Oxytocin-mediated PVN-NAcSh Pathway Regulates Food Intake And Gastric Function In CUMS Model Rats

Objective:With the acceleration of life rhythm and the increase of survival pressure,more and more people have psychological stress symptoms.Oxytocin（OT）is a short peptide containing 9amino acids,which is mainly synthesized in the paraventricular nucleus（PVN）of the hypothalamus,the supraoptic nucleus（SON）of the hypothalamus and the bed nucleus of the stria terminalis.OT neurons in PVN can emit fiber projections to the ventral tegmental area（VTA）and nucleus accumbens（NAc）of the midbrain,and participate in the regulation of various physiological functions,such as reproduction,food intake,energy metabolism,emotion and analgesia.Under stress,the mental state and physical function of the body will change significantly,such as decreased food intake and gastrointestinal dysfunction.But under stress,especially chronic stress,does the central OT neuron network pathway,such as PVN-NAc OT pathway,change?Does OT participate in the regulation of food intake and gastric movement under stress in this pathway?And potential mechanism?None of them has been reported.In this study,we plan to construct a model of chronic unpredictable mild stress（CUMS）in rats,and use the experimental methods of retrograde tracing combined with fluorescence immunohistochemical staining,single cell discharge study,in vivo gastric movement,radioimmune ligand-receptor binding activity,and neural pathway activation regulation,Observe whether there is OT nerve or functional pathway between the nucleus accumbens shell（NAcSh）of PVN,and whether OT participates in food intake,gastric movement,and gastric exhaust air conditioning in this pathway.The differences and potential mechanisms between normal and CUMS rats were analyzed and compared.Methods:（1）Healthy male SD rats,construct CUMS model rats.Rats were given seven different stimuli:（1）fasting for 24 hours;（2）No water for 24h;（3）Apply wet padding in 24h;（4）4℃cold water forced swimming for 5min;（5）45℃warm water forced swimming for5min;（6）Day and night are reversed（8:00-20:00 darkness,20:00-8:00 light）;（7）One side of the cage was raised at 45°for 24h.Each kind of stimulation was only given once a week,and the order of stimulation conditions was different.The rats were weighed after the end of stimulation in the first,third,fifth and seventh weeks.After 7 weeks,if the weight of the rats decreases and the weight loss exceeds 30%of the weight of the rats in the control group,the modeling will be considered successful.（2）The formation of PVN-NAcSh OT nerve pathway was observed by retrograde fluorogold tracing combined with fluorescence immunohistochemical staining;The expression of OT-R immunoreactive neurons in NAcSh was observed by fluorescence immunohistochemistry and Western blot,and the differences of PVN-NAcSh OT neural pathway between normal and CUMS rats were analyzed and compared.（3）The effects of electric stimulation of PVN on the change of OT content in NAcSh were observed by high-performance liquid chromatography-electrochemical method,which further confirmed that there was OT nerve pathway between PVN-NAcSh.（4）NAcSh microinjected OT to observe the changes of food intake,gastric movement and gastric emptying in rats,and analyze and compare the differences between normal and CUMS rats.（5）Using electric stimulation of PVN,NAcSh microinjection of OT receptor antagonist Atosiban and other drugs,the regulation of PVN-NAcSh OTergic pathway on feeding,gastric motility and gastric emptying in rats was observed,and the differences between normal and CUMS rats were analyzed and compared.（6）The distribution of gastric afferent neurons in NAcSh and the effect of OT between PVN-NAcSh on the discharge activity of these gastric stretch response neurons（GD-N）were observed by gastric stretch and single neuron extracellular discharge recording,and the differences between normal and CUMS rats were analyzed and compared.（7）The binding activity of OT and OT receptor（OT-R）in NAcSh of normal and CUMS rats was compared by radioligand receptor binding method.Results:1 The composition of PVN-NAcSh OT nerve pathway in normal and CUMS rats:under the fluorescence microscope,there are FG-labeled neurons in the PVN of rats,and OT fluorescence immunohistochemistry staining is performed on the same section.The results show that some FG-labeled neurons in the PVN also present OT immunoreactivity;Compared with normal rats,there was no significant difference in the number of FG labeled neurons in PVN of CUMS rats（P>0.05）,but PVNOT immunoreactive neurons（P<0.05）and OT/FG（P<0.05）were significantly increased in CUMS rats.2 Expression of NAcSh OT-R immunoreactive neurons:The results of fluorescent immunohistochemistry showed that OT-R immunoreactive neurons were expressed in NAcSh of normal rats and CUMS rats;Compared with normal rats,the expression of OT-R immunoreactive neurons in CUMS rats increased significantly（P<0.05）.3 PVN OT and NAcSh OT-R protein expression:Western blot results showed that OT protein was expressed in PVN of normal or CUMS rats,and OT-R protein was expressed in NAcSh;Compared with normal rats,the expression of OT protein in PVN（P<0.05）or OT-R protein in NAcSh（P<0.05）in CUMS rats was significantly increased.4 Effect of OT on rat food intake:NAcSh microinjection of OT significantly reduced rat food intake in 0-2 h（P<0.05）.If NAcSh was injected with the OT receptor antagonist Atosiban in advance,the OT feeding inhibition effect was significantly reduced（P<0.05）,but the injection of Atosiban alone had no significant effect on the feeding of rats（P>0.05）.Compared with normal rats,the food intake of CUMS rats was significantly reduced（P<0.05）,and the inhibition of NAcSh microinjection of OT on the 0-2 h food intake of CUMS rats was significantly stronger than that of normal rats（P<0.05）.5 The PVN-NAcSh OT pathway regulates the feeding of rats:after