Activation of ERK2 in basolateral amygdala underlies the promoting influence of stress on fear memory and anxiety: influence of midazolam pretreatment.

Exposure to emotionally arousing experiences elicits a robust and persistent memory and enhances anxiety. The amygdala complex plays a key role in stress-induced emotional processing and in the fear memory formation. It is well known that ERK activation in the amygdala is a prerequisite for fear memory consolidation. Moreover, stress elevates p-ERK2 levels in several areas of the brain stress circuitry. Therefore, given that the ERK1/2 cascade is activated following stress and that the role of this cascade is critical in the formation of fear memory, the present study investigated the potential involvement of p-ERK2 in amygdala subnuclei in the promoting influence of stress on fear memory formation and on anxiety-like behavior. A robust and persistent ERK2 activation was noted in the Basolateral amygdala (BLA), which was evident at 5min after restraint and lasted at least one day after the stressful experience. Midazolam, a short-acting benzodiazepine ligand, administered prior to stress prevented the increase in the p-ERK2 level in the BLA. Pretreatment with intra-BLA infusion of U0126 (MEK inhibitor), but not into the adjacent central nucleus of the amygdala, attenuated the stress-induced promoting influence on fear memory formation. Finally, U0126 intra-BLA infusion prevented the enhancement of anxiety-like behavior in stressed animals. These findings suggest that the selective ERK2 activation in BLA following stress exposure is an important mechanism for the occurrence of the promoting influence of stress on fear memory and on anxiety-like behavior.

The influence of stress on fear memory processes

It is well recognized that stressful experiences promote robust emotional memories, which are well remembered. The amygdaloid complex, principally the basolateral complex (BLA), plays a pivotal role in fear memory and in the modulation of stress-induced emotional responses. A large number of reports have revealed that GABAergic interneurons provide a powerful inhibitory control of the activity of projecting glutamatergic neurons in the BLA. Indeed, a reduced GABAergic control in the BLA is essential for the stress-induced influence on the emergence of associative fear memory and on the generation of long-term potentiation (LTP) in BLA neurons. The extracellular signal-regulated kinase (ERK) subfamily of the mitogen-activated protein kinase (MAPK) signaling pathway in the BLA plays a central role in the consolidation process and synaptic plasticity. In support of the view that stress facilitates long-term fear memory, stressed animals exhibited a phospho-ERK2 (pERK2) increase in the BLA, suggesting the involvement of this mechanism in the promoting influence of threatening stimuli on the consolidation fear memory. Moreover, the occurrence of reactivation-induced lability is prevented when fear memory is encoded under intense stressful conditions since the memory trace remains immune to disruption after recall in previously stressed animals. Thus, the underlying mechanism in retrieval-induced instability seems not to be functional in memories formed under stress. All these findings are indicative that stress influences both the consolidation and reconsolidation fear memory processes. Thus, it seems reasonable to propose that the emotional state generated by an environmental challenge critically modulates the formation and maintenance of long-term fear memory.

The influence of stress on fear memory processes.

It is well recognized that stressful experiences promote robust emotional memories, which are well remembered. The amygdaloid complex, principally the basolateral complex (BLA), plays a pivotal role in fear memory and in the modulation of stress-induced emotional responses. A large number of reports have revealed that GABAergic interneurons provide a powerful inhibitory control of the activity of projecting glutamatergic neurons in the BLA. Indeed, a reduced GABAergic control in the BLA is essential for the stress-induced influence on the emergence of associative fear memory and on the generation of long-term potentiation (LTP) in BLA neurons. The extracellular signal-regulated kinase (ERK) subfamily of the mitogen-activated protein kinase (MAPK) signaling pathway in the BLA plays a central role in the consolidation process and synaptic plasticity. In support of the view that stress facilitates long-term fear memory, stressed animals exhibited a phospho-ERK2 (pERK2) increase in the BLA, suggesting the involvement of this mechanism in the promoting influence of threatening stimuli on the consolidation fear memory. Moreover, the occurrence of reactivation-induced lability is prevented when fear memory is encoded under intense stressful conditions since the memory trace remains immune to disruption after recall in previously stressed animals. Thus, the underlying mechanism in retrieval-induced instability seems not to be functional in memories formed under stress. All these findings are indicative that stress influences both the consolidation and reconsolidation fear memory processes. Thus, it seems reasonable to propose that the emotional state generated by an environmental challenge critically modulates the formation and maintenance of long-term fear memory.

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.

Gabaergic modulation of the stress response in frontal cortex and amygdala.

