Cardiovascular Reactivity to a Novel Stressor: Differences on Susceptible and Resilient Rats to Social Defeat Stress.

Prolonged and heightened responses to stress are known factors that influence the development of mood disorders and cardiovascular diseases. Moreover, the coping strategies related to the experience of adverse events, i.e., resilience or the susceptibility to stress, are determinants for the individual risk of developing such diseases. Susceptible rats to the social defeat stress (SDS), identified by the social interaction test (SIT), show behavioral and cardiovascular alterations after SDS exposure that are not found in resilient rats. However, it is not elucidated yet how the cardiovascular system of susceptible and resilient phenotypes responds to a new stressor after SDS exposure. Thus, using the SDS exposure followed by the SIT, we evaluated heart rate, blood pressure (BP), tail skin temperature, and circulating corticosterone responses to an acute session of restraint stress in susceptible and resilient rats to SDS. Susceptible rats showed resting tachycardia and exaggerated BP response to restraint stress, while resilient rats did not present such alterations. In contrast, both phenotypes showed increased plasma corticosterone and a drop in tail skin temperature to restraint stress, which was similar to that observed in control animals. Our results revealed an increased cardiovascular reactivity in response to a new stressful stimulus in susceptible rats, which might be related to a greater risk for the development of cardiovascular diseases.

Susceptibility to extinction and reinstatement of ethanol-induced conditioned place preference is related to differences in astrocyte cystine-glutamate antiporter content.

Individual susceptibility to alcohol effects plays an important role in the development of alcohol addiction and studies have shown that glutamate release is altered after chronic ethanol consumption. The cystine-glutamate antiporter (xCT) is a protein that regulates glutamate release. However, little is known about the relationship between xCT levels and this individual susceptibility. Thus, this study aimed to evaluate the relationship between the extinction and stress-induced reinstatement of ethanol conditioned place preference (CPP) and xCT levels in the medial prefrontal cortex (mPFC), nucleus accumbens (NAcc) and amygdala (Amy). Male Swiss mice were submitted to a CPP procedure followed by an extinction protocol and then identified as those which extinguished the CPP and those that did not. In another cohort, mice that extinguished the CPP were submitted to a protocol of stress-induced reinstatement. Immediately after the tests, brains were removed for xCT quantification. The xCT levels were significantly lower in the mPFC and NAcc of mice that did not extinguish CPP. Moreover, mice that were susceptible to stress-induced reinstatement of CPP had lower levels of xCT in the NAcc. Our results suggest that individual susceptibility to the extinction and reinstatement of ethanol CPP is related to alterations in xCT levels.

The AT1 Receptor Antagonist Losartan Does Not Affect Depressive-Like State and Memory Impairment Evoked by Chronic Stressors in Rats.

The present study investigated the effect of the treatment with the angiotensin II type 1 receptor (AT1) antagonist losartan in the depressive-like state and memory impairment evoked by exposure to either homotypic (i.e., repeated exposure to the same type of stressor) or heterotypic (i.e., exposure to different aversive stimuli) chronic stressors in rats. For this, male Wistar rats were subjected to a 10 days regimen of repeated restraint stress (RRS, homotypic stressor) or chronic variable stress (CVS, heterotypic stressor) while being concurrently treated daily with losartan (30 mg/kg/day, p.o.). Depressive-like state was evaluated by analysis of the alterations considered as markers of depression (decreased sucrose preference and body weight and coat state deterioration), whereas cognitive non-emotional performance was tested using the novel object recognition (NOR) test. Locomotor activity was also evaluated in the open field test. Both RRS and CVS impaired sucrose preference and caused coat state deterioration, whereas only CVS impaired body weight gain. Besides, RRS impaired short-term memory (but not long-term memory) in the NOR test. Neither depressive-like state nor memory impairment evoked by the chronic stressors was affected by the treatment with losartan. Nevertheless, CVS increased the locomotion, which was inhibited by losartan. Taken together, these results provide evidence that the chronic treatment with losartan does not affect the depressive-like state and memory impairment evoked by either homotypic or heterotypic chronic stress regimens in rats.

Cardiovascular outcomes related to social defeat stress: New insights from resilient and susceptible rats.

