The role of the nucleus basalis magnocellularis in fear conditioning consolidation in the rat

The nucleus basalis magnocellularis (NBM) is known to be involved in the memorization of several conditioned responses. To investigate the role of the NBM in fear conditioning memorization, this neural site was subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that had undergone fear training to acoustic conditioned stimulus (CS) and context. TTX was stereotaxically administered to different groups of rats at increasing intervals after the acquisition session. Memory was assessed as the conditioned freezing duration measured during retention testing, always performed 72 and 96 h after TTX administration. In this way, there was no interference with normal NBM function during either acquisition or retrieval phases, allowing any amnesic effect to be due only to consolidation disruption. The results show that for contextual fear response memory consolidation, NBM functional integrity is necessary up to 24 h post-acquisition. On the other hand, NBM functional integrity was shown to be necessary for memory consolidation of the acoustic CS fear response only immediately after acquisition and not 24-h post-acquisition. The present findings help to elucidate the role of the NBM in memory consolidation and better define the neural circuits involved in fear memories.     

Disrupting reconsolidation: pharmacological and behavioral manipulations

We previously demonstrated that disrupting reconsolidation by pharmacological manipulations "deleted" the emotional expression of a fear memory in humans. If we are to target reconsolidation in patients with anxiety disorders, the disruption of reconsolidation should produce content-limited modifications. At the same time, the fear-erasing effects should not be restricted to the feared cue itself considering that fear generalization is a main characteristic of anxiety disorders. In Experiment I and Experiment I(b), we addressed these issues using a within-subject differential startle fear conditioning paradigm and a test of fear generalization. In Experiment II, we tested whether a behavioral approach targeting the reconsolidation through extinction learning was also effective in weakening the original fear memory. A behavioral procedure is evidently preferred over drug manipulations provided that similar effects can be obtained. Here, the extinction procedure subsequent to retrieval did not "erase" the emotional expression of the fear memory as the retrieval techniques (i.e., reminder shocks and reacquisition) unveiled a return of the startle fear response to the fear-relevant stimuli. In contrast, β-adrenergic receptor blockade during reconsolidation selectively deleted the fear-arousing aspects of the memory (i.e., startle fear response) along with its category-related information. The pharmacological manipulation rendered the core memory trace too weak to observe fear generalization after successful reacquisition. Hence, relearning following the disruption of reconsolidation seems to be qualitatively different from initial learning. Our findings demonstrate that disrupting reconsolidation by pharmacological manipulations, although selective, undermines the generalization of fear, a key feature of anxiety disorders.     

Distinctive roles for amygdalar CREB in reconsolidation and extinction of fear memory

Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral amygdala disrupted reconsolidation of auditory fear memories. In contrast, manipulations of CREB in the amygdala did not modify extinction of fear. These findings suggest that the role of CREB in modulation of memory after retrieval is dynamic and that CREB activity in the basolateral amygdala is involved in fear memory reconsolidation.     

Trace and contextual fear conditioning are impaired following unilateral microinjection of muscimol in the ventral hippocampus or amygdala, but not the medial prefrontal cortex

Trace fear conditioning, in which a brief empty "trace interval" occurs between presentation of the CS and UCS, differs from standard delay conditioning in that contributions from both the hippocampus and prelimbic medial prefrontal cortex (PL mPFC) are required to form a normal long term memory. Little is currently known about how the PL interacts with various temporal lobe structures to support learning across this temporal gap between stimuli. We temporarily inactivated PL along with either ventral hippocampus or amygdala in a disconnection design to determine if these structures functionally interact to acquire trace fear conditioning. Disconnection (contralateral injections) of the PL with either the ventral hippocampus or amygdala impaired trace fear conditioning; however, ipsilateral control rats were also impaired. Follow-up experiments examined the effects of unilateral inactivation of the PL, ventral hippocampus, or amygdala during conditioning. The results of this study demonstrate that unilateral inactivation of the ventral hippocampus or amygdala impairs memory, while bilateral inactivation of the PL is required to produce a deficit. Memory deficits after unilateral inactivation of the ventral hippocampus or amygdala prevent us from determining whether the mPFC functionally interacts with the medial temporal lobe using a disconnection approach. Nonetheless, our findings suggest that the trace fear network is more integrated than previously thought.     

