| Objective:Long-term and chronic stress increases the risk of neuropsychiatric disorders,such as anxiety,and intestinal dysfunction,such as irritable bowel syndrome.These conditions can have negative impacts on both physical and mental health.However,the regulatory mechanisms of the related neural circuits that contribute to stress-induced anxiety-like behaviors and intestinal dysfunction remain unclear.Melanin-concentrating hormone(MCH)is mainly expressed in the lateral hypothalamic area(LHA)and zona incerta.There is evidence that chronic and repeated stress increases MCH levels in the brains of mice.MCHergic neurons project throughout the central nervous system,including limbic nuclei such as the somatosensory cortex,caudate putamen,hippocampus,nucleus accumbens,and amygdala.The limbic system plays a key role in stress-induced anxiety and intestinal disorders,but the functional circuits associated with the limbic system involved in this process remain unclear.As an important part of the limbic system,the amygdala complex is investigated to determine the regulatory effects and underlying mechanisms of MCH neurons in LHA projecting to BLA on anxiety-like behaviors and intestinal motility in mice.Methods:1.Open field test(OFT)and elevated plus maze(EPM)tests were used to observe the effects of microinjection of MCH into the BLA on the anxiety behaviors of mice.2.Flouro-Gold(FG)retrograde tracing combined with fluorescence immunohistochemistry was used to observe the MCHergic neural pathway of LHABLA.3.The adeno-associated virus(AAV)AAV2/9-m MCHp-h M3D(Gq)-m Cherry-WPRE-PA was stereotaxically injected into the LHA of mice by chemogenetic method.After 3weeks of recovery,mice were intraperitoneally injected with Clozapine N-oxide(CNO)or normal saline(NS),and the BLA was injected MCH-R1 antagonist SNAP94847(SNAP)or NS.The mice were randomly divided into four groups: NS + NS,SNAP +NS,NS + CNO,and SNAP + CNO groups.OFT,EPM,marble burial experiment(MBT)and sucrose preference test(SPT)were used to observe the effects of chemogenetic activation of MCH neurons on anxiety-like behaviors of mice.4.Chronic acute combining stress(CACS)was used to establish anxiety model in mice.Changes in body weight,food intake,and anxiety behaviors were observed during the establishment of the model.5.Fluorescence immunohistochemical staining was used to observe the distribution of MCH receptor 1(MCH-R1)in the BLA and the co-expression of MCH and c-Fos immunoreactive neurons in the LHA of CACS model mice and normal mice.The effect of microinjection of SNAP into the BLA on the expression of 5-hydroxytryptamine(5-HT)and neural nitric oxide synthase(n NOS)in the colon of CACS model mice was also investigated.6.SNAP or NS was microinjected into the BLA of CACS model mice and normal control mice to observe the anxiety-like behaviors in mice.7.The effects of BLA microinjection of SNAP on intestinal motility and sensitivity in CACS model mice was evaluated by fecal discharge time,fecal water content,and visceral sensitivity.8.Enzyme linked immunosorbent assay(ELISA)was used to investigate the effects of BLA microinjection of SNAP on tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and 5-HT expressions in intestinal tissue of CACS model mice.9.Western Blot(WB)was employed to observe the effects of BLA microinjection of SNAP on the expressions of intestinal tight junction proteins Occludin and ZO-1 in CACS model mice.Results:1.The results of behavioral tests showed that,compared with the NS group,the MCH group spent significantly less time and distance in the central area of OFT(P < 0.05),and also had a significant reduction in both the number and time spent entering the open arm of EPM(P < 0.05).2.FG retrograde tracing combined with immunofluorescence histochemical staining showed that there were retrograde FG-labeled neurons in the LHA and were partially co-expressed with immunopositive MCH neurons.3.Behavioral results showed that,compared with the NS + NS group,the NS + CNO group spent significantly less time and distance in the central area of the OFT(P < 0.05),had a significantly reduced number and duration of entries into the open arm of the EPM(P < 0.05),buried significantly fewer beads in MBT(P < 0.05),while also exhibiting a significant decrease in sucrose preference(P < 0.05).Compared with the NS + CNO group,the SNAP + CNO group showed a significant increase in both time and distance spent in the central area of the OFT(P < 0.05).Additionally,there was a significant increase in both number and time spent entering the open arm of the EPM(P < 0.05),as well as an increase in beads buried in MBT(P < 0.05)and sucrose preference rate(P < 0.05).4.The behavioral results showed a significant reduction in movement time and distance in the central area of the open field on Day 18(P < 0.05).Additionally,the results for body weight and food intake indicated a significant decrease in mice from the 7th day of modeling(P < 0.05).5.Immunohistochemical results showed that MCH-R1 was distributed in the BLA.The number of c-Fos and MCH double-labeled cells in MCH positive neurons of anxiety mice was significantly more than that of normal mice.The expressions of 5-HT and n NOS in CACS + NS group were significantly higher than those in Control + NS group(P < 0.05);There were no significant difference in the expressions of 5-HT and n NOS between CACS + NS group and CACS + SNAP group(P > 0.05).6.Behavioral results showed that,compared with the Control + NS group,the CACS +NS group spent significantly less time and distance in the central area of the OFT(P <0.05),had a significantly reduced number and time of entering the open arm of the EPM(P < 0.05),buried significantly fewer beads in MBT(P < 0.05),and exhibited a significant decrease in sucrose preference(P < 0.05).Compared with the CACS + NS group,the CACS + SNAP group showed a significant increase in time and distance spent in the central area of the OFT(P < 0.05),as well as a significant increase in both number and duration of entries into the open arm of the EPM(P < 0.05).Additionally,there was a significant increase in the number of beads buried in MBT(P < 0.05)and an increased sucrose preference rate(P < 0.05).7.Compared with the Control + NS group,the CACS + NS group showed significantly shortened first black stool excretion time(P < 0.05),increased fecal water content(P <0.05),and significantly increased abdominal withdrawal reflex(AWR)score under 60 mm Hg pressure(P < 0.05).Furthermore,SNAP microinjection in the BLA of CACS mice significantly prolonged fecal excretion time(P < 0.05),significantly reduced fecal water content(P < 0.05),and the AWR score reduced significantly(P < 0.05).8.ELISA results showed that compared with the Control + NS group,the expressions of TNF-α,IL-6 and 5-HT in the colon tissue were significantly increased in the CACS +NS group(P < 0.05),while there was no significant difference in IL-10 expression(P >0.05).Compared to CACS + NS mice,TNF-α and IL-6 expressions were significantly decreased in the colon of CACS + SNAP mice(P < 0.05),but there was no change in IL-10 and 5-HT expressions(P > 0.05).9.The WB results showed that compared with the Control + NS group,the expressions of Occludin and ZO1 in colon tissue were significantly decreased(P < 0.05)in the CACS+ NS group,while their expressions in colon tissues of CACS + SNAP mice were significantly higher than those of CACS + NS mice(P < 0.05).Conclusions:There is a distribution of MCH-R1 in the BLA of mice,and MCH neurons in the LHA can project to the BLA.Both chemogenetic activation of MCH neurons and microinjection of MCH peptide into the BLA induced anxiety-like behaviors in mice,which were reversed by the MCH-R1 antagonist SNAP-94847.In CACS mice,SNAP administration in the BLA ameliorated anxiety-like behaviors and intestinal dysfunction by reducing intestinal permeability and inflammation.In conclusion,MCHergic circuit from the LHA to the BLA participates in the regulation of anxiety-like behaviors in mice,and this neural pathway improves intestinal dysfunction in CACS model mice by regulating intestinal permeability and inflammation. |
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