The Role Of Endogenous Orexins In The Establishment,Extinction And Recovery Of Conditioned Sucrose-seeking Reflexes In Rats

Objective:To investigate the role of endogenous orexin in the establishment,extinction and recovery of the conditioned sucrose-seeking in rats.Methods:1.The distribution of orexin-1 receptor in hypothalamus was observed by fluorescence immunohistochemistry.2.Fixed ratio responding for sucrose pellets experiment: Rats were trained to lever press for sucrose reward(45 mg sucrose pellets)on a fixed ratio(FR)(active press the 1 lever,you can obtain sucrose reward),trainig 1 hours a day.Presses on the inactive lever had no sucrose reward.Pellet delivery was accompanied by a light + tone compound cue(78 d B,2900 Hz;white stimulus light),and lasts 20 seconds.Rats were injected of the drug(such as SB-334867)into intracerebroventricular,and after 30 min,the effects of SB-334867 on the conditioned reflex were observed.All rats take conditioned reflex training daily before the 30 min intracerebroventricular injection of the drug,which lasted for 10 days.Intracerebroventricular injection of saline was used as the control group.3.Progressive ratio responding for sucrose pellets experiment: In the last 24 hours after FR training,the rats were transferred to the progressive ratio(PR)rats were trained to press a lever to get the reward of sucrose(45 mg sucrose particles),the number of press a lever to reach the required ratio will reward a sucrose.Generally,a stable baseline of PR reaction can be established after 6 tests.The rats were injected of drugs into the intracerebroventricular(such as SB-334867)30 minutes before the experiment,and then the PR test was carried out.4.The extinction and recovery of conditioned sucrose-seeking reflexes experiment: rats were trained to lever press for sucrose reward(45 mg sucrose pellets)on a fixed ratio(FR)(active press the 1 lever,you can obtain sucrose reward).After the conditioned reflex was established,the rats were placed in closed boxes with no sucrose reward and no a light + tone compound cue,namely,when the rats pressed the lever effectively,neither sucrose reward nor a light + ton ecompound cue appeared.These sessions continued until rats met the criteria oftwo consecutive sessions with <25 active lever presses each.This is defined as the extinction of the conditioned reflex.24 h conditioned reflex disappeared,after intracerebroventricular injection of drugs 30 minutes,the rats were tested for recover active lever presses get the sucrose reward by delivering the a light + tone compound cue.That is,when the rats pressed the lever,there was a a light + tone compound cue.The rats were observed to recover active press the lever for sucrose reward under the a light + tone compound cue,but no sucrose reward was provided at this time.Results:1.Immunohistochemistry staining showed that orexin-1 receptor positive cells expression in the hypothalamic arcuate nucleus(ARC),the lateral hypothalamic area(LHA),dorsomedial hypothalamic nucleus(DMD)and the hypothalamic paraventricular nucleus(Pv)so on.2.Thefixed ratio responding for sucrose result show that during the last 10 days of the reflex training,compared with the ad libitum-fed rats,the number of active lever presses,the number of inactive lever presses,and the number of sucrose pellets increased significantly in food-restricted rats(P<0.05).Compared with first days,the number of active pressing were increased on test days 2-10 in ad libitum-fed rats(P<0.05).The number of sucrose pellets were increased on test days 2-10 in food-restricted rats and ad libitum-fed(P<0.05)..The intracerebroventricular injection of SB-334867(15 nmol,25 nmol or 35 nmol)can significantly reduce the number of active lever presses in the food-restricted rats,and showed a dose dependence(P<0.05-0.001);the intracerebroventricular injection of high dose SB-334867(35 nmol),can reduce the number of sucrose pellets in the food-restricted rats(P<0.05).3.Progressive ratio responding for sucrose experiment show that there was no significant difference in PR breakpoints between the ad libitum-fed rats and the food-restricted rats(P>0.05).After the intracerebroventricular injection of SB-334867(15 nmol,25 nmol or 35 nmol),PR breakpoints in the ad libitum-fed rats and the food-restricted rats have no significantly change(P>0.05).4.The extinction and recovery of conditioned sucrose-seeking reflexes experiment show that the OX1 R receptor antagonist SB-334867 was injected into the lateral ventricles during the extinction phase,which significantly reduced the number of active lever presses in the food-restricted rats and the ad libitum-fed rats(P<0.05).However,there was no significant difference in the number of effective levers between the food-restricted rats and the ad libitum-fed rats(P>0.05).The present study found that in reinstatement training,the intracerebroventricular injection of the OX1 R receptor antagonist,SB-334867,significantly reduced the number of active lever presses in the food-restricted rats and the ad libitum-fed rats(P<0.05).Moreover,compared with ad libitum-fed rats,the number of active lever presses increased significantly in the food-restricted rats(P<0.05).Conclusion:Endogenous orexins is involved in the establishment,extinction,and recovery regulation of conditioned feeding reflex in rats,and this regulation process is related to food-restricted.The hypothalamus may be one of the sites in which orexins plays important role.