Extinction is not a sufficient condition to prevent fear memories from undergoing reconsolidation in the basolateral amygdala

Consolidated memories when reactivated may return to a state that requires protein synthesis in order to be restabilized (reconsolidation). It has been shown in a variety of systems that if reactivation induces significant extinction then extinction is the protein synthesis dependent memory state, rather than reconsolidation. Thus, extinction consolidation may prevent the memory from undergoing reconsolidation. We investigated whether such an interaction also exists between extinction and reconsolidation of fear memories within the amygdala, by using a within subjects experimental design. We found that inhibition of protein synthesis in the basolateral amygdala (BLA) impaired reconsolidation for both the briefly reactivated and extinguished fear memories suggesting that extinction is not a sufficient condition to prevent induction of reconsolidation in the amygdala. These findings demonstrate that extinction consolidation does not always interact with reconsolidation. Therefore, under these conditions, extinction is not a boundary condition on reconsolidation of fear memories in the basolateral amygdala.     

A protein synthesis inhibitor, cycloheximide does not alter cue-induced reinstatement of saccharin seeking

A large body of evidence indicates that reactivation of aversive memories leads to protein synthesis-dependent memory reconsolidation which can be disrupted by cycloheximide and other protein synthesis inhibitors. The aim of the present study was to investigate whether cycloheximide would alter reconsolidation of the associations involving discrete cues paired with a sweet reward in an appetitive instrumental task. Rats trained to lever press for 0.1% saccharin were repeatedly tested for cue-induced reinstatement of non-reinforced responding for saccharin. CHX (3 mg/kg, s.c.) or its vehicle was injected immediately after each reinstatement session. The protein synthesis inhibitor did not alter the ability of the saccharin-paired cues to reinstate saccharin seeking. The present results suggest that passive re-exposure to saccharin-paired discrete cues in the reinstatement procedure does not lead to any cycloheximide-sensitive reconsolidation of the original associations.     

Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats

The amygdala is an essential neural substrate for Pavlovian fear conditioning. Nevertheless, long-term synaptic plasticity in amygdaloid afferents, such as the auditory thalamus, may contribute to the formation of fear memories. We therefore compared the influence of protein synthesis inhibition in the amygdala and the auditory thalamus on the consolidation of Pavlovian fear conditioning in Long-Evans rats. Rats received three tone-footshock trials in a novel conditioning chamber. Immediately after fear conditioning, rats were infused intra-cranially with the protein synthesis inhibitor, anisomycin. Conditional fear to the tone and conditioning context was assessed by measuring freezing behaviour in separate retention tests conducted at least 24 h following conditioning. Post-training infusion of anisomycin into the amygdala impaired conditional freezing to both the auditory and contextual stimuli associated with footshock. In contrast, intra-thalamic infusions of anisomycin or a broad-spectrum protein kinase inhibitor [1-(5'-isoquinolinesulphonyl)-2-methylpiperazine, H7] did not affect conditional freezing during the retention tests. Pre-training intra-thalamic infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV), which blocks synaptic transmission in the auditory thalamus, produced a selective deficit in the acquisition of auditory fear conditioning. Autoradiographic assays of cerebral [14C]-leucine incorporation revealed similar levels of protein synthesis inhibition in the amygdala and thalamus following intra-cranial anisomycin infusions. These results reveal that the establishment of long-term fear memories requires protein synthesis in the amygdala, but not the thalamus, after auditory fear conditioning. Forms of synaptic plasticity that depend on protein synthesis, such as long-term potentiation, are likely candidates for the encoding and long-term storage of fear memories in the amygdala.     

Cued memory reconsolidation in rats requires nitric oxide

It is well‐known that the reactivation of consolidated fear memory under boundary conditions of novelty and protein synthesis blockade results in an impairment of memory, suggesting that the reactivated memory is destabilized and requires synthesis of new proteins for reconsolidation. We tested the hypothesis of nitric oxide (NO) involvement in memory destabilization during the reconsolidation process in rats using memory reactivation under different conditions. We report that administration of NO‐synthase selective blockers 3‐Br‐7‐NI or ARL in the conditions of reactivation of memory under a protein synthesis blockade prevented destabilization of fear memory to the conditioned stimulus. Obtained results support the role of NO signaling pathway in the destabilization of existing fear memory triggered by reactivation, and demonstrate that the disruption of this pathway during memory reconsolidation may prevent changes in long‐term memory.     

