Various auditory tones have been used as conditioned stimuli (CS) for fear conditioning, but researchers have largely neglected the effect that different types of auditory tones may have on fear memory processing. Here, we report that at lateral amygdala (LA) synapses (a storage site for fear memory), conditioning with different types of auditory CSs (2.8 kHz tone, white noise, FM tone) recruits distinct forms of long-term potentiation (LTP) and inserts calcium permeable AMPA receptor (CP-AMPAR) for variable periods. White noise or FM tone conditioning produced brief insertion (<6 hr after conditioning) of CP-AMPARs, whereas 2.8 kHz tone conditioning induced more persistent insertion (≥6 hr). Consistently, conditioned fear to 2.8 kHz tone but not to white noise or FM tones was erased by reconsolidation-update (which depends on the insertion of CP-AMPARs at LA synapses) when it was performed 6 hr after conditioning. Our data suggest that conditioning with different auditory CSs recruits distinct forms of LA synaptic plasticity, resulting in more malleable fear memory to some tones than to others.

It is generally believed that after memory consolidation, memory-encoding synaptic circuits are persistently modified and become less plastic. This, however, may hinder the remaining capacity of information storage in a given neural circuit. Here we consider the hypothesis that memory-encoding synaptic circuits still retain reversible plasticity even after memory consolidation. To test this, we employed a protocol of auditory fear conditioning which recruited the vast majority of the thalamic input synaptic circuit to the lateral amygdala (T-LA synaptic circuit; a storage site for fear memory) with fear conditioning-induced synaptic plasticity. Subsequently the fear memory-encoding synaptic circuits were challenged with fear extinction and re-conditioning to determine whether these circuits exhibit reversible plasticity. We found that fear memory-encoding T-LA synaptic circuit exhibited dynamic efficacy changes in tight correlation with fear memory strength even after fear memory consolidation. Initial conditioning or re-conditioning brought T-LA synaptic circuit near the ceiling of their modification range (occluding LTP and enhancing depotentiation in brain slices prepared from conditioned or re-conditioned rats), while extinction reversed this change (reinstating LTP and occluding depotentiation in brain slices prepared from extinguished rats). Consistently, fear conditioning-induced synaptic potentiation at T-LA synapses was functionally reversed by extinction and reinstated by subsequent re-conditioning. These results suggest reversible plasticity of fear memory-encoding circuits even after fear memory consolidation. This reversible plasticity of memory-encoding synapses may be involved in updating the contents of original memory even after memory consolidation.

The myristoylated zeta inhibitory peptide (ZIP), which was originally developed as a protein kinase C/Mζ (PKCζ/PKMζ) inhibitor, is known to produce the loss of different forms of memories. However, ZIP induces memory loss even in the absence of PKMζ, and its mechanism of action, therefore, remains elusive. Here, through a kinome-wide screen, we found that glycogen synthase kinase 3 beta (GSK-3β) was robustly activated by ZIP in vitro. ZIP induced depotentiation (a cellular substrate of memory erasure) of conditioning-induced potentiation at LA synapses, and the ZIP-induced depotentiation was prevented by a GSK-3β inhibitor, 6-bromoindirubin-3-acetoxime (BIO-acetoxime). Consistently, GSK-3β inhibition by BIO-acetoxime infusion or GSK-3β knockdown by GSK-3β shRNA in the LA attenuated ZIP-induced disruption of learned fear. Furthermore, conditioned fear was decreased by expression of a non-inhibitable form of GSK-3β in the LA. Our findings suggest that GSK-3β activation is a critical step for ZIP-induced disruption of memory.

Fear renewal, the context-specific relapse of fear following fear extinction, is a leading animal model of post-traumatic stress disorders (PTSD) and fear-related disorders. Although fear extinction can diminish fear responses, this effect is restricted to the context where the extinction is carried out, and the extinguished fear strongly relapses when assessed in the original acquisition context (ABA renewal) or in a context distinct from the conditioning and extinction contexts (ABC renewal). We have previously identified Ser831 phosphorylation of GluA1 subunit in the lateral amygdala (LA) as a key molecular mechanism for ABC renewal. However, molecular mechanisms underlying ABA renewal remain to be elucidated. Here, we found that both the excitatory synaptic efficacy and GluA2-lacking AMPAR activity at thalamic input synapses onto the LA (T-LA synapses) were enhanced upon ABA renewal. GluA2-lacking AMPAR activity was also increased during low-threshold potentiation, a potential cellular substrate of renewal, at T-LA synapses. The microinjection of 1-naphtylacetyl-spermine (NASPM), a selective blocker of GluA2-lacking AMPARs, into the LA attenuated ABA renewal, suggesting a critical role of GluA2-lacking AMPARs in ABA renewal. We also found that Ser831 phosphorylation of GluA1 in the LA was increased upon ABA renewal. We developed a short peptide mimicking the Ser831-containing C-tail region of GluA1, which can be phosphorylated upon renewal (GluA1S); thus, the phosphorylated GluA1S may compete with Ser831-phosphorylated GluA1. This GluA1S peptide blocked the low-threshold potentiation when dialyzed into a recorded neuron. The microinjection of a cell-permeable form of GluA1S peptide into the LA attenuated ABA renewal. In support of the GluA1S experiments, a GluA1D peptide (in which the serine at 831 is replaced with a phosphomimetic amino acid, aspartate) attenuated ABA renewal when microinjected into the LA. These findings suggest that enhancements in both the GluA2-lacking AMPAR activity and GluA1 phosphorylation at Ser831 are required for ABA renewal.

