Long-lasting, sex- and age-specific effects of social stressors on corticosterone responses to restraint and on locomotor responses to psychostimulants in rats

Many neural systems are undergoing marked development over adolescence, which may heighten an animal's vulnerability to stressors. One consequence may be altered sensitivity to drugs of abuse. We previously reported that social stressors in adolescence increased behavioral sensitization to nicotine in adulthood in female, but not male, rats. Here we examined whether social stressors in adolescence alter the functioning of the hypothalamic-pituitary-adrenal (HPA) axis by examining corticosterone release in response to restraint in adulthood. To further assess effects of social stressors on behavioral sensitivity to psychostimulants, we examined locomotor activity in response to nicotine and to amphetamine. In a second set of experiments, we investigated whether the same procedure of social stressors administered in adulthood produces effects similar to that observed when administered in adolescence. Rats underwent daily 1 h isolation followed by pairing with a new cage mate on either postnatal days 33-48 (pubertal stress: PS) or days 65-80 (adult stress: AS). Three weeks later rats tested for either: (a) corticosterone levels were measured in response to restraint, or (b) locomotor sensitization to nicotine (0.25 mg/kg; 5 days) followed by an amphetamine challenge (0.5 mg/kg) 24 h later. Effects of social stressors were evident only in females. PS females had increased locomotor activity to amphetamine compared to controls, and AS females had increased corticosterone release compared to controls. No effect of the social stressors was found in males at either age except for reduced weight gain during the stress procedure. Thus, females are more susceptible to the enduring effects of these moderate social stressors than are males. However, in terms of behavioral sensitivity to drugs of abuse, females may be more susceptible to stressors during adolescence than adulthood, although the reverse appears to be true for HPA function.     

Behavioral and neuroendocrine consequences of juvenile stress combined with adult immobilization in male rats

Exposure to stress during childhood and adolescence increases vulnerability to developing several psychopathologies in adulthood and alters the activity of the hypothalamic-pituitary-adrenal (HPA) axis, the prototypical stress system. Rodent models of juvenile stress appear to support this hypothesis because juvenile stress can result in reduced activity/exploration and enhanced anxiety, although results are not always consistent. Moreover, an in-depth characterization of changes in the HPA axis is lacking. In the present study, the long-lasting effects of juvenile stress on adult behavior and HPA function were evaluated in male rats. The juvenile stress consisted of a combination of stressors (cat odor, forced swim and footshock) during postnatal days 23–28. Juvenile stress reduced the maximum amplitude of the adrenocorticotropic hormone (ACTH) levels (reduced peak at lights off), without affecting the circadian corticosterone rhythm, but other aspects of the HPA function (negative glucocorticoid feedback, responsiveness to further stressors and brain gene expression of corticotrophin-releasing hormone and corticosteroid receptors) remained unaltered. The behavioral effects of juvenile stress itself at adulthood were modest (decreased activity in the circular corridor) with no evidence of enhanced anxiety. Imposition of an acute severe stressor (immobilization on boards, IMO) did not increase anxiety in control animals, as evaluated one week later in the elevated-plus maze (EPM), but it potentiated the acoustic startle response (ASR). However, acute IMO did enhance anxiety in the EPM, in juvenile stressed rats, thereby suggesting that juvenile stress sensitizes rats to the effects of additional stressors.     

An animal model of eating disorders associated with stressful experience in early life

Experience of childhood abuse is prevalent among patients with eating disorders, and dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is implicated in its pathophysiology. Neonatal maternal separation is considered as an animal model of stressful experience early in life. Many of studies have demonstrated its impact both on the activity of HPA axis and the development of psycho-emotional disorders later in life. In this paper, a series of our researches on developing an animal model of eating disorders is reviewed. An animal model of neonatal maternal separation was used; Sprague-Dawley pups were separated from dam daily for 180 min during the first 2 weeks of life (MS) or undisturbed. Anxiety-/depression-like behaviors were observed in MS rats at the age of two months with decreased serotonergic activity in the hippocampus and the raphe. Post-weaning social isolation promoted food intake and weight gain of adolescent MS pups, with impacts on anxiety-like behaviors. Sustained hyperphagia was observed in the MS pups subjected to a fasting/refeeding cycle repeatedly during adolescence, with increased plasma corticosterone levels. Anhedonia, major symptom of depression, to palatable food was observed in adolescent MS pups with blunted response of the mesolimbic dopaminergic activity to stress. Results suggest that neonatal maternal separation lead to the development of eating disorders when it is challenged with social or metabolic stressors later in life, in which dysfunctions in the HPA axis and the brain monoaminergic systems may play important roles.     