Extracellular signal‐regulated kinase activation is required for consolidation and reconsolidation of memory at an early stage of ontogenesis

The ability to form long‐term memories exists very early during ontogeny; however, the properties of early memory processes, brain structures involved and underlying cellular mechanisms are poorly defined. Here, we examine the role of extracellular signal‐regulated kinase (ERK), a member of the mitogen‐activated protein kinase/ERK signaling cascade, which is crucial for adult memory, in the consolidation and reconsolidation of an early memory using a conditioned taste aversion paradigm in 3‐day‐old rat pups. We show that intraperitoneal injection of SL327, the upstream mitogen‐activated protein kinase kinase inhibitor, impairs both consolidation and reconsolidation of early memory, leaving short‐term memory after acquisition and after reactivation intact. The amnesic effect of SL327 diminishes with increasing delays after acquisition and reactivation. Biochemical analyses revealed ERK hyperphosphorylation in the amygdala but not the hippocampus following acquisition, suggesting functional activation of the amygdala as early as post‐natal day 3, although there was no clear evidence for amygdalar ERK activation after reactivation. These results indicate that, despite an immature brain, the basic properties of memory and at least some of the molecular mechanisms and brain structures implicated in aversion memory share a number of similarities with the adult and emerge very early during ontogeny.     

Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory

Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.SIGNIFICANCE STATEMENT Coordinated communications between brain regions are thought to be essential during post-learning periods for consolidation of memories. However, the role of specific neural circuit pathways in this process has been scarcely explored. Using a precise optogenetic inhibition of auditory input pathways, either thalamic or cortical or both, to the LA during post-training periods, we here show that thalamic input is required for consolidation of both recent and remote fear memory, whereas cortical input is crucial for consolidation of remote fear memory. These results reveal a dissociated role of auditory input pathways to the LA for consolidation of long-term fear memory.     

Reconsolidation of fresh, remote, and extinguished fear memory in Medaka: old fears don't die

Long-term fear memory in the medaka fish (Oryzias latipes) regains transient sensitivity to a consolidation blocker immediately after memory reactivation in retrieval ('reconsolidation'). Here we show that reconsolidation occurs in fresh long-term memories but not in remote memories, and that the apparent amnesia induced by blockade of reconsolidation can be reinstated by an unpaired reinforcer, a procedure that has no effect on amnesia induced by blockade of consolidation. Extinction memory also undergoes post-reactivation reconsolidation, the blockade of which exposes the previously acquired fear. Hence in medaka, the process manifested in reconsolidation seems itself to consolidate; moreover, even when the post-reactivation application of the consolidation blocker is still able to disrupt the memory, the conditioned fear does not seem to go away permanently.     

Mechanisms of memory stabilization: are consolidation and reconsolidation similar or distinct processes?

Consolidation of new memories depends on a crucial phase of protein synthesis. It is widely held that, once consolidated, memories are stable and resilient to disruption. However, established memories become labile when recalled and require another phase of protein synthesis to be maintained. Therefore, it has been proposed that when a memory is reactivated it must undergo additional consolidation (reconsolidation) to persist. To determine whether reconsolidation recapitulates consolidation, in the past few years several groups have investigated whether the same molecules and pathways mediate the formation of a memory and its maintenance after reactivation. At first glance, the results appear conflicting: although both processes appear to engage the same molecules and mechanisms, brain areas involved in consolidation after initial training are not required for reconsolidation. In addition, the formation of a memory and its maintenance after reactivation seem to have distinctive temporal molecular requirements. This review concludes with a working model that could explain the apparent controversy of memory vulnerability after reactivation.     

Effects of the protein synthesis inhibitor cycloheximide on anxiety-like extinction behavior in an animal model of post-traumatic stress

The effect of cycloheximide (CXM), a protein synthesis inhibitor, on memory reconsolidation and extinction was explored in rats using a model of post-traumatic stress. Forty-two animals were exposed to predator stress followed by 1, 2, or 4 extinction trials. Saline or CXM (1 mg/kg) was administered following the last extinction trial and anxiety was measured in the elevated-plus maze (EPM) seventy-two hours later. Saline control animals exhibited elevated anxiety levels in comparison to a no stress control group. Cycloheximide appeared to maintain stress-induced anxiety responses, which otherwise declined with repeated extinction trials in the saline control groups. These findings suggest that cycloheximide may have induced amnesia for extinction, leaving the target memory of the predatory stress intact resulting in elevated levels of anxiety. The relationships between de novo protein synthesis and reconsolidation of anxiety-related memories following extinction trials may be more complex than originally thought.     

