伏隔核-下丘脑外侧区GABA和MCH神经元对肥胖大鼠奖赏性摄食的调控作用

Effects of nucleus accumbens GABA-lateral hypothalamic area MCH neural pathway on rewarding feeding

目的探讨伏隔核(the nucleus accumbens,NAc)-下丘脑外侧区(lateral hypothalamus,LHA)神经通路的γ-氨基丁酸(γ-aminobutyric acid,GABA)神经元和黑色素聚集激素(melanin-concentrating hormone,MCH)神经元对肥胖大鼠奖赏性摄食(可口食物甜炼乳)的调控作用。方法142只SPF级雄性Wistar大鼠按体质量匹配原则分为普通饮食(normal diet,ND)组(68只)和高脂饮食致肥胖(diet induced obesity,DIO)组(74只),分别给予连续8周的普通饮食和高脂饮食。8周后,随机选取6只DIO大鼠,采用荧光金逆行追踪结合荧光免疫组化方法观察NAc中GABA神经元到LHA中MCH神经元的神经投射。免疫荧光观察两组大鼠饱食状态摄取甜炼乳(奖赏性摄食)后LHA内的c-Fos和MCH表达(每组6只)。通过LHA核团微量注射GABA受体激动剂Musimol或GABA受体阻断剂Bicuculine,观察GABA在LHA对ND大鼠和DIO大鼠奖赏性摄食的影响(每组8只),并观察阻断MCH信号后奖赏性摄食量的变化(每组8只)。采用SPSS 17.0进行统计分析,多样本均数比较采用双因素方差分析及post hoc Bonferroni检验,两组间比较采用t检验。结果经8周高脂饮食造模后,DIO组大鼠饱食后对可口食物摄取量显著多于ND组[(12.52±2.29)mL,(7.45±1.23)mL,t=4.778,P<0.01]。荧光金逆行追踪结合荧光免疫组化方法结果显示,伏隔核GABA神经元发出神经纤维投射至下丘脑外侧区神经元,且下丘脑外侧区部分神经元GABAA受体与MCH共存。NAc-LHA通路对可口食物摄入量影响实验结果显示,大鼠组别和药物干预交互作用显著(F=9.869,P<0.01)。简单效应分析显示,ND大鼠LHA核团微量注射Musimol后可口食物摄取量显著少于注射生理盐水[(4.25±1.38)mL,(7.29±1.49)mL,P<0.01],而注射Bicuculine后可口食物摄取量显著性多于注射生理盐水[(10.72±2.11)mL,(7.29±1.49)mL,P<0.05]。DIO组大鼠LHA核团微量注射Musimol后可口食物摄取量显著少于注射生理盐水[(3.51±1.77)mL,(13.68±2.95)mL,P<0.01],而注射Bicuculine后可口食物摄取量与注射生理盐水差异无统计学意义[(14.83±3.44)mL,(13.68±2.95)mL,P>0.05]。阻断MCH信号对可口食物摄入量研究结果显示,ND大鼠的SNAP-94847和Bicuculine干预交互作用无显著性(F=1.468,P>0.05),SNAP-94847干预主效应显著(F=15.880,P<0.01),Bicuculine干预主效应显著(F=6.930,P<0.05)。侧脑室注射MCH受体阻断剂SNAP-94847,ND大鼠可口食物摄取量显著少于注射生理盐水[(4.78±1.72)mL,(7.63±2.77)mL,P<0.05],且不受LHA预先注射Bicuculine影响[(6.24±2.18)mL,(4.78±1.72)mL,P>0.05]。DIO大鼠的SNAP-94847和Bicuculine干预交互作用无显著性(F=0.006,P>0.05),SNAP-94847干预主效应显著(F=18.460,P<0.01),而Bicuculine干预主效应不显著(F=2.059,P>0.05)。侧脑室注射MCH受体阻断剂SNAP-94847,DIO大鼠可口食物摄取量显著少于注射生理盐水[(6.89±2.11)mL,(12.19±4.36)mL,P<0.05],且不受LHA预先注射Bicuculine影响[(8.72±2.26)mL,(6.89±2.11)mL,P>0.05]。结论伏隔核GABA能信号可调节下丘脑外侧区神经元MCH表达,肥胖大鼠LHA内MCH神经元对饱食信号敏感性下降,奖赏性摄食增多。

