It is well known that, under certain boundary conditions, the retrieval of a stable consolidated memory results into a labile one. During this unstable phase, memory can be vulnerable to interference by a number of pharmacological agents, including benzodiazepines. One of the goals of this study was to evaluate the vulnerability to midazolam (MDZ) after reactivation of recent and remote contextual fear memories in animals that experienced a stressful situation before learning. Animals were subjected to a restraint session and trained in a contextual fear paradigm the following day; consolidated memories were reactivated at different times after learning and different MDZ doses (1.5, 3.0 mg/kg) were administered to rats after reactivation. Our results show that MDZ did not affect memory reconsolidation in older-than-one-day memories of stressed animals, even after the administration of a higher MDZ dose and a longer reactivation session (5 min). In contrast, MDZ was effective in blocking reconsolidation at all memory ages in unstressed animals. In addition, the current research investigated whether activating NMDA sites before reactivation promotes the destabilization of resistant memories such as those of stressed animals. We tested the influence of pre-reactivation D-cycloserine (DCS), a partial NMDA agonist, on MDZ's effect on fear memory reconsolidation in stressed animals. Our findings indicate that DCS before reactivation promotes retrieval-induced lability in resistant memory traces, as MDZ-induced memory impairment in stressed rats became evident with pre-reactivation DCS but not after pre-reactivation sterile isotonic saline.

The present study investigates the fear memory resulting from the interaction of a stressful experience and the retrieval of an established fear memory trace. Such a combination enhanced both fear expression and fear retention in adult Wistar rats. Likewise, midazolam intra-basolateral amygdala (BLA) infusion prior to stress attenuated the enhancement of fear memory thus suggesting the involvement of a stress-induced reduction of the GABAergic transmission in BLA in the stress-induced enhancing effect. It has been suggested that, unlike the immediate-early gene Zif268 which is related to the reconsolidation process, the expression of hippocampal brain-derived neurotrophic factor (BDNF) is highly correlated with consolidation. We therefore evaluate the relative contribution of these two neurobiological processes to the fear memory resulting from the above-mentioned interaction. Intra-dorsal hippocampus (DH) infusions of either the antisense Zif268 or the inhibitor of the protein degradation (Clasto-Lactacystin b-Lactone), suggested to be involved in the retrieval-dependent destabilization process, did not affect the resulting contextual memory. In contrast, the knockdown of hippocampal BDNF mitigated the stress-induced facilitating influence on fear retention. In addition, the retrieval experience elevated BDNF level in DH at 60 min after recall exclusively in stressed animals. These findings suggest the involvement of a hippocampal BDNF sensitive mechanism in the stress-promoting influence on the fear memory following retrieval.

The present research investigated the resulting contextual fear memory and structural plasticity changes in the dorsal hippocampus (DH) following stress and fear conditioning. This combination enhanced fear retention and increased the number of total and mature dendritic spines in DH. Intra-basolateral amygdala (BLA) infusion of midazolam prior to stress prevented both the enhancement of fear retention and an increase in the density of total and mature dendritic spines in DH. These findings emphasize the role of the stress-induced attenuation of GABAergic neurotransmission in BLA in the promoting influence of stress on fear memory and on synaptic remodeling in DH. In conclusion, the structural remodeling in DH accompanied the facilitated fear memory following a combination of fear conditioning and stressful stimulation.It is well accepted that prior exposure to stressful events facilitates the emergence of fear memory (Shors et al. 1992;Beylin and Shors 1998;Shors 2001;Cordero et al. 2003;Rodriguez Manzanares et al. 2005). Consistent with this, recent data shows that the interaction of an unrelated aversive experience and an established fear memory trace results in a robust and persistent fear memory (Giachero et al. 2013). This stress-induced promoting influence was prevented by midazolam (MDZ) intra-basolateral amygdala (BLA) infusion prior to stress, indicating that such an influence is related to the modulation of the GABAergic transmission in BLA (Giachero et al. 2013).Dendritic spines represent the structural platform for excitatory synaptic contacts between neurons (Gray 1959;Chapleau and Pozzo-Miller 2007), with growing evidence suggesting that synaptic remodeling accompanies the formation of long-term memory (Restivo et al. 2009). In line with this view, the encoding of a novel contextual representation was shown to result in a rapid synaptic rearrangement through an increase in spine density, particularly in the hippocampus across multiple memory paradigms, including contextual fear memory (Leuner et al. 2003;Restivo et al. 2009;Vetere et al. 2011).The dorsal hippocampus (DH) is crucially involved in the contextual representation following fear conditioning (Kim and Fanselow 1992;Phillips and LeDoux 1995;Maren and Fanselow 1997;Fanselow and Dong 2010). Related to this, the present research investigates the structural plasticity in the DH that may underlie the contextual fear memory resulting from the interaction of fear conditioning and an unrelated stressful experience. The BLA is an essential component of the neural circuitry orchestrating emotional response to threatening stimuli (LeDoux 2000), and compelling evidence has shown that the activation of GABAa sites, specifically in BLA prior to stress, attenuates the enhancement of fear memory (Rodriguez Manzanares et al. 2005;Giachero et al. 2013). Therefore, we also explored the modulatory role of the GABAergic neurotransmission in the BLA in both fear retention and structural plasticity in the DH, which is potentially associated with the fear memory...

