The Role And Underlying Mechanism Of Autophagy Of Amygdala In Fear Memory Consolidation Of Mice

Part I The role of autophagy of amygdala in fear memory consolidation of miceObjective:Autophagy,which is a major determinant of protein turnover and highly conserved in mammals,is an essential process that takes place in the central neuron system.Autophagy could regulate memory deficiency caused by aging or some neurodegenerative diseases and also plays a role in the regulation of synaptic plasticity,such as the regulation of dopamine receptor and glutamate receptor trafficking.On this basis,we herein attempt to investigate the potential effect of autophagy on fear memory consolidation to further elucidating the mechanism of fear learning and memory.Methods:Cue fear conditioning and memory test was employed to study the expression of fear memory.We used the western blotting and Immunofluorescence of brain slices to detect the time course of autophagy related protein in 6 h following cue fear conditioning in basolateral amygdaloid（BLA）.The transmission electron microscope was used to assesse the level of autophagosome in BLA after fear conditioning.Drug microinfusion techniques and lentivirus-Atg5 shRNA（LV-Atg5 shRNA）knockdown were employed to inhibit autophagy in amygdala,combined with fear conditioning paradigm to study the involvements of autophagy in each phase of fear memory.Results:（1）Autophagy was activated after cue fear conditioning,the level of microtubule associated protein 1 light chain 3（LC3）was increased significantly at 1 h after fear conditioning in amygdala and reached baseline at 6 h(control:1.00±0.00,training 1 h:1.27±0.049;F_5,35=2.698,p=0.037).Transmission electron microscope of tissue from BLA showed the autophagosome-like organelles in fear conditioning mice were increased compared with control mice（control:1.33±0.33,training 1 h:2.50±0.19;t₉=3.166,p=0.011）.（2）p62 expression was decreased at 1.5 h after fear conditioning(control:1.00±0.11,training 1.5 h:0.77±0.11;F_5,40=3.546,p=0.009),and the fluorescence of mRFP-GFP-tagged LC3 showed that autophagy flux was activated after cue fear conditioning.（3）Treatment of 3-methyladenine（3-MA）which is an inhibitor of autophagy in BLA decreased the freezing levels of long-term memory(DMSO:61.15±5.86%,3-MA:32.73±6.13%;F_1,44=10.53,p=0.026)suggested that treatment of 3-MA in BLA impaired fear memory consolidation.（4）Treatment of wortmannin which is also an inhibitor of autophagy in mice BLA also decreased the freezing levels of long-term memory(DMSO:53.69±7.11%,wort:32.02±4.36%;F_1,44=10.53,p=0.002)suggested that treatment of wortmannin in mice BLA impaired fear memory consolidation.（5）Mice microinjected with LV-Atg5 shRNA in BLA showed decreased freezing levels in the fear memory test at24 h later after fear conditioning(control:64.42±3.89%,LV-control:65.27±2.51%,LV-Atg5 shRNA:50.25±2.83%;F_2,42=4.989,p=0.010),and the acquisition of fear memory was not affected by Atg5 shRNA,suggesting that LV-Atg5 shRNA impaired fear memory consolidation(F_2,49=2.118,p=0.171).（6）Quantitative analysis of the ratio of p-p70s6k/p70s6k was increased in fear conditioning group revealed that cue fear conditioning induced autophagy in an mTOR-independent pathway(control:1.00±0.04,training:1.19±0.05;t₁₀=2.746,p=0.021).Conclusion:The present study indicated that cue fear conditioning could induce autophagy in an mTOR-independent pathway in BLA.Inhibitors of autophagy and Atg5 knockdown in BLA both impaired long-term memory but not short-term memory which represented the impairment of fear memory consolidation.Part II The mechanism of autophagy in amygdala involved in fear memory consolidation of miceObjective: The agonist of γ-aminobutyric acid type A receptor（GABAAR）in amygdala impaired the