Activation Of Liver X Receptor β By GW3965 Alleviating Anxiety-like Behaviors Induced By Acute Stress In Mice

Background:Anxiety,also named as anxiety disorder,is a mental disease accompanied with excessive and persistent tension and worry,as well as brain dysfunction.In recent years,the prevalence of anxiety continues to increase,which leads to not only psychological discomfort of patients,but also physical dysfunction and even disability,thus affecting the physical and mental health seriously.The pathogenesis of anxiety has not been fully elucidated,and there is still a lack of effective anxiolytic target.Therefore,it is of significance to explore the pathogenesis of anxiety and find new potential anxiolytic targets for drug research and development.The normal advanced brain function is dependent on the balance between excitatory neurotransmitters glutamate(Glu),and inhibitory transmitterγ-aminobutyric acid(GABA),which refers as the balance of excitatory/inhibitory neurotransmission(E/I).Imbalance of E/I will lead to the dysregulation of neural activities,resulting in various mental diseases including anxiety.Amygdala is the key brain region for emotion regulation,especially the basolateral amygdala(BLA),and the abnormal E/I in the BLA is closely related to the occurrence of fear and anxiety.Therefore,correcting E/I imbalance in the BLA may be an effective way to treat anxiety.The improvement of E/I imbalance can be achieved through regulation of excitatory/inhibitory neurotransmitters and their receptors.Many factors affect the level of neurotransmitters in the brain.Among them,liver X receptors(LXRs),an important member of the nuclear receptor family,has attracted more and more attentions due to the important influence on neurotransmitters.LXRs,important transcriptional regulatory factors in vivo,include two subtypes LXRαand LXRβ,which play important roles in maintaining cholesterol homeostasis and regulating synaptic plasticity in the brain.It has been reported the activation of LXRs decreased the release and transportation of Glu,while loss of LXRβresulted in a reduction of key enzymes which is responsible for GABA synthesis,suggesting the effect of LXRs on the levels of Glu/GABA neurotransmitter.GW3965 is an agonist of LXRs,and a number of studies have confirmed the neuroprotective roles of GW3965 by inhibiting neuroinflammation and promoting neurogenesis in the brain.However,it is not clear whether GW3965 exerts anxiolytic effects by regulating the neurotransmission and reconciling E/I balance under stress.Objectives:To explore the role of LXRs in anxiety-like behaviors in mice induced by acute stress,and find novel targets for anxiety treatment.In addition,this study is aimed to evaluate the anxiolytic effect of GW3965 targeting LXRs and reveal the mechanism involved,providing theoretical and experimental basis for the research and development of anxiolytic drugs targeting LXRs.Methods:1 The alleviating effects of GW3965 on anxiety-like behaviors in mice induced by acute stress1.1 The anxiety model was established by forced swimming,and mice anxiety-like behaviors were detected by elevated plus maze test(EPM)and open field test(OFT)to determine whether the model was constructed successfully.1.2 The expression pattern of two subtypes of LXRs,LXRαand LXRβ,in the BLA was detected by immunofluorescence staining.1.3 The expression changes of LXRαand LXRβin the BLA were detected by Western blot(WB)method.1.4 The amelioration effects of GW3965 on the anxiety-like behaviors induced by stress were evaluated by EPM and OFT.2 To identify the receptor subtypes mediating the anxiolytic effects of GW39652.1 The lentiviruses of sh RNA(short hairpin RNA)downregulating LXRαand LXRβ(sh LXRαand sh LXRβ),labelled with GFP fluorescent,were constructed respectively.The infection efficiency of lentivirus was observed in the cultured primary neurons in vitro after transfection using fluorescence microscope,and the cells were collected to detect the downregulation effects of LXRαand LXRβusing WB analysis.sh LXRαand sh LXRβwere injected into bilateral BLA by stereotactic microinjection system.After 14 days,the expression of GFP was observed to determine the accuracy of the injection site under fluorescence microscope,and the BLA tissues were collected to evaluate the downregulation of LXRαand LXRβby WB assay.2.2 The effects of downregulation of LXRαor LXRβin the BLA on anxiety-like behaviors in mice were examined by EPM and OFT.Furthermore,the effect of downregulation of LXRαor LXRβon GW3965-mediated anxiolytic improvement was also detected.3 The mechanism of GW3965 alleviating anxiety-like behaviors in stressed mice3.1 The expression pattern of LXRβin different cell type was detected by immunofluorescence staining through co-labelling with Neu N,GFAP and Iba-1,markers for neuron,astrocyte and microglia in the BLA.3.2 The expression levels of Glu and GABA in the BLA from each group were detected by HPLC.3.3 The expression changes of Glu and GABA receptors in the BLA were detected by WB assay.3.4 Immunofluorescence staining was applied to detect the co-labeling of LXRβwith Ca MKIIαas excitatory neuronal marker,and GAD67 as inhibitory neuronal marker in the BLA,so as to further determine the expression characteristics of LXRβin neurons.3.5 The effects of GW3965 on the amplitude and frequency of m EPSC and m IPSC in the BLA from each group were detected using whole cell patch clamp record.Results:1 GW3965 alleviated anxiety-like behaviors induced by acute stress1.1 Forced swimming induced mice anxiety-like behaviors.Compared with control,the mice spent less time in the open arms,and avoided to enter the open arm in EPM,and spent less time in the central area in OFT after forced swimming,while the total distance did not change between 2 groups.These results indicated that acute stress induced mice anxiety-like behaviors by forced swimming.1.2 The reduction of LXRβexpression in the BLA of stressed mice(1)Immunofluorescence staining results showed that both LXRαand LXRβwere expressed in the BLA of mice.LXRβwas expressed highly in the BLA,whereas that of LXRαlimited.This provides the basis for LXRs affecting the BLA function.