Stress exposure is an important risk factor for psychiatric and cardiovascular disorders. Two phenotypes related to coping with stress can be observed in rodents that experience chronic social defeat stress (SDS): susceptible, showing social avoidance and behavioral changes related to depression, and resilient, showing none of these alterations. Moreover, a strong correlation exists between depression and the development of or mortality due to cardiovascular diseases. Nevertheless, little is known about cardiovascular alterations related to SDS exposure in those phenotypes or their correlation with depressive-like behaviors. Using a chronic SDS protocol followed by the social interaction test, we identified Wistar rats as resilient or susceptible to SDS. Susceptible animals showed increased depressive-like behaviors with resting tachycardia and decreased heart rate variability (HRV) due to increased sympathetic tone in the heart and a less effective baroreflex. In contrast, resilient rats were protected from these alterations by increased vagal tone, resulting in greater HRV values. To our knowledge, our study is the first to indicate that harmful cardiovascular outcomes are related to depressive-like behaviors in susceptible rats and to suggest a mechanism by which resilient rats are protected from these changes. Also, our results suggest that enhanced HRV and vagal tone may be an important trait in resilient individuals.

Sex differences in cardiovascular, neuroendocrine and behavioral changes evoked by chronic stressors in rats.

This study investigated the physiological, somatic and behavioral changes evoked by daily exposure to the same type of stressor (homotypic) or different aversive stressor stimuli (heterotypic) in male and female rats. For this, adult Wistar rats were subjected to a 10days regimen of repeated restraint stress (RRS, homotypic stressor) or chronic variable stress (CVS, heterotypic stressor). Effects evoked by CVS included: (i) adrenal hypertrophy and decreased body weight gain in male animals, (ii) a sympathetically-mediated increase in basal heart rate in males, and (iii) a rise in plasma corticosterone concentration and anxiogenic effects in female animals. The homotypic stressor RRS also induced an increase in plasma corticosterone and anxiogenic effects in females, decreased body weight gain in males and evoked a sympathetically-mediated increase in heart rate in both sexes. Changes in cardiovascular function and autonomic activity evoked by both stressors were followed by impairment of baroreflex activity in males, but not female animals. Both chronic stressors evoked changes in blood pressure responsiveness to vasoconstrictor and vasodilator agents in both sexes. Taken together, these results indicate that regardless of chronic stress regimen males are more vulnerable to somatic effects of chronic stressors, while females appear to be more susceptible to neuroendocrine and behavioral changes. Present findings also indicate that females are selectively vulnerable to cardiovascular and autonomic changes evoked by homotypic stressors. Nevertheless, homotypic and heterotypic stressors similarly affect cardiovascular function and autonomic activity in males.

Stress-Induced Locomotor Sensitization to Amphetamine in Adult, but not in Adolescent Rats, Is Associated with Increased Expression of ΔFosB in the Nucleus Accumbens.

While clinical and pre-clinical evidence suggests that adolescence is a risk period for the development of addiction, the underlying neural mechanisms are largely unknown. Stress during adolescence has a huge influence on drug addiction. However, little is known about the mechanisms related to the interaction among stress, adolescence and addiction. Studies point to ΔFosB as a possible target for this phenomenon. In the present study, adolescent and adult rats (postnatal day 28 and 60, respectively) were restrained for 2 h once a day for 7 days. Three days after their last exposure to stress, the animals were challenged with saline or amphetamine (1.0 mg/kg i.p.) and amphetamine-induced locomotion was recorded. Immediately after the behavioral tests, rats were decapitated and the nucleus accumbens was dissected to measure ΔFosB protein levels. We found that repeated restraint stress increased amphetamine-induced locomotion in both the adult and adolescent rats. Furthermore, in adult rats, stress-induced locomotor sensitization was associated with increased expression of ΔFosB in the nucleus accumbens. Our data suggest that ΔFosB may be involved in some of the neuronal plasticity changes associated with stress induced-cross sensitization with amphetamine in adult rats.

N-acetylcysteine treatment blocks the development of ethanol-induced behavioural sensitization and related ΔFosB alterations.

