Victor A. Molina

Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction

Retrieval of a consolidated memory triggers a number of processes which depend, among other factors, on the duration of the reactivation session: reconsolidation requires a brief reactivation session, and extinction, a prolonged one. The scope of this study is to explore the potential role of the hippocampal endocannabinoid system on reconsolidation and extinction processes. Bilateral infusion of the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) into the CA1 region of the dorsal hippocampus of Wistar rats after memory reactivation facilitated the reconsolidation of the contextual fear conditioning memory. The inhibition of protein synthesis with DRB in the same brain region blocked memory reconsolidation. Both effects were persistent, lasting up to 7 days after the first retrieval experience. In contrast, the local infusion of anandamide blocked memory reconsolidation, an effect that was antagonized by the combined administration of anandamide with a subthreshold dose of a CB1 antagonist, supporting a CB1-mediated role of the hippocampal endocannabinoid system in the modulation of the memory reconsolidation. Local infusion of AM251 into CA1 blocked memory extinction whereas the administration of anandamide facilitated it; however, when combined with a subthreshold concentration of the CB1 antagonist, anandamide did not affect the extinction process. The clear-cut, opposite effects observed in each situation suggest a possible role of the hippocampal endocannabinoid system as a switching mechanism deciding which processes will take place, either maintaining the original memory (reconsolidation) or promoting a new learning (extinction).

Early exposure to chronic variable stress facilitates the occurrence of anhedonia and enhanced emotional reactions to novel stressors: reversal by naltrexone pretreatment.

The present research studied the influence of an early chronic variable stress (CVS) paradigm - an animal model of depression - on behavioral responses to subsequent environmental challenges suggested to model anhedonia and emotional reactions such as anxiety and fear. In order to explore a potential involvement of an endogenous opiate mechanism - presumably activated during CVS exposure - in the development of such behavioral reactions, in all experiments rats were administered naltrexone (NAL, 2 mg/kg, i.p.) or vehicle (VH) prior to each daily stressor of the CVS procedure. Animals were exposed to CVS and 1 week later tested for sucrose preference (1%) in a free choice paradigm after the presentation or not of a 90-min restraint period. Only CVS treated animals that were later exposed to restraint showed a reduction of sucrose preference, this reduction was absent when CVS rats were pretreated previously with NAL. Moreover, CVS rats were one week later tested on the elevated plus maze (EPM) and in their conditioned and unconditioned freezing response to a single shock session. Early chronic stress resulted in an anxiogenic behavior in the EPM and in an enhanced conditioned and unconditioned freezing which were all abolished by NAL pretreatment. These behavioral findings suggest that the potential activation of an endogenous opiate mechanism during CVS participates in the development of anhedonia and exaggerated emotional reactions in response to subsequent stressful experiences.

Midazolam disrupts fear memory reconsolidation

The current research examines the influence of midazolam (MDZ) on memory reconsolidation using a contextual fear paradigm in rats, based on three context-shock training trials (0.7 mA, 3 s). First, we evaluate the effect of MDZ (1 mg/kg, i.p.) injected shortly after the training procedure. Second, we examined the influence of MDZ after a brief exposure (90 s) either in the training context (reactivation procedure) or in a neutral environment (no reactivation procedure) and one day later, freezing behavior was scored when rats were re-exposed to the training environment. Third, we investigate both the effect of MDZ administered at different times following reactivation on fear memory and the persistence of such effect 10 days after reactivation. Finally, we test whether the MDZ effect could be reverted by a single weak training trial (0.2 mA, 3 s) or by the presentation of the same unconditioned stimulus in the absence of the conditioned stimulus as a reminder which proves to induce significant freezing in rats not previously trained. Results show that MDZ interferes with the formation of a contextual fear memory only when administered after the reactivation procedure but not after the training procedure. This interference was effective up to 60 min after reactivation and not at a later time. No spontaneous recovery of freezing behavior was observed 11 days after MDZ injection which was not reverted by a weak training trial and by the unconditioned stimulus alone. All these data support the idea that stimulating GABA A receptor sites via MDZ selectively disrupts the reconsolidation process of a contextual fear memory.

Chronic stress-induced changes in locus coeruleus neuronal activity

Locus coeruleus (LC) activity was assessed in rats exposed to either acute or chronic stress. After one immobilization session, the number of spontaneously active LC neurons dramatically decreased. On the other hand, repeated restraint sessions enhanced noradrenergic (NA) transmission and the inhibitory effect of clonidine (CLON) was greater on these cells than in those of controls. These results bear on the adaptive changes in the NA system following acute or chronic stress.

Metyrapone pretreatment prevents the behavioral and neurochemical sequelae induced by stress

In the present study, we examined the effect of metyrapone, an inhibitor of corticosterone (CS) synthesis, on the behavioral and neurochemical sequelae induced by a brief restraint session. A 15-min stress period induced an anxiogenic-like behavior on the elevated plus-maze (EPM), which was reversed with metyrapone (75 mg/kg i.p.) injected 3 h prior to the stress event. It was further demonstrated that metyrapone pretreatment normalized the decrease in maximal chloride uptake following GABA stimulation observed in brain cortex tissue obtained from animals exposed to both restraint and the EPM. In addition, plasma CS levels were assessed both after restraint and following EPM exposure. Furthermore, the administration of both CS (2.5 mg/kg s.c. at a dose that mimics CS levels induced by restraint) or dexamethasone (DEXA, 1.25 microg/kg s.c) resulted in an anxiogenic response in the EPM comparable to that induced by restraint. Taken together, all these evidence suggest that CS released in response to stress seems to be associated with functional changes at the GABAergic supramolecular complex which could underlie the enhanced anxiety observed following the exposure to an aversive experience.

