Study In Mechanism Underlying Sigma-1 Receptor Deficiency-induced Depressive-like Behavior

BackgroundSigma-1 receptor(aiR)is mainly expressed in neurons of hippocampus,hypothalamus and amygdala.The activation of ?1R enhances calcium influx of N-methyl-D-aspartate receptor(NMDAr),to increase calmodulin activity and ERK1/2-CREB phosphorylation,leading to the facilitation of long-term potentiation(LTP)induction.The activation of aiR negatively modulates GABAA receptors(GABAAR).The activation of aiR promotes the calcium influx of voltage-gated calcium channels.The ?1R knockout in male mice show depressive-like phenotypes,but the underlying mechanisms have not yet been elucidated.Basolateral amygdala(BLA)neurons are thought to be one of the important brain regions regulating emotional behavior.The neurons in the BLA express aiR.BLA mainly contains two types of neurons:glutamatergic principal neurons and GABAergic interneurons.A large number of studies have shown that there are two neuronal circuits in BLA:excitatory circuits of external capsule-BLA glutamate neurons and GABAAR-mediated inhibitory local circuits.The glutamate neuron synaptic plasticity is involved in the formation of fear memory and depression-anxiety behavior.For example,LTP is the molecular basis for the formation of fear memory,and the induction of long-term depression(LTD)can eliminate the formation of fear memory.LTP induction is dependent on NMDAr-mediated calcium influx.The GABAAR-mediated inhibitory local circuits is closely related to the induction of LTD.In addition,NMDAr-mediated calcium influx can increase the production and release of NO by promoting the combination of Postsynaptic density-95(PSD-95)and Neuronal NO synthase(nNOS).Based on the above analysis,it is proposed that aiR deficiency may induce depressive-like behavior by altering the synaptic plasticity in BLA.Objective1.To clarify the influence of aiR deficiency on BLA neural circuit and synaptic plasticity.2.To elucidate the molecular mechanism of ?1R deficiency-induced depression-like behavior.Experimental Methods1.alR-KO mice were provided by Professor Zollila of California,USA.The DNA of tail was extracted and the genotype was identified by PCR.2.Behavioral examination:open field test(OFT)was performed to evaluate the spontaneous activity and exploratory behavior of mice.Tail suspension test(TST)and forced swimming test(FST)were performed to detect the depressive-like behavior.3.Morphological examination:BLA paraffin slices were prepared and stained with toluidine blue or anti-?1R antibody.4.EC-BLA synaptic properity,paired-pulse facilitation(PPF,to evaluat presynaptic glutamate release)and paired-pulse inhibition(PPI,to estimate inhibitory local circuits)were examined using field potential recording.LTP and LTD were induced by high-frequency stimulation(HFS,100 Hz,1 min x 5 times)and low-frequency stimulation(LFS,1 Hz,15 min stimulation).5.Level of NO was estimated as a formed nitrite(N02-)using a Griess reagent assay..6.The combination of nNOS and PSD-95 was detected by Coimmunoprecipitation,Co-IP);nNOS,PSD-95,calmodulin,phosphorylation of NR2A and NR2B was detected by Western blotting(WB).7.Levels of AMPAr,NMDAr and GABAAR mRNA were detected by Real-time quantitative polymerase chain reaction(RT-PCR).Results1.Using immunohistochemical staining,we observed the ?1R positive neurons in the BLA of WT mice.Compared with WT mice,the number and morphology of large neurons in ?1R-KO mice did not differ significantly.2.Compared with WT mice,the excitatory postsynaptic potential(fEPSP)slope at EC-BLA synaptic transmission was significantly decreased in ?1R-KO mice with a increase in the PPF value,indicating that ?1R deficiency reduces presynaptic glutamate release to decrease synaptic transmission.3.Levels of AMPAr(GluR1,GluR2),NMDAr(NR1,NR2B,NR2A)and GABAAR(GABAAR-?4 and GABAAR-?)mRNA in BLAhad no significant difference between WT mice and ?1R-KO mice.NMDAr NR2B subunit phosphorylation level was significantly reduced in ?1R-KO mice,while the NR2A subunit phosphorylation level was not altered,indicating that ?1R deficiency reduces the activity of NMDAr.4.Levels of nNOS,PSD-95 and calmodulin protein in BLA of ?1R-KO mice did not differ from WT mice,but the coupling of nNOS to PSD-95 was significantly decreased.The level of NO was also decreased,which could be restored by the BLA-injection of NMDAr agonist NMDA,indicating that ?1R deficiency ?reducing the activity of NMDAr? attenuating combination of nNOS and PSD-95 ? reducing NO production.5.The application of NMDA could correct the fEPSP slopes and PPF values in?1R-KO mice,which was blocked by nNOS inhibitor 7-NI.NO donor DETA/NO has a similar effect with NMDA,indicating that aiR deficiency ? reducing NO production ? inhibiting glutamate release.6.HFS induced the NMDA receptor-dependent LTP in WT mice,but not in aiR-KO mice.The application of NMDA in ?1R-KO mice restored LTP amplitude,which was blocked by 7-NI,indicating that ?1R deficiency? down-regulation of NMDAr ? impaired LTP induction.7.The PPI value in BLA of ?1R-KO mice was larger than that in WT mice,which could be corrected by the GABAAR agonist muscimol.In WT mice,the application of GABAAR blocker bicuculine caused the increase in the PPI value,indicating that ?1R deficiency reduces GABAAR-mediated inhibitory circuits.8.The application of LFS induced LTD in WT mice,but not in ?1R-KO mice.bicuculine can block LTD induction in WT mice.The application of muscimol,NMDA and DETA can restore the induction of LTD in ?1R-KO mice,indicating that ?1R deficiency ? reduced GABAAR-mediated inhibitory circuitsimpaired LTD induction.9.Compared with WT mice,?1R-KO mice showed an increase in the immobility time in FST and TST,which were improved by the BLA-injection of NMDA,DETA and muscimol.7-NI could block the effect of NMDA,indicating that aiR deficiency ? impaires LTD induction?leading to depressive-like behaviorsn.Conclusion1.aiR deficiency in BLA glutamate neurons ? down-regulation of NMDAr ?reducing combination of nNOS and PSD-95? reducing NO production.2.aiR deficiency? reducd NO release ? presynaptic glutamate release? deficits in synaptic transmission function and LTP induction;3.?1R deficiency ? reducd NO release? reduceing GABAAR-mediated inhibitory circuits ? impaired LTD induction ? depressive-like behavior.