mGluR5 in amygdala modulates fear memory generalization.

Introduction: Fear 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. Methods: Foot 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. Results: Fear 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. Discussion: These 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.

Genes and pathways associated with fear discrimination identified by genome-wide DNA methylation and RNA-seq analyses in nucleus accumbens in mice.

Fear memory is critical for individual survival. However, the maladaptive fear response is one of the hallmarks of fear-related disorders, which is characterized by the failure to discriminate threatening signals from neutral or safe cues. The biological mechanisms of fear discrimination remain to be clarified. In this study, we found that the nucleus accumbens (NAc) was indispensable for the formation of cued fear memory in mice, during which the expression of DNA methyltransferase 3a gene (DNMT3a) increased. Injection of Zebularine, a nonspecific DNMT inhibitor, into NAc immediately after conditioning induced a maladaptive fear response to neutral cue (CS-). Using whole-genome bisulfite sequencing (WGBS), differentially methylated sites and methylated regions (DMRs) were investigated. 16,226 DMRs in the genenome were identified, in which, 214 genes with significant differences in their methylation levels and mRNA expression profiles were identified through correlation analysis. Notably, 15 genes were synaptic function-related and 8 genes were enriched in the cGMP-PKG signaling pathway. Moreover, inhibition of PKG impaired fear discrimination. Together, our results revealed the profile and role of genome-wide DNA methylation in NAc in the regulation of fear discrimination.

The Molecular Gut-Brain Axis in Early Brain Development.

Millions of nerves, immune factors, and hormones in the circulatory system connect the gut and the brain. In bidirectional communication, the gut microbiota play a crucial role in the gut-brain axis (GBA), wherein microbial metabolites of the gut microbiota regulate intestinal homeostasis, thereby influencing brain activity. Dynamic changes are observed in gut microbiota as well as during brain development. Altering the gut microbiota could serve as a therapeutic target for treating abnormalities associated with brain development. Neurophysiological development and immune regulatory disorders are affected by changes that occur in gut microbiota composition and function. The molecular aspects relevant to the GBA could help develop targeted therapies for neurodevelopmental diseases. Herein, we review the findings of recent studies on the role of the GBA in its underlying molecular mechanisms in the early stages of brain development. Furthermore, we discuss the bidirectional regulation of gut microbiota from mother to infant and the potential signaling pathways and roles of posttranscriptional modifications in brain functions. Our review summarizes the role of molecular GBA in early brain development and related disorders, providing cues for novel therapeutic targets.

Deficiency of Tet3 in nucleus accumbens enhances fear generalization and anxiety-like behaviors in mice.

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.

Ketamine Alleviates Fear Generalization Through GluN2B-BDNF Signaling in Mice.

Fear memories are critical for survival. Nevertheless, over-generalization of these memories, depicted by a failure to distinguish threats from safe stimuli, is typical in stress-related disorders. Previous studies have supported a protective role of ketamine against stress-induced depressive behavior. However, the effect of ketamine on fear generalization remains unclear. In this study, we investigated the effects of ketamine on fear generalization in a fear-generalized mouse model. The mice were given a single sub-anesthetic dose of ketamine (30 mg/kg, i.p.) 1 h before, 1 week before, immediately after, or 22 h after fear conditioning. The behavioral measure of fear (indicated by freezing level) and synaptic protein expression in the basolateral amygdala (BLA) and inferior-limbic pre-frontal cortex (IL-PFC) of mice were examined. We found that only ketamine administered 22 h after fear conditioning significantly decreased the fear generalization, and the effect was dose-dependent and lasted for at least 2 weeks. The fear-generalized mice showed a lower level of brain-derived neurotrophic factor (BDNF) and a higher level of GluN2B protein in the BLA and IL-PFC, and this was reversed by a single administration of ketamine. Moreover, the GluN2B antagonist ifenprodil decreased the fear generalization when infused into the IL-PFC, but had no effect when infused into the BLA. Infusion of ANA-12 (an antagonist of the BDNF receptor TrkB) into the BLA or IL-PFC blocked the effect of ketamine on fear generalization. These findings support the conclusion that a single dose of ketamine administered 22 h after fear conditioning alleviates the fear memory generalization in mice and the GluN2B-related BDNF signaling pathway plays an important role in the alleviation of fear generalization.

Fear renewal activates cyclic adenosine monophosphate signaling in the dentate gyrus.

BACKGROUND: Fear renewal, the context-specific relapse of a conditioned fear after extinction, is a widely pursued model of post-traumatic stress disorder and phobias. However, its cellular and molecular mechanisms remain poorly understood. The dentate gyrus (DG) has emerged as a critical locus of plasticity with relevance to memory, anxiety disorders, and depression, and it contributes to fear memory retrieval. Here, we have identified the role of the DG in fear renewal and its molecular mechanism. MATERIALS AND METHODS: Muscimol (MUS), activator of cyclic adenosine monophosphate (cAMP) forskolin (FSK), inhibitor of protein kinase A (PKA), Rip-cAMP, and a phosphodiesterase inhibitor rolipram were infused into DG of standard deviation rats before renewal testing. cAMP levels after fear renewal was measured by enzyme-linked immunosorbent assay. The protein levels of phosphodiesterase 4 (PDE4) isoforms were tested by western blot. At last, the roles of cAMP signaling were also tested in the acquisition of fear conditioning, fear retrieval, and extinction. RESULTS: Intra-DG treatment of MUS and Rp-cAMP impaired fear renewal. FSK and rolipram exhibited the opposite effect, which also occurred in the retrieval of original fear memory. This change in fear renewal was regulated by PDE4 isoforms PDE4A, PDE4A5, and PDE4D. In addition, FSK and rolipram facilitated the acquisition of fear conditioning in long-term memory, but not short-term memory, while Rp-cAMP impaired long-term memory. For extinction, FSK and rolipram inhibited extinction process, while Rp-cAMP facilitated fear extinction. CONCLUSION: These findings demonstrated that fear renewal activated cAMP signaling in the DG through decreased PDE4 activity. Because of the role of cAMP signaling in the acquisition or retrieval of fear conditioning and encoding of extinction, it is speculated that initial learning and extinction may have similarities in molecular mechanism, especially fear retrieval and fear renewal may share cAMP signaling pathway in the DG.

Regulation of Fear Extinction in the Basolateral Amygdala by Dopamine D2 Receptors Accompanied by Altered GluR1, GluR1-Ser845 and NR2B Levels.

The amygdala, a critical structure for both Pavlovian fear conditioning and fear extinction, receives sparse but comprehensive dopamine innervation and contains dopamine D1 and D2 receptors. Fear extinction, which involves learning to suppress the expression of a previously learned fear, appears to require the dopaminergic system. The specific roles of D2 receptors in mediating associative learning underlying fear extinction require further study. Intra-basolateral amygdala (BLA) infusions of a D2 receptor agonist, quinpirole, and a D2 receptor antagonist, sulpiride, prior to fear extinction and extinction retention were tested 24 h after fear extinction training for long-term memory (LTM). LTM was facilitated by quinpirole and attenuated by sulpiride. In addition, A-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor glutamate receptor 1 (GluR1) subunit, GluR1 phospho-Ser845, and N-methyl-D-aspartic acid receptor NR2B subunit levels in the BLA were generally increased by quinpirole and down-regulated by sulpiride. The present study suggests that activation of D2 receptors facilitates fear extinction and that blockade of D2 receptors impairs fear extinction, accompanied by changes in GluR1, GluR1-Ser845 and NR2B levels in the amygdala.