Ethanol addiction is a serious public health problem that still needs more effective pharmacological treatment. A key factor in the development and maintenance of this disease is the advent of neuroadaptations in the mesocorticolimbic brain pathway upon chronic ethanol abuse. In general, these neuroadaptations are maladaptive and affect numerous neurotransmitter systems and intracellular molecules. One of these molecules is ΔFosB, a transcription factor that is altered after chronic drug use. Behavioural sensitization is a useful model for the study of the neuroadaptations related to addiction. Recent works have shown a role for the imbalance of glutamatergic neurotransmission in the symptoms found in addicted people. In this sense, the treatment with N-acetylcysteine, a l-cysteine prodrug that acts by restoring extrasynaptic concentrations of glutamate through the activation of cystine-glutamate antiporter, has shown promising results in the treatment of addiction. Thus, an animal model of behavioural sensitization was used to evaluate the effects of N-acetylcysteine treatment in the behavioural and molecular alterations induced by chronic ethanol administration. Swiss mice were subject to 13 days of daily ethanol administration to induce behavioural sensitization. Two hours before each ethanol administration and locomotor activity evaluation, the animals received intraperitoneally N-acetylcysteine injections. Immediately after the last test session, their brains were removed for ΔFosB and cystine-glutamate antiporter quantification. It was found that N-acetylcysteine treatment blocked ethanol-induced behavioural sensitization, the increase of ΔFosB content in the prefrontal cortex, and its reduction in the nucleus accumbens. The results suggest a possible use of N-acetylcysteine in ethanol-related disorders.

Conessine, an H3 receptor antagonist, alters behavioral and neurochemical effects of ethanol in mice.

Ethanol abuse potential is mainly due to its reinforcing properties, crucial in the transition from the recreational to pathological use. These properties are mediated by mesocorticolimbic and nigrostriatal dopaminergic pathways and neuroadaptations in these pathways seem to be responsible for addiction. Both pathways are modulated by other neurotransmitters systems, including neuronal histaminergic system. Among the histamine receptors, H3 receptor stands out due to its role in modulation of histamine and other neurotransmitters release. Thus, histaminergic system, through H3 receptors, may have an important role in ethanol addiction development. Aiming to understand these interactions, conessine, an H3 receptor antagonist, was given to mice subjected to the evaluation of ethanol-induced psychostimulation, ethanol CPP and quantification of norepinephrine, dopamine, serotonin and their metabolites in mesocorticolimbic and nigrostriatal pathways following acute ethanol treatment. Systemic conessine administration exacerbated ethanol effects on locomotor activity. Despite of conessine reinforcing effect on CPP, this drug did not alter acquisition of ethanol CPP. Ethanol treatment affects the serotoninergic neurotransmission in the ventral tegmental area, the dopaminergic neurotransmission in the pre-frontal cortex (PFC) and caudate-putamen nucleus (CPu) and the noradrenergic neurotransmission in the CPu. In the PFC, conessine blocked ethanol effects on dopaminergic and noradrenergic neurotransmission. The blockade of H3 receptors and ethanol seem to interact in the modulation of dopaminergic neurotransmission of nigrostriatal pathway, decreasing dopamine metabolites in substantia nigra. In conclusion, conessine was able to change psychostimulant effect of ethanol, without altering its reinforcing properties. This exacerbation of ethanol-induced psychostimulation would be related to alterations in dopaminergic neurotransmission in the nigrostriatal pathway.

Stress abolishes the effect of previous chronic ethanol consumption on drug place preference and on the mesocorticolimbic brain pathway.

BACKGROUND: Conditioned place preference (CPP) to ethanol (EtOH) is an important addiction-related alteration thought to be mediated by changed neurotransmission in the mesocorticolimbic brain pathway. Stress is a factor of major importance for the initiation, maintenance, and reinstatement of drug abuse and modulates the neurochemical outcomes of drugs. Thus, the aim of this study was to investigate the effects of concomitant exposure to chronic EtOH and stress on CPP to this drug and alterations of dopaminergic and serotonergic neurotransmission in mice. METHODS: Male Swiss mice were chronically treated with EtOH via a liquid diet and were exposed to forced swimming stress. After treatment, animals were evaluated for conditioning, extinction, and reinstatement of CPP to EtOH. Also, mice exposed to the same treatment protocol had their prefrontal cortex (PFC), nucleus accumbens (NAc), and amygdala dissected for the quantitation of dopamine, serotonin, and their metabolites content. RESULTS: Data showed that previous chronic exposure to EtOH potentiated EtOH conditioning and increased dopaminergic turnover in PFC. Exposure to stress potentiated EtOH conditioning and decreased dopaminergic turnover in the NAc. However, animals exposed to both chronic EtOH and stress did not display alterations of CPP and showed an elevated content of dopamine in amygdala. No treatment yielded serotonergic changes. CONCLUSIONS: The present study indicates that previous EtOH consumption as well as stress exposure induces increased EtOH conditioning, which can be related to dopaminergic alterations in the PFC or NAc. Interestingly, concomitant exposure to both stimuli abolished each other's effect on conditioning and PFC or NAc alterations. This protective outcome can be related to the dopaminergic increase in the amygdala.

