Stress during adolescence alters behavioral sensitization to amphetamine

In humans, chronic intermittent and uncontrollable stress during adolescence is viewed as a key factor for vulnerability to drug abuse and development of psychopathologies later in life. Less is known about the long-term effects of chronic stress in animals during the juvenile period. Although there is evidence of cross sensitization during prenatal period and adulthood between chronic stress and amphetamine-induced behavioral sensitization in the rat, no studies have been conducted on cross sensitization between chronic variable stress in adolescence and behavioral sensitization to amphetamine. To address this question, at the onset of adolescence (28 days) male rats were subjected to 28 days of intermittent non-habituating social stress (isolation, novel environment, crowding, litter-shifting, subordination), or physical stress (restraint, swim, cold, ether, noise), or were handled as controls. Twenty-four hours after the last stressor or handling, all groups were exposed to a novel environment for 1 h, after which they underwent a regimen of behavioral sensitization to amphetamine. Our results showed that socially stressed rats have low locomotor activity in the novel environment, when compared to the control and physical groups who were identical in the same test. Even though socially stressed rats had lower locomotor activity in response to amphetamine injections, there were no significant differences during the training phase between the three groups at this dose of amphetamine. However, when tested for behavioral sensitization to amphetamine control and physically stressed rats showed a robust sensitization, socially stressed rats were significantly inhibited. We conclude that our chronic variable social stress protocol during adolescence inhibits behavioral sensitization to amphetamine during adulthood.     

Neonatal maternal separation exacerbates the reward-enhancing effect of acute amphetamine administration and the anhedonic effect of repeated social defeat in adult rats

Early life adversity or parental neglect is linked to the development of a number of psychiatric illnesses, including major depression and substance use disorder. These two disorders are often comorbid and characterized by anhedonia, defined as the reduced ability to experience pleasure or reward. The aim of the present study was to determine the effects of neonatal maternal separation in Long Evans rats, a model of early life stress, on anhedonia under baseline conditions and in response to drug and stress exposure during adulthood. Three hours of daily maternal separation from postnatal day 1 to 14 led to marked decreases in arched-back nursing, licking, and grooming of pups by their dams. In adulthood, brain reward function was assessed using intracranial self-stimulation of the lateral hypothalamus. Lowered current thresholds derived from this procedure are interpreted as reward-enhancing effects, whereas elevations in thresholds are an operational measure of anhedonia. Maternally separated rats did not exhibit anhedonia under baseline conditions compared with non-handled controls but exhibited a greater reward-enhancing effect of acute amphetamine administration. Acute social defeat produced anhedonia in non-handled controls, but not in maternally separated rats. Conversely, control rats habituated to 7 days of repeated social defeat, whereas maternally separated rats developed an increased anhedonic response to the repeated stressor. One week after termination of stress exposure, maternally separated rats still exhibited an increased reward-enhancing effect of acute amphetamine administration compared with non-handled controls, regardless of prior social defeat experience. These data indicate that early life stress increases the reward-enhancing properties of amphetamine, protects against the anhedonic effects of acute stress exposure, and exacerbates the anhedonic response to repeated stress. Thus, early life stress may increase an individual's vulnerability to depressive or addictive disorders when confronted with stress or drug challenge in adulthood.     

Temperature-dependent effects of maternal separation on growth, activity, and amphetamine sensitivity in the rat.

This study was conducted to examine the effects of an early stressor, maternal separation, on development, arousal, and sensitivity to amphetamine in the rat. Rat pups were maternally separated at nest temperature (WARM), room temperature (COLD), or nest temperature with agitation (AGIT) for 6 hr each day from 2 to 15 days of age. A control group (CONT) remained with the mother in the nest during this time. COLD subjects were developmentally delayed and had lower body and brain weights than the other three groups into adulthood. WARM and AGIT subjects (both maternally separated at nest temperature) had significant growth delays compared to CONT, but grew more quickly than COLD subjects. COLD subjects were less active than the other maternally separated groups, and WARM and AGIT groups were more active. Activity did not differ at 28 or 75 days of age. However, adult WARM subjects were less sensitive and COLD subjects were more sensitive to amphetamine as measured by locomotor activity than CONT and AGIT subjects, who did not differ from each other. The relationship between early stress, changes in dopaminergic systems, and altered drug responsiveness are discussed in terms of the implications for the etiology of drug abuse.     