Objective To explore the effects of theγ-aminobutyric acid(GABA)neurons and melanin-concentrating hormone(MCH)neurons of the nucleus accumbens(NAc)-lateral hypothalamic area(LHA)neural pathway on the rewarding feeding(palatable food sweat condensed milk)in the obesity rats.Methods Total 142 male Wistar rats of SPF grade were divided into normal diet(ND)group(n=68)and high-fat diet induced obesity(DIO)group(n=74)according to the principle of body mass matching.The rats in the two groups were given normal diet and high-fat diet for 8 weeks.Eight weeks later,6 DIO rats were randomly selected to observe the nerve projection from GABA neurons in NAc to MCH neurons in LHA by fluorogold retrograde tracing combined fluorescence immunohistochemistry.And the expressions of c-Fos and MCH in LHA after ingestion of sweet condensed milk(rewarding feeding)were observed by fluorescence immunohistochemistry(6 rats in each group).GABA receptor agonist Musimol or GABA receptor antagonist Bicuculine was microinjected into the nucleus of LHA to observe the effect of GABA on rewarding food intake in ND and DIO rats(n=8 in each group),and the changes of rewarding food intake after blocking MCH signal(n=8 in each group).SPSS 17.0 was used for statistical analysis,two-way ANOVA and post hoc Bonferroni test were used for comparison among multiple groups,and t-test was used for comparison between two groups.Results After 8 weeks of high-fat diet modeling,the intake of delicious food in DIO rats was significantly higher than that in ND rats((12.52±2.29)mL,(7.45±1.23)mL,t=4.778,P<0.01)after satiety.The results of fluorogold retrograde tracing combined with fluorescence immunohistochemistry showed that GABA neurons in NAc projected nerve fibers to neurons in LHA,and GABAA receptors in some neurons in LHA coexisted with MCH.The results of NAc-LHA pathway on delicious food intake showed that the interaction between rat group and drug intervention was significant(F=9.869,P<0.01).Simple effect analysis showed that the intake of delicious food after microinjection of Musimol into LHA nucleus of ND rats was significantly lower than that of microinjection normal saline((4.25±1.38)mL,(7.29±1.49)mL,P<0.01),while the intake of delicious food after injection of Bicuculine was significantly higher than that of microinjection normal saline((10.72±2.11)mL,(7.29±1.49)mL,P<0.05).The intake of delicious food after microinjection of Musimol into LHA nucleus in DIO group was significantly lower than that of microinjection normal saline((3.51±1.77)mL,(13.68±2.95)mL,P<0.01),but there was no significant difference between microinjection Bicuculine and microinjection normal saline((14.83±3.44)mL,(13.68±2.95)mL,P>0.05).The results of blocking MCH signal on delicious food intake showed that the interaction effect between SNAP-94847 and Bicuculine intervention was not significant(F=1.468,P>0.05).The main effect of SNAP-94847 intervention was significant(F=15.880,P<0.01)and the main effect of Bicuculine intervention was significant(F=6.930,P<0.05).After intracerebroventricular injection of MCH receptor blocker SNAP-94847,the delicious food intake of ND rats was significantly less than that of injection normal saline((4.78±1.72)mL,(7.63±2.77)mL,P<0.05),and it was not affected by pre injection of Bicuculine in LHA((6.24±2.18)mL,(4.78±1.72)mL,P>0.05).In the DIO rats,the interaction effect between SNAP-94847 and Bicuculine intervention was not significant(F=0.006,P>0.05).The main effect of SNAP-94847 intervention was significant(F=18.46,P<0.01)and the main effect of Bicuculine intervention was not significant(F=2.059,P>0.05).After intracerebroventricular injection of MCH receptor blocker SNAP-94847,the delicious food intake of DIO rats was significantly lower than that of injection normal saline((6.89±2.11)mL,(12.19±4.36)mL,P<0.05),and it was not affected by pre injection of Bicuculine in LHA((8.72±2.26)mL,(6.89±2.11)mL,P>0.05).Conclusion GABAergic signal in NAc can regulate the expression of MCH in neurons of LHA.In the DIO rats,the sensitivity of MCH neurons in LHA to satiety signal decreases and the hedonic feeding increases.