Role of amygdala astrocytes in different phases of contextual fear memory.

Growing evidence indicates a critical role of astrocytes in learning and memory. However, little is known about the role of basolateral amygdala complex (BLA-C) astrocytes in contextual fear conditioning (CFC), a paradigm relevant to understand and generate treatments for fear- and anxiety-related disorders. To get insights on the involvement of BLA-C astrocytes in fear memory, fluorocitrate (FLC), a reversible astroglial metabolic inhibitor, was applied at critical moments of the memory processing in order to target the acquisition, consolidation, retrieval and reconsolidation process of the fear memory. Adult Wistar male rats were bilaterally cannulated in BLA-C. Ten days later they were infused with different doses of FLC (0.5 or 1 nmol/0.5 µl) or saline before or after CFC and before or after retrieval. FLC impaired fear memory expression when administered before and shortly after CFC, but not one hour later. Infusion of FLC prior and after retrieval did not affect the memory. Our findings suggest that BLA-C astrocytes are critically involved in the acquisition/early consolidation of fear memory but not in the retrieval and reconsolidation. Furthermore, the extinction process was presumably not affected (considering that peri-retrieval administration could also affect this process).

Effect of intra-BLA overexpression of IGF-1 on the expression of a contextual fear memory trace.

Growth factors, such as insulin-like growth factor 1 (IGF-1), among others are known for their critical involvement in learning and memory processes. IGF-1 regulates cognitive functions, synapse density, neurotransmission, and adult neurogenesis and induces structural and synaptic plasticity-specific changes. Although IGF-1 has been suggested to participate in different memory processes, its role in memories associated with negative emotional experiences still remains to be elucidated. The principal aim of the present study was to test whether IGF-1 overexpression using adenoviral vectors in basolateral amygdala (BLA) influences both the expression and formation of contextual fear memory, as well as the hippocampal structural plasticity associated with such memory trace. We found that IGF-1 overexpression promotes the formation and expression of a specific contextual fear memory trace, and such effect persisted at least 7 days after recall. Moreover, the overexpression of this growth factor in BLA upregulates the activation of the ERK/MAPK pathway in this brain structure. In addition, intra-BLA IGF-1 overexpression causes dorsal hippocampus (DH) structural plasticity modifications promoting changes in the proportion of mature dendritic spines in the CA1 region, after a weak conditioning protocol. The present findings contribute to the knowledge underlying BLA-DH trace memory of fear and reveal important new insights into the neurobiology and neurochemistry of fear acquisition modulated by IGF-1 overexpression. The understanding of how IGF-1 modulates the formation of a fear contextual trace may pave the way for the development of novel therapeutic strategies focused on fear, anxiety, and trauma-related disorders.

Temporal dynamic of the hippocampal structural plasticity associated with the fear memory destabilization/reconsolidation process.

Reconsolidation of a contextual fear memory is a protein synthesis-dependent process in which a previously destabilized memory returns to a stable state. This process has become the subject of many studies due to its importance in memory processing, maintenance and updating, and its potential role as a therapeutical target in fear memory disorders such as phobias and post-traumatic stress disorder. In this sense, understanding the underlying mechanisms of memory reconsolidation is paramount in developing potential treatments for such memory dysfunctions. In the present work, we studied the interaction between two key neural structures involved in the reconsolidation process: the basolateral amygdala complex of the amygdala (BLA) and the dorsal hippocampus (DH). Our results show changes in the structural plasticity of the CA1 region of the DH in the form of dendritic spines density changes associated with the destabilization/reconsolidation process. Furthermore, we demonstrate a modulatory role of BLA over such structural plasticity by infusing different drugs such as ifenprodil, a destabilization blocker, and propranolol, a reconsolidation disruptor, in this brain structure. Altogether our work shows a particular temporal dynamic in the CA1 region of DH that accompanies the destabilization/reconsolidation process and aims to provide new information on the underlying mechanisms of this process that potentially contributes for a better understanding of memory storage, maintenance, expression and updating, and its potential medical applications.