Post-session injections of a protein synthesis inhibitor,cycloheximide do not alter saccharin self-administration

A large body of evidence indicates that reactivation of aversive memories leads to protein synthesis-dependent memory reconsolidation which can be disrupted by cycloheximide (CHX) and other protein synthesis inhibitors. The aim of the present study was to investigate whether CHX would alter maintenance of well-trained instrumental responding for 0.1% saccharin. Male Wistar rats were trained to lever press for saccharin. When lever pressing stabilized, experimental self-administration sessions with CHX (3 mg/kg, s.c.) started. The animals received four experimental sessions, with each session separated by 5 days. The protein synthesis inhibitor was injected immediately after the experimental sessions 1–3. Repeated post-session injections of CHX did not alter saccharin self-administration. A two-bottle choice test conducted after the last experimental session revealed that CHX had not induced any conditioned taste aversion to 0.1% saccharin. The present results suggest that well-consolidated long-term memory of an appetitive instrumental task does not depend on de novo protein synthesis.     

Memory Destabilization and Reconsolidation Dynamically Regulate the PKMζ Maintenance Mechanism

Useful memory must balance between stability and malleability. This puts effective memory storage at odds with plasticity processes, such as reconsolidation. What becomes of memory maintenance processes during synaptic plasticity is unknown. Here we examined the fate of the memory maintenance protein PKMζ during memory destabilization and reconsolidation in male rats. We found that NMDAR activation and proteasome activity induced a transient reduction in PKMζ protein following retrieval. During reconsolidation, new PKMζ was synthesized to re-store the memory. Failure to synthesize new PKMζ during reconsolidation impaired memory but uninterrupted PKMζ translation was not necessary for maintenance itself. Finally, NMDAR activation was necessary to render memories vulnerable to the amnesic effect of PKMζ-antisense. These findings outline a transient disruption and renewal of the PKMζ memory maintenance mechanism during plasticity. We argue that dynamic changes in PKMζ protein levels can serve as an exemplary model of the molecular changes underlying memory destabilization and reconsolidation.SIGNIFICANCE STATEMENT Maintenance of long-term memory relies on the persistent activity of PKMζ. However, after retrieval, memories can become transiently destabilized and must be reconsolidated within a few hours to persist. During this period of plasticity, what happens to maintenance processes, such as those involving PKMζ, is unknown. Here we describe dynamic changes to PKMζ expression during both destabilization and reconsolidation of auditory fear memory in the amygdala. We show that destabilization induces a NMDAR- and proteasome-dependent loss of synaptic PKMζ and that reconsolidation requires synthesis of new PKMζ. This work provides clear evidence that memory destabilization disrupts ongoing synaptic maintenance processes which are restored during reconsolidation.     

Inhibition of protein synthesis or mTOR in the basolateral amygdala blocks retrieval-induced memory strengthening

Fear memory retrieval can lead to either reconsolidation (accompanied or not by strengthening of the memory trace) or extinction. Here, we show that non-reinforced retrieval of inhibitory avoidance (IA) conditioning can induce memory strengthening assessed in a subsequent retention test trial. Infusion of the protein synthesis inhibitor cycloheximide or the mTOR inhibitor rapamycin into the rat basolateral complex of the amygdala (BLA) after a reactivation (retrieval) session impaired retrieval-induced strengthening. Intra-BLA infusion of the mRNA synthesis inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole (DRB) after retrieval had no effect. These findings provide the first evidence suggesting that non-reinforced IA retrieval can lead to memory strengthening through a mechanism dependent on protein synthesis and mTOR activity in the BLA.     

Noradrenergic enhancement of reconsolidation in the amygdala impairs extinction of conditioned fear in rats--a possible mechanism for the persistence of traumatic memories in PTSD

Background: Posttraumatic stress disorder (PTSD) is associated with enhanced noradrenergic activity. Animal and human studies demonstrate that noradrenergic stimulation augments consolidation of fear learning. Retrieval of well‐established memories by presenting a learned fear cue triggers reconsolidation processes during which memories may be updated, weakened, or strengthened. We previously reported that noradrenergic blockade in the rat amygdala impairs reconsolidation of fear memories. Here we investigated the effects of noradrenergic enhancement on reconsolidation of learned fear. Methods: Using auditory fear conditioning in rats, we tested the effects of postretrieval intraamygdala infusion of the β‐adrenergic receptor agonist isoproterenol or the antagonist propranolol on conditioned fear in the amygdala. Results: A single intraamygdala infusion of isoproterenol following a retrieval of a well‐consolidated memory enhanced fear memory elicited by the learned fear stimulus and impaired extinction of this memory 48 hr later. Intraamygdala infusion of the β‐adrenergic receptor antagonist propranolol following a consecutive retrieval trial blocked the enhancing effects of isoproterenol on fear memory. Conclusions: Postretrieval β‐adrenergic stimulation in the amygdala enhances reconsolidation of fear memories, making them resistant to extinction. Noradrenergic augmentation during retrieval of fear memories may thus contribute to persistence and severity of traumatic memories. Reconsolidation may be a useful tool in understanding the pathology of PTSD and may thus help in developing new and in modifying existing treatments of traumatic memories. Depression and Anxiety 28:186–193, 2011. © 2011 Wiley‐Liss, Inc.     

