Sevil Duvarci

Characterization of Fear Memory Reconsolidation

Reactivation of consolidated memories returns them to a protein synthesis-dependent state. One interpretation of these findings is that the memory reconsolidates after use. Two alternative interpretations are that protein synthesis inhibition facilitates extinction and that postreactivation protein synthesis inhibition leads to an inability to retrieve the consolidated memory. First, using two different approaches, we report that reconsolidation cannot be reduced down to facilitated extinction. We show that the reconsolidation deficit does not show renewal after a contextual shift, whereas an extinguished auditory fear memory does under the same conditions and the deficit occurs regardless of whether the memory is reactivated with an extinction [conditioned stimulus (CS) alone] or a reinforced trial (CS-unconditioned stimulus). To address the issue of whether postreactivation anisomycin leads to an inability to retrieve the consolidated memory, we used two traditional assays for retrieval deficits. First, we demonstrate that the amnesia induced by blockade of reconsolidation does not show any spontaneous recovery. Second, we show that application of reminder shock does not result in the reinstatement of the memory. These findings support the idea that reactivation of consolidated memories initiates a second time-dependent memory formation process.

Amygdala microcircuits controlling learned fear.

We review recent work on the role of intrinsic amygdala networks in the regulation of classically conditioned defensive behaviors, commonly known as conditioned fear. These new developments highlight how conditioned fear depends on far more complex networks than initially envisioned. Indeed, multiple parallel inhibitory and excitatory circuits are differentially recruited during the expression versus extinction of conditioned fear. Moreover, shifts between expression and extinction circuits involve coordinated interactions with different regions of the medial prefrontal cortex. However, key areas of uncertainty remain, particularly with respect to the connectivity of the different cell types. Filling these gaps in our knowledge is important because much evidence indicates that human anxiety disorders results from an abnormal regulation of the networks supporting fear learning.

Glucocorticoids Enhance the Excitability of Principal Basolateral Amygdala Neurons

A large body of pharmaco-behavioral data implicates the basolateral nucleus of the amygdala (BLA) in the facilitation of memory consolidation by emotions. Overall, this evidence suggests that stress hormones released during emotional arousal increase the activity of BLA neurons. In turn, this increased BLA activity would facilitate synaptic plasticity elsewhere in the brain, to which the BLA projects. However, the direct effects of glucocorticoids on BLA neurons are incompletely understood. In the present study, we examined the direct effects of corticosterone (CORT) on principal neurons of the rat BLA in vitro using whole-cell patch-clamp recordings. We found that application of a stress level of CORT for 20 min caused significant changes in the passive properties and responsiveness of BLA cells measured 1–2 h later. Indeed, CORT application produced a depolarization of the resting potential, an increase in input resistance, and a dramatic decrease in spike-frequency adaptation. In addition, GABAA IPSPs evoked by stimulation of the external capsule were significantly reduced by CORT application. This effect of CORT was not attributable to a reduction in the amount of GABA released because GABAB IPSPs were unchanged and the resistance drop associated with GABAA IPSPs was not altered. Rather, we found that this effect of CORT resulted from a positive shift of the GABAA reversal potential. Overall, these results suggest that, in agreement with previous behavioral findings, glucocorticoids enhance the excitability of principal BLA cells by increasing their intrinsic excitability and decreasing the impact of GABAA IPSPs.

Activation of extracellular signal-regulated kinase-mitogen-activated protein kinase cascade in the amygdala is required for memory reconsolidation of auditory fear conditioning

Consolidation of new fear memories has been shown to require de novo RNA and protein synthesis in the lateral nucleus of amygdala (LA). Recently we have demonstrated that consolidated fear memories, when reactivated, return to a labile state which is sensitive to disruption by the protein synthesis inhibitor anisomycin. The specific molecular mechanisms that underlie this reconsolidation of fear memories are still largely unknown. The activation of extracellular signal‐regulated kinase–mitogen‐activated protein kinase (ERK–MAPK) pathway in the LA is required for the consolidation of auditory fear memories. In the present study, we examined the role of ERK–MAPK cascade in the LA during reconsolidation of auditory fear conditioning. We show that intra‐LA infusions of the MAPK kinase (MEK) inhibitor U0126, a manipulation which inhibits activation of ERK–MAPK, impairs postreactivation long‐term memory (PR‐LTM) but leaves the postreactivation short‐term memory (PR‐STM) intact. The same treatment with U0126, in the absence of memory reactivation, has no effect. Furthermore, we verified that reconsolidation requires translation using a second protein synthesis inhibitor, cycloheximide. Post‐reactivation infusions of cycloheximide blocked PR‐LTM but not PR‐STM and, in the absence of reactivation, had no effect. Our data show that activation of ERK–MAPK signalling pathway and protein synthesis in the LA are required for reconsolidation of auditory fear memories.

Coherent amygdalocortical theta promotes fear memory consolidation during paradoxical sleep

Brain activity in sleep plays a crucial role in memory consolidation, an offline process that determines the long-term strength of memory traces. Consolidation efficacy differs across individuals, but the brain activity dynamics underlying these differences remain unknown. Here, we studied how interindividual variability in fear memory consolidation relates to neural activity in brain structures that participate in Pavlovian fear learning. From the end of training to testing 24 h later, some rats showed increased and others decreased conditioned fear responses. We found that overnight bidirectional changes in fear memory were selectively correlated with modifications in theta coherence between the amygdala, medial prefrontal cortex, and hippocampus during paradoxical sleep. Thus, our results suggest that theta coordination in the limbic system may influence interindividual differences in memory consolidation of aversive experiences.