Repeated administration of caffeine induces either sensitization or tolerance of locomotor stimulation depending on the environmental context.

Caffeine is the psychostimulant substance consumed in greatest quantities in the world. The repeated administration of psychostimulants can either decrease or increase the drug effect, inducing tolerance or sensitization, respectively, depending on administration procedure. Not only the dose and regimen, but also the environment where drug is administered, seem to modulate the changes in locomotor activity following repeated psychostimulant administration. The purpose of the present study was to examine the influence of the environmental context on caffeine-induced psychomotor stimulation following repeated administration of this drug. Our results showed that repeated caffeine induced psychomotor sensitization when drug injections were paired with the environment in which the animals were subsequently tested, whereas tolerance occurred when the animals received repeated caffeine in an environment different from that where the tests were performed. In conclusion, the present results demonstrated that the environmental context where caffeine is administered is a key factor modulating the adaptations of the organism to drug effects.

Behavioral and neuroendocrine effects of the exposure to chronic restraint or variable stress in early adolescent rats.

Stress events during adolescence may contribute to the expression or exacerbation of physical and behavioral disorders. However, little attention has been given to the physiological and behavioral changes promoted by stress during this period of ontogeny. In the present study we investigated, in adolescent male rats, the effects of repeated exposure to restraint or variable stress on: (a) locomotor activity and corticosterone levels after exposure to a novel environment; (b) corticosterone levels in response to the exposure to restraint stress; and (c) changes in body, thymus and adrenal weights. The results demonstrated that repeated exposure to restraint or variable stress reduced the locomotor response, but did not affect corticosterone secretion, in response to a novel environment. Moreover, both chronic stress procedures did not change corticosterone secretion in response to acute restraint stress. Furthermore, our results showed that repeated restraint, but not variable stress, produced a decrease in body weight along the stress exposure. Finally, we observed that the exposure to variable stress reduced the thymus relative weight. Taken together our results suggest that behavioral and physiological changes induced by exposure to chronic stress during adolescence depend on the stress regimen.

Comparison of caffeine-induced locomotor activity between adolescent and adult rats.

Caffeine is the psychostimulant drug most consumed in the world. This drug is present in food, beverages and medicines marketed for individuals of all ages. In spite of this, caffeine effects on adolescents are poorly understood. The aim of this study was to evaluate the differences on caffeine-induced locomotor stimulant or depressant effects in adolescent and adult rats. Adolescent (37-40 days old) or adult (70-74 days old) Wistar rats were tested for stimulant and depressant caffeine effects in two different experiments. The first was designed to evaluate the locomotor effect of caffeine in habituated rats. To this end, rats were previously habituated to test environment and had their locomotor activity registered following i.p. injections of vehicle or caffeine (3, 10, 30, 60 or 120 mg/kg). In the second experiment adolescent or adult rats were not habituated to the test environment and their locomotor activity was registered following i.p. injections of vehicle or caffeine (30, 60 or 120 mg/kg). In both experiments caffeine-induced a biphasic effect, with stimulation in small to moderate drug doses and no effect or locomotor depression in higher caffeine doses. Moreover, caffeine-induced locomotor stimulation was higher in adolescent than adult rats. Also, locomotor depression was only revealed in adult rats non-habituated to the test environment. These results suggest that adult and adolescent respond differently to caffeine indicating the need of more studies on the effects of caffeine in animals' models of adolescence.

Stress-induced reinstatement of amphetamine-conditioned place preference and changes in tyrosine hydroxylase in the nucleus accumbens in adolescent rats.

