Effect Of Gaba Pathway In The Zona Incerta To The Nucleus Accumbens On Gastric Functions And Food Intake In Rats And Its Underlying Mechanism

ObjectiveEnergy metabolism and obesity related diseases seriously threaten human health,and over intake of energy is one of the main causes of obesity.The central nervous system is not only directly involved in the regulation of the peripheral digestive system,but also can further influence the feeding behavior through the feeding "reward" pathway,so as to regulate energy metabolism.Nucleus accumbens(NAc)is an important part of the central reward pathway,which can regulate many complex physiological behaviors such as stimulation motivation,learning and reward behavior.It is found that there are a large number of GABA neurons in NAc,which can regulate the energy balance in vivo,especially the feeding behavior.In addition,recent studies have found that the zona incerta(ZI)of thalamus is closely related to feeding behavior,and is another important nuclear group involved in feeding and gastric function regulation.According to the study of photogenetics,the GABA neurons in the ZI stimulated by light can lead to overeating or forced eating behavior in mice.Glucagon like peptide 1(GLP-1)can significantly inhibit gastric emptying,reduce intestinal peristalsis,and then inhibit food intake,which is also one of the important targets for the treatment of obesity.It has been reported that GLP-1 neurons project widely in the center,and their fibers can project to the whole brain,including the NAc.In this study,we intend to use the methods of retrograde tracing combined with fluorescence immunohistochemical staining,recording of single extracellular discharge,microinjection of drugs into nuclei,recording of gastric motility in vivo,to observe the GABA pathway between the zona incerta to the nucleus accumbent(ZI-NAc).The purpose of this study is to investigate whether GABA regulates gastric function and feeding through the ZI-NAc pathway and its underlying mechanism.Method 1.The male Sprague Dawley(SD)rats of 10-12 weeks old were selected;2.Five rats were randomly selected to observe the GABA pathway between ZI-NAc by retrograde tracing and fluorescence immunohistochemistry;3.The effects of microinjection of GABA or biculline(Bic)on the activity of reactive GLP-1 neurons in rats with gastric stretch(GD)were observed by single extracellular recording,and the changes of the activity of reactive GLP-1 neurons in rats with GD were observed by electrical stimulation of the ZI or pre-injection of GABA-A receptor antagonist Bic into the NAc;4.According to the brain atlas of rats,the tube was placed in the NAc of rats.The effects of microinjection of GABA or Bic into the NAc on the frequency and amplitude of gastric motility,the amount of gastric acid secretion and the 0-2h cumulative food intake were observed by the in vivo gastric motility record,the determination of gastric acid secretion and the analysis of food intake.Experimental design and grouping: 48 rats with the NAc catheterization were randomly divided into 8 groups(n = 6):(1)0.5 ?L saline group(NS group);(2)0.5 ?g GABA group;(3)2.5 ?g GABA group;(4)5 ?g GABA group;(5)10 ?g Bic group;(6)10 ?g GLP-1 receptor(GLP-1R)antagonist exendin9-39 group(10 ?g Ex9 group);(7)10 ? g Bic + 2.5 ?g GABA group;(8)10 ?g Ex9 + 2.5 ?g GABA group.The changes of gastric motility,gastric acid secretion and 0-2 h food intake were observed;In addition,10 rats were randomly divided into two groups(n = 5).At the same time,the changes of gastric motility in vivo were observed when GABA was microinjected into the NAc,and NS was injected into the NAc as the control group.5.The frequency and amplitude of gastric motility,the amount of gastric acid secretion,and the cumulative intake of 0-2 h were observed in rats with the NAc and the ZI catheterization according to the brain atlas.Experimental design and grouping: 36 rats were randomly divided into 6 groups(n = 6):(1)sham electric stimulation(SS)group;(2)electrical stimulation the ZI(ES)group;(3)sham electric stimulation + microinjection of 0.5 ?L NS(SS + NS)in the NAc group;(4)electrical stimulation the ZI + microinjection of 0.5 ?L NS(ES + NS)in the NAc group;(5)sham electric stimulation + microinjection of 0.5 ?L 10 ?g GABA-AR antagonist Bic in the NAc group(SS + Bic);(6)electrical stimulation the ZI + microinjection of 0.5 ?L 10 ?g GABA-AR antagonist Bic in the NAc group(ES + Bic).Result 1.The results of retrograde tracing with Fluorogold(FG)and immunocytochemistry showed that 0.2 ?L 3% Fluorogold was microinjected into the NAc.After 7 days,FG labeled neurons in the ZI were observed under fluorescence microscope.GABA immunocytochemistry was performed on all the slices.Under the microscope,GABA immunoreactive neurons were expressed in the ZI,and some of them coexisted with FG.It is suggested that part of GABA immunoreactive neurons in the ZI can project nerve fibers to the NAc,that is,there may be GABA pathway between the ZI and the NAc.2.The effect of GABA microinjection into the NAc on the discharge activity of GLP-1 / GD-R neurons showed that there were GD neurons in the NAc;most of GLP-1 / GD-E or GLP-1 / GD-I neurons decreased significantly after GABA