The Bed Nucleus of the Stria Terminalis Mediates Inter-individual Variations in Anxiety and Fear

While learning to fear stimuli that predict danger promotes survival, the inability to inhibit fear to inappropriate cues leads to a pernicious cycle of avoidance behaviors. Previous studies have revealed large inter-individual variations in fear responding with clinically anxious humans exhibiting a tendency to generalize learned fear to safe stimuli or situations. To shed light on the origin of these inter-individual variations, we subjected rats to a differential auditory fear conditioning paradigm in which one conditioned auditory stimulus (CS+) was paired to footshocks whereas a second (CS−) was not. We compared the behavior of rats that received pretraining excitotoxic lesions of the bed nucleus of the stria terminalis (BNST) to that of sham rats. Sham rats exhibit a continuum of anxious/fearful behaviors. At one end of the continuum were rats that displayed a poor ability to discriminate between the CS+ and CS−, high contextual freezing, and an anxiety-like trait in the elevated plus maze (EPM). At the other end were rats that display less fear generalization to the CS−, lower freezing to context, and a nonanxious trait in the EPM. Although BNST-lesioned rats acquired similarly high levels of conditioned fear to the CS+, they froze less than sham rats to the CS−. In fact, BNST-lesioned rats behaved like sham rats with high discriminative abilities in that they exhibited low contextual fear and a nonanxious phenotype in the EPM. Overall, this suggests that inter-individual variations in fear generalization and anxiety phenotype are determined by BNST influences on the amygdala and/or its targets.

Amygdala microcircuits mediating fear expression and extinction.

This review summarizes the latest developments in our understanding of amygdala networks that support classical fear conditioning, the experimental paradigm most commonly used to study learned fear in the laboratory. These recent advances have considerable translational significance as congruent findings from studies of fear learning in animals and humans indicate that anxiety disorders result from abnormalities in the mechanisms that normally regulate conditioned fear. Because of the introduction of new techniques and the continued use of traditional approaches, it is becoming clear that conditioned fear involves much more complex networks than initially believed, including coordinated interactions between multiple excitatory and inhibitory circuits within the amygdala.

CENTRAL AMYGDALA ACTIVITY DURING FEAR CONDITIONING

The central amygdala (Ce), particularly its medial sector (CeM), is the main output station of the amygdala for conditioned fear responses. However, there is uncertainty regarding the nature of CeM control over conditioned fear. The present study aimed to clarify this question using unit recordings in rats. Fear conditioning caused most CeM neurons to increase their conditioned stimulus (CS) responsiveness. The next day, CeM cells responded similarly during the recall test, but these responses disappeared as extinction of conditioned fear progressed. In contrast, the CS elicited no significant average change in central lateral (CeL) firing rates during fear conditioning and a small but significant reduction during the recall test. Yet, cell-by-cell analyses disclosed large but heterogeneous CS-evoked responses in CeL. By the end of fear conditioning, roughly equal proportions of CeL cells exhibited excitatory (CeL+) or inhibitory (CeL−) CS-evoked responses (∼10%). The next day, the proportion of CeL− cells tripled with no change in the incidence of CeL+ cells, suggesting that conditioning leads to overnight synaptic plasticity in an inhibitory input to CeL− cells. As in CeM, extinction training caused the disappearance of CS-evoked activity in CeL. Overall, these findings suggest that conditioned freezing depends on increased CeM responses to the CS. The large increase in the incidence of CeL− but not CeL+ cells from conditioning to recall leads us to propose a model of fear conditioning involving the potentiation of an extrinsic inhibitory input (from the amygdala or elsewhere) to CeL, ultimately leading to disinhibition of CeM neurons.

The fear circuit revisited: contributions of the basal amygdala nuclei to conditioned fear.

The lateral nucleus (LA) is the input station of the amygdala for information about conditioned stimuli (CSs), whereas the medial sector of the central nucleus (CeM) is the output region that contributes most amygdala projections to brainstem fear effectors. However, there are no direct links between LA and CeM. As the main target of LA and with its strong projection to CeM, the basomedial amygdala (BM) constitutes a good candidate to bridge this gap. Consistent with this notion, it was reported that combined posttraining lesions of the basal nuclei [BM plus basolateral nucleus (BL)] abolish conditioned fear responses, whereas selective BL inactivation does not. Thus, we examined the relative contribution of BM and BL to conditioned fear using unit recordings and inactivation with muscimol microinfusions in rats. Approximately 30% of BM and BL neurons acquired robust responses to auditory CSs predicting footshocks. While most BL cells stopped firing at CS offset, BM responses typically outlasted the CS by ≥40 s, paralleling the persistence of conditioned fear after the CS. This observation suggests that BM neurons are not passive relays of rapidly adapting LA inputs about the CS. Surprisingly, independent inactivation of either BM or BL with muscimol did not cause a reduction of conditioned freezing even though an extinction recall deficit was seen the next day. In contrast, combined BL–BM inactivation did. Overall, there results support the notion that the basal nuclei are involved in conditioned fear expression and extinction but that there is functional redundancy between them.

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.