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Objective adenosine (adenosine, AD) and 5 - hydroxytryptamine (5-hydroxytryptamine ,5-HT) is now known to sleep - wake cycle regulation neurohumoral factors, the median preoptic nucleus (median preoptic nucleus, MnPN) is preoptic area brain regions important sleep regulation. In this study, stereotaxic, nuclei intubation, microdialysis, microinjection, meter, etc. polysomnography method MnPN District AD and 5-HT on rat sleep - wake cycle. Method 1. Modeling male adult SD rats of clean grade, weighing 280-320 g. Sodium pentobarbital (40 mg / kg) were anesthetized by intraperitoneal injection, installation EEG and EMG electrodes for polysomnography account will microdialysis or microinjection with a guide tube into the rat MnPN area (AP: 0.24 mm, L / R: 0.0 mm, H: 7.8 mm) for nuclei within the administration to use. Cannula and recording electrodes are fixed on the skull of dental cement and guide the EEG and EMG electrodes connected by wires to a miniature socket. Postsurgical week recovery placed soundproof chamber, the day before the connection record EEG / EMG leads to adapt to the recording state. (2) Animal grouping animals were randomly divided into four groups: artificial cerebrospinal fluid (artificial cerebrospinal fluid, ACSF) group, AD group nitrothiophenol purine nucleoside [S-(4-nitrobenzyl)-6-thioinosine, NBTI] group, 5 -HTP (5-hydroxytryptophan) group. 3 microdialysis infusion administered 1 week after the microdialysis probe into the guide tube, place the probe one days after infusion administration. 2 h before administration of pre-infusion artificial cerebrospinal fluid, administration time is 21:00 ~ 23:00. ACSF group prefilled ACSF, AD group prefilled 300μmol / L of AD, NBTI group prefilled 20μmol / L of NBTI, perfusion rate 2μl/min. 4 Drug microinjection through the guide tube to MnPN injection of 5-HTP (5-HTP group) or ACSF (control group), 1 min injection is completed, the needle 1 min to prevent liquid overflow. Before embarking on pseudo-injection experiment (only analog injection operation) two days. Delivery time is 08:50-09:00 5. Polysomnography data analysis taking into account both time tracings from 07:00, continuous recording of 24 h. To 10 s as a split time, the sleep - wake cycle is divided into three: (1) Awakening (W): with low amplitude fast wave EEG and EMG activity is characterized by distinct; (2) non-rapid eye movement sleep (NREM): to sleep spindle wave and the wave is characterized by high amplitude δ, EMG activity was significantly reduced; (3) rapid eye movement sleep (REM): with low amplitude fast wave is characterized, in addition to occasional muscle tics, but no obvious EMG activity. Total sleep time (TST) is the sum of NREM and REM. 6 Histological identification of the end of experiment, through the guide tube inserted to MnPN area unipolar stainless steel electrodes. Through the electrode to the microdialysis or microinjection sites through to 2.5 mA DC 10 s, line anode damage, to determine whether it is located in the bottom of the probe trajectory MnPN area, only the accurate positioning data for statistical rats. 7. Statistical analyzes were performed using SPSS 12.0 software for statistical analysis, data (x | ˉ) ± S that was used to compare two sample t-test, P lt; 0.05 was considered statistically significant. Results 1. MnPN District AD and NBTI microdialysis perfusion in rats significantly increased NREM sleep time, reduce the time of awakening. 2 sleep time after infusion AD in the second three hours, increased sleep time after perfusion NBTI in the third four-hour increases. 3. MnPN microinjection of 5-HTP can significantly increase the time of awakening, reducing time NREM sleep. Conclusions 1. Adenosine is a physiological sleep regulator, MnPN District adenosine with the promotion of the role of sleep, its role may be through its A2A receptor mediated. 2. MnPN area NBTI can be given to increasing concentrations of extracellular adenosine play a sleep-promoting effect. 3. 5-HT in the sleep - wake cycle regulation, MnPN District 5-HT has a role in promoting awakening, its effect may be via its 5-HT1B receptor-mediated.
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