The Effect And Mechanism Of Metformin On Anxiety-like Behavior And Contextual Fear Conditioning

Part ? The effect and mechanism of metformin on anxiety-like behaviorObjective:Metformin,as a drug for the treatment of type II diabetes,has been found to ameliorate anxiety-like symptoms associated with cerebral ischemia.Our previous work has confirmed the mechanism of anxiolytic effects of metformin at the molecular level.However,in vivo study is needed to elucidate the potential anxiolytic effct and mechanism of metformin.Furthermore,most of the previous pharmacokinetic studies focused on peripheral tissues,and little is known in the central nervous system.Based on this,we aimed to reveal the mechanism of anxiolytic action and its pharmacokinetics in the central nervous system.Methods:Open field test?OFT?and elvated plus maze?EPM?were used to evaluate the anxiolytic effects of metformin in different strains of animals.Diazepam was used in OFT to determine wether there is tolerance in the anxiolytic effects of metformin.LC-MS/MS was used to analyze the pharmacokinetics of metformin intraperitoneally?100 mg/kg?.Brain microtomy patch clamp electrophysiology and molecular biology techniques were used to test the effect of metformin on miniature inhibitory postsynaptic currents?mIPSCs?and the surface expression of?-aminobutyric acid type A receptors?GABA_ARs?subunit.Results:?1?LC-MS/MS analysis found that metformin?100 mg/kg,i.p.?could rapidly pass through the blood-brain barrier,the important pharmacokinetic parameters in the hippocampus were:C_max:7.4±0.8?M,MRT:4.0±0.5 h,and T_max:15 minutes.?2?Metformin significantly increased the amplitude?1.21±0.03 vs saline-treated:1.00±0.12,p<0.05?and frequency?1.26±0.17 vs saline-treated:1.00±0.13?of mIPSCs in hippocampal CA1 region.?3?Metformin increased the suface expression of GABA_ARs subunits?1,?2,but had no effect on the espression of total protein.?4?During OFT,both acute?1 day?and chronic?21 days?treatment?100 mg/kg,i.p.?of metformin had anxiolytic effect?center duration:Day 1:2.29±0.45 vs saline-treated:1.00±0.15,p<0.01;Day 21:2.03±0.37 vs saline-treated:1.00±0.15,p<0.05?.The anxiolytic effect of chronic treatment with diazepam?1mg/kg,i.p.?significantly decreased compared to acute-treated group?1.44±0.45 vs acute diazepam-treated:2.57±0.21.p<0.05?.During EPM,both acute?1 day?and chronic?21 days?treatment of metformin?100 mg/kg,i.p.?had anxiolytic effect?open arm duration:Day 1:2.43±0.32 vs saline-treated:1.00±0.26.p<0.05 and Day 21:2.23±0.52 vs saline-treated:1.00±0.26.p<0.05?.?5?Both acute and chonic treatment of metformin had no effect on fasting blood glucose?Day 1:5.33±0.11;Day 21:5.54±0.30 vs saline-treated:5.03±0.13?and locomotor activity?Day 1:1.06±0.02;Day 21:1.07±0.13 vs saline-treated:1.00±0.02?.?6?Metformin?150mg/kg,i.p.for 30 min?produced rapid anxiolytic effect in OFT?center time?sec?:45.29±4.67 vs saline-treated:28.87±4.16,p<0.05?and EPM?open arm time?sec?:94.38±8.50 vs saline-treated:73.10±7.90,p<0.05?,but had no effect on locomotor activity?total distance?m?:29.55±2.05 vs saline-treated:31.46±2.59?in mice.Conclusion:Compared with diazepam,chronic treatment of metformin had no tolerance to anxiolytic effects.Metformin?100 mg/kg,i.p.?rapidly passed through the blood-brain barrier,increased the frequency and amplitude of mIPSCs and the surface expression of the GABA_ARs in the hippocampal CA1 region.Part ? The effect and mechanism of metformin on learning and memory of mice in contextual fear conditioningObjective: The impact of metformin on cognition and memory is controversial.Some studies have found that metformin has an improving effect;however,other studies have reported that metformin is ineffective or damaging.Most of the current studies on the effects of metformin on learning and memory are based on disease models with impaired cognition and memory.Few studies have directly explored the effects of metformin on learning and memory in normal.Sirtuin 1?SIRT1?and brain-derived neurotrophic factor?BDNF?play a key role in learning