Network-level changes in the brain underlie fear memory strength.

The strength of a fear memory significantly influences whether it drives adaptive or maladaptive behavior in the future. Yet, how mild and strong fear memories differ in underlying biology is not well understood. We hypothesized that this distinction may not be exclusively the result of changes within specific brain regions, but rather the outcome of collective changes in connectivity across multiple regions within the neural network. To test this, rats were fear conditioned in protocols of varying intensities to generate mild or strong memories. Neuronal activation driven by recall was measured using c-fos immunohistochemistry in 12 brain regions implicated in fear learning and memory. The interregional coordinated brain activity was computed and graph-based functional networks were generated to compare how mild and strong fear memories differ at the systems level. Our results show that mild fear recall is supported by a well-connected brain network with small-world properties in which the amygdala is well-positioned to be modulated by other regions. In contrast, this connectivity is disrupted in strong fear memories and the amygdala is isolated from other regions. These findings indicate that the neural systems underlying mild and strong fear memories differ, with implications for understanding and treating disorders of fear dysregulation.

Remembering the fear that arose during a dangerous experience is important as it teaches us to avoid similar circumstances in the future. The intensity of the initial experience will often influence the strength of the memory. Milder memories often lead to responses that protect individuals from harm (known as adaptive behaviors). However, stronger memories of more traumatic experiences can sometimes trigger disproportionate responses to a situation (known as maladaptive behaviors), such as in individuals with phobias or post-traumatic stress disorder (PTSD). Forming and retrieving fear memories requires different parts of the brain to work together and send signals to one another. At the core of this network is the amygdala (also known as the fear center of the brain), which other brain regions then feed into to modulate the fear response to ensure it is appropriate and manageable. However, it remained unclear whether neurons in these brain regions wire together differently when recalling mild or more severe fear memories. Identifying these differences may help explain why certain fear memories lead to adaptive behaviors, while others result in maladaptive ones. To investigate this question, Haubrich and Nader generated fear memories in rats that triggered either mild fear responses or strong responses akin to trauma. Imaging tools were then used to measure the activity and connections between neurons across 12 regions of the brain known to be involved in remembering fearful experiences. This revealed that recalling mild fear memories resulted in a well-coordinated network of neurons which could effectively send information between the different brain regions. In contrast, severe fear memories led to disrupted overall connectivity, with the amygdala becoming disconnected from the other brain regions. The results reveal stark contrasts in the pattern of neuronal connections formed by mild and severe fear memories. Investigating the specific pathways involved in these differences will allow scientists to gain a better understanding of why memories of traumatic experiences can lead to maladaptive behaviors, including those formed as a result of PTSD.

Dorsal Hippocampal ß-Adrenergic System Modulates Recognition Memory Reconsolidation.

Targeting reconsolidation with propranolol, a blocker of ß-adrenergic receptors (ß-ARs), emerged as a potential treatment for maladaptive memories such as those involved in posttraumatic stress disorder (PTSD). Reconsolidation targeting treatments for PTSD are becoming a common practice in the clinic and it is important to unveil any side effects upon 'non-targeted' memories. While previous studies have focused on propranolol's effects on the reconsolidation of emotional/distressful memories, the present study asked whether propranolol is involved in the reconsolidation of recognition memories - by assessing its effects on distinct memory components and the role of the dorsal hippocampus. Rats performed an object recognition (OR) task where they were exposed to different objects: A and B presented during the sample phase; A and C presented during the reactivation phase; and D in combination of either A, B, or C during a final test. Intra-hippocampal injections of propranolol (5 µg or 10 µg) were conducted immediately after the reactivation session. Propranolol infusions consistently impaired the addition of novel information to the previously consolidated memory trace regardless of dose, and the retention of familiar objects was not affected. Higher doses of propranolol also hindered memory of a familiar object that was not presented during the reactivation session, but was previously placed at the same location where novel information was presented during reactivation. The present results shed light on the role of ß-ARs on the reconsolidation of different memory components and argue for the need for further studies examining possible recognition memory deficits following propranolol treatment.

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.

Noradrenergic projections from the locus coeruleus to the amygdala constrain fear memory reconsolidation.

