No abstract

Stress-induced neuroepigenetic programming gains growing more and more interest in the studies of the etiology of posttraumatic stress disorder (PTSD). However, seldom attention is focused on DNA demethylation in fear memory generalization, which is the core characteristic of PTSD. Here, we show that ten-eleven translocation protein 3 (TET3), the most abundant DNA demethylation enzyme of the TET family in neurons, senses environmental stress and bridges neuroplasticity with behavioral adaptation during fear generalization. Foot shock strength dependently induces fear generalization and TET3 expression in nucleus accumbens (NAc) in mice. Inhibition of DNA demethylation by infusing demethyltransferase inhibitors or AAV-Tet3-shRNA virus in NAc enhances the fear generalization and anxiety-like behavior. Furthermore, TET3 knockdown impairs the dendritic spine density, PSD length, and thickness of neurons, decreases DNA hydroxymethylation (5hmC), reduces the expression of synaptic plasticity-related genes including Homer1, Cdkn1a, Cdh8, Vamp8, Reln, Bdnf, while surprisingly increases immune-related genes Stat1, B2m, H2-Q7, H2-M2, C3, Cd68 shown by RNA-seq. Notably, knockdown of TET3 in NAc activates microglia and CD39-P2Y12R signaling pathway, and inhibition of CD39 reverses the effects of TET3 knockdown on the fear memory generalization and anxiety. Overexpression of TET3 by Crispr-dSaCas9 virus delivery to activate endogenous Tet3 in NAc increases dendritic spine density of neurons in NAc and reverses fear memory generalization and anxiety-like behavior in mice. These results suggest that TET3 modulates fear generalization and anxiety via regulating synaptic plasticity and CD39 signaling pathway.

IntroductionFear memory generalization is regarded as the core characteristic of posttraumatic stress disorder (PTSD) development. However, the mechanism that contributes to the generalization of conditioned fear memory is still unclear. The generalization is generally considered to be a mismatch that occurs during memory consolidation.MethodsFoot shocks and tones were given as unconditioned stress and conditioned stress, respectively for fear conditioning training. Immunofluorescence staining, western blotting and qPCR were performed to determine the expression of different genes in amygdala of mice after fear conditioning training. Cycloheximide was used as a protein synthesis inhibitor and 2-methyl-6-phenylethynyl-pyridine was injected for mGluR5 inhibition.ResultsFear conditioning using caused incremental generalization, which was clearly observed during training. The density of c-Fos+ cells or the synaptic p-NMDAR expression did not differ with stress intensities. Strong-shock fear conditioning could induce significant mGluR5 de novo synthesis in the amygdala, which was not observed in the weak-shock group. Inhibition of mGluR5 impaired fear memory generalization induced by strong-shock fear conditioning, but the generalization level induced by weak-shock training was enhanced.DiscussionThese results indicated that mGluR5 in the amygdala is critical to the function of inappropriate fear memory generalization and suggested that this may be a potential target for the treatment of PTSD.