Stress-induced resistance to fear memory destabilization is associated with an impairment of Lys-48-linked protein polyubiquitination in the Basolateral Amygdala: Influence of D-cycloserine.

The destabilization/reconsolidation process can be triggered by memory recall, allowing consolidated memories to be modified. We have previously reported that stress prior to fear conditioning induces memories that exhibit resistance to the engagement of some molecular events associated with the destabilization/reconsolidation process. Here, we evaluated whether stress could affect the expression of Lys-48 polyubiquitinated proteins within the basolateral amygdala complex, a phenomenon crucially linked to memory destabilization. As expected, a post-recall increase of Lys-48 polyubiquitinated proteins in control animals was observed; however, this phenomenon was prevented by stress exposure before fear conditioning. On the other hand, pre-recall administration of D-cycloserine -a positive modulator of NMDA sites capable of reverting memory resistance to pharmacological interference-, facilitated the increase of Lys-48 polyubiquitinated proteins in stressed animals. In conclusion, the protein polyubiquitination-dependent destabilization is impaired after the recall of stress-induced resistant memories, with D-cycloserine restoring such molecular event. Hence, the present report contributes to further characterize the neurobiological events associated with stress-induced memory resistance as well as to corroborate the connection between glutamatergic signaling, protein degradation and memory destabilization in stress-induced resistant memories.

Taking advantage of fear generalization-associated destabilization to attenuate the underlying memory via reconsolidation intervention.

Upon retrieval, an aversive memory can undergo destabilization and reconsolidation. A traumatic-like memory, however, may be resistant to this process. The present study sought to contribute with a strategy to overcome this potential issue by investigating whether generalized fear retrieval is susceptible to destabilization-reconsolidation that can be pharmacologically modified. We hypothesized that exposure to a context that elicits moderate generalization levels would allow a malleable memory state. We developed a fear conditioning protocol in context A (cxt-A) paired with yohimbine administration to promote significant fear to a non-conditioned context B (cxt-B) in rats, mimicking the enhanced noradrenergic activity reported after traumatic events in humans. Next, we attempted to impair the reconsolidation phase by administering clonidine (CLO) immediately after exposure to cxt-A, cxt-B, or a third context C (cxt-C) neither conditioned nor generalized. CLO administered post-cxt-B exposure for two consecutive days subsequently resulted in decreased freezing levels in cxt-A. CLO after cxt-B only once, after cxt-A or cxt-C in two consecutive days, or independently of cxt-B exposures did not affect fear in a later test. A 6-h-delay in CLO treatment post-cxt-B exposures produced no effects, and nimodipine administered pre-cxt-B exposures precluded the CLO action. We then quantified the Egr1/Zif268 protein expression following cxt-B exposures and CLO treatments. We found that these factors interact to modulate this memory destabilization-reconsolidation mechanism in the basolateral amygdala but not the dorsal CA1 hippocampus. Altogether, memory destabilization can accompany generalized fear expression; thus, we may exploit it to potentiate reconsolidation blockers' action.

Resilience and Vulnerability to Trauma: Early Life Interventions Modulate Aversive Memory Reconsolidation in the Dorsal Hippocampus.