Memory reconsolidation is a fundamental plasticity process in the brain that allows established memories to be changed or erased. However, certain boundary conditions limit the parameters under which memories can be made plastic. Strong memories do not destabilize, for instance, although why they are resilient is mostly unknown. Here, we investigated the hypothesis that specific modulatory signals shape memory formation into a state that is reconsolidation-resistant. We find that the activation of the noradrenaline-locus coeruleus system (NOR-LC) during strong fear memory encoding increases molecular mechanisms of stability at the expense of lability in the amygdala of rats. Preventing the NOR-LC from modulating strong fear encoding results in the formation of memories that can undergo reconsolidation within the amygdala and thus are vulnerable to post-reactivation interference. Thus, the memory strength boundary condition on reconsolidation is set at the time of encoding by the action of the NOR-LC.

New memories must go through a period of consolidation to become stable and long-lasting in the brain. Recalling memories can make them unstable again, so that they need reconsolidating. Treatments in which the reconsolidation process is interrupted have been used to help weaken traumatic fear memories. However, memories of severe trauma, such as in post-traumatic stress disorder, are particularly resistant to reconsolidation treatments. Haubrich et al. used rats to study how trauma shapes memory formation and what biological mechanisms are involved in preventing the destabilization/reconsolidation cycle. The rats were exposed to a sound at the same time as receiving a mild electric shock. Half of the rats experienced the shock once, creating a 'weak' memory. The other half experienced it ten times, creating a 'strong' memory. The rats' memory of the electric shock was measured by seeing how they responded when they heard the sound again without the shock. Some of the rats were given the drug anisomycin, an antibiotic that stops cells from making new proteins and is known for producing amnesia, to block reconsolidation of the memory after hearing the sound again. Treatment with the drug reduced future responses in the rats that had experienced the shock once, but had no effect on the rats that had experienced it ten times, demonstrating that the stronger memories were resistant to reconsolidation therapy. The rats with the strong memories also had lower levels of proteins in the brain that are involved in plasticity ­ the ability of the brain to change and adapt. Haubrich et al. hypothesized that the stability of the strong memories could be caused by signaling from the locus coeruleus, a region of the brainstem involved in the response to stress. When the signaling from the locus coeruleus was blocked in the strong-memory rats, they became responsive to reconsolidation therapy with anisomycin. These results help to better understand how traumatic memories become engrained, potentially leading to new treatment options for people with post-traumatic stress disorder.

Impairments to Consolidation, Reconsolidation, and Long-Term Memory Maintenance Lead to Memory Erasure.

An enduring problem in neuroscience is determining whether cases of amnesia result from eradication of the memory trace (storage impairment) or if the trace is present but inaccessible (retrieval impairment). The most direct approach to resolving this question is to quantify changes in the brain mechanisms of long-term memory (BM-LTM). This approach argues that if the amnesia is due to a retrieval failure, BM-LTM should remain at levels comparable to trained, unimpaired animals. Conversely, if memories are erased, BM-LTM should be reduced to resemble untrained levels. Here we review the use of BM-LTM in a number of studies that induced amnesia by targeting memory maintenance or reconsolidation. The literature strongly suggests that such amnesia is due to storage rather than retrieval impairments. We also describe the shortcomings of the purely behavioral protocol that purports to show recovery from amnesia as a method of understanding the nature of amnesia.

Synaptic consolidation as a temporally variable process: Uncovering the parameters modulating its time-course.

Memories are not instantly created in the brain, requiring a gradual stabilization process called consolidation to be stored and persist in a long-lasting manner. However, little is known whether this time-dependent process is dynamic or static, and the factors that might modulate it. Here, we hypothesized that the time-course of consolidation could be affected by specific learning parameters, changing the time window where memory is susceptible to retroactive interference. In the rodent contextual fear conditioning paradigm, we compared weak and strong training protocols and found that in the latter memory is susceptible to post-training hippocampal inactivation for a shorter period of time. The accelerated consolidation process triggered by the strong training was mediated by glucocorticoids, since this effect was blocked by pre-training administration of metyrapone. In addition, we found that pre-exposure to the training context also accelerates fear memory consolidation. Hence, our results demonstrate that the time window in which memory is susceptible to post-training interferences varies depending on fear conditioning intensity and contextual familiarity. We propose that the time-course of memory consolidation is dynamic, being directly affected by attributes of the learning experiences.

Hippocampal plasticity mechanisms mediating experience-dependent learning change over time.