Differential effects of periodic maternal separation on adult stress coping in a rat model of extremes in trait anxiety

We studied interactions of genetic and environmental factors shaping adult emotionality and stress coping, and tested the hypothesis that repeated periodic maternal deprivation (PMD) exerts differential effects on adult behavioral and neuroendocrine stress responsiveness in dependence on the genetic predisposition to either hyper- or hypo-anxiety. Exposure of male Wistar rats bidirectionally bred for either high (HAB) or low (LAB) anxiety-related behavior to PMD between postnatal days 2 and 15 resulted in a behavioral approximation of the selected lines. This was reflected by test-dependent signs of reduced anxiety-related behavior in adult HAB rats and of enhanced levels of anxiety in LAB rats compared with their corresponding unstressed controls. In addition to behavioral parameters, differential effects of PMD were also seen with respect to the responsiveness of the hypothalamo-pituitary-adrenocortical axis to acute stressor exposure (novel environment) in adulthood. The corticotrophin (ACTH) and corticosterone hyper-responses seen in control rats of the HAB line compared with those of the LAB line became attenuated in PMD-HAB rats, whereas PMD did not significantly alter neuroendocrine responses in LAB rats. Thus, as a result of PMD, both ACTH and corticosterone responses became indistinguishable between HAB and LAB rats. Although HAB dams spent more time on the nest with the litter compared with LAB dams during the first 5 days postpartum, licking and grooming behavior did not differ between the lines prior to separation, and was found to be increased to the same extent in both HAB and LAB dams during the first hour immediately after reunion with the pups. In contrast to early life stress, exposure of adult HAB and LAB rats to a 10-day unpredictable stress schedule failed to alter their emotional measures. The mitigating effect of PMD on both behavioral and neuroendocrine parameters in rats representing extremes in trait anxiety might reflect an evolutionary benefit as the genetic variability among individuals of a species is sustained while allowing adequate responses to potentially dangerous stimuli in adulthood dependent on early life conditions.     

The serotonin transporter gene linked polymorphic region is associated with the behavioral response to repeated stress exposure in infant rhesus macaques

The short allele of the serotonin transporter linked polymorphic region (5-HTTLPR) moderates the effects of stress on vulnerability to mood and anxiety disorders. The mechanism by which this occurs may relate to differential sensitivity to stressful life events. Here we explored whether 5-HTTLPR and sex affected behavioral responses to repeated maternal separation in infant rhesus macaques. Behaviors were collected during the acute (Day 1) and the chronic (Days 2-4) phases of the separation, and the effects of duration of separation (acute vs. chronic), genotype (long/long vs. short allele), and sex (male vs. female) on behavioral responses were analyzed across four successive separations. Males increased their levels of locomotion with repeated maternal separation, whereas females exhibited an increase in frequency of self-directed behavior, a measure of "depression-like" behavior. The short-allele predicted increased environmental exploration, particularly during the chronic phase of social separation, indicative of higher arousal. In addition, the short-allele carriers were more likely to increase their levels of self-directed behavior during the chronic phase of separation, as a function of repeated exposures. These findings suggest that the short allele may increase reactivity to repeated, chronic stressors, leaving them more vulnerable to affective psychopathology, with females particularly vulnerable.     

