Ghrelin/GHS-R1a Exacerbates Anxiety-related Behaviors In Mice Exposed To Repeated Restraint Stress

Recent studies have revealed that the peptide hormone ghrelin and its receptor GHS-R1 a,which are well-known to control food intake and energy balance,play dual roles in modulation of anxiety-related behaviors,but the underlying mechanism remains unclear.Environmental stress is an important predisposition of mood disorders such as anxiety and depression.Therefore,in this study,we proposed to apply both GHS-R1 a knockout and virus-mediated GHS-R1 a expression in specific brain regions,to investigate the possible effect of endogenous ghrelin and its receptor GHS-R1 a on anxiety-related behaviors in mice exposed to repeated restraint stress,and to explore the possible neuronal circuit mechanisms.The behavioral paradigms used includes elevated plus maze test(EPM),forced swimming test(FS),tail suspension test(TST)and social interaction test(SI).We found that:1.Chronic repeated restraint stress led to elevated m RNA expression of endogenous GHS-R1 a in multiple brain regions participating in neural circuit of anxiety,including the medial prefrontal cortex(mPFC),the ventral hippocampus(v HPC)and the hypothalamus(HY).2.GHS-R1 a KO alleviated anxiety deficits observed in mice exposed to repeated restraint stress,while the basal level of anxiety of GHS-R1 a KO mice were comparable to that of WT littermates.The elevated plus maze results showed that WT and GHS-R1 a KO mice decreased in the open arm time,indicating that both anxiety levels were increased,but the anxiety behavior of WT mice was more obvious.Consistent with its results,social experiments showed that the social activity time of WT mice after chronic repeated restraint stress was significantly reduced(p<0.01),mice showed significant social deficits;and the social deficits of GHS-R1 a KO mice was not obvious.In addition,the results of forced swimming test showed that the immobility time of WT mice in the chronic repeated restraint stress was significantly higher than that in the ground state(p<0.01),showing obvious hopeless,and the immobility time of GHS-R1 a KO mice in water was not significantly different from that of ground state.3.Overexpression of human GHS-R1a(h GHS-R1a)in ?Ca MKII-expressing,excitatory neurons in the mPFC did not change the basal anxiety level of WT mice.The overexpression of h GHS-R1 a mice in the elevated plus maze open arm exploration time?forced swimming desperate time and social activity time compared with the control virus mice,there was no significant difference(p>0.05).4.Overexpression of hGHS-R1 a in ?CaMKII-expressing excitatory neurons in the mPFC exacerbated anxiety deficits caused by repeated restraint stress in WT mice.The results showed that overexpression of h GHS-R1 a in ?Ca MKII-expressing excitatory neurons in the mPFC mice had significantly increased compared with control virus mice in the tail suspension test(p<0.05)after repeated restraint stress.After repeated restraint stress,overexpression of h GHS-R1 a in ?Ca MKII-expressing excitatory neurons in the mPFC of mice in the elevated plus maze open arm exploration time compared with the control mice,there is a decreasing trend,but no statistically significant(p>0.05).5.Overexpression of hGHS-R1 a in the basal lateral amygdala(BLA)excitatory neurons did not affect the anxiety-related behaviors in WT mice.The results showed that there was no significant difference in the elevated plus maze open arm exploration time?forced swimming desperate time and social activity time between overexpression of h GHS-R1 a in ?Ca MKII-expressing excitatory neurons in the BLA mice and control virus mice(P>0.05).In summary,we found that GHS-R1 a KO alleviates,while hGHS-R1 a overexpression in the mPFC excitatory neurons deteriorates,anxiety deficits in mice exposed to repeated restraint stress,indicating that increased expression of GHS-R1 a in the mPFC due to repeated restraint stress is anxiogenic.Our results also indicate that the mPFC may be an important node in the neural network for ghrelin/GHS-R1 a to modulate anxiety-related behaviors.To be noted,the modulatory effect of ghrelin/GHS-R1 a on neural circuit of anxiety may be brain-region and neuron-type specific,and also depend on the animal's stress state.Further studies are required to dissect the underlying cellular and circuit mechanisms.Nevertheless,our study not only deepens the understanding of multiple physiological functions and underlying mechanisms of ghrelin and its receptor,but also provides possible guidance for the treatment of neuropsychiatric disorders associated with mood and cognitive impairment.