GABAergic neurotransmission is thought to play an important role in the modulation of the central response to stress. In the present study we evaluate the influence of a brief restraint exposure on GABA-stimulated chloride influx in diverse brain areas presumed to have a major role in the mediation of emotional behaviors following aversive stimulation. A reduced chloride uptake after stress exposure was only observed in frontal cortex and amygdala. Moreover, rats subjected to such stressor performed an anxiogenic behavior when exposed later to the elevated plus-maze. A comparable behavior in the elevated plus-maze was observed between animals that were allowed to chew during the restraint experience and those without any stressful manipulation, suggesting that chewing served as an efficient coping behavioral strategy during such threatening situations. In order to explore if chewing during the restraint experience could suppress the reduction in GABA-stimulated chloride uptake induced by this stressor, rats were allowed or not to chew during restraint and in both cases GABA-stimulated chloride influx was assayed in frontal cortex and amygdala. The finding of this experiment showed that restrained rats that have the possibility to chew exhibited a similar GABA-stimulated chloride uptake in cortical tissue to that shown by control, unstressed rats. Moreover, chewing in response to restraint attenuated the reduction of GABA-stimulated chloride uptake in amygdala, supporting the notion that chewing is an effective coping response to restraint. These experiments suggest that a reduced GABAergic inhibitory control in these areas could be implicated in the emotional sequelae generated by this uncontrollable stressor and that the suppression of this reduction seems to be associated with the occurrence of coping behavioral response to such fear-inducing stimulus.

A dopaminergic mechanism is involved in the 'anxiogenic-like' response induced by chronic amphetamine treatment: a behavioral and neurochemical study.

The purpose of this study was to examine the influence of chronic d-amphetamine (AMPH) treatment (2 mg/kg i.p., for 9 consecutive days) on behavioral and neurochemical responses to a subsequent exposure - 4 days after the last AMPH injection--to the elevated plus-maze (EPM), as well as to determine the involvement of a dopaminergic mechanism in that influence. Results showed that chronic AMPH treatment induced an 'anxiogenic-like' response when animals were evaluated in the EPM test. Pretreatment with either haloperidol (HAL, 1 mg/kg i.p., 20 min prior to each injection) or SCH-23390 (0.1 mg/kg i.p., 10 min prior to each injection) completely abolished the chronic AMPH-induced 'anxiogenic-like' effect displayed in the EPM test. However, sulpiride pretreatment (60 mg/kg i.p., 10 min prior to each AMPH injection) did not modify such effect. In addition, rats treated with AMPH and subsequently exposed to the EPM, showed a decrease in the maximal GABA-stimulated chloride uptake in cortical microsacs. HAL pretreatment restored the maximal chloride uptake induced by chronic AMPH. Altogether, these results suggest that: (1) previous exposure to chronic AMPH treatment induces an increased emotional response following a conflict situation, (2) dopamine D(1) receptors are mainly involved in chronic AMPH-induced changes in the behavior displayed in EPM test, and (3) an interaction between GABAergic and dopaminergic mechanisms may be implicated in neurochemical and behavioral changes induced by chronic AMPH treatment.

Chronic benzodiazepine administration facilitates the subsequent development of ethanol dependence.

This study investigated the effect of chronic benzodiazepine (BZD) administration and its abrupt discontinuation on later subsequent ethanol consumption employing a free choice paradigm between water and increasing ethanol concentrations. In addition, we also studied the anxiolytic and reinforcing properties of ethanol assessed in the elevated plus maze (EPM) and in the conditioned place preference paradigm, respectively. Adult male Wistar rats were subjected to a chronic diazepam (DZM) treatment (2 mg/kg/day, i.p.) during 21 days. Twenty-four hours after that treatment and, in another experiment, 10 days after the last DZM injection, rats were subjected to an oral ethanol self-administration procedure (ethanol was increased in concentration (v/v) on 4 consecutive days as follows: 2%, 4%, 6%, 8% followed by an additional period of 8 days in which animals were offered a 10% (v/v) ethanol solution. Diazepam treated rats showed a higher ethanol intake and spontaneous signs of ethanol withdrawal when the access to ethanol was discontinued. These results were observed when ethanol was available at day 1 of withdrawal but not when DZM treated rats were initiated in the ethanol choice test 10 days after BDZ withdrawal. Furthermore, DZM treated rats exhibited an increased anxiolytic ethanol induced effect (1 g/kg, i.p.) in the EPM and a significant ethanol-induced conditioned place preference (1 g/kg, i.p.). These data suggest that early DZM treatment facilitates ethanol consumption and the development of ethanol dependence.