The reinstatement of amphetamine-induced place preference is long-lasting and related to decreased expression of AMPA receptors in the nucleus accumbens

A great deal of effort has been devoted to elucidating the psychopharmacology underlying addiction and relapse. Long-term neuroadaptations in glutamate transmission seem to be of great relevance for relapse to stimulant abuse. In this study, we investigated amphetamine-induced conditioned place preference during adolescence and the reinstatement of the conditioned behavior following a priming injection of the drug 1 day (adolescence), 30 days (early adulthood) and 60 days (adulthood) after the extinction test. The nucleus accumbens was dissected immediately after the reinstatement test to examine alterations in GluR1 and NR1 subunits of glutamatergic receptors. Our results showed that a priming injection of amphetamine was able to reinstate the CPP 1 and 30 days after extinction. However, it failed to reinstate the conditioned response after 60 days. GluR1 levels were decreased on days 1 and 30 but not on day 60 while NR1 levels were unaltered in the reinstatement test. Using a relapse model we found that reinstatement of amphetamine-induced conditioning place preference during adolescence is long lasting and persists through early adulthood. Decreased levels of GluR1 in the nucleus accumbens might be related to the reinstatement of amphetamine-induced conditioning place preference.     

Selective Breeding for Divergence in Novelty-seeking Traits: Heritability and Enrichment in Spontaneous Anxiety-related Behaviors

Outbred Sprague–Dawley rats can be classified as high responders (HR) or low responders (LR) based on their levels of exploratory locomotion in a novel environment. While this novelty-seeking dimension was originally related to differential vulnerability to substance abuse, behavioral, neuroendocrine and gene expression studies suggest a fundamental difference in emotional reactivity between these animals. Here, we report the first study to selectively breed rats based on this novelty-seeking dimension. Response to novelty was clearly heritable, with a >2-fold difference in behavior seen after eight generations of selection. Three tests of anxiety-like behavior consistently showed significantly greater anxiety in LR-bred rats compared to HR-bred animals, and this difference was diminished in the open field test by administration of the anxiolytic benzodiazepine drug, chlordiazepoxide. Cross-fostering revealed that responses to novelty were largely unaffected by maternal interactions, though there was an effect on anxiety-like behavior. These selected lines will enable future research on the interplay of genetic, environmental and developmental variables in controlling drug seeking behavior, stress and emotional reactivity.John H. Stead and Sarah Clinton contributed equally to this research.     

Gender differences in the regulation of 3 alpha-hydroxysteroid dehydrogenase in rat brain and sensitivity to neurosteroid-mediated stress protection

The enzyme 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) is involved in the generation of neuroactive steroids through ring-A-reduction of hormonal precursors. We examined the developmental regulation of, gender differences in, and effects of hormonal manipulations on the expression of 3 alpha-HSD in the rat hippocampus. High levels of 3 alpha-HSD mRNA were found on postnatal day 7, coinciding with the stress hyporesponsive period in the rat. Gender differences in 3 alpha-HSD expression were documented during puberty, but not in adulthood. Adrenalectomy and gonadectomy, and supplementation with individual steroid hormones influenced 3 alpha-HSD expression in a gender-specific mode. We also demonstrate that the manifestation of behavioral and endocrine consequences of early life stress depends on the individual's gender and gonadal status. Males are liable to aftereffects of neonatal maternal deprivation, regardless of their adult gonadal status. In females, however, anxiogenic aftereffects of neonatal stress become apparent only after gonadectomy. These data suggest that (i) transient increase of neurosteroid biosynthesis may contribute to stress hyporesponsiveness during early infancy; (ii) gonadal steroids regulate 3 alpha-HSD expression in the hippocampus in a sex-specific mode; (iii) physiological sex steroid secretions in females may mask behavioral consequences of adverse early life events, and (iv) concomitant treatment with the neurosteroid THP counteracts behavioral and endocrine dysregulation induced by neonatal stress in both genders.     

Within-litter variance in early rat pup-mother interactions and adult offspring responses to novelty

Siblings share similar genetics and environments, however, their behavior can be quite different. To determine if within-litter variance in neonatal-maternal interactions predict adult sibling behavioral variance, we observed mother-pup interactions during postnatal days 1-8 in four Sprague-Dawley rat litters and measured adult offspring behavioral responses to social and physical novelty. Our results indicate that pup and maternal behavior varied by at least twofold within each litter, and that specific pup behaviors within each litter (perioral contact) were associated with increased maternal licking. Furthermore, siblings that received more licks and made more perioral contact during postnatal days 1-8 had longer latencies to approach novel objects in adulthood than siblings that received less licking and made less perioral contact. This within-litter variance in postnatal mother and pup behavior and offspring adult behavior indicates that early social dynamics within families are an important area to examine to understand the development of sibling variance.     

