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Previously, we demonstrated that auditory fear conditioning produces presynaptic potentiation at lateral to basal amygdala (LA-BA) synapses, which occludes high-frequency stimulation (HFS)-induced ex-vivo LTP. We also found that the HFS-induced ex-vivo LTP requires presynaptic hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity. In this study, we investigated whether HCN channels are necessary for auditory fear conditioning in vivo. Our results show that ZD7288, an HCN channel blocker, reduced synaptic transmission and decreased the paired pulse ratio (PPR) only in slices from rats that underwent auditory fear conditioning, but not from naïve rats. This indicates that fear conditioning involves HCN channel-dependent presynaptic potentiation at LA-BA synapses. Importantly, injecting ZD7288 into the basal amygdala (BA) before auditory fear conditioning significantly impaired long-term fear memory formation. Since HCN channel activity is necessary for LTP at LA-BA synapses but not at cortico-BA, cortico-LA, or thalamo-LA synapses, HCN channel-dependent presynaptic potentiation at LA-BA synapses appears to be crucial for auditory fear conditioning., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The lateral amygdala (LA) is a main sensory input site from the cortical and thalamic regions. In turn, LA glutamatergic pyramidal neurons strongly project to the basal amygdala (BA). Although it is well known that auditory fear conditioning involves synaptic potentiation in the LA, it is not clear whether the LA-BA synaptic transmission is modified upon auditory fear conditioning. Here we found that high-frequency stimulation ex vivo resulted in long-term potentiation (LTP) with a concomitant enhancement of neurotransmitter release at LA-BA synapses. Auditory fear conditioning also led to the presynaptic facilitation at LA-BA synapses. Meanwhile, AMPA/NMDA current ratio was not changed upon fear conditioning, excluding the involvement of postsynaptic mechanism. Notably, fear conditioning occluded electrically induced ex vivo LTP in the LA-BA pathway, indicating that the conditioning and electrically induced LTP share common mechanisms. Our findings suggest that the presynaptic potentiation of LA-BA synapses may be involved in fear conditioning., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

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.

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.

Various subtypes of metabotropic glutamate receptors (mGluRs) have been implicated in fear extinction, but mGluR2/3 subtype has not been tested. Here, we found that microinjection of an mGluR2/3 antagonist, LY341495, into the lateral amygdala (LA), but not into the adjacent central amygdala (CeA), impaired extinction retention without affecting within-session extinction. In contrast, we failed to detect any significant changes in motility and anxiety during a period when extinction training or retention was performed after LY341495 injection, suggesting that the effect of LY341495 is specific to conditioned responses. Subsequently, on the basis of a previous finding that a long-term potentiation of presynaptic efficacy at cortical input synapses onto the lateral amygdala (C-LA synapses) supports conditioned fear, we tested the hypothesis that activation of mGluR2/3 leads to fear extinction via a long-term weakening of presynaptic functions at C-LA synapses. Fear extinction produced a decrease in C-LA synaptic efficacy, whereas LY341495 infusion into the LA blocked this extinction-induced C-LA efficacy decrease without altering synaptic efficacy at other LA synapses. Furthermore, extinction enhanced paired pulse ratio (PPR) of EPSCs, which inversely correlates with presynaptic release probability, whereas LY341495 infusion into the LA attenuated the extinction-induced increase in PPR, suggesting the presence of mGluR2/3-dependent presynaptic changes after extinction. Consistently, extinction occluded a presynaptic form of depression at C-LA synapses, whereas the LY341495 infusion into the LA rescued this occlusion. Together, our findings suggest that mGluR2/3 is required for extinction retention and that the mGluR2/3 action is mediated by the long-term weakening of release probability at C-LA synapses.

Fear renewal, a widely pursued model of post-traumatic stress disorder and phobias, refers to the context-specific relapse of conditioned fear after extinction. However, its molecular mechanisms are largely unknown. We found that renewal-inducing stimuli, generally believed to be insufficient to induce synaptic plasticity, enhanced excitatory synaptic strength, activity of synaptic GluA2-lacking AMPA receptors and Ser831 phosphorylation of synaptic surface GluA1 in the lateral nucleus of the amygdala (LAn) of fear-extinguished rats. Consistently, the induction threshold for LAn synaptic potentiation was considerably lowered after extinction, and renewal occluded this low-threshold potentiation. The low-threshold potentiation (a potential cellular substrate for renewal), but not long-term potentiation, was attenuated by dialysis into LAn neurons of a GluA1-derived peptide that competes with Ser831-phosphorylated GluA1. Microinjections of the same peptide into the LAn attenuated fear renewal, but not fear learning. Our findings suggest that GluA1 phosphorylation constitutes a promising target for clinical treatment of aberrant fear-related disorders.

Late-phase long-term potentiation (L-LTP) of excitatory synaptic transmission at thalamic input synapses onto the lateral amygdala (T-LA synapses) has been proposed as a cellular substrate for long-term fear memory. This notion is evidenced primarily by previous reports in which the same pharmacological treatments block both T-LA L-LTP and the consolidation of fear memory. In this study, we report that fear conditioning occludes L-LTP at T-LA synapses in brain slices prepared after fear memory consolidation. L-LTP was restored either when synaptic depotentiation was induced prior to L-LTP induction in brain slices prepared from conditioned rats or when brain slices were prepared from conditioned rats that had been exposed to subsequent fear extinction, which is a behavior paradigm known to induce in vivo synaptic depotentiation at T-LA synapses. These results suggest that fear conditioning recruits L-LTP-like mechanisms that are reversible and saturable at T-LA synapses., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Auditory fear memory is thought to be maintained by fear conditioning-induced potentiation of synaptic efficacy, which involves enhanced expression of surface AMPA receptor (AMPAR) at excitatory synapses in the lateral amygdala (LA). Depotentiation, reversal of conditioning-induced potentiation, has been proposed as a cellular mechanism for fear extinction; however, a direct link between depotentiation and extinction has not yet been tested. To address this issue, we applied both ex vivo and in vivo approaches to rats in which fear memory had been consolidated. A unique form of depotentiation reversed conditioning-induced potentiation at thalamic input synapses onto the LA (T-LA synapses) ex vivo. Extinction returned the enhanced T-LA synaptic efficacy observed in conditioned rats to baseline and occluded the depotentiation. Consistently, extinction reversed conditioning-induced enhancement of surface expression of AMPAR subunits in LA synaptosomal preparations. A GluR2-derived peptide that blocks regulated AMPAR endocytosis inhibited depotentiation, and microinjection of a cell-permeable form of the peptide into the LA attenuated extinction. Our results are consistent with the use of depotentiation to weaken potentiated synaptic inputs onto the LA during extinction and provide strong evidence that AMPAR removal at excitatory synapses in the LA underlies extinction.