Prior exposure to a brief restraint session facilitates the occurrence of fear in response to a conflict situation: behavioral and neurochemical correlates.

The influence of two different stressors on the behavioral and neurochemical responses to a subsequent exposure to the elevated plus maze (EPM) was examined. Rats were submitted to either a 15-min restraint period or to a 15-min forced swimming test (FS) and one day later exposed to the EPM. Animals with early restraint exhibited a significant decrease in the percent time spent and in the number of entries on the open arms. In addition, restraint induced a reduction in the total number of entries. An identical behavior in the EPM was observed between unstressed rats and those exposed to a previous swimming experience. As a humoral index of stress, corticosterone (CS) secretion in response to each stressor was evaluated. A similar increase of CS release was observed following each aversive stimulus. Exposure to both restraint and EPM decreased the cortical chloride uptake following GABA stimulation. Similar values of chloride flux were obtained from animals submitted to either restraint but without subsequent exposure to the EPM, exposed only to the EPM, or without any manipulation (controls). These findings are discussed in terms of a facilitated behavioral and neurochemical response to a fearful situation following an early and brief restraint experience.

SHUTTLE-BOX DEFICITS INDUCED BY CHRONIC VARIABLE STRESS : REVERSAL BY IMIPRAMINE ADMINISTRATION

Escape performance in a shuttle-box task was evaluated in rats chronically exposed to a series of unpredictable stressors either during 14 or 7 consecutive days. Failure in escape responses was observed when animals were subjected to both regimes of variable aversive situations. The association between chronic exposure to unpredictable stressors with imipramine resulted in a significant reversal of escape deficits. Furthermore, animals submitted to repeated immobilization sessions during 7 days presented similar escape response to control rats. A possible involvement of beta-adrenergic sites on this behavioral response is discussed.

Influence of ethanol withdrawal on fear memory: Effect of d-cycloserine

Animals made dependent via an ethanol (ETOH) -containing liquid diet (6% v/v) for 14 days were subjected to a contextual fear conditioning paradigm 3 days after the last consumption day. After conditioning, rats were subjected to four extinction trials by exposing the animals to the conditioned context and their freezing was evaluated for each trial. Immediately after the first extinction trial, animals were injected with D-cycloserine (DCS) 5 mg/kg i.p., a dose that did not influence the extinction in control rats. Spontaneous recovery of learned fear was tested seven days after the last extinction trial. The following day, animals were subjected to a reacquisition or a reinstatement procedure and their freezing responses evaluated 24 h later. The present study shows that: 1. discontinuation from chronic ETOH administration facilitated the formation of a new fear memory concomitant with a marked resistance to being extinguished, 2. administration of DCS (5 mg/kg) facilitated the extinction process only in ETOH withdrawn rats, 3. both reinstatement and reacquisition procedures restored the increased freezing in ETOH withdrawn animals after extinction, 4. DCS administered immediately after the first extinction trial prevented the increase in freezing following both reacquisition and reinstatement. The enhanced sensitivity to the facilitatory effect of DCS in ETOH withdrawn animals may be mediated by adaptive changes in N-methyl-D-aspartate (NMDA) receptor provoked by ETOH dependence.

Effect of different restraint schedules on the immobility in the forced swim test: Modulation by an opiate mechanism

The present research was conducted to evaluate the influence of different stress schedules on behaviors displayed during both phases of the forced swim test (FST). In addition, the involvement of an opiate mechanism in the behavioral consequences of chronic restraint was investigated. Exposure to a single, but not to chronic, restraint event induced an increase in the immobility score obtained during the 10-min initial swimming exposure (initial test) of the FST. Animals submitted to a previous regime of repeated restraint showed a significant increase in immobility during the 5-min second swimming exposure (retest period) of this behavioral task. However, naloxone (NAL) administered before each of the seven restraint events, blocked the higher immobility observed in chronically stressed rats during the retest period suggesting the involvement of an opiate mechanism. Results concerning the effect of chronic stress on the behavior displayed during the FST were discussed with reference to previous reports which have proposed that immobility performed during the retest period of the FST represents an efficient adaptive response in this inescapable aversive experience.

Chronic stress sensitizes frontal cortex dopamine release in response to a subsequent novel stressor: reversal by naloxone

The present study examined the influence of an early chronic variable stress procedure with or without concurrent naloxone administration at different doses (1, 2 or 3 mg/kg, i.p.) on stress (restraint)-induced dopamine release in the frontal cortex in vivo. A higher increase in cortical dopamine release in response to a subsequent restraint event was observed in chronically stressed rats as compared with those without chronic stress exposure. Naloxone pretreatment normalized this sensitized response only at the higher dose (3 mg/kg, i.p.). The present results indicate that cortical dopamine response to a novel and uncontrollable stressor sensitizes after exposure to a chronic variable stress procedure and that an endogenous opiate mechanism, presumably activated during chronic stress, may be involved in the development of such a sensitization process.