Benzodiazepine withdrawal facilitates the subsequent onset of escape failures and anhedonia: influence of different antidepressant drugs.

The effect of benzodiazepine (BDZ) withdrawal on escape acquisition and on the behavioral response to two different reinforcing stimuli was investigated. In addition, the influence of antidepressant drugs (AD) differing in their mechanism of action on these behavioral outputs was also evaluated. Rats subjected to withdrawal from a chronic treatment with diazepam (DZM; 2 mg/kg per day, i.p.) during 21 days were subsequently exposed to a brief inescapable shock session (IS) and 48 h later to an active avoidance test. Only withdrawn animals exposed to the IS exhibited enhanced escape failures. In an additional experiment, withdrawn rats were repeatedly administered with vehicle (VEH), desipramine (DMI; 5 mg/kg, i.p.), fluoxetine (FLU; 5 mg/kg, i.p.) or phenelzine (PHEN; 5 mg/kg, i.p.) and subsequently exposed to IS and to active avoidance task. A significant reversal of escape deficit was only observed following DMI and PHEN but not after FLU. Furthermore, withdrawn rats showed a reduced preference for a sexually relevant olfactory cue, this reduced sensitivity was only normalized following DMI but not after the administration of FLU or PHEN. Finally, rats exposed to abrupt cessation of chronic BDZ administration did not exhibit preference for a context previously associated with amphetamine (AMP) under the conditioned place preference (CPP) procedure. All these findings are indicative that BDZ withdrawal facilitates the subsequent occurrence of behavioral changes-escape failures and reduced behavioral response to rewarding stimuli-suggested to parallel important symptoms of human depression. In addition, DMI seems to be much more effective in restoring such behavioral abnormalities as compared to a MAO inhibitor and to a inhibitor of 5-HT uptake.

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.

Anxiogenic-like effects of Tagetes minuta L essential oil on T-maze and tonic immobility behaviour in domestic chicks.

In a first experiment, four doses (ranging between 0.04 and 0.45 mg/kg of body weight) of the essential oil from Tagetes minuta L. were subcutaneously injected in two-day-old chicks and a dose-response curve assessed for escape performance in a T-maze test. The 0.1, 0.25 and 0.45 mg/kg doses impaired the first escape performance suggesting an anxiogenic-like effect of the essential oil. After 3 h the same chicks were tested for a second escape performance, without being injected again, and no differences were observed compared to controls, suggesting that the essential oil did not affect retention. Furthermore, the effects of the essential oil were observed in the three sections of the T-maze apparatus. So, the performance was impaired in the isolation chamber section, suggesting the induction of increased anxiogenic behaviour, and also in the mirror section, suggesting that the social reinstatement behaviour was modified by an increased anxiety level. Changes in the principal corridor section were not observed, suggesting that the locomotor activity was not affected by these oil doses. The second escape performance was not affected in any of the T-maze sections, confirming that these doses did not affect learning ability. In a second experiment, a middle dose of the essential oil (0.25 mg/kg) increased the tonic immobility reaction in 15 days old chicks similarly to an anxiogenic dose of FG 7142 (1 mg/kg), while an anxiolytic dose of diazepam (0.08 mg/kg) did not affect this behaviour. Taken together, the present results suggest that the essential oil from Tagetes minuta L. may exert a negative modulation on the GABAergic function without affecting the learning ability.

Carbamazepine normalizes the altered behavioral and neurochemical response to stress in benzodiazepine-withdrawn rats.

Rats chronically treated with diazepam (2 mg/kg per day, i.p.) for 21 days were tested 96 h after the last injection in both the forced swim test (inescapable stress) and in an active avoidance test (escapable stress). The influence of carbamazepine (7.5 mg/kg, i.p.) administered 25 min prior to each behavioral task was investigated. Withdrawn animals showed a reduced time spent in immobility in the forced swim test and an enhanced latency to escape in the active avoidance test. Both behavioral effects were normalized by a single carbamazepine administration. An additional experiment was performed to investigate the effect of a forced swim experience on cortical chloride uptake following GABA (gamma-aminobutyric acid) stimulation 96 h after diazepam withdrawal, and the influence of a single administration of carbamazepine on these effects. An increased chloride uptake was observed in vehicle-treated rats but not in diazepam-withdrawn animals following the swimming experience. Carbamazepine pretreatment enhanced chloride uptake after diazepam withdrawal but did not modify chloride flux in stressed or unstressed vehicle-treated rats. These results support the hypothesis that diazepam withdrawal affects the ability to develop adaptive responses to stress and that carbamazepine can normalize such an alteration.