Endocrine and behavioral effects of neuromedin S

The present experiments focused on the effects of neuromedin S on hypothalamic–pituitary–adrenal (HPA) activation and behavior. The peptide (0.25–1 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open field observations and an elevated plus-maze (EPM) test. Autonomic functions such as the temperature and the heart rate were recorded by telemetry. The action on the HPA axis was assessed via measurements of the plasma corticosterone and ACTH levels. To reveal the transmission of the endocrine responses, animals were pretreated with corticotrophin releasing hormone receptor (CRHR) antagonists (1 nmol). In the open field test, the animals were pretreated with either a CRHR₁ antagonist (antalarmin) or haloperidol (10 μg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). The dopamine release from striatal and amygdala slices after peptide treatment was measured with a superfusion apparatus. Neuromedin S exerted dose-dependent effects on the HPA system, which were inhibited by antalarmin. It also activated grooming and decreased the entries to and time spent in the open arms during the EPM test. The grooming response was abolished by haloperidol and antalarmin pretreatment, while diazepam and antalarmin showed a tendency to attenuate the response evoked in the EPM test. In the superfusion studies, neuromedin S enhanced the dopamine release from the amygdala slices. These results demonstrate that neuromedin S stimulates the HPA axis through the CRHR₁ pathway and evokes stereotyped behavior and anxiety through mesolimbic dopamine and corticotrophin releasing hormone release.     

Influence of prenatal stress on the rat hypothalamic-pituitary-adrenal axis activity: the role of the brain glycocorticoid receptors

The effects of prenatal stress on the hypothalamic-pituitary-adrenal (HPA) axis activity and brain glycocorticoid receptors were studied in neonatal male and female offspring, as well as the influence of neonatal glycocorticoid receptors blockade on hormonal stress reactivity of adult rats. The results showed that there were sexual differences in plasma corticosterone level and corticosteroid binding in the cortex and hypothalamus of 5-day old control rats. Prenatal stress increased basal level of corticosterone in female rats, decreased corticosterone binding in hypothalamus and hippocampus of male and female rats, and increased corticosteroid receptor level in the male cortex. Neonatal administration of glycocorticoid receptor antagonist did not change plasma corticosterone level in 5-day old rats, but prolonged hormonal stress response of the HPA axis in adult male rats and increased hormonal stress response in female ones. The character of the IIPA axis activity of male and female rats with neonatal blockade of glycocorticoid receptors correspond to hormonal stress response of prenatal stressed rats. These data suggest that change of brain glycocorticoid receptors function in neonatal period of development might be one of the mechanisms of prenatal stress influence on the HPA axis activity in the adulthood.     

The synthesis and biological evaluation of 2-(3-methyl or 3-phenylisoxazol-5-yl)-3-aryl-8-thiabicyclo[3.2.1]octanes

Cocaine, a potent stimulant of the central nervous system, owes its reinforcing and stimulant properties to its ability to inhibit monoamine uptake systems such as the Dopamine Transporter (DAT), and the Serotonin Transporter (SERT) located on presynaptic neurons in the striatum. The search for pharmacotherapies for cocaine addiction has focused on the design of compounds that bind selectively to the DAT and manifest slow onset of stimulatory action with long duration of action. We had reported that 3-aryl-2-carbomethoxy-8-thiabicyclo[3.2.1]octanes are potent and selective inhibitors of the DAT. In this Letter we report on the effects of replacement of the 2-carbomethoy group by a 2-isoxazole. This new class of 8-thiabicyclo[3.2.1]octanes provides potent and selective inhibitors of the DAT. The 3β-aryl compounds are particularly potent inhibitors of DAT (IC₅₀ = 7-43 nM) with substantial selectivity versus inhibition of SERT.     

Discovery of N-methyl-1-(1-phenylcyclohexyl)methanamine, a novel triple serotonin, norepinephrine, and dopamine reuptake inhibitor

The current work discloses a novel cyclohexylarylamine chemotype with potent inhibition of the serotonin, norepinephrine, and dopamine transporters and potential for treatment of major depressive disorder. Optimized compounds 1 (SERT, NET, DAT, IC₅₀ = 169, 85, 21 nM) and 42 (SERT, NET, DAT IC₅₀ = 34, 295, 90 nM) were highly brain penetrant, active in vivo in the mouse tail suspension test at 30 mpk po and were not general motor stimulants.     

