The Study On The Mechanism Of ?-GABA_AR In Depressive-like Behavior Of Adolescent Female Mice

ObjectiveDepression(Major Depressive Disorder,MDD)is an emotional mental disorder with“anhedonia”as the core symptom,accompanied by impaired cognitive function and physical changes such as weight fluctuations and sleep disorders.The incidence of depression in adolescent females is two to three higher than in males,reaching the peak of depression incidence.The depressive symptoms are more serious and suicidal tendencies are more obvious.Those seriously damage the physical and mental health of depression adolescent and poses a serious family and social problems.For the confirmed MDD adolescent patients,the current clinical medication is single and has a slow onset of action.Psychotherapy does not have a long-term therapeutic effect on moderate to severe adolescent depression.There is a lack of research on the pathogenesis of adolescent depression.The decrease of neurosteroid 3?,5?-Allopregnanolone(THP)leads to a compensatory increase in the expression of?₄??-GABA_A receptor subunit of hippocampal pyramidal neurons,which leads to anxiety in adolescent female mice.The pathological of postpartum depression is the long-term mismatch between neurosteroid levels and GABA_AR subunit(such as?-GABA_AR)levels.However,the reasons for the sharp increase in the incidence of depression in adolescent women are currently unclear.There is an urgent need to investigate the susceptibility factors of young women and the specific physiological changes in adolescent female depression.Whether the?₄??-GABA_A receptor subunit plays a role in the onset of adolescent female depression.Therefore,we observe the changes of the structure and function on hippocampal CA1 pyramidal neurons in female mice caused by prepubertal and early puberty stress in cellular,subcellular and receptor levels.In addition,we observe the effect of?-GABA_AR activator Gaboxadol(THIP)on these changes.We explore the mechanism of the drug in adolescent female depression,and provide a new way for the prevention and treatment of depression in adolescent women.Methods1.The role of?-GABA_AR in adolescent female depression.(1)A three-week modified chronic unpredictable mild stress(CUMS)and a THP(10mg/kg)injection once a day for 10 consecutive days from the time P33 day were used to construct a depression model of adolescent female mice.(2)We used sucrose preference,forced swimming and tail suspension experiments to conduct behavioral tests.The mice were successfully modeled will be included in subsequent studies.2.The effect of?-GABA_AR on pyramidal neuron morphology in hippocampal of depressed adolescent mice.(1)Golgi stain and two-photon laser scanning microscopy imaging techniques were used to study dendritic spine density at the apical dendritic spines of CA1hippocampal pyramidal neurons..3.The effect of?-GABA_AR on pyramidal neuron function in hippocampal of depressed adolescent mice.(1)With patch clamp whole-cell recording techniques,we recorded the GABAergic tonic and spontaneous post-synaptic current(s IPSC).(2)With patch clamp whole-cell recording techniques,we assayed NMDAR-mediated synaptic transmission using the ratios of NMDA EPSC to AMPAR EPSC.(3)With patch clamp whole-cell recording techniques,we recorded paired-pulse ratios(PPRs)to measure the presynaptic release probability of glutamate and GABAergic.(4)We measured the total and surface expression of?-GABA_AR,Glu A1 and Glu A2by western blot assays.4.The changes and significance of?-GABA_AR in PV neurons of depressed adolescent mice.(1)Immunofluorescence colocalization staining was used to detect the changes of?-GABA_AR subunits in hippocampal CA1parvalbumin intermediate neurons(PV).Results1.The role of?-GABA_AR in adolescent female depression.(1)Adolescent wild-type mice were able to successfully establish a depression-like behavior,and?knock female adolescent mice were susceptible to the CUMSmodel.Moreover,It is also possible to successfully induce depression-like behavior in adolescent female mice by intraperitoneal injecting THP for 10 consecutive days.(2)THIP rapidly improves depression-like behavior in adolescent female mice by activating?-GABA_Areceptor subunits.2.The effect of?-GABA_AR on pyramidal neuron morphology in hippocampal of depressed adolescent mice.(1)we found the chronic stress in prepubertal and early adolescence causes a significant decrease in the density of apical dendritic spines on hippocampal CA1 pyramidal neurons.(2)THIP can rapidly increase the density of apical dendritic spines on hippocampal CA1 pyramidal neurons of adolescent female mice with depression-like behavior by activating?-GABA_Areceptor subunits.3.The effect of?-GABA_AR on pyramidal neuron function in hippocampal of depressed adolescent mice.(1)The effects of CUMS and long-term administration of THP on glutamate receptors in hippocampal CA1 pyramidal neurons. CUMS and long-term administration of THP caused no changes in NMDAR stoichiometry.There also no change in presynaptic release probability.But the amplitude or frequency of AMPAR had a significant reduction.Meanwhile,there was a greatly decrease in the surface expression of Glu A1 and Glu A2 by western blot assays.(2)The effects of CUMS and long-term administration of THP on GABA_AR in hippocampal CA1 pyramidal neurons. CUMS and long-term administration of THP lead to a significant increase in presynaptic release probability of GABAergic and GABA_A receptor s IPSC current. There was a decrease in GABA_AR tonic current.These indirectly reflect the increased excitability of interneurons.(3)THIP could improve the damage of AMPA receptor caused by CUMS and THP. After injected THIP,it can be seen that the amplitude and frequency of the GABA_AR s IPSC current decrease greatly but there was no significantly change in GABA_AR tonic current.In addition,the amplitude and frequency of the AMPAR are significantly increased.The activation of?-GABA_AR could improve the damage of AMPA receptor caused by CUMS and THP.4.The changes and significance of?-GABA_AR in PV neurons of depressed adolescent mice.(1)The effects of CUMS and long-term administration of THP on the?-GABA_AR of PV interneuron in hippocampal CA1. It was found that long-term administration of THP and CUMS lead to a significant decrease in the fluorescence density of the?-GABA_AR subunit of PV interneuron.(2)THIP could improve the damage of?-GABA_AR in PV interneuron caused by CUMS and THP. After injected THIP,it can be seen that the fluorescence density of?-GABA_AR of PV interneuron in hippocampal is significantly increased.The activation of ?-GABA_AR could improve the damage of the?-GABA_AR of PV interneuron caused by CUMS and THP.Conclusion1.The activation of?-GABA_AR subunit can ameliorate CUMS and THP-induced depression-like behavior,but it has no effect on depressed mice with delta subunit deletion.The female adolescent mice knocked with?genes are susceptible to the CUMS model,and the application of THIP has protective effects on adolescent depressed mice.These indicating that the?-GABA_AR subunit plays an important role in the behavior of adolescent female depression.2.CUMS and THP lead to a decrease in the synaptic plasticity of hippocampal CA1 pyramidal neurons in adolescent female mice.Activation of the?-GABA_AR subunit can enhance pyramidal neuronal synaptic plasticity.3.CUMS and THP led to increased excitability of the hippocampal CA1 PV interneurons in adolescent female mice,thereby suppressing the excitability and synaptic plasticity of pyramidal neurons.4.THIP reduces the excitability of interneurons by activating the?-GABA_AR subunit.This weakens the inhibitory effect of interneurons on pyramidal neurons to quickly improve depression-like behavior.This study confirmed the rapid antidepressant effect of THIP in the female mouse depression model caused by the stress before and during puberty,and provided new ideas for the treatment of adolescent women depression in clinical.