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.

Effect of diazepam and A beta-carboline on open-field and T-maze behaviors in 2-day-old chicks.

The effects of diazepam and the beta-carboline FG 7142 in chicks were examined on several behavioral measures in open-field and T-maze tasks. In the open field, only the higher doses of diazepam affected behavior, suggesting a sedative-like effect, while FG 7142 influenced behavior as would a fear-inducing manipulation. After a low dose of either drug was injected, testing in a T-maze showed that diazepam improved and FG 7142 impaired the escape performance from the isolation chamber, without affecting the time spent in the T-corridor. In three groups of chicks selected on the basis of their first escape performance, only lower performance chicks were affected by an anxiolytic dose of diazepam. T-maze results suggest that: (a) T-maze is more sensitive than open-field test to behavior changes induced by anxiolytic doses of diazepam; (b) isolation chamber behavior could be an index of general emotionality in young chicks; (c) diazepam and FG 7142 do not modify the social motivation to escape the maze; (d) higher performance chicks present an escape behavior of a less anxious type than lower performance chicks. The results suggest that the GABAergic system is involved in the behavioral expression of fear and anxiety in young chicks.

Recruitment of peripheral-type benzodiazepine receptors after acute stress in chick forebrain membranes: action of Triton X-100.

A significant increase in the number of measurable [3H]Ro 5-4864 receptors was found in forebrain membranes of chicks submitted to 15 min of acute swim stress compared to non-stressed chicks. In addition, low subsolubilizing concentrations of Triton X-100 caused a significant increase in the measurable [3H]Ro 5-4864 receptor number in forebrain membranes from non-stressed chicks. However, this increase caused by Triton X-100 was not observed when tested in forebrain membranes from stressed chicks. In all cases the affinity remained unchanged. These results suggest that: (1) acute stress and Triton X-100 induce receptor increase by enhancing [3H]Ro 5-4864 accessibility to a pool of receptors not detected before stress or in the absence of detergent; (2) the pool of non-measured receptors represents about a third of the total in control chicks; (3) the increments are not additive and could involve receptors coming from the same non-measured pool; (4) the receptor increase during a short time of stress could be explained by recruitment of receptors but not by an increase in the receptor protein biosynthesis; (5) stress induces a maximal recruitment of measurable [-3H]Ro 5-4864 receptors.

Altered behavioral and neurochemical response to stress in benzodiazepine-withdrawn rats.

Rats chronically treated with diazepam (DZM) (2 mg/kg/day i.p.) for 21 days, were subsequently tested at 24, 48, 72 and 96 h after the last injection in the elevated plus-maze. When compared with their respective controls, withdrawn animals showed an anxiogenic response since they exhibited a significant decrease in the % of time spent in the open arms, and a reduction in open and total arms entries at 24 and 48 h following the last benzodiazepine administration. No other behavioral differences were observed in the remaining groups. Ninety-six hours following DZM withdrawal, when the anxiogenic response was no longer evident, withdrawn rats showed a significant decrease in immobility time when exposed to a forced-swim test (FST). GABA stimulation of chloride uptake in cortical tissue was measured in BDZ-withdrawn rats or vehicle-treated animals with or without exposure to the FST. An enhanced chloride uptake following GABA stimulation was observed in vehicle-treated rats following the swimming trial. However, similar values of chloride uptake were found among rats withdrawn from DZM at 96 h either exposed or not to the FST and vehicle-treated animals without prior stress exposure. These findings show that BDZ withdrawal alters the neurochemical and behavioral response to a subsequent stressful experience. These lines of evidence may indicate that BZD withdrawal reduced the ability to develop an adaptive response to stress.

The essential oil from Tagetes minuta L. modulates the binding of [3H]flunitrazepam to crude membranes from chick brain.