Neonatal handling enhances contextual fear conditioning and alters corticosterone stress responses in young rats

Previous studies have indicated that neonatal handling influences development of hypothalamic-pituitary-adrenal (HPA) control of corticosterone. In addition, corticosterone influences memory consolidation processes in contextual fear conditioning. Therefore, neonatal handling may affect hippocampal-dependent memory processes present in contextual fear conditioning by influencing the development of HPA control of corticosterone. To investigate the effects of neonatal handling on early learning, rat pups were either handled (15-min removal from home cage) on the first 15 days after birth or left undisturbed in their home cage. Handled rats and nonhandled rats were fear conditioned at 18, 21, or 30 days of age and then tested at two time points--24 h following conditioning and at postnatal day 45. Subsequently, at approximately postnatal day 60, rats were exposed to restraint stress and corticosterone levels were assessed during restraint and recovery. Handled and nonhandled rats did not differ significantly in their freezing response immediately following footshock on the conditioning day. However, when tested for contextual fear conditioning at 24 h following conditioning and at postnatal day 45, handled rats showed more freezing behavior than nonhandled rats. When exposed to restraint stress, handled rats had a more rapid return of corticosterone to basal levels than nonhandled rats. These results indicate that neonatal handling enhances developmentally early memory processes involved in contextual fear conditioning and confirms previously reported effects of neonatal handling on HPA control of corticosterone.     

Adult Consequences of Post-weaning High Fat Feeding on the Limbic–HPA Axis of Female Rats

The peripubertal period is critical for the final maturation of circuits controlling energy homeostasis and stress response. However, the consequence of juvenile fat consumption on adult physiology is not clear. This study analyzed the adult consequences of post-weaning fat feeding on limbic–hypothalamic–pituitary–adrenal (HPA) axis components and on metabolic regulators of female rats. Wistar rats were fed either a high fat (HF) diet or the normal chow from weaning to puberty or to 3 months of age. Additional groups crossed their diets at puberty onset. Plasma leptin, insulin, and corticosterone levels were determined by radioimmunoassay and their brain receptors by western blot analysis. Adult HF-fed animals though not overweight, had higher corticosterone and reduced glucocorticoid receptor levels in the hypothalamus and hippocampus, compared to the controls. The alterations in HPA axis emerged already at puberty onset. Leptin receptor levels in the hypothalamus were reduced only by continuous fat feeding from weaning to adulthood. The pre-pubertal period appeared more vulnerable to diet-induced alterations in adulthood than the post-pubertal one. Switching from fat diet to normal chow at puberty onset restored most of the diet-induced alterations in the HPA axis. The corticosteroid circuit rather than the leptin or insulin system appears as the principal target for the peripubertal fat diet-induced effects in adult female rats.     

Japanese quail selected for high plasma corticosterone response deposit high levels of corticosterone in their eggs

Poor habitat quality or body condition often correlates with high responsiveness of the hypothalamo-pituitary-adrenal (HPA) axis rather than with elevated baseline levels of glucocorticoids. We hypothesized that, for egg-laying vertebrates, high responsiveness of the HPA axis would correspond to high concentrations of corticosterone in yolk. We tested the prediction that Japanese quail (Coturnix coturnix japonica) selected for high plasma corticosterone response to brief immobilization (HS quail) would lay eggs with higher yolk corticosterone concentrations than birds selected for low response (LS quail). Quail from both lines were left undisturbed, outside of the stressors associated with daily management, before a first round of egg collection. In a second experiment, quail of both lines were experimentally stressed during the week before egg collection. In both cases we found quail from the HS line to lay eggs with significantly higher yolk corticosterone concentrations than quail of the LS line. After exposure to added experimental stressors, the line difference was more pronounced (increasing from 62% to 96%). There was no line difference in concentrations of yolk testosterone. Our results suggest that (1) genetic differences underly differences in the transfer of maternal corticosterone to yolk and (2) females may be able to control deposition of corticosterone into yolk through a mechanism independent of baseline corticosterone titers.     

Synthesis and evaluation of novel N-fluoropyridyl derivatives of tropane as potential PET imaging agents for the dopamine transporter

A series of novel N-fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT (K_i = 4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [¹⁸F]6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [¹⁸F]6d may be useful as a potential radioligand for imaging DATs with PET.     

