GABA Neurons In The Central Amygdala Regulate Mood And Food Intake Through The Paraventricular Nucleus In Mice

Objective:Anxiety and depression-like mood disorders are common mental diseases,which cause great pain to patients and have poor effect due to many side effects of treatment.Mood disorders are also strongly associated with manifestations such as weight loss and decreased appetite.Therefore,in-depth exploration of the regulatory mechanism of anxiety and depression-like mood disorders will help to find precise targets and treat the disease more effectively.The central amygdala(Ce A)is the main output region of the amygdala complex,which plays an important role in regulating a variety of physiological and behavioral responses such as emotional state,reward processing,energy balance,and feeding behavior.The Ce A contains a large number of γ-aminobutyric acid(GABA)neurons.Inhibitory gabaergic neurons project from the Ce A to the paraventricular nucleus(PVN)of the hypothalamus,but the role and mechanism involved in the regulation of this pathway are still unclear.In addition,nucleobindin 2(NUCB2)and its processing product nesfatin-1(NUCB2/nesfatin-1)are localized in the paraventricular nucleus(PVN)of the hypothalamus and participate in the regulation of food ingestion.Therefore,the aim of this study is to investigate the gabaergic neural pathway of Ce A-PVN and its regulatory effects on mood and food intake in mice,as well as the underlying mechanism related to nesfatin-1.Methods:1.Chronic restraint stress(CRS)was used to establish a mouse model of anxiety and depression.CRS and normal control(NOR)mice were injected with 0.3 μL of normal saline(NS)or GABAA receptor antagonist bicuculline(BIC)into the PVN,and the mice were divided into four groups(n=10): NOR+NS;NOR + BIC;CRS + NS;CRS+ BIC.Open field test(OFT),elevated cross maze(EPM),marble burial test(MBT)and forced swimming test(FST)were used to observe the effect of microinjection of BIC into the PVN on the behavior of anxiety and depression-like mice.2.Food intake analysis was used to observe the effect of microinjection of BIC into the PVN on food intake of anxiety model mice.3.Fluorogold retrograde tracing combined with fluorescence immunohistochemistry was used to observe the distribution of fluorogold and GABA immunoreactive neurons in Ce A.4.Adeno-associated viral(AAV)vectors(AAV2/9-m Dlx-h M3D(Gq)-EGFP-ER2-WPRE-PA)were directed into Ce A of mice for binding to GABA neurons by chemogenetic method.The mice were divided into two groups according to intraperitoneal injection of Clozapine N-oxide(CNO,0.3 mg kg-1)or NS: NS group and CNO group.Fluorescent immunohistochemical staining was used to observe the co-expression of GABAAR and c-Fos immunoreactive neurons in the PVN of the two groups(n = 6).5.Using chemogenetic methods and behavioral tests,NS or BIC was injected into the PVN of mice injected with AAV within the Ce A,followed by an intraperitoneal injection of CNO(0.3 mg kg-1)or NS.Mice were divided into four groups: NS(intraperitoneal injection)+ NS(PVN microinjection),NS + BIC,CNO + NS,and CNO + BIC(n = 10).Behavioral experiments were performed to observe the effect of gabaergic neural pathway of Ce A-PVN on the emotional behavior of mice.6.The 24-hour food intake of mice in NS+NS,NS+BIC,CNO+NS and CNO+BIC(n =10)groups was measured by chemogenetic method and food intake analysis.The effect of gabaergic pathway of Ce A-PVN on food intake in mice was observed.7.Fluorescent immunohistochemical staining was used to observe the co-expression of GABAA receptor(GABAAR)and NUCB2/nesfatin-1 immunoreactive neurons in PVN.Results:1.The behavioral results showed that compared with the NOR+NS group,the CRS+NS group had a significant increase in the time spent in the open field center(P < 0.01)and distance(P < 0.05),and the number and time of entering the open arm of the elevated maze were also significantly reduced(P < 0.05).The number of beads buried by mice was significantly increased in the marble burying experiment(P < 0.05),and the immobility time of the mice was significantly increased in the forced swimming test(P < 0.01);Compared with the CRS+NS group,the time spent in the center of the open field(P < 0.05)and increased distance(P < 0.05),and the number and time of entering the open arm of the elevated maze were also significantly increased(P <0.05),the number of beads buried by mice was significantly reduced in the marble burying experiment(P < 0.05),and the immobility time of the mice was significantly reduced in the forced swimming test(P < 0.05).2.Compared with NOR+NS group,the food intake decreased significantly at 6-12 h and12-24 h(P < 0.01).Compared with CRS+NS group,the food intake increased significantly at 6-12 h and 12-24 h(P < 0.05).3.FG retrograde tracking combined with fluorescent immunohistochemical staining showed that after microinjection of FG into one side of the PVN,FG-labeled neurons and GABA immunopositive neurons in the ipsilateral Ce A could be observed under the microscope after GABA immunofluorescence staining,and some GABA immunopositive cells were co-labeled with FG.4.The results of fluorescent immunohistochemical staining showed that compared with the NS group,the co-expression of c-Fos and GABAAR positive neurons in the PVN was significantly reduced after the activation of GABA neurons in the Ce A in the CNO group(P < 0.01).5.The results of chemogenetic stimulation method and behavioral test showed that compared with the NS+NS group,the CNO+NS group had a significant increase in the time spent in the open field center(P < 0.05)and distance(P < 0.05),and the number and time of entering the open arm of the elevated maze were also significantly reduced(P < 0.05),and the number of beads buried by mice significantly increased in the marble burying experiment(P < 0.05),and the immobility time of the mice was significantly increased in the forced swimming test(P < 0.01);Compared with the CNO+NS group,the time(P < 0.05)and distance(P < 0.05)of staying in the center of the open field in the CNO+BIC group were significantly increased(P <0.05),and the number and time of entering the open arm of the elevated maze were also significantly increased(P < 0.05),the number of beads buried by mice was significantly reduced in the marble burying experiment(P < 0.05),and the immobility time of the mice was significantly reduced in the forced swimming test(P < 0.05).6.Compared with the NS+NS group,the food intake decreased significantly at 6-12 h and 12-24 h(P < 0.01).Compared with CNO+NS group,the food intake increased significantly at 6-12 h and 12-24 h(P < 0.05).7.The results of fluorescent immunohistochemical staining showed that NUCB2/nesfatin-1 co-expressed with GABAAR in the PVN.Conclusions:Microinjection of GABAA receptor antagonist BIC into the PVN can improve the anxiety-depression-like behavior and increase the food intake of mice with chronic restraint stress.Fluorogold retrograde tracing revealed that GABA neurons of Ce A could project to the PVN.The chemogenetic method activated GABA neurons in the Ce A,and the activation of GABAAR in the PVN was reduced,and the mice showed anxiety-depressive-like behaviors and decreased food intake.Microinjection of BIC into the PVN could reverse these effects.NUCB2/ nesfatin-1-ergic neurons in the PVN express GABAAR.Therefore,our study suggests that the gabaergic pathway of Ce A-PVN is involved in the emotional behavior and feeding regulation of mice,and it may act by regulating nesfatin-1 neurons in the PVN.