and memory,synaptic morphological structure and function regulation.Metformin has been shown to increase BDNF and SIRT1 expression.Thus,our experimental aimed to clarify the effects of metformin on normal learning and memory,and its effects on synaptic structure and function during learning and memory.Methods: Contextual fear conditioning and novel object cognitive exprimental were used to test the effect of metformin on learning and memory.Western blotting used to detect the expression of SIRT1,BDNF and Glu A1.Immunofluorescence technique was used to observe the effect of metformin on dendritic spine.Field potential recording technique was used to detect the effect of metformin on the LTP of the Schaffer collateral-CA1 pathway.Results:?1?Metformin increased contextual fear memory?rat: freezing: 74.78 ± 5.57% vs saline-treated: 55.96 ± 6.43%,p < 0.05;mice: freezing: 40.89 ± 4.27% vs saline-treated: 28.48 ± 2.83%,p < 0.05?,but had no effect on cue fear memory in both of rat?freezing: 89.63 ± 6.31% vs saline-treated: 87.72 ± 3.14%?and mice?freezing: 52.16 ± 3.84% vs saline-treated: 54.21 ± 5.22%?.?2?Metformin?150 mg/kg,i.p.?treated 30 min prior to test,showed fast acquisition of contextual fear memory of mice?trail 1: 19.38 ± 2.50% vs saline-treated: 10.93 ± 2.52%,p < 0.05;trail 2: 40.94 ± 3.39% vs saline-treated: 27.67 ± 3.22%,p < 0.01;trail 3: 55.39 ± 3.74% vs saline-treated: 32.73 ± 3.71%,p < 0.0001;trail 4: 58.35 ± 3.52%,vs saline-treated: 42.78 ± 4.90%,p < 0.05?and slightly increased LTM?52.88 ± 3.70% vs saline-treated: 39.47 ± 4.08%,p < 0.05?,but had no effect on STM?50.72 ± 4.41% vs saline-treated:46.37 ± 4.06%?.?3?Metformin was administered immediately after contextual fear training and did not affect the expression of STM?66.84 ± 3.61% vs saline-treated: 66.64 ± 3.52%?,but slightly reduced the retention of LTM?57.97 ± 3.16% vs saline-treated: 69.21 ± 3.73%,p < 0.05?.?4?Metformin?150 mg/kg,i.p.,30 min?impaired the contextual fear extinction during the chronic short term extinction memory?day 2: 46.84 ± 4.03% vs saline-treated: 35.45 ± 4.03%,p < 0.05;day 3: 43.77 ± 3.56% vs saline-treated: 30.20 ± 3.67%,p < 0.05;day 4: 42.37 ± 3.81% vs saline-treated: 26.95 ± 3.00%,p < 0.01?and single long term extinction memory expression?68.46 ± 4.86% vs saline-treated: 51.34 ± 4.66%,p < 0.05?.?5?Metformin Increased the ability of novel object recognition?NOR?at 3 h NOR test?0.76 ± 0.02 vs saline-treated: 0.64 ± 0.04,p < 0.05?and 24 h test?0.56 ± 0.032 vs saline-treated: 0.47 ± 0.03,p < 0.05?.?6?Metformin did not affect the locomotor activity?total distance: 29.55 ± 2.05 vs saline-treated: 31.46 ± 2.59?,but showed rapid anxiolytic effect?center time: 45.29 ± 4.67 vs saline-treated: 28.87 ± 4.16,p<0.05?.?7?Metformin had no effect on blood glucose?6.91 ± 0.21 vs saline-treated: 6.62 ± 0.39?and the threshold of pain in mice?jumping: 0.99 ± 0.07,vocalizing: 0.87 ± 0.09,vs saline-treated: jumping: 1.00 ± 0.07,vocalizing: 1.00 ± 0.09?.?8?Metformin increased the amplitude of LTP in contextual fear training mice?1.26 ± 0.10 vs saline-treated: 1.00 ± 0.004?.?9?Metformin increased the surface expression of Glu A1 in mice exposed to contextual fear training?1.32 ± 0.09 vs saline-treated: 1.00 ± 0.07,p < 0.05?.?10?Metformin increased the expression of pro-BDNF?1.27 ± 0.09 vs saline-treated: 1.00 ± 0.034,p < 0.05?,BDNF?1.18 ± 0.04 vs saline-treated: 1.00 ± 0.02,p < 0.01?and SIRT1?1.22 ± 0.06 vs saline-treated: 1.00 ± 0.04,p < 0.05?in mice exposed to contextual fear training.Conclusion: Metformin facilitated the acquisition of contextual fear memory,increased the retention of LTM and the ability of cognition,but had no effect on locomotor activity,fasting blood glucose,and pain threshold in mice.The effect of metformin on contextual fear memory was related to the enhanced LTP in the hippocampal Schaffer collateral-CA1 pathway,which may be induced by the increase of Glu A1 membrane expression via activation of the SIRT1-BDNF pathway.Besides,metformin had dynamic regulation of hippocampal dendritic spine subtypes.