retention of fear memory,and the antagonist of GABAAR could promote fear memory.GABARAP could bind the γ2 subunit of GABAAR to transport the GABAAR to the cell membrane.At the same time,GABAR-associated protein（GABARAP）is a member of Atg8 family,and plays a key role in the late phase of the autophagosome formation.In our early study we found that fear conditioning induced autophagy was essential for memory consolidation,here we conducted studies aimed at figuring out the role of GABARAP and GABAAR in the memory consolidated regulated by fear conditioning induced autophagy.Methods: We used western blotting analysis and biotinylation experiments to detect surface expression of GABAAR.Immunofluorescence was used to test whether autophagy had an effect on membrane trafficking GABAAR in BLA.Whole-cell patch-clamp technique was employed to test the GABAergic synaptic transmission in the slices of amygdala.Co-immunoprecipitation was used to detect the interaction of GABARAP with the GABAAR γ2 subunit.We used the LV-Atg5 sh RNA knockdown and autophagy inhibitors to evaluate the effect of autophagy on the GABAAR trafficking and the interaction of GABARAP with the γ2 subunit.GABARAP-peptide that can interrupt the binding of GABARAP and the γ2 subunit was used to test the fear memory.Results:（1）Surface expression of GABAAR γ2 subunit was reduced by fear conditioning（control: 1.00±0.03,training: 0.78±0.04;t27=4.314,p=0.000）,Inhibition of autophagy by 3-MA and wortmannin reversed the decrease of surface GABAAR γ2 subunit（control: 1.00±0.05,training: 0.72±0.03,wort: 0.99±0.10,3-MA: 1.05±0.07;F3,20=6.052,p=0.003）and the fluorescence of surface GABAAR γ2 subunit after fear conditioning（control: 3.48±0.37,training: 2.10±0.38,3-MA: 3.8±0.34;F2,56=3.866,p=0.027）.Inhibition of autophagy by LV-Atg5 sh RNA also reversed the decrease of surface GABAAR γ2 subunit（control: 1.00±0.01,training: 0.80±0.04,LV-control: 0.88±0.05,LV-Atg5 sh RNA: 1.04±0.05;F3,18=7.822,p=0.002）and the fluorescence of surface GABAAR γ2 subunit after fear conditioning（control: l4.32±0.34,LV-control: 2.87±0.46,LV-Atg5 sh RNA: 4.05±0.30;F2,53=4.115,p=0.022）.（2）Inhibition of autophagy by LV-Atg5 sh RNA increased the m IPSC amplitude（LV-control: 17.69±1.39,LV-Atg5 sh RNA: 24.21±1.76;t9=2.812,p=0.020）and frequency（LV-control: 1.67±0.41,LV-Atg5 sh RNA: 2.94±0.24;t6=2.928,p=0.026）.（3）The expression of GABARAP was increased in amygdala following fear conditioning（control: 1.00±0.14,training: 1.20±0.17;t23=3.142,p=0.005）,so was the interaction between GABARAP and GABAAR γ2 subunit（control: 1.00±0.14,training: 1.49±0.14;t10=2.958,p=0.016）.3-MA decreaseed the interaction of GABARAP and GABAAR γ2 subunit（control: 1.00±0.09,DMSO: 1.49.4±0.14,3-MA: 0.99±0.08;F2,15=7.486,p=0.006）.Inhibition of autophagy by LV-Atg5 sh RNA also decreased the interaction between GABARAP and GABAAR γ2 subunit（control: 1.00±0.03,training+LV-control: 1.37±0.08,training+LV-Atg5 sh RNA: 1.07±0.06;F2,12=9.813,p=0.003）.（4）GABARAP-peptide blocked the trafficking of surface GABAAR（control: 1.00±0.08,Scramble-peptide: 0.64±0.08,GABARAP-peptide: 1.15±0.10;F2,18=8.583,p=0.003）.Mice microinjected with GABARAP-peptide in BLA decreased the freezing levels of long-term memory（training: 59.72±3.02%,Scramble-peptide: 59.54±2.35%,GABARAP-peptide: 47.33±2.31%;F2,48=5.968,p=0.005）.Conclusion: Activated autophagy degraded the surface GABAAR through the interaction of GABARAP and GABAAR γ2 subunit,the downregulation of the surface GABAAR made the unstable short-term memory stabilized to long-term memory,which was essential for fear memory consolidation.Inhibition of autophagy interrupted the interaction of GABARAP and GABAAR γ2 subunit and blocked the trafficking of surface GABAAR,leading to the impairment of fear memory consolidation.