Effects of intra-amygdala infusion of CB1 receptor agonists on the reconsolidation of fear-potentiated startle

The cannabinoid CB1 receptor has been shown to be critically involved in the extinction of fear memory. Systemic injection of a CB1 receptor antagonist prior to extinction training blocked extinction. Conversely, administration of the cannabinoid uptake inhibitor AM404 facilitated extinction in a dose-dependent manner. Here we show that bilateral infusion of CB1 receptor agonists into the amygdala after memory reactivation blocked reconsolidation of fear memory measured with fear-potentiated startle. The effect was dose-dependent and could be blocked by AM251, a specific CB1 receptor antagonist. In contrast, the effect of CB1 agonists on reconsolidation was no longer seen if memory reactivation was omitted. Concomitant with block of reconsolidation, CB1 agonist-treated animals did not exhibit shock-induced reinstatement or spontaneous recovery of fear. The absence of recovery was not attributable to permanent damage to the amygdala in WIN-treated rats, nor did the effect result from alteration of baseline startle or shock reactivity. These results suggest that CB1 agonists could impair fear memory via blocking reconsolidation.     

Early growth response gene 1 (Egr-1) is required for new and reactivated fear memories in the lateral amygdala

The immediate-early gene early growth response gene-1 (EGR-1, zif-268) has been extensively studied in synaptic plasticity and memory formation in a variety of memory systems. However, a convincing role for EGR-1 in amygdala-dependent memory consolidation processes has yet to emerge. In the present study, we have examined the role of EGR-1 in the consolidation and reconsolidation of amygdala-dependent auditory Pavlovian fear conditioning. In our first series of experiments, we show that EGR-1 is regulated following auditory fear conditioning in the lateral nucleus of the amygdala (LA). Next, we use antisense oligodeoxynucleotide (ODN) knockdown of EGR-1 in the LA to show that training-induced expression of EGR-1 is required for memory consolidation of auditory fear conditioning; that is, long-term memory (LTM) is significantly impaired while acquisition and short-term memory (STM) are intact. In a second set of experiments, we show that EGR-1 is regulated in the LA by retrieval of an auditory fear memory. We then show that retrieval-induced expression of EGR-1 in the LA is required for memory reconsolidation of auditory fear conditioning; that is, post-retrieval (PR)-LTM is significantly impaired while memory retrieval and PR-STM are intact. Additional experiments show these effects to be restricted to the LA, to be temporally graded, and unlikely to be due to nonspecific toxicity within the LA. Collectively, our findings strongly implicate a role for EGR-1 in both the initial consolidation and in the reconsolidation of auditory fear memories in the LA.     

Epigenetic alterations in the lateral amygdala are required for reconsolidation of a Pavlovian fear memory

Epigenetic mechanisms have been widely implicated in synaptic plasticity and in memory consolidation, yet little is known about the role of epigenetic mechanisms in memory reconsolidation processes. In the present study, we systematically examine the role of histone acetylation and DNA methylation in the reconsolidation of an amygdala-dependent Pavlovian fear memory. We first show that the acetylation of histone 3 (H3), but not histone 4 (H4), is regulated following auditory fear memory retrieval in the lateral nucleus of the amygdala (LA). We next show that histone deacetylase (HDAC) inhibition in the LA enhances both retrieval-induced histone acetylation and reconsolidation of an auditory fear memory. Conversely, inhibition of DNA methytransferase (DNMT) activity in the LA significantly impairs both retrieval-related H3 acetylation and fear memory reconsolidation. The effects of HDAC and DNMT inhibitors on fear memory reconsolidation were observed to be time-limited and were not evident in the absence of memory reactivation. Further, memories lost following DNMT inhibition were not observed to be vulnerable to spontaneous recovery, reinstatement, or to a shift in testing context, suggesting that memory impairment was not the result of facilitated extinction. Finally, pretreatment with the HDAC inhibitor was observed to rescue the reconsolidation deficit induced by the DNMT inhibitor. These findings collectively suggest that histone acetylation and DNA methylation are critical for reconsolidation of fear memories in the LA.     

