| With the increase of aging population in China, prevention and treatment of geriatric disease, particularly Alzheimer's disease (AD), has become one of the most challenging problems in life sciences. AD is a degenerative disease happened in central nervous system in the old people, characterized by a progressive impairment of learning, memory and unrelenting cognitive decline. AD not only seriously affects the health, life expectancy and the quality of life in the aged, but also brings a huge burden to the family of AD patients and the society.The cholinergic neurotransmitter system in brain is critical for the processing of information related to cognitive function. The nearly complete destruction of cholinergic neurons located within the nucleus basalis of Meynert (NBM) in AD has led many investigators to think that cholinergic dysfunction is a primary cause of the memory decline associated with AD. In addition, the results from pathological examination of AD patients have shown that there are high density of senile plaques and neurofibrillary tangles, two typical pathological characteristics of AD, within the basal forebrain. These findings indicate that dysfunction of cholinergic projection neurons of NBM correlates with cognitive deficits in AD. So, further acquaintancing the correlation between NBM-lesion and AD should be very critical in clarifying the pathogenesis and unlocking the mystery of AD.The neurons in hippocampus express nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors (mAChRs), which play important regulatory roles in hippocampal functional activities. It has been proved that nAChRs are involved in various physiological processes, including learning and memory. An important finding in recent year is that the acetylcholine (ACh) contents and AChRs have a serious reduction in the cortex and hippocampus of AD patients apart from the deficits of cholinergic neurons in NBM. Therefore, the cholinesterase inhibitors in the treatment of AD is just based on the reduction of ACh and trying to increase synaptic levels of ACh in cholinergic synapses. However, most drugs, including cholinesterase inhibitors, can not alter or reverse the progression of AD. One of the possible reasons is that the mechanisms by which the destruction of cholinergic system causes cognitive dysfunction is unclear. Therefore, to explore the cholinergic mechanism in pathogenesis of AD and to understand the role of the cholinergic system in neural network activity, the present study, by using in vivo behavioral and electrophysiological techniques, observed the changes of spontaneous discharges in the CA1 area of hippocampus, as well as the alteration of spatial learning and memory after NBM-lesion and thereafter application of nAChRs agonists. Experiments were divided into two parts as follows:Aim:The nucleus basalis of meynert (NBM) in the cholinergic basal forebrain plays a key role in the activities of learning and memory, which undergoes some degree of degeneration in AD. To investigate the relationship between the decline in cognitive function in AD and the cholinergic system dysfunction, the present study, by using Morris water maze test, observed the changes of spatial learning and memory after NBM-lesion in rats, and further investigated its mechanisms by intracerebroventricular injection of Choline.Method:Adult male SD rats were used in the experiment. After anesthetized with chloral hydrate, rats were placed in a stereotaxic instrument and injected bilaterally with KA or normal saline as a vehicle control by a micro-syringe into the NBM. With another micro-syringe, choline or saline was injected into the right cerebral ventricle slowly. One week after operation, Morris water maze test, a popular and classic technique used to test learning and memory behavior of animals, was performed. The main parameters include the escape latencies, swim distance in hidden platform tests, and target quadrant preference in probe trialsResults:(1) Intracerebroventricular (i.c.v.) injection of KA (0.5%,2μl) into the NBM resulted in a significant decline in spatial learning. The average escape latencies for searching for the platform under water were 101.0±5.3 s,79.5±4.3 s,55.9±3.7 s,45.3±3.5 s, and 37.2±6.3 s and escape distances were 2059.8±137.4cm,1654.4±139.3cm,1146.1±153.1cm,871.6±81.7cm, 740.6±72.1cm at training day 1-5, respectively. These values were significantly (p<0.01) larger than that in control (67.3±5.8s,47.3±3.5s,25.9±3.3s,12.0±2.8s,10.9±2.0 s and 1479.9±125.2 cm,1043.9±53.4 cm,582.6±90.4 cm,266.8±64.1 cm,242.1±53.4 cm).In probe trials, the percentage of total time elapsed and distance swam in right quadrant was 33%±1.1 and 33.5%±1.0, significantly lower than 48%±0.6 and 50.8%±0.3 in control group (p<0.01). (2) After i.c.v. injection of 500 u M (2μl) Choline, the escape latencies were 57.8±3.8s 38.3±4.2 s, 25.9±3.6 s,9.8±2.1s,8.8±2.3 s and distances swam were 1271.0±90.6 cm,827.5±83.5 cm, 576.4±80.4 cm,228.3±54.8 cm, and 194.4±50.9 cm, remarkably shorter than that in control at training day 1-5 (p<0.01), respectively. The percentage