The Regulation Of Calcium-sensitive Receptors In Basolateral Nucleus Of Amygdala On Feeding And Gastrointestinal Motility Of Mice And Its Mechanism

Objective:With the acceleration of the rhythm of life and the change of people’s diet,metabolic diseases have become a serious topic worldwide.Therefore,in-depth exploration of the regulatory mechanism of food intake and gastrointestinal function will help us to find targets to treating metabolic diseases more effectively.calcium sensing receptor(CaSR)is abundantly expressed in gastrointestinal mucosa and enteric nervous system and participates in the regulation of feeding and gastrointestinal motility by affecting hormone secretion and neurotransmitter release.The following studies demonstrate that CaSR is also expressed in feeding related brain areas,such as the hypothalamus and limbic system,but the effect of central CaSR on feeding and gastrointestinal motility has not been reported.Therefore,this study was to explore the effect of CaSR in the basolateral amygdala(BLA)on feeding and gastrointestinal movement and the potential mechanism was also studied.Methods:1.To observe the effect of microinjection of R568 in the basolateral amygdala(BLA)on the food intake of mice;2.Open field test,elevated plus maze test,forced swimming test,tail suspension test,and sucrose preference test were used to observe the changes in the anxiety and depression-like behavior in mice injected with R568 into the BLA;3.Enzyme-linked immunosorbent assay(ELISA)was used to observe the effect of microinjection of R568 on the synthesis of neuropeptide cholecystokinin(CCK)、neuropeptide Y(NPY)and neurotransmitters glutamate(Glu)、y-a minobutane(GABA)in BLA.4.Fluorescence immunohistochemical staining was used to observe the co-expression of CaSR/NPY or CaSR/VGLUT1 in BLA.5.Fluorescence immunohistochemical staining was used to observe the expression of cFos immunopositive neurons in paraventricular nucleus(PVN),lateral area(LHA)and arcuate nucleus(ARC)of hypothalamus after R568 microinjected into BLA.6.ELISA was used to observe the content of neuropeptides ghrelin,orexin,cholecystokinin(CCK),nesfatin-1,neurotransmitter serotonin(5-HT)and dopamine(DA)in hypothalamus after BLA microinjection of R568.7.ELISA was used to observe the content of DA in ventral tegmental area(VTA)and 5HT in dorsal raphe nucleus(DRN)after BLA microinjection of R568.8..ELISA was used to observe the effect of R568 microinjected into BLA on the synthesis of dopamine in hypothalamus and VTA after microinjection of ionic Glu receptor blockerMK-801 in ARC or VTA.9.To observe the effect of BLA microinjection of R568 on the intake of palatable food in mice.10.Fluorescence immunohistochemical staining was used to observe the co-expression of Dynorphin/c-Fos in ARC or GABA/c-Fos in VTA.11.To study the effect of R568 on the firing frequency of glucose sensitive neurons(GS)in the BLA,single-cell extracellular discharge recording was used.12.The effect of microinjection of R568 in BLA on gastric emptying and small intestinal propulsion rate was observed by the method of phenol red excretion experiment and small intestinal propulsion with carbon powder.Results:1.The food intake results showed that,compared with the DMSO group,the food intake decreased in 0-lh and 1-2 h in the R568 treated group with normal food(P<0.05),and the food intake in 2-4 h and 4-24 h was no significant difference(P>0.05).Food intake of delicious food decreased significantly in 0-2 h(P<0.05),but there was no significant difference in 2-4 h and 4-24 h(P>0.05).2.The behavioral test results showed that compared with the DMSO group,the mice in the R568 group had a significant reduction in the central movement distance in the open field,the dwell time in the central grid was significantly reduced(P<0.05)and the number of times and the residence time in the open arms of the elevated plus maze were significantly reduced(P<0.05);The immobility time of mice was significantly increased in forced swimming and tail suspension experiments(P<0.05);The percentage of sucrose preference decreased significantly in 0-2 h in the sucrose preference experiment(P<0.05).3.ELISA results showed that compared with the DMSO group,after microinjection of R568 into the BLA,NPY was significantly decreased(P<0.05),Glutamate was significantly increased(P<0.01),and there was no significant difference in CCK and GABA(P>0.05);5-HT was significantly increased,DA was significantly decreased(P<0.05),but there was no difference in ghrelin,orexin,CCK,and nesfatin-1(P>0.05)in the hypothalamus;DA was significantly decreased in VTA(P<0.05),but 5-HT had no obvious change in DRN(P>0.05);While pre-microinjection of Glutamate NMDA ionotropic receptor blocker MK-801 in ARC or VTA could block the effect of R568 on the synthesis of DA(P<0.05).4.The results of fluorescence immunohistochemistry showed that there was co-expression of CaSR/NPY and CaSR/Glu positive neurons in BLA.Compared with DMSO group,the c-Fos positive neurons increased in the PVN,LHA and ARC after microinjection of R568 in BLA.In addition,the co-expression of c-Fos positive neurons with dynorphin neurons in ARC,and GABA neurons in VTA were significantly increased in R568 treated group(P<0.05).5.Electrophysiological results showed that after microinjection of R568 in BLA,the firing frequency of glucose excitatory neurons(GS-EXC)was significantly increased,and the firing frequency of glucose inhibitory neurons(GS-INH)was significantly decreased(P<0.05).6.The results of gastrointestinal motility experiments showed that compared with the control group,there was no significant difference in gastric emptying and small intestine propulsion rate after BLA microinjection of R568(P>0.05),but R568 gavage could significantly inhibit gastric emptying and small intestine propulsion rate.Conclusion:microinjection of R568 in the BLA inhibited feeding in mice in a short period of time.The anorexia induced by R568 is related to cause anxiety and depression-like emotions and reducing the secretion of NPY in BLA.R568 administration activates Dynorphin and GABA neurons respectively via NMDA ion channel receptors,thereby indirectly inhibiting the release of DA in hypothalamus and VTA and then reducing food intake.R568 enhances the firing activity of glucose excitatory neurons and attenuates the firing activity of glucose inhibitory neurons,which suggests that R568 may be involved in the regulation of feeding through the activity of glucose-sensitive neurons.R568 administrated in BLA has no significant effect on gastrointestinal motility,while peripheral administration of R568 can significantly reduce gastrointestinal motility,suggesting that R568 has a role in regulating gastrointestinal function,but BLA is not the target of action.Our study can further improve the theory of energy metabolism balance and provide a research basis for clinical treatment of diseases related to energy metabolism disorders.