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

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. OBJECTIVE: 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 1997 to 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. ① Learning ability test: The learning ability of rats was tested by trisection Y-type maze and figured as attempting times. ② Expression 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. RESULTS: The twelve rats were all involved in the final analysis. ① Learning ability test: The learning abilities 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 operation [(13.83±8.33) times, P < 0.01]. ② Results of AchE-positive fibers density in cerebral cortex of rats: 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), (18 911±1 048), (24 108±1 671), (22 917±1 909) μm2, P < 0.01]. ③ Expression of nNOS in various cerebral 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.

Age-dependent loss of cholinergic neurons in learning and memory-related brain regions and impaired learning in SAMP8 mice with trigeminal nerve damage

The tooth belongs to the trigeminal sensory pathway. Dental damage has been associated with impairments in the central nervous system that may be mediated by injury to the trigeminal nerve. In the present study, we investigated the effects of damage to the inferior alveolar nerve, an important peripheral nerve in the trigeminal sensory pathway, on learning and memory be-haviors and structural changes in related brain regions, in a mouse model of Alzheimer’s disease. Inferior alveolar nerve transection or sham surgery was performed in middle-aged （4-month-old） or elderly （7-month-old） senescence-accelerated mouse prone 8 （SAMP8） mice. When the middle-aged mice reached 8 months （middle-aged group 1） or 11 months （middle-aged group 2）, and the elderly group reached 11 months, step-down passive avoidance and Y-maze tests of learn-ing and memory were performed, and the cholinergic system was examined in the hippocampus （Nissl staining and acetylcholinesterase histochemistry） and basal forebrain （choline acetyltrans-ferase immunohistochemistry）. In the elderly group, animals that underwent nerve transection had fewer pyramidal neurons in the hippocampal CA1 and CA3 regions, fewer cholinergic ifbers in the CA1 and dentate gyrus, and fewer cholinergic neurons in the medial septal nucleus and vertical limb of the diagonal band, compared with sham-operated animals, as well as showing impairments in learning and memory. Conversely, no signiifcant differences in histology or be-havior were observed between middle-aged group 1 or group 2 transected mice and age-matched sham-operated mice. The present ifndings suggest that trigeminal nerve damage in old age, but not middle age, can induce degeneration of the septal-hippocampal cholinergic system and loss of hippocampal pyramidal neurons, and ultimately impair learning ability. Our results highlight the importance of active treatment of trigeminal nerve damage in elderly patients and those with Alzheimer’s disease, and indicate that tooth extraction should be avoided in these populations.

Innervation of Cholinergic Vestibular Efferent System in Vestibular Periphery of Rats

The innervation of cholinergic efferent fibers in the vestibular endorgans of the rat was investigated using a modified preembedding immunostaining technique of immunoelectron microscopy. A monoclonal antibody to choline acetyltransferase (ChAT) was used as a marker of cholinergic fibers. It was found that there were four types of cholinergic innervation in the vestibular endorgans of the rat: (1) cholinergic nerve endings formed axo-dendritic synapses with afferent chalice surrounding the type I sensory hair cells; (2) cholinergic nerve endings formed axo-somatic synapses with type Ⅱ hair cells; (3) cholinergic fibers synapse with afferent nerve fibers and (4) a synaptic contact developed between cholinergic nerve endings. The results demonstrated that a multiform innervation of the cholinergic efferents exists in the rat vestibular periphery.