睡眠剥夺影响恐惧情绪加工的认知神经机制

睡眠剥夺与恐惧情绪加工的各个过程息息相关。睡眠剥夺损害了恐惧的习得过程, 而且影响着杏仁核、内侧前额叶的活动及它们之间的功能连接; 睡眠剥夺削弱了恐惧记忆的巩固和再巩固过程, 不仅破坏了恐惧记忆再巩固过程相关蛋白质和酶的合成, 同时也影响着海马、杏仁核、内侧前额叶的活动以及它们之间的功能连接; 睡眠剥夺损害了恐惧的消退, 同时也改变了海马、杏仁核等相关脑区的活动模式。未来的研究应从睡眠剥夺影响恐惧情绪加工的认知神经机制、睡眠剥夺与恐惧情绪相关障碍的关系等角度展开, 力图深入理解睡眠剥夺影响恐惧情绪加工的认知神经机制。     

Late expression of brain-derived neurotrophic factor in the amygdala is required for persistence of fear memory

In many instances, increase in neuronal activity can induce biphasic secretion of a modulator. The initial release of the modulator triggers the induction of synaptic plasticity, whereas the second-phase release reinforces the efficacy of synaptic transmission and growth of dendrites and axons. In this study, we showed that fear conditioning not only induced the first but also a second peak of brain-derived neurotrophic factor (BDNF) expression. Fluorescent immunohistostaining confirmed that BDNF expression increased at 1 and 12 h after conditioning and returned to baseline at 30 h after conditioning. Mature BDNF expression increased in a similar manner. TrkB-IgG or K252a infusion before training impaired fear memory on days 1 and 7 after training. In contrast, TrkB-IgG or K252a infusion 9 h after fear conditioning did not affect memory retention on day 1 after training but impaired fear memory on day 7 after training. Fear conditioning significantly enhanced Zif268 expression in the amygdala at 12 h after training; this enhanced expression was completely inhibited by TrkB-IgG infusion 9 h after training. The level of growth-associated protein 43 (GAP-43), a marker of newly formed synapses, in the amygdala increased 7 days after fear conditioning. Moreover, conditioned rats had higher AMPA/NMDA ratio than unpaired rats. These results suggest that consolidated memory could be continuously modulated by previous molecular changes produced during memory acquisition.     

The central nucleus of the amygdala is essential for acquiring and expressing conditional fear after overtraining

The basolateral complex of the amygdala (BLA) is critical for the acquisition and expression of Pavlovian fear conditioning in rats. Nonetheless, rats with neurotoxic BLA lesions can acquire conditional fear after overtraining (75 trials). The capacity of rats with BLA lesions to acquire fear memory may be mediated by the central nucleus of the amygdala (CEA). To examine this issue, we examined the influence of neurotoxic CEA lesions or reversible inactivation of the CEA on the acquisition and expression of conditional freezing after overtraining in rats. Rats with pretraining CEA lesions (whether alone or in combination with BLA lesions) did not acquire conditional freezing to either the conditioning context or an auditory conditional stimulus after extensive overtraining. Similarly, post-training lesions of the CEA or BLA prevented the expression of overtrained fear. Lastly, muscimol infusions into the CEA prevented both the acquisition and the expression of overtrained fear, demonstrating that the effects of CEA lesions are not likely due to the destruction of en passant axons. These results suggest that the CEA is essential for conditional freezing after Pavlovian fear conditioning. Moreover, overtraining may engage a compensatory fear conditioning circuit involving the CEA in animals with damage to the BLA.     