Early life experiences program lifelong responses to stress. In agreement, resilience and vulnerability to psychopathologies, such as posttraumatic stress disorder (PTSD), have been suggested to depend on the early background. New therapies have targeted memory reconsolidation as a strategy to modify the emotional valence of traumatic memories. Here, we used animal models to study the molecular mechanism through which early experiences may later affect aversive memory reconsolidation. Handling (H)-separation of pups from dams for 10 min-or maternal separation (MS) - 3-h separation-were performed from PDN1-10, using non-handled (NH) litters as controls. Adult males were trained in a contextual fear conditioning (CFC) task; 24 h later, a short reactivation session was conducted in the conditioned or in a novel context, followed by administration of midazolam 3 mg/kg i.p. (mdz), known to disturb reconsolidation, or vehicle; a test session was performed 24 h after. The immunocontent of relevant proteins was studied 15 and 60 min after memory reactivation in the dorsal hippocampus (dHc) and basolateral amygdala complex (BLA). Mdz-treated controls (NH) showed decreased freezing to the conditioned context, consistent with reconsolidation impairment, but H and MS were resistant to labilization. Additionally, MS males showed increased freezing to the novel context, suggesting fear generalization; H rats showed lower freezing than the other groups, in accordance with previous suggestions of reduced emotionality facing adversities. Increased levels of Zif268, GluN2B, ß-actin and polyubiquitination found in the BLA of all groups suggest that memory reconsolidation was triggered. In the dHc, only NH showed increased Zif268 levels after memory retrieval; also, a delay in ERK1/2 activation was found in H and MS animals. We showed here that reconsolidation of a contextual fear memory is insensitive to interference by a GABAergic drug in adult male rats exposed to different neonatal experiences; surprisingly, we found no differences in the reconsolidation process in the BLA, but the dHc appears to suffer temporal desynchronization in the engagement of reconsolidation. Our results support a hippocampal-dependent mechanism for reconsolidation resistance in models of early experiences, which aligns with current hypotheses for the etiology of PTSD.

Resistance to fear memory destabilization triggers exaggerated emotional-like responses following memory reactivation.

Fear memory reactivation does not always lead to memory destabilization-reconsolidation. For instance, fear memories formed following withdrawal from chronic ethanol consumption or a stressful event are less likely to become destabilized after reactivation, with the effect of recall of these memories on the affective state still requiring elucidation. Here, we investigated the negative emotional-like responses following fear memory reactivation in ethanol-withdrawn (ETOH) rats by focusing on the possible role played by destabilization. Our findings indicated that ETOH rats displayed an increased freezing in a novel context and an anxiogenic-like response in the elevated plus maze (EPM) following memory reactivation, whereas the behavior of CON animals was not affected. The destabilization blockade by pre-reactivation nimodipine (16 mg/kg, s.c) administration promoted in CON animals a similar behavior in the EPM and in a novel environment as that exhibited by ETOH rats after the reminder. Moreover, facilitating destabilization by pre-reactivation d-cycloserine (5 mg/kg, i.p) administration prevented the emotional-like disturbances observed in ETOH rats. Finally, using restraint stress, which is also an inductor of a fear memory resistant to destabilization, an increased fear response in an unconditioned environment and an anxiogenic-like state was also found after the presentation of the fear reminder in stressed rats. Our results suggest that, in the context of resistant fear memories, the occurrence of destabilization influences how animals respond to subsequent environmental challenges following reactivation.

Ethanol withdrawal limits fear memory reactivation-induced molecular events associated with destabilization phase: Influence of d-cycloserine.

A 1-day fear memory in ethanol withdrawn (ETOH) rats is resistant to destabilization-reconsolidation process. However, d-cycloserine (DCS) reverts this disturbance. Considering that the formation of pathological fear memories in humans often occurs long time before the requirement of an intervention, the study of older memories is relevant in ETOH rats. In addition, the resistance to destabilization and DCS effect on this memory phase at molecular level in ETOH rats have not been corroborated yet. Firstly, we examined the effect of a pharmacological intervention after reactivation on reconsolidation of a 7-day fear memory in ETOH rats. Then, and considering that enhanced GluN2B expression and ubiquitin-proteasome system (UPS) activity are involved in destabilization, we evaluated them following reactivation in ETOH rats. Furthermore, DCS effect on such destabilization markers was examined. It was found that the pharmacological intervention after reactivation did not affect the 7-day fear memory in ETOH rats with DCS reversing this resistance. Memory reactivation increased GluN2B expression, polyubiquitination levels and proteasome activity in the basolateral amygdala complex (BLA) of control (CON) rats only; without affecting these molecular events in ETOH rats. Finally, ETOH rats treated with DCS and CON animals displayed elevated and similar UPS activities in the BLA after reactivation. In conclusion, the reactivation of an older fear memory formed during ethanol withdrawal does not trigger the molecular events associated with destabilization, and DCS facilitates this memory phase by enhancing the UPS activity.