The requirement of NMDA receptor (NMDAR) activity for memory formation is well described. However, the plasticity mechanisms for memory can be modified by experience, such that a future similar learning becomes independent of NMDARs. This effect has often been reported in learning events conducted with a few days interval. In this work, we asked whether the NMDAR-independency is permanent or the brain regions and plasticity mechanisms of experience-dependent learning may change over time. Considering that contextual memories undergo a gradual reorganization over time, becoming progressively independent from the hippocampus and dependent upon cortical regions, we investigated the brain regions mediating a new related learning conducted at a remote time-point, when the first memory was already cortically established. First, we demonstrated that anterior cingulate cortex was not able to support a learning subsequent to a previous systems-level consolidated memory; it did require at least one functional subregion of the hippocampus (ventral or dorsal). Moreover, after replicating findings showing that a few days interval between trainings induces a NMDAR-independent learning, we managed to show that a learning following a longer interval once again becomes dependent on NMDARs in the hippocampus. These findings suggest that while the previous memory grows independent from the hippocampus over time, an experience-dependent learning following a systems-consolidated memory once again engages the hippocampus and a NMDAR-dependent plasticity mechanism.

Limits on lability: Boundaries of reconsolidation and the relationship to metaplasticity.

Reconsolidation, a process by which long-term memories are rendered malleable following retrieval, has been shown to occur across many different species and types of memory. However, there are conditions under which memories do not reconsolidate, and the reasons for this are poorly understood. One emerging theory is that these boundary conditions are mediated by a form of metaplasticity: cellular changes through which experience can affect future synaptic plasticity. We review evidence that N-methyl-D-aspartate receptors (NMDARs) might contribute to this phenomenon, and hypothesize that resistance to memory destabilization may be mediated by the ratio of GluN2A/GluN2B subunits that make up these receptors. Qualities such as memory strength and the age of the memory may increase the GluN2A/GluN2B ratio, reducing the ability of reactivation cues to induce destabilization, thereby preventing reconsolidation. Other examples of experience-dependent learning and evolutionary perspectives of reconsolidation are also discussed.

Memory Reconsolidation.

Scientific advances in the last decades uncovered that memory is not a stable, fixed entity. Apparently stable memories may become transiently labile and susceptible to modifications when retrieved due to the process of reconsolidation. Here, we review the initial evidence and the logic on which reconsolidation theory is based, the wide range of conditions in which it has been reported and recent findings further revealing the fascinating nature of this process. Special focus is given to conceptual issues of when and why reconsolidation happen and its possible outcomes. Last, we discuss the potential clinical implications of memory modifications by reconsolidation.

Enhancement of extinction memory by pharmacological and behavioral interventions targeted to its reactivation.

Extinction is a process that involves new learning that inhibits the expression of previously acquired memories. Although temporarily effective, extinction does not erase an original fear association. Since the extinction trace tends to fade over time, the original memory can resurge. On the other hand, strengthening effects have been described in several reconsolidation studies using different behavioral and pharmacological manipulations. In order to know whether an extinction memory can be strengthened by reactivation-based interventions in the contextual fear conditioning task, we began by replicating the classic phenomenon of spontaneous recovery to show that brief reexposure sessions can prevent the decay of the extinction trace over time in a long-lasting way. This fear attenuation was shown to depend both on L-type calcium channels and protein synthesis, which suggests a reconsolidation process behind the reactivation-induced strengthening effect. The extinction trace was also susceptible to enhancement by a post-reactivation infusion of a memory-enhancing drug (NaB), which was also able to prevent rapid fear reacquisition (savings). These findings point to new reactivation-based approaches able to strengthen an extinction memory to promote its persistence. The constructive interactions between extinction and reconsolidation may represent a promising novel approach in the realm of fear-related disorder treatments.

Reconsolidation-induced rescue of a remote fear memory blocked by an early cortical inhibition: Involvement of the anterior cingulate cortex and the mediation by the thalamic nucleus reuniens.

Systems consolidation is a time-dependent reorganization process involving neocortical and hippocampal networks underlying memory storage and retrieval. The involvement of the hippocampus during acquisition is well described; however we know much less about the concomitant contribution of cortical activity levels to the formation of stable remote memories. Here, after a reversible pharmacological inhibition of the anterior cingulate cortex (ACC) during the acquisition of a contextual fear conditioning, retrieval of both recent and remote memories were impaired, an effect that was reverted by a single memory reactivation session 48 h after training, through a destabilization-dependent mechanism interpreted as reconsolidation, that restored the normal course of systems consolidation in order to rescue a remote memory. Next we have shown that the integrity of both the anterior cingulate cortex and the thalamic nucleus reuniens (RE) were required for this reactivation-induced memory rescue. Because lidocaine infused into the RE inhibited LTP induction in the CA1-anterior cingulate cortex pathways, it seems that RE is a necessary component of the circuit underlying systems consolidation, mediating communication between dorsal hippocampus and cortical areas. To our notice, this is the first demonstration of the rescue of remote memories disrupted by ACC inhibition during acquisition, via a reconsolidation-driven mechanism. We have also shown the importance of RE to ensure the interconnection among brain areas that collectively seem to control the natural course of systems consolidation and allow the persistence of relevant emotional engrams. © 2017 Wiley Periodicals, Inc.