The central amygdala nucleus via corticotropin-releasing factor is necessary for time-limited consolidation processing but not storage of contextual fear memory

The central nucleus of the amygdala (CeA) is traditionally portrayed in fear conditioning as the key neural output that relays conditioned information established in the basolateral amygdala complex to extra-amygdalar brain structures that generate emotional responses. However, several recent studies have questioned this serial processing view of the amygdalar fear conditioning circuit by showing an influence of the CeA on memory consolidation. We previously reported that inhibition of endogenous CeA secretion of corticotropin-releasing factor (CRF) at the time of contextual training effectively impaired fear memory consolidation. However, the time-dependent range of CeA CRF secretion in facilitating consolidation processing has not been examined. Therefore, to address this issue, we performed CeA site-specific microinjections of CRF antisense oligonucleotides (CRF ASO) at several post-training time intervals. Rats microinjected with CRF ASO at post-training intervals up to 24-h subsequently exhibited significant impairments in contextual freezing retention in contrast to animals treated 96-h after training. To further establish the validity of the results, CeA fiber-sparing lesions were made at two distinct post-training periods (24-h and 96-h), corresponding respectively to the temporal intervals when CeA CRF ASO administration disrupted or had no significant effects on memory consolidation. Similar to the CeA CRF ASO results, CeA lesions made 24-h, but not 96-h, after training induced significant freezing deficits in the retention test. In conclusion, the current results demonstrate: (1) an extended involvement of CeA CRF in contextual memory consolidation and (2) that contextual fear memory storage is not dependent on a functional CeA.     

Phthalic acid amygdalopetal lesion of the nucleus basalis magnocellularis induces reversible memory deficits in rats

The basolateral amygdala (BLA) is extensively implicated in emotional learning and memory. The current study investigated the contribution of cholinergic afferents to the BLA from the nucleus basalis magnocellularis in influencing aversive learning and memory. Sprague-Dawley rats were given permanent unilateral phthalic acid (300 ng) lesions of the nucleus basalis magnocellularis and were chronically implanted with cannulas aimed at the ipsilateral BLA. Lesioned rats showed a pronounced inhibitory avoidance task retention deficit that was attenuated by acute posttraining infusions of the muscarinic cholinergic agonist oxotremorine (4 ng) or the indirect agonist physostigmine (1 microg) into the BLA. Continuous multiple-trial inhibitory avoidance training and testing revealed that lesioned rats have a mild acquisition deficit, requiring approximately 1 additional shock to reach the criterion, and a pronounced consolidation deficit as indicated by a shorter latency to enter the shock compartment on the retention test. Because lesioned rats did not differ from sham-operated controls in performance on a spatial water maze task or in shock sensitivity, it is not likely that the memory impairments produced by the phthalic acid lesions are due to any general sensory or motor deficits. These findings suggest that the dense cholinergic projection from the nucleus basalis magnocellularis to the BLA is involved in both the acquisition and the consolidation of the aversive inhibitory avoidance task.     

Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala

Pavlovian fear conditioning is a robust and enduring form of emotional learning that provides an ideal model system for studying contextual regulation of memory retrieval. After extinction the expression of fear conditional responses (CRs) is context-specific: A conditional stimulus (CS) elicits greater conditional responding outside compared with inside the extinction context. Dorsal hippocampal inactivation with muscimol attenuates context-specific CR expression. We have previously shown that CS-elicited spike firing in the lateral nucleus of the amygdala is context-specific after extinction. The present study examines whether dorsal hippocampal inactivation with muscimol disrupts context-specific firing in the lateral amygdala. We conditioned rats to two separate auditory CSs and then extinguished each CS in separate and distinct contexts. Thereafter, single-unit activity and conditional freezing were tested to one CS in both extinction contexts after saline or muscimol infusion into the dorsal hippocampus. After saline infusion, rats froze more to the CS when it was presented outside of its extinction context, but froze equally in both contexts after muscimol infusion. In parallel with the behavior, lateral nucleus neurons exhibited context-dependent firing to extinguished CSs, and hippocampal inactivation disrupted this activity pattern. These data reveal a novel role for the hippocampus in regulating the context-specific firing of lateral amygdala neurons after fear memory extinction.     

