The Regulation Of The LHA-NAcSh Orexinergic Neural Pathway On The Gastric Function And Reward-based Feeding In The High-Fat Diet Rats

Objective:To observe the orexinergic neural pathway from the lateral hypothalamic area(LHA)to the nucleus accumbens shell(NAcSh),and to explore the effects of the orexin-A neural pathway on the discharge of NAcSh gastric distension(GD)neurons,gastric function and reward-based feeding in rats and its potential mechanisms,and to compare the differences in the regulatory effects of the orexinergic neural pathway on the normal diet and high-fat diet rats.Methods:To establish the high-fat diet model,healthy male Wistar rats(180-200g)were selected and fed with high-fat diet for 6 weeks.The high-fat diet is composed of 20% protein,20%carbohydrate and 60% fat.The orexin-A neurons projection from the LHA to the NAcSh were observed via fluorescent gold retrograde tracing combined with fluoro-immunohistochemical staining.The expression of orexin-A immunopositive cells in LHA and OX1 R immunopositive cells in NAcSh of the normal diet and high-fat diet rats were observed and analyzed.The brain cannulae implantation and microinjection in the NAcSh were conducted,to observe the effects of orexin-A on the firing rates of GD reactive neurons,gastric motility,gastric secretion function and reward-based feeding behaviors in the normal diet and high-fat diet rats.Through the LHA electrical stimulation,the effects of endogenous orexin-A on the firing rates of GD neurons in the NAcSh,gastric motility,gastric secretion function and reward-based feeding behaviors in the normal diet and high-fat diet rats were monitored,and the effects of the pretreatment of orexin-A receptor 1(OX1R)inhibitor SB334867 were observed.Results:Fluorescent gold(FG)retrograde tracing combined with fluoro-immunohistochemical staining showed that FG labeled neurons can be found in the LHA 7 days after the FG microinjection into the NAcSh.Fluoro-immunohistochemical staining results showed that the orexin-A immunopositive neurons expressed in the same slice of the LHA and part of the orexin-A were co-labeled with FG.In addition,compared with the normal diet rats,there was no significant difference in the expression of orexin-A immunopositive cells in the LHA of the high-fat diet rats(P > 0.05),while the expression of OX1 R cells in the NAcSh was increased significantly(P < 0.05).The results of electro-physiological studies indicated that after NAcSh microinjection of orexin-A the firing frequency of gastric distraction excitatory neurons(GD-E)were significantly increased(P < 0.05),and the firing frequency of gastric distraction inhibitory neurons(GD-I)also had a marked increase(P < 0.05).Besides,the rates of change of the discharge frequency in the high-fat diet rats were significantly higher than the normal diet rats(P < 0.05).If the Orexin-1 receptor(OX1R)antagonist SB334867 was injected into NAcSh before administration of the orexin-A,the excitatory effect of orexin-A on GD neurons could be completely blocked(P < 0.05).Electrical stimulation of LHA could also enhance the firing frequency of the GD-E neurons and the GD-I neurons significantly(P < 0.05),and pre-administration of SB334867 could partly block the effect of the LHA electrical stimulation on the GD reactive neurons(P < 0.05).The results of gastric motility in vivo showed that orexin-A microinjection into the NAcSh or the LHA electrical stimulation could increase the amplitude and frequency of gastric contraction significantly(P < 0.05),and the effects could be blocked by SB334867 microinjection in the NAcSh(P < 0.05).Compared with the normal diet rats,the rate of change of the amplitude and frequency of gastric contraction in the high-fat diet rats were increased markedly(P < 0.05).The gastric emptying of the normal diet and high-fat diet rats significantly increased after orexin-A microinjection or the LHA electrical stimulation(P < 0.05),and the rate of change of gastric emptying of the high-fat diet rats was significantly more than that of the normal diet rats(P < 0.05).The positive effects of orexin-A microinjection or the LHA electrical stimulation could be blocked by the pretreatment of SB334867 in the NAcSh(P< 0.05).Gastric acid secretion had a marked increase after the orexin-A microinjection in the NAcSh or the LHA electrical stimulation(P < 0.05),while there was no significant difference in the gastric acid secretion between the normal diet rats and the high-fat diet rats(P > 0.05).Similarly,these effects could also be blocked by OX1 R blocker SB334867(P < 0.05).The results of conditioned place preference(CPP)related to reward-based feeding behaviors showed that the rats spent significantly more time in the palatable food compartment(P < 0.05)after microinjection of orexin-A or electrical stimulation of the LHA.In addition,after orexin-A microinjection,the time was significantly longer in the high-fat diet rats,while electrical stimulation of LHA could not cause significant difference between the normal diet rats and the high-fat diet rats(P > 0.05).Based on an analysis of the sweetened condensed milk(SCM)intake,it was concluded that the SCM consumption was significantly increased in rats by administration of orexin-A into the NAcSh or the LHA electrical stimulation(P < 0.05),especially in the high-fat diet rats,the increase of SCM intake was more significant(P < 0.05).These positive effects could also be blocked by pretreatment of SB334867 in the NAcSh(P < 0.05).Conclusion:There is an orexinergic pathway from LHA to NAcSh,which may have potential regulatory effects on the excitability of GD reactive neurons in the NAcSh,gastric motility,gastric acid secretion and reward-based feeding behaviors of the normal diet and high-fat diet rats,and these effects might be realized through the OX1 R signaling pathway in the NAcSh.The effects of the orexinergic pathway on the gastric motility and the palatable food intake were more significant in the high-fat diet rats,which might be related to the increased expression of OX1 R in the NAcSh.