electrical stimulation of PVN(PVN_ES),the feeding amount of normal or CUMS rats in 0-2 h decreased significantly（P<0.05）,but the feeding amount in 2-4 h did not change significantly（P>0.05）.If the PVN(PVN_ES+NAcSh _Atosiban)was electrically stimulated after the microinjection of Atosiban with NAcSh,compared with the control group(PVN_ES+NAcSh _NS),the 0-2 h food intake of normal or CUMS rats was significantly increased（P>0.05）.It is suggested that PVN-NAcSh has direct or indirect OT pathway involved in acute feeding regulation of rats.The analysis of change rate of food intake showed that in CUMS rats,the inhibitory effect of electrical stimulation of PVN on food intake was significantly stronger than that of normal rats（P<0.05）.6 The effect of OT on gastric motility in rats:compared with NS control group,the amplitude of gastric motility（P<0.05）and the frequency of gastric motility（P<0.05）in normal or CUMS rats were significantly decreased after microinjection of OT with NAcSh.If NAcSh was injected with Atosiban,the inhibitory effect of OT on gastric motility could be significantly reduced（P<0.05）.The results of gastric motility index analysis also showed that NAcSh microinjection of OT could significantly reduce the MI%of rats（P<0.05）,but NAcSh alone injection of Atosiban had no significant effect on the MI%of rats（P>0.05）.Compared with normal rats,OT in NAcSh has a more significant inhibitory effect on gastric motility in CUMS rats（P<0.05）.7 The regulation of PVN-NAcSh OT pathway on gastric motility in rats:the amplitude（P<0.05）or frequency（P<0.05）of gastric motility in normal or CUMS rats were significantly decreased by electrical stimulation of PVN.Similarly,if NAcSh was pre-injected with Atosiban and then electrostimulated PVN,the inhibitory effect of electric stimulation on gastric motility was significantly reduced,which showed that the amplitude of gastric motility（P<0.05）or the frequency of gastric motility（P<0.05）was significantly higher than that of NS control group.The results of MI%analysis showed that the MI%of normal（P<0.05）or CUMS（P<0.05）rats significantly decreased after electrical stimulation of PVN;Similarly,the MI%of normal（P<0.05）or CUMS P<0.05)rats was significantly increased after pre-NAcSh injection of Atosiban and electrical stimulation of PVN.It is suggested that PVN-NAcSh OT pathway may also participate in the regulation of gastric motility in rats.The analysis of the change rate of MI%showed that the inhibitory effect of electrical stimulation of PVN on the MI%of CUMS rats was more significant than that of normal rats（P<0.05）.8 Effect of OT on gastric emptying in rats:compared with NS control group,the gastric emptying rate of normal or CUMS rats was significantly decreased by microinjection of OT with NAcSh（P<0.05）;Similarly,Atosiban could weaken the inhibition of gastric emptying induced by OT（P<0.05）.NAcSh alone injected with Atosiban had no significant effect on gastric emptying in rats（P>0.05）.The analysis of the change rate of gastric emptying showed that the inhibitory effect of OT on gastric emptying in CUMS rats was significantly stronger than that in normal rats（P<0.05）.9 PVN-NAcSh OT pathway controlled gastric emptying in rats:compared with sham electric stimulation group,electric stimulation of PVN could significantly reduce gastric emptying in normal（P<0.05）or CUMS（P<0.05）rats.Pre-injection of NAcSh Atosiban could attenuate the effect of gastric emptying induced by electrical stimulation（P<0.05）.It is suggested that PVN-NAcSh OT pathway may also be involved in the control of gastric discharge in rats.The analysis of the change rate of gastric emptying showed that the inhibitory effect of electrical stimulation of PVN on gastric emptying in CUMS rats was significantly stronger than that in normal rats（P<0.05）.10 Effect of OT on the excitability of gastric stretch afferent neurons:The above results show that NAcSh OT is involved in the regulation of feeding or gastric motility in rats.Does NAcSh have gastric afferent information-related neurons?Therefore,this study further used single neuron extracellular discharge recording to observe whether there are gastric stretch response（GD）neurons in NAcSh and the regulation of OT on its discharge activity.The results showed that there were GD neurons in NAcSh of normal and CUMS rats.After microinjection of OT,the excitability of most GD-E neurons（70.53%,normal rats;75.47%,CUMS rats）increased significantly（P<0.05）,and the discharge frequency of GD-I neurons（68.49%,normal rats;75.00%,CUMS rats）decreased significantly（P<0.05）.If NAcSh was injected with Atosiban in advance and then OT was administered,the excitatory/inhibitory effect of OT on the discharge activity of GD neurons could be significantly reduced（P<0.05）,but the excitability of GD neurons was not significantly affected by injection of Atosiban alone（P<0.05）.Compared with normal rats,OT had significantly stronger excitatory/inhibitory effects on the discharge activity of GD-E/GD-I neurons in NAcSh than CUMS rats（P<0.05）.11 Effect of electrical stimulation of PVN on the expression of NAcSh OT content:The results of high-performance liquid chromatography-electrochemical method showed that electrical stimulation of PVN significantly increased the content of OT in rat NAcSh（P<0.05）.12 Comparison of OT receptor and OT ligand affinity between normal and CUMS rats:The study of receptor-ligand binding activity showed that the binding activity of NAcSh OT and its specific OT-R in CUMS rats was significantly higher than that in normal rats（P<0.05）.Conclusion:There are OT neural and functional pathways between PVN-NAcSh.In this pathway,OT can inhibit food intake,gastric motility and gastric emptying by activating its receptor signal system;This regulatory effect is more significant in CUMS rats,which may be related to the increase of OT receptor expression and OT receptor-ligand binding capacity in NAcSh.