of total time elapsed and distance swam in the target quadrant were 58%±1.0 and 54%±1.5, also longer than those in control group (p<0.05). (3) In Choline (5μM,2μl) plus KA group, the escape latencies was 93.0±8.2 s, 74.9±7.0 s,52.6±6.0 s,39.6±3.6 s, and 33.8±4.0s; the escape distances swam was 2026.3±158.9 cm,1503.6±102.7 cm,1019.3±67.8 cm,783.6±81.0 cm, and 606.6±140.7 cm at training day 1-5, respectively, shorter than those in NBM-lesion group (p<0.05); the percentages of total time elapsed and distance swam in the target quadrant was 37.25%±0.9 and 37.25%±0.75, with mild differences compared with those of NBM-lesion group (p<0.05). After co-application of 500 u M Choline and KA, the escape latencies were further reduced, being 79.9±3.6 s,55.0±3.3 s, 33.2±4.0 s,19.4±3.5 s and 15.4±1.4 s; the escape distances were further shortered, being 1742.8±115.6 cm,1200.9±76.0 cm,703.6±58.5 cm,402.1±65.3 cm and 345.6±34.0 cm attraining day 1-5 (p<0.01); the percentages of total time elapsed and distance swam in the target quadrant were 49.6%±0.7 and 46.0%±1.1, with significant differences compared with those in the NBM-lesion group (p<0.01).4) All drugs in the present experiment did not affect the vision and movement of rats. The average escape latency and swimming speed in five groups had little difference with each other, being 10.1±1.2 s, 11.1±0.9 s,10.4±1.2 s,10.4±1.3 s,10.8±10 s and 23.1±2.3 cm/s,22.8±1.4 cm/s,23.6±3.1 cm/s,23.5±1.8 cm/s,23.4±2.6 cm/s (p>0.05).Conclusion:These results indicated that NBM-lesion induced by KA resulted in a significant decline in spatial learning and memory, and the larger dosage of Choline could reverse KA-induced impairment of spatial learning and memory, suggesting that cholinergic system in NBM and nAChs in the brain are involved in the maintaining of cognitive activity; the up-regulation of cholinergic transmitter might be beneficial in the prevention and treatment of AD demential.Aim:In order to explore the electrophysiological mechanisms by which NBM-lesion impaired spatial learning and memory in the experiment above, the present study, by using multielectrode extracellular recording technique, investigated the effects of NBM-lesion and acute intracerebroventricular injection of Choline on the spontaneous discharges in hippocampal CA1 area of rats.Method:Adult male SD rats were used in this study. All rats were anesthetized with 3% chloral hydrate (400 mg/kg) and placed in a stereotaxic instrument. A sterilized needle connected a 20 ul syringe was sterotaxically placed into the NBM. KA (0.5%,2 ul) or normal saline were bilaterally injected to chemically destroy the neurons in NBM. Electrophysiological experiments were carried out one week after operation. The rats was anesthetized by urethane, then fixed in the three-dimensional stereotaxic instrument. A stainless steel cannula was implanted into the right lateral ventricle of the rat in order to injection saline/Choline. Meanwlile, a self-made bound recording electrode was inserted into the hippocampal CA1 region. When closed to the target region, the electrode moveed downward slowly at 30 um every time, until spontaneous discharge appeared. The ratio of amplitude for discharge signal and noise was required at least more than 4:1. The threshold level of action potentials was carefully chosen to make sure that the signals for spike classification had no noise mixed in. Electrical signals were amplified by a multi-channel microelectrode amplifier, and filtered (HF 1K Hz, LF 300 Hz). All the discharges were on-line real-time processed by a spike sorting technique.Results:(1)Three types of discharge pattern were found, including regular, irregular and bursting; the average discharge frequency in control group was 5.55±0.64 Hz (n=49). (2) The average frequency of the spontaneous discharges in the CA1 area in NBM-lesion rats induced by KA infusion was significantly lower than that in control rats, being 3.15±1.09 Hz (n=50, p<0.05), which was observed not only in regular firing, but also in burst firing and irregular firing. (3) In the NBM-lesion plus 500μM (2μl) Choline group, the average frequency of the spontaneous discharges in the CA1 area significantly increased, being 4.34±0.87 Hz (n=50, p<0.05), larger than that in the NBM-lesion alone group, but without change in firing pattern. (4) The percentage of burst firing in NBM-lesion rats, however, had a significantly increase compared to control, from 21% to 46%(p<0.05).Conclusion:NBM-lesion decreased the average frequency and changed the firing pattern of spontaneous discharges in the hippocampal CA1 area. Acute intracerebroventricular (i.c.v.) injection of Choline in NBM-lesion rats significantly increased the spike frequency of neurons in CA1 area but without altering its firing pattern. These results suggest that cholinergic system in NBM is involved in the control of neuronal activities in hippocampal circuits, and the NBM-lesion-induced alteration in hippocampal discharges may be linked to the impairment of learning and memory in AD.
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