The hypothesis that the essential oil from Tagetes minuta L. can interact with biological membranes was investigated by assessing its ability of perturbing the binding of a benzodiazepine [flunitrazepam (FNTZ)] to crude members from chick brains. The essential oil from T. minuta L. inhibited [3H]FNTZ specific binding to chick brain members. These values were obtained from the analysis of the saturation curve for the kinetic parameters: dissociation equilibrium constant (Kd) = 2.47 +/- 0.32 nM, maximal binding (Bmax) = 556 +/- 5 fmoles/mg protein, and Hill coefficient (n) = 1.00 +/- 0.07 in the absence, and Kd = 6.73 +/- 1.4 nM, Bmax = 583 +/- 69 fmoles/mg protein, and n = 1.02 +/- 0.08 in the presence of 29 microgram/mL of essential oil. The essential oil could self-aggregate with a critical micellar concentration (CMC) of 60 microgram/mL. The marked increase in [3H]FNTZ nonspecific binding starting at 60 micrograms of essence per mL was due to that phenomenon and revealed the ability of self-aggregated structures to interact with members. [3H]FNTZ specific binding decrement as a function of essence concentration cannot be ascribed merely to oil's micelles ability of trapping the lipophilic radioligand molecules, because the discontinuous behavior that characterizes a monomer-aggregate phase transition was not shown. Oil's components might behave as competitive inhibitors or allosteric modulators of FNTZ specific binding. However, their ability to increase FNTZ nonspecific binding at concentrations below oil's CMC suggests that this effect may be due to oil's partitioning into the lipid bilayer. This latter phenomenon would induce an increment in membrane fluidity and a change on FNTZ binding site toward a lower affinity conformation. Therefore, the essential oil components can interact with brain membranes either as monomers, by partitioning into the lipid bilayer, or as self-aggregated structures, through an adsorption to the membrane surface.

Chick imprinting performance and susceptibility to acute stress associated to flunitrazepam receptor increase.

One-day-old chicks were selected on their performance in imprinting behaviour and termed high-imprinted (H-I), partially imprinted (P-I) and low-imprinted (L-I) chicks. Then, H-I and L-I chicks were submitted to acute handling stress and [3H]flunitrazepam receptor-binding was performed on synaptosomal membranes from forebrain at various times after handling. The receptor number significantly increased in L-I but not in H-I chicks at 30 min after handling while the affinity remained unchanged at all times. In addition, when the three selected groups were maintained to reach 15 days of age and then they were submitted to acute swimming stress, the degree of receptor increase was also inversely related to the degree of imprinting performance. The receptor increase associated to swimming stress was higher in the left hemisphere, suggesting an interhemispheric asymmetry of stress effects. The results suggest that more-imprinted chicks are less susceptible than less-imprinted chicks to acute stress associated to central benzodiazepine receptor increase, probably due to differences in the degree of endogenous emotionality.

Transient benzodiazepine-GABAA receptor increase after a passive avoidance learning in synaptosomal membranes from chick forebrain.

One-day-old chicks were exposed to a one-time passive avoidance learning task. After chicks peak a bead dipped in a bitter-tasting liquid, they learn to stop pecking the bead. Radioligand binding analysis of [3H]flunitrazepam was performed on crude synaptosomal membranes from forebrains, at 10, 30, and 60 min post-training. Water-trained chicks (control) pecked a bead dipped in water, and they did not learn to stop pecking the bead. The water control was complemented with a methyl anthranilate fed control chick to demonstrate that taste per se does not affect the [3H]flunitrazepam binding. At 30 min in relation to 10 min post-training, the Bmax increased 31% in water-trained chicks and 56% in taste-trained chicks, with Bmax of the taste-trained chicks reaching a value 22% higher than that in water-trained chicks. The difference, attributable to the learning, disappeared at 60 min post-training, and at all times the affinity remained unchanged. The Bmax increase in water-trained chicks might be attributable to psychological stress accompanying the task and the Bmax increase in taste-trained chicks attributable to the learning in addition to the stress accompanying the task. The results suggest that the receptor increase associated with learning is involved in early stages of memory formation.

Benzodiazepine receptor recruitment after acute stress in synaptosomal membranes from forebrain of young chicks: action of Triton X-100.

In young chicks submitted to acute stress by forced swimming there was a significant increase in the number of the measurable [3H]-flunitrazepam receptors in synaptosomal membranes from forebrain. In addition, low subsolubilizing concentrations of Triton X-100 caused a significant increase in the measurable [3H]-flunitrazepam receptor number in synaptosomal membranes from non-stressed chicks. However, this Triton X-100 stimulatory effect was not observed when tested in synaptosomal membranes from stressed chicks. In all cases the affinity remained unchanged. This result suggest that: (i) acute stress and Triton X-100 induce receptor recruitment by enhancing [3H]-flunitrazepam accessibility to a pool of receptors which is unmeasurable either before stress or in absence of detergent; (ii) neither recruitment types are additive and they involve receptors coming from the same nonmeasurable pool; (iii) stress induces a maximal recruitment of existing benzodiazepine receptors; (iiii) the pool of nonmeasurable receptors represents about a quarter of the total in control chicks. The recruitment at a short time of stress could be interpreted in terms involving internalization; recycling or modulation of receptors but not its biosynthesis or degradation.