Hormonal and behavioral responses to stress in lactating and non-lactating female common marmosets (Callithrix jacchus)

In several mammalian species, hypothalamic-pituitary-adrenal (HPA) and behavioral responses to stressors are down-regulated in lactating females, possibly preventing stress-induced disruptions of maternal care. Experimental elevations of HPA axis hormones have been found to inhibit maternal behavior in lactating common marmoset monkeys (Callithrix jacchus), raising the question of whether lactating female marmosets also have blunted endogenous responses to stress. Therefore, we compared HPA and behavioral responses to standardized stressors in reproductively experienced female common marmosets that were undergoing ovulatory cycles and that either were (N = 7) or were not lactating (N = 8). Each marmoset underwent (1) a restraint stressor during the early follicular phase of the ovarian cycle (approximately 5 weeks postpartum for lactating females) and (2) exposure to a simulated hawk predator during the early to mid-luteal phase (approximately 7 weeks postpartum for lactating females). Lactating females were tested in the presence of one of their infants. Blood samples were collected before, during, and immediately after each test for determination of plasma adrenocorticotropic hormone (ACTH) and cortisol concentrations. Both stressors caused significant elevations in plasma ACTH and cortisol levels, and significant decreases in cortisol:ACTH ratios; however, lactating and non-lactating females showed no significant differences in their endocrine or behavioral responses to either stressor, or in baseline ACTH or cortisol levels. These findings suggest that in contrast to several other mammalian species, lactating female marmosets maintain full behavioral and HPA responsiveness to stress, at least in the presence of their infants.     

Early life experience alters behavior during social defeat: focus on serotonergic systems

Early life experience can have prolonged effects on neuroendocrine, autonomic, and behavioral responses to stress. The objective of this study was to investigate the effects of early life experience on behavior during social defeat, as well as on associated functional cellular responses in serotonergic and non-serotonergic neurons within the dorsal raphe nucleus, a structure which plays an important role in modulation of stress-related physiology and behavior. Male Long Evans rat pups were exposed to either normal animal facility rearing or 15 min or 180 min of maternal separation from postnatal days 2-14. As adults, these rats were exposed to a social defeat protocol. Differences in behavior were seen among the early life treatment groups during social defeat; rats exposed to 180 min of maternal separation from postnatal days 2-14 displayed more passive-submissive behaviors and less proactive coping behaviors. Analysis of the distribution of tryptophan hydroxylase and c-Fos-like immunoreactivity in control rats exposed to a novel cage and rats exposed to social defeat revealed that, independent of the early life experience, rats exposed to social defeat showed an increase in the number of c-Fos-like immunoreactive nuclei in serotonergic neurons in the middle and caudal parts of the dorsal dorsal raphe nucleus and caudal part of the ventral dorsal raphe nucleus, regions known to contain serotonergic neurons projecting to central autonomic and emotional motor control systems. This is the first study to show that the dorsomedial part of the mid-rostrocaudal dorsal raphe nucleus is engaged by a naturalistic stressor and supports the hypothesis that early life experience alters behavioral coping strategies during social conflict; furthermore, this study is consistent with the hypothesis that topographically organized subpopulations of serotonergic neurons principally within the mid-rostrocaudal and caudal part of the dorsal dorsal raphe nucleus modulate stress-related physiological and behavioral responses.     

Amphetamine effects in microtine rodents: a comparative study using monogamous and promiscuous vole species

We compared amphetamine-induced dopamine release in the nucleus accumbens of vole species that exhibit differing mating systems to examine potential interactions between social organization and substance abuse. We found no species or regional differences in basal extracellular dopamine, however, monogamous voles had greater and longer-lasting increases in extracellular dopamine after amphetamine treatment than did promiscuous voles. We then examined whether amphetamine-induced increase in extracellular dopamine could induce pair bonds in monogamous voles. We found that, despite increasing dopamine in the nucleus accumbens, amphetamine administration did not induce pair-bonds in male prairie voles unless the animals were pretreated to preclude D1 receptor activation, which is known to inhibit pair-bond formation. These results support suggestions that social attachment and substance abuse share a common neural substrate.     

