32) times, P ＞ 0.05], the learning abilities in the experimental group at 40 days after pinealectomy[(28.67±2.42) times] were obviously more than those before pinealectomy and those in the control group after The AChE-positive fibers densities in motor and somatosensory cortex, CA1, CA2 and CA3 areas of hippocampus and in lamina multiforms of dentate gyrus in the experimental group were obviously lower than those in the control group [experimental group: (15 244±1 339), (14 764±1 391), (12 991±970), (15 077±1 020),(19 546±1 489), (19 337±1 378) μm2; control group: (21 001±1 021), (17 930±2 225), (17 260±1 342),areas: nNOS-positive cells in cerebral cortex of rats of the experimental group were more, furthermore the ones in somatosensory cortex were slightly more in motor cortex and the number (5.90±0.68) was more than that in the control group (3.68±0.39,P ＜ 0.05). The nNOS-positive cells in SM-DB (16.21 ±2.03) were markedly more than those in the control group (9.32±1.05,P＜ 0.01). The nNOS-positive cells in hippocampus (4.27±0.75) and in corpus striatus (9.35±2.58) were not different with those in the control group (3.94±0.53, 8.96±2.31, P＞ 0.05).CONCLUSION: Decrease of melatonin due to pinealectomy of rats can result in learning disorder, which may be related to trauma of cholinergic neuron in cerebral cortex which were caused by nitric oxide neurotoxicity arose from the overexpression of nNOS in cerebral neocortex and SM-DB.