| Sleep plays an indispensable physiological role in lots of aspects of our lives,such as growth and development, learning and memory, metabolism, survival adaptation, immune, restoration of energy, etc. Yet, the underlying mechanism of initiation or maintenance of sleep has not been clarified clearly. While on the whole,with advances in science and technology, the research of sleep mechanism has gained a lot of achievement and researchers established many significant theories, such as passive sleep, sleep center, sleep factor, and so on. The related researches show that neural mechanism is the mainstream of research orientation. Its content mainly involves activities of neural network and biochemical small molecules, in which activities of neural center are the top priority. As a result, the central mechanism of sleep is always taken as a hot spot of research. And in recent years, many researches found that the ventrolateral preoptic area(VLPO) in hypothalamus is vital to the initiation and maitenance of sleep. VLPO is located in the hypothalamic preoptic area(POA), which is adjacent to the basal forebrain(BF). Neurons in VLPO are mainly?-GABAergic and galaninergic. Most of these neurons cluster together to form the VLPO cluster, and other neurons are scattered dorsally and medially to the VLPO cluster to form the extended VLPO(eVLPO). There are complicated reciprocal projections between VLPO and the ascending arousal system(AAS), and VLPO also receives few projections from the suprachiasmatic nucleus(SCh), which is the control center of circadian rhythm. In early electrophysiological study, electrical stimulation to the basal forebrain synchronizing area as the close neighbour of VLPO in behaving cats resulted in behavioral and electroencephalographic manifestations of sleep.Cell-specific damage to VLPO of rats by ibotenic acid caused severe insomnia for at least 3 weeks persistently. Neuronal activities of VLPO become stronger during sleeping time and become weaker during waking time in rats. Fragmented sleep in older adults has a significant correlation with the decrease of neuron numbers of VLPO. On the basis, Clifford B. Saper et al. put forward the “flip-flop switch”hypothesis that VLPO functions in the onset and maintenance of sleep as the mode of“flip-flop switch” in electrical engineering. However, the causal role of VLPO in sleep implied in the hypothesis is still uncertain. In the natural process, whether stronger activities of VLPO neurons triggered the sleep or sleep activated VLPO? Or,all collected data about activities of VLPO when sleeping are just the manifestation of sleeping? Questions above need to be studied and discussed further. In this study, we stimulated and damaged VLPO by electric in rats, using the method with chronic electrode. The data of electrocorticogram(ECoG) and animal behaviors were collected to analyze the relationship between VLPO and sleep, so as to provide some basic research data to reveal the neural mechanism of sleep.260-360 g adult male SD rats were used as experimental subjects, and they were divided randomly into control group and experimental group. Animal feeding,experimental operations, and data recording were all finished in the quiet, clean and light-controlled basement(lights on from 08:00:00-20:00:00 and off from20:00:00-08:00:00). Experimental chronic electrodes were self-designed metal eclectrodes. Stimulating electrodes were made of 180 ?m stainless steel acupuncture needles after the needle body were insulated by insulating varnish except for the tip,and recording electrodes of ECoG were made of stainless steel flat-head spectacle screws. In surgery, anesthetized rats(intraperitoneal injection of pentobarbital sodium,40 mg/kg) were fixed with stereotaxic apparatus. Four screw electrodes were implanted into the skull to touch the dura mater over the frontal cortex( × 2) and parietal cortex( × 2) to record the ECoG, and a screw electrode was threaded into nasal bone as the reference electrode for ground connection. According to the grouping design(table 1 and table 2), a pair of stimulating electrodes were implanted into brain parenchyma. All electrodes were anchored to the skull with dental cement.Implanted electrodes were connected with self-made lead connector, and both were fixed with dental cement. After 3 days of recovery, the lead connector was connected with physiological signal collecting and processing system of RM6280 c when the rats were anaesthetized lightly. The rats were in the shielding cabinet for adaptation for 1day, then the ECoGs of rats were recorded for 12 h-24 h, and the animal behaviors were videoed simultaneously. The next day, the rats were stimulated with “pattern 1”(pulses of unidirectional positive current; amplitude, 0.02 mA; pulse width, 0.5 ms;pulse interval, 200 ms; pulse number per cycle, 8; major cycle, 2s; cycle number, 3-6)when the animals showed the most active behaviors such as drinking during22:00:00-23:00:00, and ECoGs with real-time videoing of behaviors were collected all the time. When the stimulating experiment was finished, the stimulation of electrical damage was performed with “pattern 2”(pulses of unidirectional positive current; amplitude, 0.2 mA; pulse width, 1000 ms; pulse interval, 0.1 ms; pulse number per cycle, 10; major cycle, 2s; cycle number, 1) to the rats, and the recording of ECoG with real-time video of behaviors was collected for 12 h-24 h.After the data collecting was finished, the rat was perfused with 4%paraformaldehyde for fixation in deep anesthesia. And the brain was taken out for frozen section. Sections were stained with Nissl's method to examine or measure positions of stimulating eclectrodes and damaged areas by electric. ECoG data was processed with program of physiological signal collecting and processing system of RM6280 c, and the processed data was analyzed by statistical analysis software STATISTICA 6.0.The results of electrical stimulating experiment were as follows. 1. When the tips of bilateral stimulating electrodes were located in or very close to VLPOs, stimulation in the behaving rats resulted in reposing for at least 3 minutes after an action of stretching, and ECoG changed from low amplitude/ high frequency in waking to high amplitude/ low frequency in sleeping. 2. When the tip positions of bilateral stimulating eclectrodes were out of VLPOs at a distance, stimulation in the behaving rats resulted in no difference of behaviors, also there was no difference in ECoG.The results of electrical damage experiment were as follows. 1. In ECoGs in 12 h after the left VLPO was damaged, ? wave indicated deep sleep was decreased highly significantly, and ? wave indicated cortex excitation was increased highly significantly. 2. When there was no electrical damage area in the brain, there was no significant difference in ?/?/?/? wave within 48 h. 3. When the electrical damage area was out of VLPO, in 24 h, ? wave was increased highly significantly, and there was no significant difference in ? wave, and ? /? wave was decreased highly significantly.Through analyzing these results above, conclusions were drawn as follows:1. It is very likely that stronger activities of VLPO initiate the sleep, in other words, the activated VLPO is a cause of sleeping, and sleeping is the result.2. VLPO not only participates directly in the onset of sleep, but also has the vital role in the maintenance of deep sleep in rats.The results of this study provide some basic data for further understanding the neural mechanism of sleep activity. |
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