Drug abuse among humans often begins during adolescence. Exposure to psychostimulants during this age period may have long-term consequences which can render the organism more susceptible to drug abuse and relapse later in life. It has been demonstrated that exposure to stress can promote relapse to drug use even after long periods of withdrawal. The reinstatement of conditioned place preference (CPP) is a useful animal model for studying relapse. In humans and animals, changes in tyrosine hydroxylase (TH) have been related to drug addiction. Our study examined whether amphetamine-induced CPP during adolescence could be reinstated by exposure to stress 1 (adolescence) and 30 (adulthood) days after the extinction test. We also investigated TH levels following the reinstatement of CPP. Our results showed that amphetamine-induced CPP during adolescence can be reinstated by stress exposure 1day (P42, end of adolescence) but not 30days after extinction (P71, adulthood). Moreover the reinstatement of AMPH-induced CPP by stress exposure occurred in the presence of decreased TH in the nucleus accumbens. In conclusion, our data add new evidence that neuroadaptations on TH may mediate relapse to drug-seeking behavior induced by stress within adolescence.

Context-specific modulation of cocaine-induced locomotor sensitization and ERK and CREB phosphorylation in the rat nucleus accumbens.

Learned associations are hypothesized to develop between drug effects and contextual stimuli during repeated drug administration to produce context-specific sensitization that is expressed only in the drug-associated environment and not in a non-drug-paired environment. The neuroadaptations that mediate such context-specific behavior are largely unknown. We investigated context-specific modulation of cAMP-response element-binding protein (CREB) phosphorylation and that of four upstream kinases in the nucleus accumbens that phosphorylate CREB, including extracellular signal-regulated kinase (ERK), cAMP-dependent protein kinase, calcium/calmodulin-dependent kinase (CaMK) II and CaMKIV. Rats received seven once-daily injections of cocaine or saline in one of two distinct environments outside their home cages. Seven days later, test injections of cocaine or saline were administered in either the paired or the non-paired environment. CREB and ERK phosphorylation were assessed with immunohistochemistry, and phosphorylation of the remaining kinases, as well as of CREB and ERK, was assessed by western blotting. Repeated cocaine administration produced context-specific sensitized locomotor responses accompanied by context-specific enhancement of the number of cocaine-induced phosphoCREB-immunoreactive and phosphoERK-immunoreactive nuclei in a minority of neurons. In contrast, CREB and CaMKIV phosphorylation in nucleus accumbens homogenates were decreased by cocaine test injections. We have recently shown that a small number of cocaine-activated accumbens neurons mediate the learned association between cocaine effects and the drug administration environment to produce context-specific sensitization. Context-specific phosphorylation of ERK and CREB in the present study suggests that this signal transduction pathway is selectively activated in the same set of cocaine-activated accumbens neurons that mediate this learned association.

Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.

Learned associations between effects of abused drugs and the drug administration environment are important in drug addiction. Histochemical and electrophysiological studies suggest that these associations are encoded in sparsely distributed nucleus accumbens neurons that are selectively activated by drugs and drug-associated cues. Although correlations have been observed between nucleus accumbens neuronal activity and responsivity to drugs and drug cues, no technique exists for selectively manipulating these activated neurons and establishing their causal role in behavioral effects of drugs and drug cues. Here we describe a new approach, which we term the 'Daun02 inactivation method', that selectively inactivates a minority of neurons previously activated by cocaine in an environment repeatedly paired with cocaine to demonstrate a causal role for these activated neurons in context-specific cocaine-induced psychomotor sensitization in rats. This method provides a new tool for studying the causal roles of selectively activated neurons in behavioral effects of drugs and drug cues and in other learned behaviors.

Amphetamine- and nicotine-induced cross-sensitization in adolescent rats persists until adulthood.

Nicotine and psychostimulants are often abused in combination and drug abuse often begins during adolescence and can have long-term consequences. Behavioral sensitization has been suggested as an animal model of neuroplasticity implicated in the development of drug addiction. We evaluated whether the pretreatment with nicotine (0.4 mg/kg; s.c.) or amphetamine (5.0 mg/kg; i.p.) in adolescent rats [from postnatal day (P) 28 to P34] could induce cross-sensitization to nicotine and amphetamine when animals were challenged during both adolescence (P37) and adulthood (P70), in separate groups of animals. Adolescent animals pretreated with amphetamine displayed behavioral sensitization to nicotine, which persisted until adulthood. Moreover, adolescent animals pretreated with nicotine showed sensitized locomotor response to amphetamine in the adulthood. These data suggest that adolescents who abuse nicotine may be particularly susceptible to the effects of amphetamine and vice versa. Moreover, this increased vulnerability may persist through their development until adulthood.