microinjection into the NAc(P < 0.05),while the inhibition of GABA on the discharge activity of GLP-1 / GD-R neurons could be significantly reduced by injection of Ex9,an antagonist of GLP-1R in the NAc in advance(P < 0.05).It is suggested that there are GD neurons receiving gastric stretch stimulation in the NAc,most of them respond to GLP-1(GLP-1 / GD-R neurons),GABA is involved in the excitability regulation of GLP-1 / GD-R neurons,and GLP-1 receptor signaling pathway is involved in the regulation of GABA.3.The effects of electrical stimulation ZI on the firing activity of NAc GLP-1 / GD-R neurons showed that most of the GLP-1 / GD-E and GLP-1 / GD-I neurons in the NAc could be excited by electrical stimulation ZI(P < 0.05).NAc Pre-injection of GABA-AR antagonist Bic could significantly inhibit the excitation effect of electrical stimulation ZI on these GLP-1 / GD-R neurons(P < 0.05).It is suggested that there may be a direct or indirect GABA pathway between the ZI and the NAc,which may be involved in the regulation of gastric afferent information.4.Compared with NS group,the gastric motility amplitude(P < 0.05-0.01)and frequency(P < 0.05-0.01)of rats increased significantly after 3 minutes of NAc microinjection of GABA.The effect of GABA on the amplitude and frequency of gastric motility lasted about 10 min.If the NAc was injected with Bic or Ex9,the promoting effect of GABA on gastric contractile amplitude(P < 0.05)and contractile frequency(P < 0.05)could be blocked or significantly weakened.However,the amplitude(P > 0.05)or frequency(P > 0.05)of gastric motility did not change significantly when the NAc was injected with Bic or Ex9 alone.It is suggested that exogenous GABA of the NAc can promote gastric motility in rats,which may be realized by activating GABA receptor signaling pathway,and GLP-1 receptor signaling pathway may also participate in the regulation of this process.5.The results showed that the frequency of GABA,GLP-1 / GD-E neurons(P < 0.05)or GLP-1 / GD-I neurons(P < 0.05)was significantly decreased after the NAc microinjection,and it was also found that after about 4 min,the amplitude of gastric motility(P < 0.05)and frequency of gastric motility(P < 0.05)also increased significantly,and the peak value lasted about 10 min.It is suggested that GLP-1 / GD-R neurons in the NAc may be involved in the regulation of GABA induced gastric motility enhancement.6.The effect of GABA microinjection into the NAc on gastric acid secretion in rats was studied,compared with NS group,the NAc microinjection of GABA can significantly increase the gastric acid secretion of rats(P < 0.05-0.01),and has a significant dose-dependent relationship;compared with GABA group,the NAc injection of GABA + Bic mixture or GABA + Ex9 mixture can block(P < 0.01)or reduce(P < 0.05)the gastric acid secretion of rats.However,when the NAc was injected with Bic or Ex9 alone,there was no significant change in gastric acid secretion(P > 0.05).It is suggested that exogenous GABA of NAc can significantly promote gastric acid secretion in rats,which may be related to the activation of GABA receptor signaling pathway,and GLP-1 receptor signaling pathway may also participate in the regulation of this process.7.The effect of microinjection of GABA into NAC on the 0-2 hour intake of rats was studied,compared with NS group,the NAc microinjection of GABA significantly increased the food intake of rats at 0-2 h(P < 0.05).Compared with GABA group,the NAc injection of GABA + Bic mixture or GABA + Ex9 mixture significantly reduced the food intake of rats at 0-2 h(P < 0.05).However,when the NAc was injected with Bic or Ex9 alone,there was no significant change in 0-2 h food intake(P>0.05).It is suggested that exogenous GABA of the NAc can significantly promote the feeding of rats at acute stage,and the activation of GABA and GLP-1 receptor signaling pathway is closely related to the feeding effect of GABA.8.The effect of the NAc injection Bic on the induction of gastric motility in vivo by electrical stimulation ZI in rats showed that compared with the sham electrical stimulation group(SS),the amplitude of gastric motility(P < 0.05)and frequency of gastric motility(P < 0.05)were significantly increased by electrical stimulation ZI,and the promoting effect lasted for about 10 minutes.However,the amplitude of gastric contraction(P < 0.05)and frequency of gastric contraction(P < 0.05)were significantly inhibited by pre injection of Bic into the NAc and electrical stimulation of the ZI(Bic + ES).Bic alone had no significant effect on gastric motility(P > 0.05).It is suggested that there may be GABAergic pathways involved in gastric motility regulation between the ZI and NAc.ConclusionIn the NAc,there are GD neurons which receive the information of gastric mechanical stretch stimulation.The excitatory changes of GD neurons may be related to the changes of gastric motility.There are GABA nerves and functional pathways between the ZI and the NAc,which can participate in the regulation of gastric motility,gastric acid secretion and food intake.The activation of GABA-A receptor is an important factor to achieve the regulation,and GLP-1 receptor signaling pathway also participates in the regulation of this process.