Neonatal maternal separation is a widely used method to construct an early-life stress model in rodents. In this method, pups are separated from their mothers for several hours every day during the first 2 weeks of life, which results in adverse early-life events. It is a known fact that maternal separation can exert a significant impact on the behavior and psychological health, such as anxiety and depression, in adolescent offspring. However, environmental conditions during maternal separation can differ such as the presence of other animals or by placing pups in a different dam. To investigate the differential effects of various conditions of maternal separation on the behavior of adolescent mice, we created the following groups: (1) iMS group: pups were moved to an isolated room with no other adult mice in a nearby cage, (2) eDam group: the pups randomly exchanged their dams, (3) OF group: pups were shifted to another cage with the bedding material containing maternal odor (olfactory stimulation), and (4) MS group: pups were shifted to another vivarium. From postnatal day (PND) 2-20 (i.e., 19 consecutive days), pups were separated from the dam daily for 4 h and exposed to various environments (MS, iMS, eDam, and OF) or were left undisturbed [control (CON) group]. A series of behavioral assessments were conducted to evaluate locomotion, anxiety, recognition, learning, and memory in adolescent offspring. The results showed that neonatal maternal separation led to impaired recognition memory, motor coordination, and motor skill learning across all groups. However, the iMS group exhibited anxiety-like behavior in the elevated plus maze test and enhanced the extinction of fear memory in the auditory fear conditioning test. The OF and eDam groups displayed partially recovered short-term working memory in the Y-maze test but exhibited opposite exploratory behaviors. The OF group spent more time in the center, while the eDam group spent less time. These findings demonstrated that exposure to different environmental conditions during maternal separation causes behavioral alterations in adolescent offspring, providing a potential explanation for the variation in behavioral phenotypes observed in the early-life stress models.

The return of fear following extinction therapy is an important issue associated with the treatment of many fear-related disorders. Fear renewal is a suitable model, with which context-dependent modulation of the fear response can be examined. In this model, any context outside of an extinction context (e.g., novel or familiar contexts) could evoke relapse of the fear response. However, brain regions associated with context-dependent modulation are not fully understood. The posterior parietal cortex (PPC) is considered a center for integrating multisensory information and making decisions. To study its role in the contextual modulation of fear relapse, we reversibly inactivated the PPC in mice before they were exposed to various contexts after extinction training. When muscimol was infused into the PPC, fear renewal was impaired in a novel context, but not in a familiar context. Fear relapses were blocked during optogenetic inhibition of the PPC, only when animals were placed in a novel context. We propose that the neural activity of the PPC is necessary for the relapse of a precise response to an extinguished conditioned stimulus in a novel context.

There has been a longstanding debate on whether original fear memory is inhibited or erased after extinction. One possibility that reconciles this uncertainty is that the inhibition and erasure mechanisms are engaged in different phases (early or late) of extinction. In this study, using single-session extinction training and its repetition (multiple-session extinction training), we investigated the inhibition and erasure mechanisms in the prefrontal cortex and amygdala of rats, where neural circuits underlying extinction reside. The inhibition mechanism was prevalent with single-session extinction training but faded when single-session extinction training was repeated. In contrast, the erasure mechanism became prevalent when single-session extinction training was repeated. Moreover, ablating the intercalated neurons of amygdala, which are responsible for maintaining extinction-induced inhibition, was no longer effective in multiple-session extinction training. We propose that the inhibition mechanism operates primarily in the early phase of extinction training, and the erasure mechanism takes over after that.

Auditory fear conditioning is a well-characterized rodent learning model where a neutral auditory cue is paired with an aversive outcome to induce associative fear memory. The storage of long-term auditory fear memory requires long-term potentiation (LTP) in the lateral amygdala and de novo protein synthesis. Although many studies focused on individual proteins have shown their contribution to LTP and fear conditioning, non-biased genome-wide studies have only recently been possible with microarrays, which nevertheless fall short of measuring changes at the level of proteins. Here we employed quantitative proteomics to examine the expression of hundreds of proteins in the lateral amygdala in response to auditory fear conditioning. We found that various proteins previously implicated in LTP, learning and axon/dendrite growth were regulated by fear conditioning. A substantial number of proteins that were regulated by fear conditioning have not yet been studied specifically in learning or synaptic plasticity.

There is conflicting evidence regarding whether calcium-permeable receptors are removed during group I mGluR-mediated synaptic depression. In support of this hypothesis, AMPAR rectification, a correlative index of the synaptic expression of GluA2-lacking calcium-permeable AMPARs (CP-AMPARs), is known to decrease after the induction of several types of group I mGluR-mediated long-term depression (LTD), suggesting that a significant proportion of synaptic CP-AMPARs is removed during synaptic depression. We have previously demonstrated that fear conditioning-induced synaptic potentiation in the lateral amygdala is reversed by group 1 mGluR-mediated depotentiation. Here, we examined whether CP-AMPARs are removed by mGluR1-mediated depotentiation of fear conditioning-induced synaptic potentiation. The synaptic expression of CP-AMPARs was negligible before, increased significantly 12 h after, and returned to baseline 48 h after fear conditioning, as evidenced by the changes in the sensitivity of lateral amygdala synaptic responses to NASPM. Importantly, the sensitivity to NASPM was not altered after induction of depotentiation. Our findings, together with previous results, suggest that the removal of CP-AMPARs is not required for the depotentiation of fear conditioning-induced synaptic potentiation at lateral amygdala synapses.