Post-weaning housing conditions influence freezing during contextual fear conditioning in adult rats.

The present study aimed to investigate the influence of housing conditions on contextual fear memory malleability. Male Wistar rats were housed in enriched, standard, or impoverished conditions after weaning and remained in these conditions throughout the entire experiment. After six weeks into those housing conditions, all animals underwent a 3-day protocol including contextual fear conditioning (day 1), memory reactivation followed by systemic administration of midazolam or vehicle (day 2), and a retention test (day 3). Percentage freezing was used as a behavioral measure of contextual fear. There was no evidence for an effect of housing conditions on the sensitivity of contextual fear memory to amnestic effects of post-reactivation midazolam administration, and no indication for amnestic effects of post-reactivation midazolam overall (including in the standard group). The inability to replicate previous demonstrations of post-reactivation amnesia using the same protocol underscores the subtle nature of post-reactivation pharmacological memory interference. Notably, impoverished housing resulted in a decrease in contextual freezing during contextual fear conditioning, reactivation and retention testing, compared to enriched and standard housing conditions. This observation warrants caution when interpreting the results from experiments regarding effects of housing on fear memory processes, particularly when freezing is used as a measure of fear.

Stress influences the dynamics of hippocampal structural remodeling associated with fear memory extinction.

Fear extinction is defined as a decline in fear-conditioned responses following non-reinforced exposure to a fear conditioned stimulus, therefore the conditioned stimulus gains new predictive properties. Patients with anxiety related disorders (e.g.: PTSD) subjected to extinction-like exposure treatments often experience a relapse of symptoms. Stress is a risk factor for those psychiatric disorders and a critical modulator of fear learning that turns the memory resistant to the extinction process. Dendritic spines are the anatomical sites where neuronal activity reshapes brain networks during learning and memory processes. Thus, we planned to characterize the dynamics of synaptic remodeling before and after contextual fear extinction in the dorsal hippocampus (DH), and how this process is affected by a previous stress experience. Animals with or without previous stress were contextually fear conditioned and one day later trained in an extinction paradigm. Rats were sacrificed one day after conditioning (pre-extinction) or one day after extinction for spine density analysis in the DH. We confirmed that stress exposure induced a deficit in extinction learning. Further, a higher density of dendritic spines, particularly mature ones, was observed in the DH of non-stressed conditioned animals at pre-extinction. Interestingly, after extinction, the spine levels returned to the control values. Conversely, stressed animals did not show such spines boost (pre-extinction) or any other change (post-extinction). In contrast, such standard dynamics of dendritic changes as well as the behavioral extinction was recovered when stressed animals received an intra-basolateral amygdala infusion of midazolam prior to stress. Altogether, these findings suggest that stress hinders the normal dynamic of dendritic remodeling after fear extinction and this could be part of the neurobiological substrate that makes those memories resistant to be extinguished.

GABAergic signaling within the Basolateral Amygdala Complex modulates resistance to the labilization/reconsolidation process.

It is well known that stress can affect mnemonic processes. In particular, stress before contextual fear conditioning induces a memory which exhibits resistance to being interfered with by Midazolam (MDZ) when applied after memory retrieval. Moreover, stress exposure strongly affects GABAergic transmission within the Basolateral Amygdala Complex (BLA), a brain structure critically involved in fear memory processing. The present study evaluated the involvement of GABAergic signaling within the BLA on the induction of resistance to memory reconsolidation interference. Results showed that MDZ administered intra-BLA before stress prevented the induction of resistance to the interfering effect of systemic administration of both MDZ and Propranolol on fear memory reconsolidation, when both applied after memory retrieval. The blockade of amygdala GABA-A receptors by the antagonist Bicuculline (BIC) before memory encoding induced resistance to interference by post-recall MDZ administration, similarly to that observed with stress exposure. Additionally, the systemic administration of d-cycloserine, a positive allosteric modulator of NMDA receptor, reverted the BIC-induced resistance to the MDZ interfering effect, in the same manner as that reported with stress-induced resistance. In summary, these results suggest that the GABAergic signaling in the BLA at the moment of memory encoding is determinant for the induction of fear memory resistance to the onset of the labilization/reconsolidation process.