An animal model of social instability stress in adolescence and risk for drugs of abuse

There is increasing evidence that adolescence, like early life, is a sensitive period in which ongoing brain development can be influenced by environmental factors. This review describes our use of social instability as a model of mild adolescent social stress, its effects on social interactions and on hypothalamic-pituitary-adrenal function over the course of the procedure and in response to new stressors. The effects of social instability are sex-specific, with qualitative differences between the sexes on HPA function over the course of the stressor procedure, and with greater effects in males on behaviour observed during the social instability and greater effects in females on behavioural responses to drugs of abuse into adulthood, long after the stress exposure. The results from investigations with this model of adolescent social stress are discussed in relation to those of studies using other stressor procedures. Elevated exposure to glucocorticoids over the course of adolescence confers sex-specific changes in behavioural responses to drugs of abuse, which may be of relevance for understanding risk factors in people.     

Early adverse experience as a developmental risk factor for later psychopathology: evidence from rodent and primate models

Increasing evidence supports the view that the interaction of perinatal exposure to adversity with individual genetic liabilities may increase an individual's vulnerability to the expression of psycho- and physiopathology throughout life. The early environment appears to program some aspects of neurobiological development and, in turn, behavioral, emotional, cognitive, and physiological development. Several rodent and primate models of early adverse experience have been analyzed in this review, including those that "model" maternal separation or loss, abuse or neglect, and social deprivation. Accumulating evidence shows that these early traumatic experiences are associated with long-term alterations in coping style, emotional and behavioral regulation. neuroendocrine responsiveness to stress, social "fitness,' cognitive function, brain morphology, neurochemistry, and expression levels of central nervous system genes that have been related to anxiety and mood disorders. Studies are underway to identify important aspects of adverse early experience, such as (a) the existence of "sensitive periods" during development associated with alterations in particular output systems. (b) the presence of "windows of opportunity" during which targeted interventions (e.g., nurturant parenting or supportive-enriching environment) may prevent or reverse dysfunction, (c) the identity of gene polymorphisms contributing to the individual's variability in vulnerability, and (d) a means to translate the timing of these developmental "sensitive periods" across species.     

Effects of maternal separation on hypothalamic-pituitary-adrenal responses, cognition and vulnerability to stress in adult female rats

We studied the long term effects of neonatal stress in female rats and subsequent responses to stress when adults. Female rats that experienced maternal separation (MS) showed in adulthood depressive-like behavior in the forced swimming test and cognitive impairments in the novel object recognition test, which were reverted by the glucocorticoid receptor antagonist mifepristone or the beta-adrenoceptor antagonist propranolol. Markers of HPA axis (corticosterone levels, CRF mRNA levels in the paraventricular nucleus and glucocorticoid receptor density in the hippocampus) were altered by MS, suggesting that an altered HPA axis function may be associated to behavioral and cognitive deficits in MS female rats. In addition, MS rats were found to be more vulnerable to chronic stress than controls as shown by decreases in open field activity, increases in immobility time in the forced swim test, and changes in markers of HPA axis (decreases in the density of glucocorticoid receptors). These present findings are discussed in terms of gender differences in adulthood.     

Maternal separation during a specific postnatal time window prevents reinforcement of hippocampal long-term potentiation in adolescent rats

In an attempt to develop an animal model to study the etiology of brain dysfunction in relation to early life experience, we tested the hypothesis that early-life stress during specific postnatal time windows affects long-term potentiation (LTP) reinforcement in adolescence. Male Wistar rat pups were stressed by separation from their dams for 24 h at postnatal day (PND) 4, 9, or 18. The animals were tested for reinforcement of LTP at adolescence (9 weeks old) by exposing them to a 2-min swim-stress. Here, we show that maternal separation during (at PND9) but not at the beginning (at PND4) or after (at PND18) the stress-hyporesponsive-period of the hypothalamic-pituitary-adrenal-axis impairs emotional LTP-reinforcement in adolescent animals. Thus, this in vivo model allows the investigation of physiological and pathophysiological emotional information processing at the cellular level in freely behaving adolescent animals.     

