| Background:Schizophrenia(SZ)is one of the most common severe mental disorders,characterized by delusions,hallucinations,thought disorder,anhedonia,and cognitive deficits.Because of its high recurrence rate and unpredictability of attack,as well as long-term medication and high unemployment rate,it has caused serious burden to patients,patients’ families and society.Untill now,the pathomechanism of SZ is not fully understood.A wide variety of clinical evidence have shown that sexual dysfunction is also a comorbidity of SZ.However,the underlying pathological mechanisms are largely unknown.The process of sexual behavior consists of sexual motivation and mating performance.Sexual motivation is the premise and beginning of sexual behavior.Accumulative evidence indicate that the medial amygdala(MeA)and medial preoptic area(mPOA)plays an important role in reproductive behavior.It has been reported that the inhibitory neurons in MeA brain area stimulated by low intensity photostimulation can induce mounting behavior,which suggests that the inhibitory neurons in MeA brain area directly participate in the regulation of mice sexual behavior.However,the molecular mechanism of the regulatory effect of MeA brain area on the level of sexual motivation is not clear.Numerous research had shown that both neuregulin 1(NRG1)and its receptor gene ErbB4 are susceptible genes of schizophrenia.It is known that NRG1 belongs to the family of neurotrophic factors.NRG1 plays an indispensable role in the development of central and peripheral neurons including migration,synapsis and myelination by acting on the receptor ErbB4 protein.In the cerebral cortex,hippocampus and amygdala,ErbB4 is specifically expressed in interneurons.Moreover,NRG1 can regulate the release of inhibitory neurotransmitters and synaptic plasticity by activating ErbB4 receptor.However,it is not clear whether NRG1-ErbB4 signaling pathway is involved in regulation of sexual motivation.Objectives:The present project aims to investigate whether NRG1-ErbB4 signaling pathway could regulate the level of sexual motivation in mice,and explore the underlying molecular mechanisms.Our finds will provide a framework to elucidate the underlying molecular mechanisms of SZ-sexual dysfunction comorbidity and identify a potential cellular target for exploring therapeutic interventions.Methods:Immunohistochemical staining of brains from genetically modified mice is employed to observe the expression and cell properties of ErbB4 positive neurons in MeA region.We knocked down ErbB4 in MeA of mice by stereotical cre virus injection or enhanced/blocked NRG1-ErbB4 signaling pharmacologically,and examined their level of sexual motivation,motor activity,anxiety and fear memory in uncondition palace preference test(UPP),open field test(OF),elevated plus maze test(EPM)and fear conditioning(FC)test respectively.The expression of NRG1,ErbB4 and p-ErbB4 protein in MeA regions were detected by Western blot.Finally,the mechanism of NRG1-ErbB4 signaling pathway regulating sexual motivation is studied by means of pharmacology,photogenetics and electrophysiological recording.Results:Part ⅠThe expression pattern of ErbB4 positive neurons in mouse brain was characterized in ErbB4-Cer ER;tdtomato mice.We found that ErbB4 positive neurons were highly expressed in the MEA region.By staining the brain slices of ErbB4-Cre ER;tdtomato;GAD67-GFP mice,we found that the majority of ErbB4 positive neurons were GAD67 positive neurons.Meanwhile,the results of Western blot in MeA region showed that the expression level of NRG1 and p-ErbB4 protein in high level group was significantly lower than that in low level group,but the expression level of ErbB4 protein showed no significant change.In addition,the expression of NRG1,ErbB4 and p-ErbB4 in high level group had no significant change compared with low level group in the hippocampus.Part ⅡCompared with the control group mice,male mice with ErbB4 knocked down in MeA via cre virus(cre mice in short,hereafter)spent significantly longer time in the room of the receptive female mice in the UPP test.In another set of control experiment,cre mice spent similar in either rooms compared with the control mice.Furthermore,The results from OF test showed that there was no significant changes in total travelled distance,movement speed,the time spent in the center and number of entries to the center between the two group;The results from EPM test showed that there is no significant changes in the time spent in each area,number of entries to the open arms,total travelled distance and movement speed between the two group;The results from FC test showed that there were no significant changes in the freezing time in the contextual and tone-cued periods test between the two group.The results showed that,compared with the control group mice,mice injected with NRG1 spent significantly less time in the room of the receptive female mice.And the results of the control experiment showed that,compared with the control group mice,mice injected with NRG1 spent similar time in either rooms.Furthermore,The results from OF test showed that there was no significant changes in total travelled distance,movement speed,the time spent in the center and number of entries to the center between the two group;The results from EPM test showed that there is no significant changes in the time spent in each area,number of entries to the open arms,total travelled distance and movement speed between the two group.Part ⅢWe injected AAV-DIO-Ch R2-e GFP into the bilateral MeA regions of ErbB4-Cre ER;td Tomato mice.Three weeks later,the staining results showed that green fluorescence signal was founds in mPOA region.Moreover,green fluorescence signal was also found in MeA region after the injection of green retrobeads into mPOA region.The postsynaptic current induced by 473 nm blue light wassuccessfully recorded in the neurons of mPOA region by using photogenetics and electrophysiological recording of brain slices.In addition,we completely blocked postsynaptic current by bicuculline methiodide(BMI),GABA receptor atagonist,suggesting that the current was mediated by GABAergic receptor.Moreover,spontaneous inhibitory postsynaptic currents(s IPSC)of ErbB4 positive neurons projecting to mPOA neurons were recorded after knock down of ErbB4 in MeA region.The results showed that,compared with the control group,the frequency of s IPSC was decreased significantly,with no change in the amplitude of s IPSC.The results showed that,compared with the control group mice,mice with injection of AG1478 in MeA region spent significantly longer time in the room of the receptive female mice,but the results of the control experiment showed that,compared with the control group mice,mice injected with AG1478 spent similar time in either rooms.Furthermore,The results from OF test showed that there was no significant changes in total travelled distance,movement speed,the time spent in the center and number of entries to the center between the two group;The results from EPM test showed that there is no significant changes in the time spent in each area,number of entries to the open arms,total travelled distance and movement speed between the two group.Conclusion:1)NRG1 level in MeA is negatively correlated to the level of sexual motivation.2)Elevated NRG1-ErbB4 signaling in MeA region can decrease the level of sexual motivation.3)Blockade of NRG1-ErbB4 signaling in MeA region can increase the level of sexual motivation.4)ErbB4 kinase activity is required for regulating the level of sexual motivation. |
PDF Full Text Request