The Role And Mechanism Of YTHDF1 On Mild Exercise Induced Facilitation Of Fear Extinction In Mice

Post-traumatic stress disorder(PTSD)is a long-term and persistent mental disorder that caused by major traumatic events,which is mainly manifested by repeated traumatic events,avoidance of stimuli related traumatic events,mood and cognitive changes.At present,exposure therapy is the main clinical treatment of PTSD,which is to achieve the extinction or reduction of fear by repeated exposure to fear-inducing stimuli without the presence of any aversive event.Fear extinction is not erasing the previously acquired fear memories,but a new learning.However,the clinical effect of exposure therapy is not effective,especially for remote fear memory extinction.Therefore,it is very urgent to seek timely,effective and economical therapeutic strategies for the treatment of fear memory extinction.In this study,we first established mild wheel exercise pattern in mice to observe the effects of mild wheel exercise prior to extinction training on cued fear memory extinction in mice,and then explored the role and possible mechanism of YTHDF1 in the medial prefrontal cortex.Methods:(1)Setting up the different intensities of exercise(5/10/30minutes)by the given wheel speed and different time duration to achieve different intensities of exercises.Open field tests and elevated plus maze tests were employed to observe emotion alterations of mice after exercise.The enzyme-linked immunosorbent experiment was used for the assessment of peripheral blood lactic acid content.According to the classical Pavlovian fear conditioning model,the cued fear memory was established in mice and the effects of different intensities of exercises prior to extinction training was investigated on fear extinction.(2)The expression of YTHDF1 and synaptic plasticity related proteins(NR2A,NR2B,GluR1,GluR2,PSD95 and Synaptophysin)were determined by the mothods of RT-q PCR,WB and immunohistochemistry in the medial prefrontal cortex and hippocampus of mice after mild wheel exercise.(3)Construction of sh RNA interfering adeno-associated virus of YTHDF1 and its control virus.The interference virus or scramble virus was bilaterally injected into PL or IL by stereotaxic injection after fear memory conditioning in mice.After 28 days of fear conditioning,the mice were underwent mild wheel exercise 2 hours prior to the fear extinction training and the extinction memory retention was examined 24 hours later.The silence efficiency of YTHDF1 was verified by RT-q PCR,WB and immunofluorescence,and the expression of synaptic plasticity related proteins was also examined.Dendrite arborization and dendrite spines of pyramidal neurons in IL subregion were examined by Golgi staining.(4)The expression of GFAP,p ERK1/2 and p Brd4 in hippocampus were examined by immunohistochemistry.Results:(1)The results of open field tests showed that time spent in the central area of the 10-minute and 30-minute exercise groups was significantly reduced compared with its sedentary group,while the differences of time spent in the central area between 5-minute exercise group and its sedentary group was not significant.(2)Mice exposed to an acute mild exercise 1 h or 2 h,but not 0.5 h or 4 h,prior to extinction training exhibited significantly augmented recent fear extinction retention.The mice exposed to an acute mild exercise 2 h,but not 0.5 h,1 h or 4 h,prior to extinction training exhibited significantly augmented remote fear extinction retention.The above results showed that compared with the exercise intensity of 10 minutes and 30 minutes,the 5-minute wheel exercise was defined as mild exercise,and the mild exercise could facilitate the extinction of recent and remote fear memory in a time dependent manner.(3)RT-q PCR and WB results showed that the expression of YTHDF1 in the medial prefrontal cortex of mice was significantly increased 2 h after mild wheel exercise,but the expression of YTHDF1 in the hippocampus was not affected.(4)Immunohistochemical results showed that YTHDF1 expression was significantly increased in the medial prefrontal cortex(including prelimbic regions(PL)and sublimbic regions(IL))at 1 and 2 h after mild wheel exercise.(5)Mild wheel exercise combined with extinction training can significantly increase the NR2B and GluR1 expression.(6)The expression levels of YTHDF1 were significantly decreased after YTHDF1 interference.Interference with YTHDF1 in PL region did not affect fear extinction learning in mice,nor did it affect the extinction memory retention in the mild wheel exercise mice,either in the sedentary mice.(7)IL-region interference with YTHDF1 did not affect fear extinction learning in mild exercise mice,but significantly blocked the facilitating effects of mild wheel exercise on the remote fear extinction in mild exercise mice,but did not affect extinction learning or fear extinction retention in the sedentary mice.(8)The expression of NR2B and GluR1 were significantly decreased in IL region after YTHDF1 interference in mild wheel exercise mice.The number of dendritic arborization and the density of dendritic spines decreased obviously in mild wheel exercise mice.(9)The expression of NR2B and GluR1 were significantly increased in IL region after YTHDF1 interference in the sedentary mice.The number of dendritic arborization and the density of dendritic spines were not affected in the sedentary mice.(10)Immunohistochemical results showed that the number of GFAP positive cells,the expression of pERK1/2 and pBrd4 in hippocampal CA3 region were significantly decreased after YTHDF1 interference in IL region.Conclusions: Mild wheel exercise prior to extinction training could significantly facilitate the extinction of remote auditory fear memory in mice.The mechanism may be related to the increased expression of YTHDF1 in IL region of medial prefrontal cortex,which affected synaptic plasticity related proteins related to fear memory extinction and changes in neuronal structural plasticity.Figures:18,tables:9,References:198...