Amygdala upregulation of NCAM polysialylation induced by auditory fear conditioning is not required for memory formation, but plays a role in fear extinction

There is much interest to understand the mechanisms leading to the establishment, maintenance, and extinction of fear memories. The amygdala has been critically involved in the processing of fear memories and a number of molecular changes have been implicated in this brain region in relation to fear learning. Although neural cell adhesion molecules (NCAMs) have been hypothesized to play a role, information available about their contribution to fear memories is scarce. We investigate here whether polysialylated NCAM (PSA-NCAM) contributes to auditory fear conditioning in the amygdala. First, PSA-NCAM expression was evaluated in different amygdala nuclei after auditory fear conditioning at two different shock intensities. Results showed that PSA-NCAM expression was increased 24 h post-training only in animals subjected to the highest shock intensity (1mA). Second, PSA-NCAM was cleaved in the basolateral amygdaloid complex through micro-infusions of the enzyme endoneuraminidase N, and the consequences of such treatment were investigated on the acquisition, consolidation, remote memory expression, and extinction of conditioned fear memories. Intra-amygdaloid cleavage of PSA-NCAM did not affect acquisition, consolidation or expression of remote fear memories. However, intra-amygdaloid PSA-NCAM cleavage enhanced fear extinction processes. These results suggest that upregulation of PSA-NCAM is a correlate of fear conditioning that is not necessary for the establishment of fear memory in the amygdala, but participates in mechanisms precluding fear extinction. These findings point out PSA-NCAM as a potential target for the treatment of psychopathologies that involve impairment in fear extinction.     

A role for ERK2 in reconsolidation of fear memories in mice

Recent studies have shown that consolidated fear memories, when reactivated, return to a labile state that requires a new protein synthesis for reconsolidation. Post-retrieval infusion of an inhibitor of protein synthesis blocks memory reconsolidation processes. In a previous research, the role of MAPKs in memory consolidation has been shown in emotional tasks, such as passive and active avoidance. In particular, mice knockout for ERK1 had a better performance in comparison to wild type mice in both passive and active avoidance tasks. In the present study, in order to investigate the involvement of MAPKs in memory reconsolidation processes we administered immediately after retrieval, different doses of SL327 (an inhibitor of MEK, a kinase that activates both ERK1 and ERK2) both in C57BL/6 (C57) mice and ERK1 mutant mice tested in a fear conditioning task. Systemic administration of SL327 dose-dependently reduced the memory reconsolidation of fear memories in C57 mice. Moreover, SL327 administration impaired memory reconsolidation also in ERK1 mutant mice. Altogether, these results clearly indicate a central role for ERK2 protein in memory reconsolidation processes in mice.     

Inhibition of matrix metalloproteinase activity disrupts reconsolidation but not consolidation of a fear memory

The reconsolidation hypothesis posits that memories that have been reactivated can be either enhanced or disrupted by pharmacological manipulation. Synaptic plasticity is presumed to underlie the reconsolidation process. Matrix metalloproteinases are proteins that regulate the extracellular matrix involved in plasticity events, and these proteins have recently been shown to influence learning and memory. However, all studies on the role of matrix metalloproteinases in learning and memory have employed tasks that rely on contextual cues. The goal of this study was to determine the extent to which FN-439 would disrupt the consolidation and/or reconsolidation of a fear memory associated with a conditioned stimulus that signaled tone-shock pairings and that was independent of contextual cues. Male Sprague-Dawley rats were given infusions of FN-439 (35 microg intracerebroventricular) 30 min prior to conditioning (tone-shock paired association) or 30 min prior to a single reactivation session given 24h after conditioning. Administration of FN-439 did not disrupt consolidation of the freezing response when the tone (conditioned stimulus) was presented. In contrast, FN-439 infusion disrupted reconsolidation of the fear memory in a reactivation-dependent manner. The reduced freezing behavior was not due to a decrease in general anxiety levels, since FN-439 had no effect on the percent of open-arm time or open-arm entries in an elevated-plus maze task. Thus, we demonstrated for the first time that matrix metalloproteinase inhibition in the brain is capable of disrupting the reconsolidation of a tone-shock association memory that does not depend on contextual cues. The finding that a fear response to a previously paired conditioned stimulus can be disrupted by treatment with an MMP inhibitor during a single reactivation session suggests that this class of compounds may have therapeutic potential for posttraumatic stress disorder and/or simple phobias.     

