Studying The Role And Underlying Mechanism Of VMH Ghrelin/GHS-R1a Signal In Regulating Anxiety-related Behaviors

Recent studies suggested that acyl-ghrelin is not only a hunger hormone but also a persistent biomarker for chronic stress exposure since chronic stressors persistently elevate circulating acyl-ghrelin.Ghrelin functions by binding to its receptor,the growth hormone secretagogue receptor 1a(GHS-R1a).In previous study,we found that Ghsr1a expression was significantly upregulated after repeated restraint stress(RRS)exposure in brain regions involved in anxiety,including medial prefrontal cortex,hypothalamus,ventral hippocampus,amygdala etc.We also found that Ghsr1a deficiency alleviated anxiety-like behaviors in mice exposed to RRS,suggesting that ghrelin/GHS-R1a is anxiogenic however the underlying mechanism is uncertain yet.Ventromedial hypothalamus(VMH)plays an important role in both energy balance and mood regulation.It has the most abundant expression of GHS-R1a in the central nervous system.Therefore,based on previous findings,our current study was designed to further investigated the effect of VMH ghrelin/GHS-R1a signaling on anxiety-related behaviors,and explored the underlying mechanism.Here we combined transgenic mice with Cre recombinase expression driven by calcium/calmodulin-dependent protein kinase II?(?CaMKII)or Dlx5/6 promoter and virus-mediated,Cre-dependent Ghsr1a expression technique,to investigate the effect of selective Ghsr1a overexpression in?CaMKII~+excitatory neurons or GABAergic inhibitory neurons in the VMH on anxiety-related behaviors both at baseline state and after RRS.Supportively,we also investigated whether interfering with endogenous Ghsr1a expression in VMH neurons affected basal and stress-induced anxiety-related behaviors.Designer receptors exclusively activated by designer drugs(DREADDs)are a powerful chemogenetic tool to reversibly control neuronal excitability.With virus-mediated hM3Dq expression or hM3Dq and hGhsr1a co-expression in VMH?CaMKII~+neurons,we could activate infected neurons by i.p.injection of CNO and detect the effect of selective activation of these VMH?CaMKII~+neurons on anxiety-related behaviors.The behavioral assays used includes an elevated plus maze(EPM)test,a social interaction(SI)test,a tail suspension(TST)test and a forced swim(FS)test.Our main findings were listed as follows:1)Cre-dependent Ghsr1a overexpression in VMH?CaMKII~+neurons of Camk2a-Cre mice increased basal anxiety-like behaviors.In details,Camk2a-Cre mice with virus-mediated Ghsr1a overexpression in VMH?CaMKII~+neurons spent less time exploring open arms than control mice in an EPM test(P<0.05).However,two groups of Camk2a-Cre mice exhibited similar immobility time in both a TST and a FS test(P>0.05).2)Cre-dependent Ghsr1a overexpression in VMH?CaMKII~+neurons of Camk2a-Cre mice exacerbated anxiety-like behavioral deficit induced by RRS exposure.Specifically,Ghsr1a-overexpressed mice exhibited less open-arm exploration time than control mice in an EPM test after RRS exposure(P<0.01).However,there was no difference between two groups of mice in immobility time in a TST and a FS(P>0.05).3)Cre-dependent Ghsr1a overexpression in GABAergic inhibitory neurons in the VMH promoted basal despair-like behaviors in Dlx5/6-Cre mice,while it had no effect on basal anxiety-like behaviors.Ghsr1a-overexpressed mice displayed longer immobility time than control mice in a TST test(P<0.05).However,the two groups of mice spent similar time exploring open arms in an EPM test(P>0.05).And no significant difference was observed in social interaction time in a SI test and immobility time in a FS test(P>0.05).4)Cre-dependent Ghsr1a overexpression in GABAergic inhibitory neurons in the VMH of Dlx5/6-Cre mice exacerbated despair-like behavioral deficit induced by RRS exposure,while it had no effect on RRS-induced anxiety-like behavioral deficits.In details,Ghsr1a-overexpressed mice showed longer immobility time than control mice in a TST test(P<0.05).However,the two groups of mice displayed similar exploration time in the open arms in an EPM,social interaction in a SI and immobility time in a FS(P>0.05).5)Virus-mediated interference with endogenous Ghsr1a expression in VMH neurons reduced basal anxiety-related behaviors.Mice receiving Ghsr1a-shRNA virus injection in the VMH displayed increased open-arm exploration time in an EPM test(P<0.01),and increased social interaction time in a SI test in comparison to control mice(P<0.05).6)Virus-mediated interference with endogenous Ghsr1a expression in VMH neurons rescued anxiety-related behavioral deficit induced by exposure to RRS.Specifically,Ghsr1a-interference mice spent more time exploring the open arms than control mice in an elevated plus maze test(P<0.01).In addition,Ghsr1a-interference mice showed less immobility time than control mice in a TST test(P<0.05).7)Chemogenetic activation of?CaMKII~+VMH neurons by CNO injection reduced anxiety-like behaviors at baseline state,not after RRS exposure.In an EPM test,mice receiving CNO injection before behavioral testing showed increased open-arm exploration time compared to control mice at baseline state(P<0.0001),but not after RRS exposure(P>0.05).However,CNO injection decreased the immobility time both at baseline state(P<0.0001)and after exposure to RRS(P<0.0001).8)Chemogenetic activation of Ghsr1a-overexpressed?CaMKII~+neurons in the VMH did not change anxiety-and despair-like behaviors,neither at baseline state nor after RRS exposure.Mice receiving CNO injection and mice receiving normal saline injection displayed similar open-arm exploration time in an EPM test and similar immobility time in a TST test,both at baseline state and after RRS exposure(P>0.05).In summary,we found that Ghsr1a overexpression in subpopulation of excitatory or inhibitory neurons in the VMH promoted anxiety-like or despair-like behaviors respectively.Interference with Ghsr1a expression in VMH neurons produced both anxiolytic and anti-despair responses.These findings thus indicate that ghrelin/GHS-R1a signaling in the VMH promotes anxiety-related behaviors and its regulation is neuron-type specific.Moreover,we found that chemogenetic activation of VMH?CaMKII~+neurons reduced basal anxiety-related behaviors.This finding indicates that activation of VMH?CaMKII~+neurons relieves anxiety,an opposite effect of ghrelin/GHS-R1a signal in VMH?CaMKII~+neurons.The fact that chemogenetic activation of VMH?CaMKII~+neurons failed to rescue RRS exposure-induced anxiety-like behavioral deficit,neither can it reduce anxiety-like behaviors when VMH?CaMKII~+neurons were overexpressed with Ghsr1a,further indicates that ghrelin/GHS-R1a signaling in VMH?CaMKII~+neurons plays an anxiogenic effect by inhibiting the activity of?CaMKII~+neurons.Based on all these findings,we reached the following conclusions:(1)Ghrelin/GHS-R1a signaling in VMH?CaMKII~+neurons promotes anxiety-like behavior,Ghsr1a up-regulation contributes to stress-induced anxiety-like behavioral deficit.(2)Ghrelin/GHS-R1a signaling in VMH GABAergic neurons promotes despair-like behavior.(3)Ghrelin/GHS-R1a signaling in the VMH promotes anxiety-like behavior by inhibiting the activity of?CaMKII~+neurons.Our study not only deepens understanding for multiple physiological functions and underlying mechanisms of ghrelin and its receptor GHS-R1a,it also provides a potential target for the drug development to treat anxiety-related disorders,like PTSD.