Cocaine-induced behavioral sensitization in adolescent rats endures until adulthood: lack of association with GluR1 and NR1 glutamate receptor subunits and tyrosine hydroxylase.

Exposure to repeated cocaine induces enduring behavioral sensitization, which has been implicated in the psychostimulant-induced craving and psychosis. Adaptations in dopamine and glutamate neurotransmission in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) seem to mediate psychostimulant-induced behavioral sensitization. The abuse of drugs often begins during adolescence; however few studies have been devoted to study the effects of drugs of abuse at this age. The aim of our study was to examine whether repeated cocaine during adolescence could induce behavioral sensitization that endures into adulthood. Moreover, the protein levels of Tyrosine Hydroxylase (TH) and the glutamate receptor subunits GluR1 and NR1 in the NAc and mPFC were measured following the behavioral tests. Adolescent rats were treated with cocaine from postnatal day (PND) 30 to PND34 and behavioral sensitization was verified recording locomotor activity after cocaine challenge injection to adolescent (PND37) or adult (PND64 or 94) rats in separate groups at each time point. TH, GluR1, and NR1 protein levels were measured by Western blotting. Rats exposed to cocaine during adolescence expressed behavioral sensitization when tested on PND37 and PND64. In cocaine sensitized rats GluR1 protein was increased in the mPFC on PND37 but not in other ages. Thus, cocaine-induced behavioral sensitization during adolescence endures into early adulthood. However, cocaine pretreatment during adolescence induced a transient increase of GluR1 in the mPFC only when animals were challenged in the same age.

Chronic restraint or variable stresses differently affect the behavior, corticosterone secretion and body weight in rats.

Organisms are constantly subjected to stressful stimuli that affect numerous physiological processes and activate the hypothalamo-pituitary-adrenal (HPA) axis, increasing the release of glucocorticoids. Exposure to chronic stress is known to alter basic mechanisms of the stress response. The purpose of the present study was to compare the effect of two different stress paradigms (chronic restraint or variable stress) on behavioral and corticosterone release to a subsequent exposure to stressors. Considering that the HPA axis might respond differently when it is challenged with a novel or a familiar stressor we investigated the changes in the corticosterone levels following the exposure to two stressors: restraint (familiar stress) or forced novelty (novel stress). The changes in the behavioral response were evaluated by measuring the locomotor response to a novel environment. In addition, we examined changes in body, adrenals, and thymus weights in response to the chronic paradigms. Our results showed that exposure to chronic variable stress increased basal plasma corticosterone levels and that both, chronic restraint and variable stresses, promote higher corticosterone levels in response to a novel environment, but not to a challenge restraint stress, as compared to the control (non-stressed) group. Exposure to chronic restraint leads to increased novelty-induced locomotor activity. Furthermore, only the exposure to variable stress reduced body weights. In conclusion, the present results provide additional evidence on how chronic stress affects the organism physiology and point to the importance of the chronic paradigm and challenge stress on the behavioral and hormonal adaptations induced by chronic stress.

Maternal separation affects cocaine-induced locomotion and response to novelty in adolescent, but not in adult rats.

Maternal separation is known to exert long-term effects on both behavior and the neuroendocrine system. We investigated cocaine-induced locomotor activation as well as the locomotor and corticosterone response to forced novelty in maternally separated adolescent and adult rats. Maternal separation consisted of separating litters from their dams daily during 5 h from postnatal days 2 to 6. Control animals were subjected only to regular cage changes. Cocaine- (10 mg/kg, i.p.) and novelty-induced locomotion were recorded in an activity cage. After the animals were tested for behavioral response to novelty, trunk blood samples were collected and plasma corticosterone levels were determined by radioimmunoassay. Adolescent rats exposed to maternal separation exhibited an increased locomotor response to novelty and cocaine; corticosterone levels were lower in these adolescent animals, after exposure to the novel environment. These effects of maternal separation were not observed in rats that were tested as adults. Thus the maternal separation protocol produced enduring but transient changes in the behavioral response to cocaine and in the stress response to novelty.