Retrieval under stress decreases the long-term expression of a human declarative memory via reconsolidation.

Acute stress impairs memory retrieval of several types of memories. An increase in glucocorticoids, several minutes after stressful events, is described as essential to the impairing retrieval-effects of stressors. Moreover, memory retrieval under stress can have long-term consequences. Through what process does the reactivated memory under stress, despite the disrupting retrieval effects, modify long-term memories? The reconsolidation hypothesis proposes that a previously consolidated memory reactivated by a reminder enters a vulnerability phase (labilization) during which it is transiently sensitive to modulation, followed by a re-stabilization phase. However, previous studies show that the expression of memories during reminder sessions is not a condition to trigger the reconsolidation process since unexpressed memories can be reactivated and labilized. Here we evaluate whether it is possible to reactivate-labilize a memory under the impairing-effects of a mild stressor. We used a paradigm of human declarative memory whose reminder structure allows us to differentiate between a reactivated-labile memory state and a reactivated but non-labile state. Subjects memorized a list of five cue-syllables associated with their respective response-syllables. Seventy-two hours later, results showed that the retrieval of the paired-associate memory was impaired when tested 20min after a mild stressor (cold pressor stress (CPS)) administration, coincident with cortisol levels increase. Then, we investigated the long-term effects of CPS administration prior to the reminder session. Under conditions where the reminder initiates the reconsolidation process, CPS impaired the long-term memory expression tested 24h later. In contrast, CPS did not show effects when administered before a reminder session that does not trigger reconsolidation. Results showed that memory reactivation-labilization occurs even when retrieval was impaired. Memory reactivation under stress could hinder -via reconsolidation- the probability of the traces to be expressed in the long term.

Effect of a positive reinforcing stimulus on fear memory reconsolidation in ethanol withdrawn rats: Influence of d-cycloserine.

The pharmacological blockade of memory reconsolidation has been suggested as a potential treatment to the attenuation of maladaptive memories associated to psychiatric disorders and drug addiction. To interfere with the process of fear memory reconsolidation using a manipulation safer than pharmacological interventions, here we examined whether a positive reinforcing stimulus (non-alcoholic beer, NB) post-memory retrieval can decrease the fear response in ethanol withdrawn (ETOH) animals. We first evaluated the potential interfering effect of NB on memory reconsolidation in non-ethanol dependent (control, CON) rats. Non-alcoholic beer intake shortly after memory retrieval attenuated the fear response in CON rats. A resistance to destabilization/reconsolidation process was previously observed in ETOH rats, which was reversed by the activation of NMDA receptor induced by pre-retrieval d-cycloserine (DCS) administration. Therefore, the influence of DCS (5mg/kg; i.p.) to facilitate the disruptive effect of NB on fear memory was examined in ETOH animals. As expected, NB was ineffective to attenuate the fear response in ETOH rats, with DCS being necessary to promote the disruptive effect of NB on the reconsolidation in these animals. Hence, DCS/reinforcing stimulus in combination with memory reactivation can be considered as an alternative approach for disrupting resistant fear memories.

Stress-induced resistance to the fear memory labilization/reconsolidation process. Involvement of the basolateral amygdala complex.

