| Previous studies have shown that ghrelin,a brain-gut peptide hormone regulating food intake and energy metabolism,and its functional receptor GHS-R1 a play complicated roles in regulating anxiety-and depression-related behaviors,but the underlying mechanism remains uncertain.Stress is an important environmental factor to evoke emotion disorders such as post-tramatic stress disorders(PTSD).In previous study,we found that:(1)Repeated restraint stress for 4 days led to up-regulated expression of endogenous Ghsr1 a in multiple brain regions associated with anxiety,including the medial prefrontal cortex(m PFC),the hypothalamus(HY)and the ventral hippocampus(v HPC).(2)Ghsr1a knock out(Ghsr1a KO)mice showed normal anxiety-related behaviors at baseline state,but alleviated anxiety-related behavioral deficits after exposure of repeated restraint stress.(3)Virus-mediated Ghsr1 a overexpression selectively in ?Ca MKII+ neurons in the m PFC did not affect the basal anxiety,but facilitated anxiety-related behaviors induced by exposure to repeated restraint stress in mice.Based on repeated restraint stress model,we planned to study the effect and underlying cellular mechanism of ghrelin/ GHS-R1 a signaling in the m PFC to regulate anxiety-related behaviors.GHS-R1 a expression in the m PFC was manipulated by genetic knock out(Ghsr1a KO mice),virus-mediated Ghsr1 a overexpression or interference mainly in the prelimbic cortex(Pr L).Anxiety-related behaviors were evaluated with combined behavioral paradigms,including elevated plus maze(EPM),tail suspension test(TST),forced swimming(FS),and social interaction(SI).Our main results are listed as follows:1.Virus-mediated Ghsr1 a overexpression selectively in ?Ca MKII+ neurons of the m PFC did not affect the basal anxiety of wild-type mice.Ghsr1a-overexpressed mice and control mice showed similar open-arm exploration time in a EPM test,similar immobility time in a FS test,and similar social interaction time in a SI test(p>0.05).2.Virus-mediated Ghsr1 a overexpression selectively in ?Ca MKII+ neurons of the m PFC aggravated anxiety-related behavioral deficit induced by repeated restraint stress.Specifically,we found that,after being exposure to repeated restraint stress,the Ghsr1a-overexpressed mice displayed less open-arm exploration time than the control mice in a EPM test(p <0.05).Meanwhile,the Ghsr1a-overexpressed mice exhibited longer immobility time than the control mice in a FS test(p <0.05).3.Virus-mediated Ghsr1 a overexpression selectively in ?Ca MKII+ neurons of the mPFC did not affect anxiety-related behaviors of Ghsr1 a KO mice,both at baseline state and after exposure to repeated restraint stress.Two groups of mice showed similar open-arm exploration time in a EPM test,similar immobility time in a TST and a FS test,and similar social time in a SI test(p> 0.05).4.Virus-mediated Ghsr1 a mRNA interference(Ghsr1a-sh RNA)in the m PFC relieved basic anxiety.Mice receiving Ghsr1a-sh RNA virus injection in the m PFC showed longer open-arm exploration time than mice that received control virus injection in a EPM test(p <0.01).In addition,mice receiving Ghsr1a-sh RNA virus injection in the m PFC exhibited less immobility time than control mice in a TST test(p <0.05).5.Virus-mediated Ghsr1 a mRNA interference in the m PFC relieved anxiety-related behavioral deficit induced by repeated restraint stress exposure.We found that stressed mice receiving Ghsr1a-sh RNA virus pre-injection in the m PFC displayed less immobility time in a TST test than stressed mice receiving control virus injection(p <0.05).Meanwhile,Ghsr1 a mRNA interference in the m PFC also tended to increase the open-arm exploration time tested in a EMP after repeated restraint stress exposure,although the difference between two groups of mice was not significant yet(p> 0.05).6.In comparison to baseline state,wild-type mice after repeated restraint stress exposure exhibited enhanced both excitatory and inhibitory synaptic transmission in the m PFC neurons,mainly the 2/3 layer pyramidal neurons within the Pr L.In details,we found that the frequency of miniature excitatory postsynaptic currents(m EPSCs)in 2/3 layer pyramidal neurons of