Reactivation with a simple exposure to the experimental environment is sufficient to induce reconsolidation requiring protein synthesis in the hippocampal CA3 region in mice

Our understanding of the memory reconsolidation process is at an earlier stage than that of consolidation. For example, it is unclear if, as for memory consolidation, reconsolidation of a memory trace necessitates protein synthesis. In fact, conflicting results appear in the literature and this discrepancy may be due to differences in the experimental reactivation procedure. Here, we addressed the question of whether protein synthesis in the CA3 hippocampal region is crucial in memory consolidation and reconsolidation of allocentric knowledge after reactivation in different experimental conditions in the Morris water maze. We showed (1) that an injection of the protein synthesis inhibitor anisomycin in the CA3 region during consolidation or after a single reactivation trial disrupted performance and (2) that protein synthesis is required even after a simple contextual reactivation without any learning trial and independently of the presence of the reinforcement. This work demonstrates that a simple exposure to the spatial environment is sufficient to reactivate the memory trace, to make it labile, and that reconsolidation of this trace requires de novo protein synthesis.     

Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.     

On the dynamic nature of the engram: evidence for circuit-level reorganization of object memory traces following reactivation

Research has implicated the perirhinal cortex (PRh) in several aspects of object recognition memory. The specific role of the hippocampus (HPC) remains controversial, but its involvement in object recognition may pertain to processing contextual information in relation to objects rather than object representation per se. Here we investigated the roles of the PRh and HPC in object memory reconsolidation using the spontaneous object recognition task for rats. Intra-PRh infusions of the protein synthesis inhibitor anisomycin immediately following memory reactivation prevented object memory reconsolidation. Similar deficits were observed when a novel object or a salient contextual change was introduced during the reactivation phase. Intra-HPC infusions of anisomycin, however, blocked object memory reconsolidation only when a contextual change was introduced during reactivation. Moreover, disrupting functional interaction between the HPC and PRh by infusing anisomycin unilaterally into each structure in opposite hemispheres also impaired reconsolidation when reactivation was done in an altered context. These results show for the first time that the PRh is critical for reconsolidation of object memory traces and provide insight into the dynamic process of object memory storage; the selective requirement for hippocampal involvement following reactivation in an altered context suggests a substantial circuit level object trace reorganization whereby an initially PRh-dependent object memory becomes reliant on both the HPC and PRh and their interaction. Such trace reorganization may play a central role in reconsolidation-mediated memory updating and could represent an important aspect of lingering consolidation processes proposed to underlie long-term memory modulation and stabilization.     

The striatal-enriched protein tyrosine phosphatase gates long-term potentiation and fear memory in the lateral amygdala.

BACKGROUND: Formation of long-term memories is critically dependent on extracellular-regulated kinase (ERK) signaling. Activation of the ERK pathway by the sequential recruitment of mitogen-activated protein kinases is well understood. In contrast, the proteins that inactivate this pathway are not as well characterized. METHODS: Here we tested the hypothesis that the brain-specific striatal-enriched protein tyrosine phosphatase (STEP) plays a key role in neuroplasticity and fear memory formation by its ability to regulate ERK1/2 activation. RESULTS: STEP co-localizes with the ERKs within neurons of the lateral amygdala. A substrate-trapping STEP protein binds to the ERKs and prevents their nuclear translocation after glutamate stimulation in primary cell cultures. Administration of TAT-STEP into the lateral amygdala (LA) disrupts long-term potentiation (LTP) and selectively disrupts fear memory consolidation. Fear conditioning induces a biphasic activation of ERK1/2 in the LA with an initial activation within 5 minutes of training, a return to baseline levels by 15 minutes, and an increase again at 1 hour. In addition, fear conditioning results in the de novo translation of STEP. Inhibitors of ERK1/2 activation or of protein translation block the synthesis of STEP within the LA after fear conditioning. CONCLUSIONS: Together, these data imply a role for STEP in experience-dependent plasticity and suggest that STEP modulates the activation of ERK1/2 during amygdala-dependent memory formation. The regulation of emotional memory by modulating STEP activity may represent a target for the treatment of psychiatric disorders such as posttraumatic stress disorder (PTSD), panic, and anxiety disorders.