Consolidated memories can enter into a labile state after reactivation followed by a restabilization process defined as reconsolidation. This process can be interfered with Midazolam (MDZ), a positive allosteric modulator of the GABA-A receptor. The present study has evaluated the influence of prior stress on MDZ's interfering effect. We also assessed the influence of both systemic and intra-basolateral amygdala (BLA) infusion of d-cycloserine (DCS), a partial agonist of the NMDA receptors, on the MDZ effect in previously stressed rats. Furthermore, we analyzed the effect of stress on the expression of Zif-268 and the GluN2B sites, two molecular markers of the labilization/reconsolidation process, following reactivation. The results revealed that prior stress resulted into a memory trace that was insensitive to the MDZ impairing effect. Both systemic and intra-BLA DCS administration previous to reactivation restored MDZ's disruptive effect on memory reconsolidation in stressed animals. Further, reactivation enhanced Zif-268 expression in the BLA in control unstressed rats, whereas no elevation was observed in stressed animals. In agreement with the behavioral findings, DCS restored the increased level of Zif-268 expression in the BLA in stressed animals. Moreover, memory reactivation in unstressed animals elevated GluN2B expression in the BLA, thus suggesting that this effect is involved in memory destabilization, whereas stressed animals did not reveal any changes. These findings are consistent with resistance to the MDZ effect in these rats, indicating that stress exposure prevents the onset of destabilization following reactivation. In summary, prior stress limited both the occurrence of the reactivation-induced destabilization and restabilization.

The effect of Midazolam and Propranolol on fear memory reconsolidation in ethanol-withdrawn rats: influence of d-cycloserine.

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.

Facilitating influence of stress on the consolidation of fear memory induced by a weak training: reversal by midazolam pretreatment.

It is well known that an emotionally arousing experience usually results in a robust and persistent memory trace. The present study explored the potential mechanisms involved in the influence of stress on the consolidation of a contextual fear memory in animals subjected to a weak fear training protocol, and whether pretreatment with intra-basolateral amygdala or systemic administration of midazolam (MDZ) prevents the potential stress-induced influence on fear memory formation. A previous restraint session facilitated fear retention, this effect was not due to a sensitized effect of restraint on the footshock experience. MDZ, both systemically or intra-basolateral amygdala infusion prior to the restraint, attenuated the stress-induced promoting influence on fear memory formation. In addition, stress exposure activated the ERK1/2 pathway in basolateral amygdala (BLA) after the weak training procedure but not after the immediate footshock protocol. Similar to our behavioral findings, MDZ attenuated stress-induced elevation of phospho-ERK2 (p-ERK2) in BLA following the acquisition session. Given that the activation of ERK1/2 pathway is essential for associative learning, we propose that stress-induced facilitation of p-ERK2 in BLA is an important mechanism for the promoting influence of stress on the consolidation of contextual fear memory.

ERK activation in the amygdala and hippocampus induced by fear conditioning in ethanol withdrawn rats: modulation by MK-801.

The extracellular signal-regulated kinase (ERK) pathway, which can be activated by NMDA receptor stimulation, is involved in fear conditioning and drug addiction. We have previously shown that withdrawal from chronic ethanol administration facilitated the formation of contextual fear memory. In order to explore the neural substrates and the potential mechanism involved in this effect, we examined: 1) the ERK1/2 activation in the central (CeA) and basolateral (BLA) nuclei of the amygdala and in the dorsal hippocampus (dHip), 2) the effect of the NMDA receptor antagonist MK-801 on fear conditioning and ERK activation and 3) the effect of the infusion of U0126, a MEK inhibitor, into the BLA on fear memory formation in ethanol withdrawn rats. Rats made dependent via an ethanol-containing liquid diet were subjected to contextual fear conditioning on day 3 of ethanol withdrawal. High basal levels of p-ERK were found in CeA and dHip from ethanol withdrawn rats. ERK activation was significantly increased both in control (60min) and ethanol withdrawn rats (30 and 60min) in BLA after fear conditioning. Pre-training administration of MK-801, at a dose that had no effect on control rats, prevented the increase in ERK phosphorylation in BLA and attenuated the freezing response 24h later in ethanol withdrawn rats. Furthermore, the infusion of U0126 into the BLA, but not the CeA, before fear conditioning disrupted fear memory formation. These results suggest that the increased fear memory can be linked to changes in ERK phosphorylation, probably due to NMDA receptor activation in BLA in ethanol withdrawn rats.