Forgetting of what was once learned: Exploring the role of postsynaptic ionotropic glutamate receptors on memory formation, maintenance, and decay.

Over the past years, extensive research in experimental cognitive neuroscience has provided a comprehensive understanding about the role of ionotropic glutamate receptor (IGluR)-dependent signaling underpinning postsynaptic plasticity induced by long-term potentiation (LTP), the leading cellular basis of long-term memory (LTM). However, despite the fact that iGluR-mediated postsynaptic plasticity regulates the formation and persistence of LTP and LTM, here we discuss the state-of-the-art regarding the mechanisms underpinning both LTP and LTM decay. First, we review the crucial roles that iGluRs play on memory encoding and stabilization. Second, we discuss the latest findings in forgetting considering hippocampal GluA2-AMPAR trafficking at postsynaptic sites as well as dendritic spine remodeling possibly involved in LTP decay. Third, on the role of retrieving consolidated LTMs, we discuss the mechanisms involved in memory destabilization that occurs followed reactivation that share striking similarities with the neurobiological basis of forgetting. Fourth, since different AMPAR subunits as well as postsynaptic scaffolding proteins undergo ubiquitination, the ubiquitin-proteasome system (UPS) is discussed in light of memory decay. In conclusion, we provide an integrated overview revealing some of the mechanisms determining memory forgetting that are mediated by iGluRs. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Novel learning accelerates systems consolidation of a contextual fear memory.

After initial encoding memories may undergo a time-dependent reorganization, becoming progressively independent from the hippocampus (HPC) and dependent on cortical regions such as the anterior cingulate cortex (ACC). Although the mechanisms underlying systems consolidation are somewhat known, the factors determining its temporal dynamics are still poorly understood. Here, we studied the influence of novel learning occurring between training and test sessions on the time-course of HPC- and ACC-dependency of contextual fear conditioning (CFC) memory expression. We found that muscimol was disruptive when infused into the HPC up to 35 days after training, while the ACC is vulnerable only after 45 days. However, when animals were subjected to a series of additional, distinct tasks to be learned within the first 3 weeks, muscimol became effective sooner. Muscimol had no effect in the HPC at 20 days after training, exactly when the ACC becomes responsive to this treatment. Thus, our data indicates that the encoding of new information generates a tight interplay between distinct memories, accelerating the reorganization of previously stored long term memories between the hippocampal and cortical areas. © 2016 Wiley Periodicals, Inc.

The dynamic nature of systems consolidation: Stress during learning as a switch guiding the rate of the hippocampal dependency and memory quality.

Memory fades over time, becoming more schematic or abstract. The loss of contextual detail in memory may reflect a time-dependent change in the brain structures supporting memory. It has been well established that contextual fear memory relies on the hippocampus for expression shortly after learning, but it becomes hippocampus-independent at a later time point, a process called systems consolidation. This time-dependent process correlates with the loss of memory precision. Here, we investigated whether training intensity predicts the gradual decay of hippocampal dependency to retrieve memory, and the quality of the contextual memory representation over time. We have found that training intensity modulates the progressive decay of hippocampal dependency and memory precision. Strong training intensity accelerates systems consolidation and memory generalization in a remarkable timeframe match. The mechanisms underpinning such process are triggered by glucocorticoid and noradrenaline released during training. These results suggest that the stress levels during emotional learning act as a switch, determining the fate of memory quality. Moderate stress will create a detailed memory, whereas a highly stressful training will develop a generic gist-like memory.

Involvement of the infralimbic cortex and CA1 hippocampal area in reconsolidation of a contextual fear memory through CB1 receptors: Effects of CP55,940.