(2)The BLA tissues were collected immediately after behavioral tests and subjected to WB analysis.Compared with control mice,forced swimming stress induced the downregulation of LXRβin the BLA,while the change of LXRαwas not obvious.This linked the abnormal expression of LXRβto anxiety like behaviors,and LXRβmight play a much more critical role during the process.1.3 GW3965 alleviated mice anxiety-like behaviors induced by forced swimmingGW3965 alleviated anxiety-like behaviors in model mice,which was manifested as more time spent along with more entries in open arms in EPM,and more time in the central area in OFT.2 The subtype LXRβwas the target of GW3965 to exert anxiolytic effect2.1 LV-sh LXRαand LV-sh LXRβto knockdown LXRαand LXRβwere constructed respectively,and LV-sh NC used as a negative control.It was observed that LV-sh RNA infected neurons efficiently in vitro under immunofluorescence microscope.The WB data indicated that the expression leves of LXRαor LXRβwere downregulated after the infection with sh LXRαor sh LXRβin cultured primary neurons,respectively.LV-sh RNAs were microinjected into the bilateral BLA and the injection sites were confirmed accurately after14 days.Meanwhile,LV-sh LXRαcould effectively downregulate LXRαexpression without affecting the level of LXRβ,and LV-sh LXRβdecreased the expression of LXRβ,while LXRαexpression kept unchanged in the BLA using WB analysis.2.2 Specific downregulation of LXRβin the BLA induced anxiety-like behaviors in normal mice,which further indicated that the decreased expression of LXRβwas linked to anxiety-like behaviors.Injection of sh LXRαinto the BLA did not affect GW3965-mediated anxiolytic effect,while sh LXRβabolished that of GW3965 in stressed mice,suggesting that GW3965 ameliorated anxiety-like behaviors through LXRβsubtype.3 GW3965 alleviated anxiety-like behaviors in stressed mice by restoring excitatory/inhibitory neurotransmission in the BLA.3.1 LXRβwas mainly expressed in neurons in the BLA,while less in astrocytes and microglia using immunofluorescence staining.3.2 The changes of neurotransmitter content in the BLA were detected by high performance liquid chromatography.The results showed that Glu level in the BLA of model mice increased and GABA level decreased compared with control.GW3965 administration decreased Glu level in the BLA,while increased GABA content from stressed mice.3.3 The expression levels of Glu and GABA receptors in mice BLA were detected by WB assay.Compared with control,the phosphorylation levels of Glu R1-831 and Glu R1-845 increased,as well as NR2B expression in the BLA,while the expression of GABA receptors,GABA_A-α2 and GABA_A-γ2,decreased after forced swimming.GW3965administration corrected the abnormal changes above.3.4 Immunofluorescence staining data showed that LXRβwas co-labeled with CAMKIIa and GAD67 in the BLA,indicating that LXRβwas expressed in both excitatory and inhibitory neurons.3.5 Data from whole cell patch clamp showed that compared with control,forced swimming stress increased the amplitude and frequency of m EPSC and decreased the amplitude and frequency of m IPSC,resulting in the imbalance of E/I in the BLA of mice.GW3965 perfusion decreased the amplitude and frequency of m EPSC,and increased the amplitude and frequency of m IPSC,thus corrected the imbalance of E/I.Conclusion:Forced swimming induced mice anxiety-like behaviors and decreased LXRβexpression in the BLA.The anxiety-like behaviors were also observed in mice after LXRβdownregulation using sh LXRβinfection in the BLA,suggesting that the decreased LXRβexpression may be a key factor associated with anxiety-like behaviors.The anxiety-like behaviors of model mice were ameliorated after GW3965 administration,indicating activation of LXRs offered the anxiolytic effect to the stressed mice.The anxiolytic effect of GW3965 was abolished after the knockdown of LXRβin the BLA,suggesting that GW3965 ameliorated anxiety-like behaviors through LXRβsubtype.The excitatory neurotransmission was enhanced,while the inhibitory neurotransmission was weakened,resulted in the impaired E/I balance in the BLA of model mice.GW3965 administration alleviated anxiety-like behaviors by regulating Glu/GABA and their receptors in the stressed BLA,manifested as decreasing the enhanced Glu neurotransmission and promoting the decreased GABA neurotransmission,thus restoring the E/I balance in the BLA.In conclusion,LXRβmay represent a potential novel target for anxiety treatment.GW3965,targeting LXRβ,exerted anxiolytic effects by regulating E/I,which provided new approaches for the mechanism research of anxiety disorder and the development of anxiolytic drug.