Deprivation of maternal care has long-lasting consequences for the hypothalamic-pituitary-adrenal axis of zebra finches

Early-life stress caused by the deprivation of maternal care has been shown to have long-lasting effects on the hypothalamic-pituitary-adrenal (HPA) axis in offspring of uniparental mammalian species. We asked if deprivation of maternal care in biparental species alters stress responsiveness of offspring, using a biparental avian species--the zebra finch, Taeniopygia guttata. In our experiment, one group of birds was raised by both male and female parents (control), and another was raised by males alone (maternally deprived). During adulthood, offspring of both groups were subjected to two stressors (restraint and isolation), and corticosterone concentrations were measured. Additionally, we measured baseline levels of the two corticosteroid receptors--glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)--in the hippocampus, hypothalamus and cerebellum. Our results suggest that maternally deprived offspring are hyper-responsive to isolation in comparison with controls. Furthermore, mRNA levels of both GR and MR receptors are altered in maternally deprived offspring in comparison with controls. Thus, absence of maternal care has lasting consequences for HPA function in a biparental species where paternal care is available.     

Persistent effects of prenatal, neonatal, or adult treatment with flutamide on the hypothalamic-pituitary-adrenal stress response of adult male rats

To explore the role of androgens in early development on adult hypothalamic-pituitary-adrenal (HPA) function in males, we administered flutamide or vehicle injections: (1) to pregnant dams on embryonic days 15-20; (2) to neonatal pups on days 0-5; or (3) to adults on days 55-60. At approximately 70 days of age, trunk blood was collected to determine corticosterone levels (1) upon removal from the home cage, (2) immediately after 30 min of restraint stress, or (3) 60 min after return to home cage following the stressor. Flutamide treatment resulted in higher basal levels of testosterone and stress levels of corticosterone compared to vehicle treatment, and there was no interaction of treatment with age at time of treatment. This suggests that testosterone is less effective at inhibiting HPA function in flutamide-treated males. In addition, prenatally treated males had higher stress levels of corticosterone than neonatally and adult-treated males, regardless of the type of treatment. There were no differences in CBG levels among the groups. The results suggest that, in males, flutamide treatment has a long-lasting effect on HPA function. These results are consistent with our previous research on neonatally gonadectomized males and the hypothesis of organizational effects of sex hormones on HPA function.     

Extracellular loops of the serotonin transporter act as a selectivity filter for drug binding

The serotonin transporter (SERT) shapes serotonergic neurotransmission by retrieving its eponymous substrate from the synaptic cleft. Ligands that discriminate between SERT and its close relative, the dopamine transporter DAT, differ in their association rate constant rather than their dissociation rate. The structural basis for this phenomenon is not known. Here we examined the hypothesis that the extracellular loops 2 (EL2) and 4 (EL4) limit access to the ligand-binding site of SERT. We employed an antibody directed against EL4 (residues 388–400) and the antibody fragments 8B6 scFv (directed against EL2 and EL4) and 15B8 Fab (directed against EL2) and analyzed their effects on the transport cycle of and inhibitor binding to SERT. Electrophysiological recordings showed that the EL4 antibody and 8B6 scFv impeded the initial substrate-induced transition from the outward to the inward-facing conformation but not the forward cycling mode of SERT. In contrast, binding of radiolabeled inhibitors to SERT was enhanced by either EL4- or EL2-directed antibodies. We confirmed this observation by determining the association and dissociation rate of the DAT-selective inhibitor methylphenidate via electrophysiological recordings; occupancy of EL2 with 15B8 Fab enhanced the affinity of SERT for methylphenidate by accelerating its binding. Based on these observations, we conclude that (i) EL4 undergoes a major movement during the transition from the outward to the inward-facing state, and (ii) EL2 and EL4 limit access of inhibitors to the binding of SERT, thus acting as a selectivity filter. This insight has repercussions for drug development.     

LPS Exposure Increases Maternal Corticosterone Levels,Causes Placental Injury and Increases IL-1Β Levels in Adult Rat Offspring: Relevance to Autism

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.     