The endocannabinoid system (ECS) has a pivotal role in different cognitive functions such as learning and memory. Recent evidence confirm the involvement of the hippocampal CB1 receptors in the modulation of both memory extinction and reconsolidation processes in different brain areas, but few studies focused on the infralimbic cortex, another important cognitive area. Here, we infused the cannabinoid agonist CP55,940 either into the infralimbic cortex (IL) or the CA1 area of the dorsal hippocampus (HPC) of adult male Wistar rats immediately after a short (3min) reactivation session, known to labilize a previously consolidated memory trace in order to allow its reconsolidation with some modification. In both structures, the treatment was able to disrupt reconsolidation in a relatively long lasting way, reducing the freezing response. To our notice, this is the first demonstration of ECS involvement in reconsolidation in the Infralimbic Cortex. Despite poorly discriminative between CB1 and CB2 receptors, CP55,940 is a potent agent, and these results suggest that a similar CB1-dependent circuitry is at work both in HPC and in the IL during memory reconsolidation.

Memory reconsolidation may be disrupted by a distractor stimulus presented during reactivation.

Memories can be destabilized by the reexposure to the training context, and may reconsolidate into a modified engram. Reconsolidation relies on some particular molecular mechanisms involving LVGCCs and GluN2B-containing NMDARs. In this study we investigate the interference caused by the presence of a distractor - a brief, unanticipated stimulus that impair a fear memory expression - during the reactivation session, and tested the hypothesis that this disruptive effect relies on a reconsolidation process. Rats previously trained in the contextual fear conditioning (CFC) were reactivated in the presence or absence of a distractor stimulus. In the test, groups reactivated in the original context with distractor displayed a reduction of the freezing response lasting up to 20 days. To check for the involvement of destabilization / reconsolidation mechanisms, we studied the effect of systemic nimodipine (a L-VGCC blocker) or intra-CA1 ifenprodil (a selective GluN2B/NMDAR antagonist) infused right before the reactivation session. Both treatments were able to prevent the disruptive effect of distraction. Ifenprodil results also bolstered the case for hippocampus as the putative brain structure hosting this phenomenon. Our results provide some evidence in support of a behavioral, non-invasive procedure that was able to disrupt an aversive memory in a long-lasting way.

Reconsolidation allows fear memory to be updated to a less aversive level through the incorporation of appetitive information.

The capacity to adapt to new situations is one of the most important features of memory. When retrieved, memories may undergo a labile state that is sensitive to modification. This process, called reconsolidation, can lead to memory updating through the integration of new information into a previously consolidated memory background. Thus reconsolidation provides the opportunity to modify an undesired fear memory by updating its emotional valence to a less aversive level. Here we evaluated whether a fear memory can be reinterpreted by the concomitant presentation of an appetitive stimulus during its reactivation, hindering fear expression. We found that memory reactivation in the presence of appetitive stimuli resulted in the suppression of a fear response. In addition, fear expression was not amenable to reinstatement, spontaneous recovery, or rapid reacquisition. Such effect was prevented by either systemic injection of nimodipine or intra-hippocampal infusion of ifenprodil, indicating that memory updating was mediated by a reconsolidation mechanism relying on hippocampal neuronal plasticity. Taken together, this study shows that reconsolidation allows for a 're-signification' of unwanted fear memories through the incorporation of appetitive information. It brings a new promising cognitive approach to treat fear-related disorders.

Reconsolidation may incorporate state-dependency into previously consolidated memories.

Some memories enter into a labile state after retrieval, requiring reconsolidation in order to persist. One functional role of memory reconsolidation is the updating of existing memories. There are reports suggesting that reconsolidation can be modulated by a particular endogenous process taking place concomitantly to its natural course, such as water or sleep deprivation. Here, we investigated whether an endogenous process activated during a natural/physiological experience, or a pharmacological intervention, can also contribute to memory content updating. Using the contextual fear conditioning paradigm in rats, we found that the endogenous content of an aversive memory can be updated during its reconsolidation incorporating consequences of natural events such as water deprivation, transforming a previously stored memory into a state-dependent one. This updating seems to be mediated by the activation of angiotensin AT1 receptors in the dorsal hippocampus and local infusion of human angiotensin II (ANGII) was shown to mimic the water deprivation effects on memory reconsolidation. Systemic morphine injection was also able to turn a previously acquired experience into a state-dependent memory, reproducing the very same effects obtained by water deprivation or local angiotensin II infusion, and suggesting that other state-dependent-inducing protocols would also be able to contribute to memory updating. These findings trigger new insights about the influence of ordinary daily life events upon memory in its continuing reconstruction, adding the realm of reconsolidation to the classical view of endogenous modulation of consolidation.