Chronic social stress in adolescence influenced both amphetamine conditioned place preference and locomotor sensitization

We previously reported that chronic social stress (SS) in adolescence, but not in adulthood, increased the locomotor-activating effects of nicotine in females, and not males, when tested in adulthood. However, SS rats had decreased locomotor response to nicotine when tested in adolescence. Here, we investigated age-related changes in the effects of SS on both conditioned place preference (CPP) and locomotor sensitization to amphetamine. In the CPP experiment, SS females tested in adolescence had increased preference for the 1.0 mg/kg dose of amphetamine, whereas SS rats of both sexes showed a decrease in CPP for the 0.5 mg/kg dose when tested as adults. Irrespective of time of testing, SS males and females had enhanced locomotor sensitization compared to controls. Thus, adolescent SS produced both immediate and enduring effects on behavioral responses to amphetamine, likely by altering the development of the mesocorticolimbic dopamine system, which holds implications for vulnerability to addiction.     

Litter size reduction accentuates maternal care and alters behavioral and physiological phenotypes in rat adult offspring

Maternal behavior has a substantial impact on the behavioral, endocrine, and neural development of the pups. This study investigated the effect of altering the neonatal nutritional environment by modifying the litter size on maternal care and anxiety- and fear-like behaviors in rats during adulthood. On postnatal day (PND) 2, litters were adjusted to a small litter (SL) size of three pups per dam or normal litter (NL) size of 12 pups per dam. Maternal behaviors were scored daily during lactation (PND2-21). The weight gain, food intake, adiposity, and biochemical landmarks of offspring rats were evaluated. On PND60, performances in the open field, elevated plus-maze (EPM), and fear conditioning test were measured. The reduction of the litter size enhanced maternal care in lactating rats, increasing the arched-back posture and licking pups. SL offspring exhibited accelerated weight gain, hyperphagia, increased visceral fat mass, dyslipidemia, and hyperleptinemia in adulthood. The SL offspring of both sexes showed an increase in the anti-thigmotactic effect in the open field, an intact anxious-phenotype in the EPM, and a decrease in the time spent freezing during the fear-conditioning test, compared to NL. The neonatal environment as determined by litter size plays a crucial role in programming the adult metabolic phenotype as well as behavioral responses to stressful stimuli, with an impact on anxiety-like and fear behaviors. These behavioral changes in offspring may be, at least in part, a result of increased maternal care.     

Effects of gestational stress and neonatal handling on pain,analgesia, and stress behavior of adult mice

Stressors presented during the late prenatal and early postnatal periods can have long-term effects on offspring behavior, due to the sensitive periods in the formation of brain circuitry associated with early development. This study investigated the long-term effects of prenatal (restraint during the last week of gestation) and postnatal (daily handling for 14 days postnatal) stress, alone and in combination, on adulthood pain behavior, analgesic responses to stress and morphine, and on behavioral indices of stress reactivity. We found that all of the adult responses measured were altered by perinatal manipulations. Nociceptive thresholds were increased by prenatal or by postnatal stress in males and females; application of both stressors in combination negated these effects. Elevations in morphine analgesia were also observed in animals undergoing either perinatal stressor, but not in those who received both stressors. Behavioral and analgesic responses to stress were consistent with previous observations of reduced stress responsiveness following neonatal handling, with some sex-specific findings. Male and female handled subjects exhibited decreases in stress behavior, and both groups of female handled subjects (regardless of prenatal stress [PS] condition) exhibited decreases in stress-induced analgesia (SIA). Males, on the other hand, exhibited decreases in SIA only if they were prenatally stressed (regardless of handling condition). Thus, prenatal and postnatal stressors have differing effects on the neural circuitry underlying pain, pain inhibition, and stress behavior.