The Regulation Of Ventrolateral Preoptic And Tuberomammillary On Sleep-wake Cycle And Its Receptor Passways In Rats

ObjectiveVentrolateral preoptic area(VLPO) and tuberomammillary nucleus(TMN) are acknowledged sleep-promoting and wake-promoting centrals, respectively. They regulate the sleep-wake cycle in a mutual restriction way similar to a flip-flop. Gamma-aminobutyric acid(GABA) and glutamic acid are critical neurotransmitte in central nervous system. The aim of this study was to further explore and interpret the mechanisms of VLPO-TMN in regulating sleep. Some techniques and methods were used to observe sleep-wake cycle and its receptor passway in the rat, including polysomnography, brain stereotaxic, nucleus spile, microinjection technologies, and immunohistochemistry.Methods1. GroupingSprague-Dawley rats were used in three parts according to the overall experimental design.1.1 Polysomnography recording and analysisRats were randomly divided into control group [Both VLPO and TMN were microinjected with artificial cerebrospinal fluid(ACSF), called VLPO+ACSF & TMN+ACSF group], experimental group ?(L-Glu was microinjected into VLPO while ACSF was microinjected into TMN, called VLPO+L-Glu & TMN+ACSF group) and experimental group П(L-Glu was microinjected into VLPO while Bic was microinjected into TMN, called VLPO+L-Glu & TMN+Bic group).1.2 Detecting the expressions of m Glu R5 in VLPO and GABAARb1 in TMN by immunostaining.1.3 Observing the changes of c-Fos expression in TMN after VLPO was excited.Rats were randomly grouped control treatment(VLPO was microinjected with ACSF, VLPO+ACSF treatment) and experimental treatment(VLPO was microinjected with L-Glu, VLPO+L-Glu treatment).2. Animal model establishmentHealthy adult Sprague-Dawley rats(weighted 290±10 g) were selected. Reagents and apparatus needed in the operation were prepared before the start of the experiment. Head of the rat was fixed onto stereotaxic apparatus after the pentobarbital(50 mg/kg,i.p.) getting anaesthesia effect, then the skull was exposed. Two hollow stainless steel catheters were respectively inserted into VLPO(AP:-0.36 mm; R: 1.30 mm; H:-7.00 mm) and TMN(AP:-3.96 mm; R: 1.50 mm; H:-7.70 mm) for the administration under the instrument of Paxinos and Waston atlas. The guide cannulas and the recording electrodes were fixed by Dental cement, while copper line was used to connect the mini-socket and electrodes which lead electroencephalogram(EEG) and electromyography(EMG). PS: the above operations were relatively simple, since only a stainless steel guide cannula was needed to insert into VLPO and the other steps can be left out, including the electrodes connection. The rats were then given a dose of antibiotic(penicillin) for recovery in a soundproof recording room after the wound had been sutured. Animals were provided with adequate food and water and the recovery situation was observed at any moment.3. Microinjection of drugsDrugs/ACSF(1 μl) was microinjected into VLPO/TMN via stainless steel guide cannulas at the speed of 1 μl/min with Hamilton micro syringe(tip diameter 26-gauge), therefore, the syringe needle should be left in nuclei for 1 min to make sure the physic liquor overflow would not be happened. Drug administration was performed between 22:00 to 22:20 for polysomnography and the c-Fos expression changes were observed between 20:00 to 20:20.4. PolysomnographyThe recording started at 2 hours before administration and then sustained for 24 hours. Ten seconds were regarding as one epoch to separate sleep-wake cycle of the rats into 3 stages: wakefulness(W), non-rapid eye movement(NREM) sleep, and rapid eye movement(REM) sleep. Total sleep time(TST) was defined as the sum of NREM and REM.5. MaterialsAfter the rat was anaesthetized intraperitoneally, the chest was opened and the heart was explored. The perfusion needle from the apex into the heart through the left ventricle to aorta, and then cut auricula dextra, rapidly infuse physiological saline about 250 ml until the liver became white. Slowly infuse 4% paraformaldehyde about 600 ml, then cut off the head and break out the brain with forceps hamostatic after strip off the skull carefully. Cut the brain into pieces of 0.5 cm with blade and put it into 4% paraformaldehyde for 6 hours.6. ImmunohistochemistryBrain parafin sections of 4 mm thickness were made which contain the objective nuclei, dewaxed with Xylene and hydrating with ethyl alcohol after roasting.Immunohistochemical kits were used for testing and the staining results were watched under a microscope.7. emi-quantitative analysisIntegrated optical density(IOD) of TMN reflects the c-Fos protein expression level.8. Statistical analysisSPSS software(version 17.0) was used for the statistics analysis. The data was presented as mean ± SEM(x ± s). Multiple comparisons between each group were analyzed in one-way ANOVA and T test. The differences at P< 0.05 level were considered in significance.Results1. The effects of microinjecting ACSF/L-Glu into VLPO and microinjecting ACSF/Bic into TMN on electroencephalogram in rats1.1 Compared with the VLPO+ACSF & TMN+ACSF group(n = 8), wakefulness amount was declined about 42.2%(38.13 ± 3.17, P < 0.01) along with the enhancement of NREM sleep about 89.5%(52.90 ± 2.98, P <0.01) and enhancement of TST about 80.5%(69.33 ± 3.63, P < 0.01) within 2 hours after microinjected L-Glu in VLPO while no significant change in REM sleep in VLPO+L-Glu & TMN+ACSF group(n = 7).1.2 Compared with the VLPO+L-Glu & TMN+ACSF group(n = 7), the amount of wakefulness was increased about 81.1%(66.12 ± 3.26, P < 0.01); the amounts of REM, NREM and TST were declined about 58.9%(4.94 ± 1.02, P < 0.05), 50.2%(39.62 ± 2.93, P < 0.01) and 56.1%(47.11 ± 4.14, P < 0.01) respectively, in the second and third hours after microinjected L-Glu in VLPO of and Bic in TMN in the VLPO+L-Glu & TMN+Bic group.1.3 Compared with the VLPO+ACSF & TMN+ACSF group, the wakefulness and REM/NREM sleep of the rats in VLPO+L-Glu & TMN+Bic group had no significant changes.2. Immunohistochemistry results2.1 The expression of mGluR5 in VLPOCompared with the control group treated with PBS, m Glu R5 positive expression in VLPO was increased significantly: the brown immunostaining and positive protein located in cytoplasmic membrane with the blue nucleus. Cell structure is clear and positive control is obvious.2.2 The expression of GABAAR?1 in TMNGABAAR?1 in TMN expressed strongly with the brown or yellow immunostaining and positive protein located in cell membrane with the blue nucleus, compared with the PBS control group. Cell structure is clear and positive control is obvious.3. c-Fos expression in TMN after VLPO neuron excitedIn comparison with the VLPO+ACSF group(53.98 ± 2.02), the integrated optical density of c-Fos was significantly decreased in the VLPO+L-Glu group(21.82 ± 1.22) after VLPO neuron excited( t =11.10, P < 0.01).ConclusionThe metabotropic glutamate receptor in VLPO and GABA receptor in TMN mediate the regulation of VLPO-TMN passway. VLPO inhibits the TMN neuronal activity via GABAAR.