the Pr L was increased after repeated restraint stress exposure(p<0.01),while the amplitude of m EPSCs did not change(p> 0.05).The frequency and amplitude of spontaneous excitatory postsynaptic currents(s EPSCs)were not changed by stress exposure(p> 0.05).The amplitude of miniature inhibitory postsynaptic currents(mIPSCs)in Pr L 2/3 layer pyramidal neurons was increased after restraint stress exposure(p <0.05),while the frequency was unchanged(p> 0.05).The amplitude and frequency of spontaneous inhibitory postsynaptic currents(s IPSCs)were not changed after stress exposure(p> 0.05).7.In comparison to baseline state,repeated restraint stress exposure enhanced excitatory synaptic transmission meanwhile suppressed inhibitory synaptic transmission in Pr L 2/3 layer pyramidal neurons of Ghsr1 a KO mice.The frequency of s EPSCs and m EPSCs in Pr L 2/3 layer pyramidal neurons was increased in Ghsr1 a KO mice(p <0.05,p <0.001),while the amplitude was unchanged(p> 0.05).The amplitude of s IPSCs and mIPSCs were both decreased(p <0.05),while the frequency was unchanged in Ghsr1 a KO mice(p> 0.05).8.In comparison to baseline state,exposure to repeated restraint stress inhibited the excitatory synaptic transmission in Pr L 2/3 layer pyramidal neurons of mice receiving the control virus injection.Specifically,after exposure to repeated restraint stress,the amplitude and frequency of m EPSCs in Pr L 2/3 layer pyramidal neurons were decreased(p <0.001,p <0.01),while the amplitude and frequency of s EPSCs were unchanged(p> 0.05);The amplitude and the frequency of s IPSCs and mIPSCs did not change(p> 0.05).9.In comparison to baseline state,restraint-stressed mice with Ghsr1 a overexpression in ?Ca MKII + neurons in the m PFC showed decreased excitatory synaptic transmission and increased inhibitory synaptic transmission in Pr L 2/3 layer pyramidal neurons.In details,we found that the amplitude and frequency of both s EPSCs and m EPSCs in Pr L 2/3 layer pyramidal neurons of Ghsr1a-overexpressed mice were decreased after repeated restraint stress exposure(p <0.001 to p <0.01).Meanwhile,the frequency of s IPSCs was increased(p <0.05)while the amplitude remained unchanged(p> 0.05).The amplitude and frequency of mIPSCs were unchanged(p> 0.05).In summary,we found that Ghsr1 a overexpression in m PFC ?Ca MKII+ neurons aggravated anxiety-related behavioral deficit induced by repeated restraint stress,while Ghsr1 a deficiency relieves such anxiety-related behavioral deficit.Consistently,we also found that interference with endogenous Ghsr1 a expression in the m PFC alleviated both basal anxiety and anxiety-related behavior deficit induced by repeated restraint stress exposure in mice.These findings thus indicate that ghrelin/GHS-R1 a signaling in the m PFC is anxiogenic.Our whole-cell patch clamp recordings revealed that,after repeated restraint stress,GHS-R1a-deficient pyramidal neurons in Pr L 2/3 layer displayed enhanced excitatory synaptic transmission and decreased inhibitory synaptic transmission;In contrast,Ghsr1a-overexpressed pyramidal neurons in Pr L 2/3 layer showed enhanced inhibitory synaptic transmission and decreased excitatory synaptic transmission.These results thus suggest that the loss or overexpression of Ghsr1 a has reverse effect on synaptic transmission in m PFC pyramidal neurons,which may be closely associated with anxiety-related behavioral phenotypes after repeated restraint stress exposure.In conclusion,our results together with previous studies provide strong evidence that stress-induced Ghsr1 a elevation in the m PFC is anxiogenic.m PFC is an important node in neural circuit of anxiety,where ghrelin/GHS-R1 a signaling palys an important role in regulating anxiety-related behaviors.The effect of ghrelin/GHS-R1 a signaling on anxiety-related behaviors may be brain-region and neuron-type specific,and depend on the state of animals during the experiment.Our study thus not only enriched knowledge of the multiple physiological functions and underlying mechanisms of Ghrelin/GHS-R1 a signaling,but also provided a potential target for the treatment of affective disorders,such as PTSD. |
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