De novo mRNA synthesis is required for both consolidation and reconsolidation of fear memories in the amygdala

Memory consolidation is the process by which newly learned information is stabilized into long-term memory (LTM). Considerable evidence indicates that retrieval of a consolidated memory returns it to a labile state that requires it to be restabilized. Consolidation of new fear memories has been shown to require de novo RNA and protein synthesis in the lateral nucleus of the amygdala (LA). We have previously shown that de novo protein synthesis in the LA is required for reconsolidation of auditory fear memories. One key question is whether protein synthesis during reconsolidation depends on already existing mRNAs or on synthesis of new mRNAs in the amygdala. In the present study, we examined the effect of mRNA synthesis inhibition during consolidation and reconsolidation of auditory fear memories. We first show that intra-LA infusion of two different mRNA inhibitors dose-dependently impairs long-term memory but leaves short-term memory (STM) intact. Next, we show that intra-LA infusion of the same inhibitors dose-dependently blocks post-reactivation long-term memory (PR-LTM), whereas post-reactivation short-term memory (PR-STM) is left intact. Furthermore, the same treatment in the absence of memory reactivation has no effect. Together, these results show that both consolidation and reconsolidation of auditory fear memories require de novo mRNA synthesis and are equally sensitive to disruption of de novo mRNA synthesis in the LA.     

Retrieval per se is not sufficient to trigger reconsolidation of human fear memory

Ample evidence suggests that consolidated memories, upon their retrieval, enter a labile state, in which they might be susceptible to change. It has been proposed that memory labilization allows for the integration of relevant information in the established memory trace (memory updating). Memory labilization and reconsolidation do not necessarily occur when a memory is being reactivated, but only when there is something to be learned during memory retrieval (prediction error). Thus, updating of a fear memory trace should not occur under retrieval conditions in which the outcome is fully predictable (no prediction error). Here, we addressed this issue, using a human differential fear conditioning procedure, by eliminating the very possibility of reinforcement of the reminder cue. A previously established fear memory (picture-shock pairings) was reactivated with shock-electrodes attached (Propranolol group, n=18) or unattached (Propranolol No-Shock Expectation group, n=19). We additionally tested a placebo-control group with the shock-electrodes attached (Placebo group, n=18). Reconsolidation was not triggered when nothing could be learned during the reminder trial, as noradrenergic blockade did not affect expression of the fear memory 24h later in the Propranolol No-Shock Expectation group. Only when the outcome of the retrieval cue was not fully predictable, propranolol, contrary to placebo, reduced the startle fear response and prevented the return of fear (reinstatement) the following day. In line with previous studies, skin conductance response and shock expectancies were not affected by propranolol. Remarkably, a double dissociation emerged between the emotional (startle response) and more cognitive expression (expectancies, SCR) of the fear memory. Our findings have important implications for reconsolidation blockade as treatment strategy for emotional disorders. First, fear reducing procedures that target the emotional component of fear memory do not necessarily affect the cognitive component and vice versa. Second, mere retrieval of the fear memory is not sufficient to induce its labilization and reconsolidation.     

Amygdala or hippocampus inactivation after retrieval induces temporary memory deficit

The hypothesis that memory is stored through a single stage of consolidation that results in a stable and lasting long-term memory has been challenged by the proposition that reactivation of a memory induces reconsolidation of the memory. The reconsolidation hypothesis is supported by evidence that, under some conditions, post-retrieval treatments affecting amygdala and hippocampus functioning impair subsequent retention performance. We now report that repeated retention testing attenuates the performance impairment induced by post-retrieval reversible inactivation of the amygdala and hippocampus of rats induced by tetrodotoxin. These findings challenge the reconsolidation hypothesis and suggest that the post-retrieval retention performance impairment is best explained as due to temporary retrieval failure.