Increased voluntary ethanol consumption and c-Fos expression in selected brain areas induced by fear memory retrieval in ethanol withdrawn rats.

Withdrawal from chronic ethanol administration facilitated the formation of contextual fear memory. The effect of fear memory retrieval on subsequent ethanol consumption, by employing a two-bottle free-choice procedure with either water or ethanol (2-8% v/v), was investigated in ethanol withdrawn rats. The effect of fear memory extinction with or without d-cycloserine (DCS, 5mg/kgi.p.) on subsequent ethanol consumption was also evaluated. In addition, we examined c-Fos expression in different brain areas following the fear memory recall. The retrieval of such fear memory induced a significant increase in ethanol consumption in ethanol withdrawn but not in control animals. Regardless of DCS treatment, this increase was attenuated by extinction training. In ethanol withdrawn rats, context-dependent memory retrieval was accompanied by an increased c-Fos expression in the basolateral amygdala, ventrolateral periaqueductal gray, dentate gyrus and dorsomedial periaqueductal gray. Among these brain areas suggested to be implicated in the modulation of motivation and of emotional states, the basolateral amygdala has a crucial role in the emergence of negative affective state during ethanol withdrawal. These data suggest that retrieval of fear memory in ethanol withdrawn rats affected ethanol consumption and that amygdala may be involved in this effect.

Previous stress exposure enhances both anxiety-like behaviour and p35 levels in the basolateral amygdala complex: modulation by midazolam.

Stress exposure induces long lasting neurobiological changes in selected brain areas, which could be associated with the emergence of negative emotional responses. In the present study, previously restrained animals exhibited excessive anxiety one day later in the elevated plus maze. We explore whether stress exposure affects the expression levels of cyclin-dependent kinase 5 (Cdk5) and of its activator protein p35, in diverse amygdaloid nuclei. Stress exposure enhanced p35 levels selectively in the basolateral amygdala (BLA). This up-regulation might be functionally associated with the occurrence of exaggerated anxiety since such emotional response was selectively reversed by an intra-BLA infusion of olomoucine, a Cdk5 inhibitor, 15 min prior to the restraint session. Moreover, pre-treatment with midazolam, a benzodiazepine ligand, not only prevented the excessive anxiety but also attenuated the p35 increase in the BLA of stressed rats. In conclusion, we suggest a pivotal role of the Cdk5/p35 complex, specifically in BLA in the excessive anxiety induced by a previous stressful experience.

Stress response recruits the hippocampal endocannabinoid system for the modulation of fear memory.

The modulation of memory processes is one of the several functions of the endocannabinoid system (ECS) in the brain, with CB1 receptors highly expressed in areas such as the dorsal hippocampus. Experimental evidence suggested an important role of the ECS in aversively motivated memories. Similarly, glucocorticoids released in response to stress exposure also modulates memory formation, and both stress and dexamethasone activate the ECS. Here, we investigate the interaction between the ECS and glucocorticoids in the hippocampus in the modulation of fear memory consolidation. Two protocols with different shock intensities were used in order to control the level of aversiveness. Local infusion of AM251 into the hippocampus immediately after training was amnestic in the strong, but not in the weak protocol. Moreover, AM251 was amnestic in animals stressed 0, but not 30-min prior to the weak protocol, reverting the stress-induced facilitatory effect. Finally, intrahippocampal AM251 infusion reduced memory in animals that received dexamethasone immediately, but not 30 min before training. These results are (1) consistent with the view that the dorsal hippocampus ECS is activated on demand, in a rapid and short-lived fashion in order to modulate the consolidation of an aversive memory, and (2) show that this recruitment seems to be mediated by glucocorticoids, either in the hippocampus or in other brain regions functionally associated with the hippocampus.