A Preliminary Study On The Role Of Hippocampal TRPC6 On Conditioning And Extinction In Contextual Fear Memory

Post-traumatic stress disorder（PTSD）is a type of severe anxiety disorder that occurs after a traumatic event.It is characterized by persistent invasion and re-experience of traumatic memory,with a lifetime prevalence of approximately 6.1%.Contextual fear conditioning provides researchers with an excellent opportunity to observe the conditioning and extinction of fear memory.The difficulty of the latter is one of the main causes of PTSD symptoms.The structural basis of memory is closely related to synaptic plasticity.A large number of studies have also shown that the conditioning and extinction of contextual fear conditioning is accompanied by some corresponding changes in dendritic spines.For example,Lai et al.has found that in the conditional fear model,the process of fear conditioning increased the elimination rate of neuronal dendritic spines in the frontal association cortex（FrA）and preliminary cortex（PL）,while the extinction process increased the rate of formation of dendritic spines.These studies confirmed that the dynamic changes of dendritic spines are closely related to the conditioning and extinction of fear memory,but the mechanism is still unclear.TRPC6 is a member of the C subfamily of transient receptor potential channels（TRP channels）and is widely expressed in the brain.TRPC6 is a non-selective cation channel that participates in a variety of physiological and pathological processes by changing the permeability to Ca²⁺and Na⁺and membrane potentials and activating downstream signaling pathways.TRPC6 has been found to have important regulatory effects on synaptic plasticity and learning and memory in the nervous system,and is closely related to the pathological mechanisms and treatment of many Central Nervous System（CNS）diseases.However,it has not been reported whether TRPC6 participates in the process of contextual fear conditioning.This study verified whether TRPC6 had an impact on the conditioning and extinction of fear memory by establishing a conditioning fear memory model,and various morphology and molecular biology methods were used to explore possible morphologies and molecules changes to provide new ideas for exploring the related neuromolecular mechanisms and therapeutic targets of post-traumatic stress disorder.Main methods:1.Immunohistochemical staining was used to detect TRPC6 protein expression in various brain regions of mice;2.Western blotting was used to detect the effect of conditioning and extinction of contextual fear memory on the expression of TRPC6 in hippocampus;3.RNA interference was used to inhibit the expression of TRPC6 protein in hippocampus,and the effects of open field experiment and elevated cross maze experiment combined with the conditioning and extinction of contextual fear memory were used to detect its effects;4.After RNA interference inhibited the expression of hippocampal TRPC6 protein,Western blotting was used to detect the change of phosphorylation status of CaMKIV-CREB pathway during the process of the conditioning and extinction of contextual fear memory,and Golgi staining was used to detect the density change of hippocampal dendritic spines;Main results:1.TRPC6 immunopositive products are widely expressed in various brain regions of adult male mice,mainly localized on the cell membrane.After CFC conditioning and extinction,the expression of TRPC6 protein in mouse hippocampus increased significantly.2.Stereotactic injection of TRPC6 shRNA adeno-associated virus can significantly inhibit the expression level of TRPC6 protein in the hippocampus,and cause significantly slow down the conditioning and extinction rate of CFC.The conditioning memory and extinction memory in the test after 24 hours were also significantly reduced.3.After suppressing the expression of TRPC6 protein in mouse hippocampus and conducting the conditioning and extinction of CFC,the phosphorylation levels of CaMKIV and CREB were significantly reduced,and the dendritic spine density in CA1 region of hippocampus was significantly reduced.Main conclusions:1.TRPC6 is widely expressed in various brain regions of adult male mice,suggesting that it may be involved in many physiological processes in CNS.Changes in the expression of TRPC6 protein in the hippocampus of mice after CFC conditioning and extinction suggest that TRPC6 channels may be involved in the regulation of fear memory.2.Inhibition of TRPC6 protein expression in mouse hippocampus slowed down the conditioning and extinction rate of mouse CFC,and reduced conditioning memory and extinction memory,respectively,indicating that hippocampal TRPC6 participates in the conditioning and extinction of CFC and inhibiting hippocampal TRPC6 expression will destroy the conditioning and extinction of fear memory.3.Inhibition of TRPC6 protein expression in mouse hippocampus reduced the CaMKIV-CREB phosphorylation level and dendritic spine density induced by CFC,indicating that the hippocampal TRPC6 channel may regulate dendritic spine changes through the CaMKIV-CREB pathway and thus participate in CFC.4.The regulation of hippocampal TRPC6 protein expression may be a new target for studying PTSD mechanisms and therapeutic methods.