Chronic Stress Suppresses the Expression of Cutaneous Hypothalamic–Pituitary–Adrenocortical Axis Elements and Melanogenesis

Chronic stress can affect skin function, and some skin diseases might be triggered or aggravated by stress. Stress can activate the central hypothalamic–pituitary–adrenocortical (HPA) axis, which causes glucocorticoid levels to increase. The skin has HPA axis elements that react to environmental stressors to regulate skin functions, such as melanogenesis. This study explores the mechanism whereby chronic stress affects skin pigmentation, focusing on the HPA axis, and investigates the role of glucocorticoids in this pathway. We exposed C57BL/6 male mice to two types of chronic stress, chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS). Mice subjected to either stress condition showed reduced melanogenesis. Interestingly, CRS and CUMS triggered reductions in the mRNA expression levels of key factors involved in the HPA axis in the skin. In mice administered corticosterone, decreased melanin synthesis and reduced expression of HPA axis elements were observed. The reduced expression of HPA axis elements and melanogenesis in the skin of stressed mice were reversed by RU486 (a glucocorticoid receptor antagonist) treatment. Glucocorticoids had no significant inhibitory effect on melanogenesis in vitro. These results suggest that, high levels of serum corticosterone induced by chronic stress can reduce the expression of elements of the skin HPA axis by glucocorticoid-dependent negative feedback. These activities can eventually result in decreased skin pigmentation. Our findings raise the possibility that chronic stress could be a risk factor for depigmentation by disrupting the cutaneous HPA axis and should prompt dermatologists to exercise more caution when using glucocorticoids for treatment.     

Influence of housing conditions from weaning to adulthood on the ventilatory, thermoregulatory, and endocrine responses to hypoxia of adult female rats

Housing conditions affect animal physiology. We previously showed that the hypoxic ventilatory and thermoregulatory responses to hypoxia of adult male rats housed in triads during the juvenile period (postnatal day 21 to adulthood) were significantly reduced compared with animals housed in pairs. Because sex hormones influence development and responsiveness to environmental stressors, this study investigated the impact of housing on the respiratory and thermoregulatory physiology of female rats. Since neonatal stress attenuates the hypoxic ventilatory response (HVR) of female rats at adulthood, experiments were performed both on "control" (undisturbed) animals and rats subjected to neonatal maternal separation (NMS; 3 h/day, postnatal days 3-12). At adulthood, ventilatory activity was measured by whole body plethysmography under normoxic and hypoxic conditions [fraction of inspired oxygen (Fi(O(2))) = 0.12; 20 min]. The ventilatory and body temperature responses to hypoxia of female rats raised in triads were reduced compared with rats housed in pairs. Housing female rats in triads did not affect basal or hypoxic plasma corticosterone levels but did increase levels of estradiol significantly. We conclude that modest changes in housing conditions (pairs vs. triads) from weaning to adulthood does influence basic homeostatic functions such as temperature and respiratory regulation. Triad housing can reverse the manifestations of respiratory instability at adulthood induced by stressful neonatal treatments. This should raise awareness of the benefits of increasing social interactions in clinical settings but also caution researchers of the potential impact of such subtle changes on experimental protocols and interpretation of results.     

Neonatal immune challenge exacerbates experimental colitis in adult rats: potential role for TNF-alpha

Early life events and childhood infections have been associated with the development and onset of inflammatory bowel disease in adulthood. However, the consequences of neonatal infection in the development and severity of colitis are not established. We investigated the effects of a neonatal (postnatal day 14) or juvenile (postnatal day 28) immune challenge with LPS on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced damage and weight loss, as well as on food intake and body temperature in adult rats. Neonatally (n)LPS-treated rats developed more severe colitis than control animals, reflected in a greater loss of weight and a significantly increased macroscopic tissue damage score. These findings were associated with a hypothermic response after TNBS treatment in nLPS rats, but not in neonatally saline-treated rats receiving TNBS. These differences were not seen after TNBS in rats that had received LPS on postnatal day 28. Plasma corticosterone was measured as an index of adult hypothalamic-pituitary-adrenal (HPA) axis activation as was TNF-alpha, a proinflammatory cytokine associated with inflammatory bowel disease. Four days after TNBS treatment, plasma corticosterone was unaltered in all groups; however, TNF-alpha was significantly increased in adult TNBS-treated rats that had LPS as neonates compared with all other groups. In conclusion, neonatal, but not later, exposure to LPS produces long-term exacerbations in the development of colitis in adults. This change is independent of HPA axis activation 4 days after TNBS treatment but is associated with increased circulating TNF-alpha, suggestive of an exaggerated immune response in adults exposed to neonatal infection.