Background:Withdrawal from chronic ethanol facilitates the formation of contextual fear memory and delays the onset to extinction, with its retrieval promoting an increase in ethanol consumption. Consequently, manipulations aimed to reduce these aversive memories, may be beneficial in the treatment of alcohol discontinuation symptoms. Related to this, pharmacological memory reconsolidation blockade has received greater attention due to its therapeutic potential.Methods:Here, we examined the effect of post-reactivation amnestic treatments such as Midazolam (MDZ, 3 mg/kg i.p) and Propranolol (PROP, 5 mg/kg i.p) on contextual fear memory reconsolidation in ethanol- withdrawn (ETOH) rats. Next, we examined whether the activation of N-methyl-D-aspartate (NMDA) receptors induced by d-cycloserine (DCS, 5 mg/kg i.p., a NMDA partial agonist) before memory reactivation can facilitate the disruptive effect of PROP and MDZ on fear memory in ETOH rats.Results:We observed a resistance to the disruptive effect of both MDZ and PROP following memory reactivation. Although intra-basolateral amygdala (BLA; 1.25 ug/side) and systemic PROP administration attenuated fear memory in DCS pre-treated ETOH rats, DCS/MDZ treatment did not affect memory in these animals. Finally, a decrease of both total and surface protein expression of the α1 GABAA receptor (GABAA-R) subunit in BLA was found in the ETOH rats.Conclusions:Ethanol withdrawal facilitated the formation of fear memory resistant to labilization post-reactivation. DCS administration promoted the disruptive effect of PROP on memory reconsolidation in ETOH rats. The resistance to MDZ’s disruptive effect on fear memory reconsolidation may be, at least in part, associated with changes in the GABAA-R composition induced by chronic ethanol administration/withdrawal.

GABAergic signaling in the basolateral amygdala complex (BLA) plays a crucial role on the modulation of the stress influence on fear memory. Moreover, accumulating evidence suggests that the dorsal hippocampus (DH) is a downstream target of BLA neurons in contextual fear. Given that hippocampal structural plasticity is proposed to provide a substrate for the storage of long-term memories, the main aim of this study is to evaluate the modulation of GABA neurotransmission in the BLA on spine density in the DH following stress on contextual fear learning. The present findings show that prior stressful experience promoted contextual fear memory and enhanced spine density in the DH. Intra-BLA infusion of midazolam, a positive modulator of GABAa sites, prevented the facilitating influence of stress on both fear retention and hippocampal dendritic spine remodeling. Similarly to the stress-induced effects, the blockade of GABAa sites within the BLA ameliorated fear memory emergence and induced structural remodeling in the DH. These findings suggest that GABAergic transmission in BLA modulates the structural changes in DH associated to the influence of stress on fear memory.

It is known that a consolidated memory can return to a labile state and become transiently malleable following reactivation. This instability is followed by a restabilization phase termed reconsolidation. In this work, we explored whether an unrelated appetitive experience (voluntary consumption of diluted sucrose) can affect a contextual fear memory in rats during the reactivation-induced destabilization phase. Our findings show that exposure to an appetitive experience following reactivation can diminish fear retention. This effect persisted after 1 wk. Importantly, it was achieved only under conditions that induced fear memory destabilization. This result could not be explained as a potentiated extinction, because sucrose was unable to promote extinction. Since GluN2B-containing NMDA receptors in the basolateral amygdala complex (BLA) have been implicated in triggering fear memory destabilization, we decided to block pharmacologically these receptors to explore the neurobiological bases of the observed effect. Intra-BLA infusion with ifenprodil, a GluN2B-NMDA antagonist, prevented the fear reduction caused by the appetitive experience. In sum, these results suggest that the expression of a fear memory can be dampened by an unrelated appetitive experience, as long as memory destabilization is achieved during reactivation. Possible mechanisms behind this effect and its clinical implications are discussed.

Fear memory overgeneralization contributes to the genesis and persistence of anxiety disorders and is a central hallmark in the pathophysiology of post-traumatic stress disorder (PTSD). Recent findings suggest that fear generalization is closely related to hippocampal dependency during retrieval. The selective serotonin reuptake inhibitor (SSRI) fluoxetine has been used as a first-line treatment for PTSD; however, how it exerts its therapeutic effect remains a matter of debate. Here, using contextual fear conditioning in rats, we show that chronic fluoxetine treatment prevents fear generalization and enhances subsequent extinction. Moreover, fluoxetine treatment after extinction prevents spontaneous recovery. The mechanism through which fluoxetine affects generalization and extinction seems to be through the postponement of systems consolidation, thereby maintaining hippocampal involvement during retrieval. Such an effect relies on a remodeling of dendritic spines in the hippocampus, as well as the number of mature, mushroom-type spines promoted by fluoxetine treatment. In order to further investigate whether fear generalization is a potential predictor of extinction effectiveness, we categorized a large naive population according to their generalization rate. We found that discriminator rats showed a better extinction profile compared to generalizers, suggesting that the generalization rate predicts extinction effectiveness. Hence, we propose that the therapeutic strategy of choice should take into account the extension of memory generalization, in which therapies based on extinction could induce a better outcome in patients who present less fear overgeneralization. These results open new avenues for the development of interventions that prevent fear generalization by maintaining memory dependency of the hippocampus.

No abstract