Age-related decline in social interaction is associated with decreased c-Fos induction in select brain regions independent of oxytocin receptor expression profiles.

Social behavior decreases with aging, and we have previously found a substantial decline in social investigative behavior of old female rats. In this study we examined the neural activation pattern (c-Fos mRNA) of young (3 month) and old (18 month) female rats after brief 10 min exposure to a novel female rat in order to identify forebrain regions that show selective age-related alterations in their neural response to social investigation. We also measured relative oxytocin receptor expression (Oxtr mRNA) as a possible factor in age-related declines in c-Fos induction after social interaction. Young rats exposed to a social partner had a greater c-Fos mRNA response than those exposed to novel context alone in the lateral septum and septohypothalamic area, with blunted increases evident in old rats. In addition, c-Fos mRNA levels in the lateral septum were positively correlated with social investigative behavior. Interestingly, age-related differences in c-Fos gene induction were unrelated to the local amount of Oxtr expression within specific brain regions, although we found an age-related decline in Oxtr expression in the ventromedial hypothalamus. This functional neuroanatomical characterization may point to certain brain regions that are especially sensitive to age-related declines associated with social interaction behavior.

Circadian Rhythms in Fear Extinction Recall Depend on the Time of Day of Extinction Recall, Not the Time of Day of Extinction Learning.

The recall of conditioned fear extinction exhibits a circadian rhythm in humans and rodents, with optimal extinction recall occurring during the early active phase. However, it remains unclear whether this rhythm depends on the circadian modulation of mechanisms supporting memory consolidation versus memory maintenance and retrieval. Here, adult male rats underwent conditioned fear extinction at one of four times throughout the day and then, starting 24 h after extinction, were repeatedly tested for extinction recall over the next 24 h. Rats undergoing extinction learning during the early active phase tended toward accelerated extinction learning compared with rats in other groups, pointing to rhythms in mechanisms that support extinction memory encoding. The next day, the strength of extinction recall followed a 24-h cycle that depended not on the time of day of extinction learning but, instead, on the time of day of extinction recall. This latter finding indicates a rhythm in mechanisms supporting extinction memory maintenance and/or retrieval. Subsequent testing for fear relapse in the conditioning context suggested reduced fear in rats tested during the early active phase. These results lay the groundwork for mechanistic investigations of circadian rhythms in fear extinction memory.

Development of the circadian system in early life: maternal and environmental factors.

In humans, an adaptable internal biological system generates circadian rhythms that maintain synchronicity of behavior and physiology with the changing demands of the 24-h environment. Development of the circadian system begins in utero and continues throughout the first few years of life. Maturation of the clock can be measured through sleep/wake patterns and hormone secretion. Circadian rhythms, by definition, can persist in the absence of environmental input; however, their ability to adjust to external time cues is vital for adaptation and entrainment to the environment. The significance of these external factors that influence the emergence of a stable circadian clock in the first years of life remain poorly understood. Infants raised in our post-modern world face adverse external circadian signals, such as artificial light and mistimed hormonal cues via breast milk, which may increase interference with the physiological mechanisms that promote circadian synchronization. This review describes the very early developmental stages of the clock and common circadian misalignment scenarios that make the developing circadian system more susceptible to conflicting time cues and temporal disorder between the maternal, fetal, infant, and peripheral clocks.

Association between Altered Cortisol Profiles and Neurobehavioral Impairment after Mild Traumatic Brain Injury in College Students.

Mild traumatic brain injury (mTBI) is the most common form of TBI, with more than 2.5 million TBI cases in the United States annually. Identification of easily obtainable biomarkers that track strongly with mTBI symptoms may improve our understanding of biological factors that contribute to mTBI symptom profiles and long-term outcomes. Notably, some individuals with mTBI exhibit circadian disruptions and elevated stress sensitivity, which in other clinical groups often correlate with disrupted secretion of cortisol, a glucocorticoid hormone that coordinates circadian and stress physiology. Here, we examined whether cortisol profiles could serve as a biomarker to complement the assessment of neurobehavioral sequelae after mTBI. We partnered with our on-campus health clinic to recruit college students seeking medical care after mTBI (n = 46) and compared this population to a well-matched non-injured student control group (n = 44). We collected data at an initial visit (shortly after injury in mTBI subjects) and one week later. At each visit, we evaluated neurobehavioral function using the Automated Neuropsychological Assessment Metric (ANAM). The subjects also provided cortisol samples through at-home saliva collection. We observed strong coherence between ANAM subjective and objective measures, indicating significant multi-dimensional impairment in subjects with mTBI. Further, female mTBI subjects exhibited diminished neurobehavioral function compared with males. Regardless of sex, decreased amplitude of diurnal cortisol and a blunted cortisol awakening response were associated with mTBI symptom severity and neurobehavioral impairment. Taken together, these findings suggest that salivary cortisol profiles may be a sensitive biomarker for studying underlying biological factors that impact mTBI symptoms and outcomes.

Iterative Metaplasticity Across Timescales: How Circadian, Ultradian, and Infradian Rhythms Modulate Memory Mechanisms.

Work in recent years has provided strong evidence for the modulation of memory function and neuroplasticity mechanisms across circadian (daily), ultradian (shorter-than-daily), and infradian (longer-than-daily) timescales. Despite rapid progress, however, the field has yet to adopt a general framework to describe the overarching role of biological rhythms in memory. To this end, Iyer and colleagues introduced the term iterative metaplasticity, which they define as the "gating of receptivity to subsequent signals that repeats on a cyclic timebase." The central concept is that the cyclic regulation of molecules involved in neuroplasticity may produce cycles in neuroplastic capacity-that is, the ability of neural cells to undergo activity-dependent change. Although Iyer and colleagues focus on the circadian timescale, we think their framework may be useful for understanding how biological rhythms influence memory more broadly. In this review, we provide examples and terminology to explain how the idea of iterative metaplasticity can be readily applied across circadian, ultradian, and infradian timescales. We suggest that iterative metaplasticity may not only support the temporal niching of neuroplasticity processes but also serve an essential role in the maintenance of memory function.

Acute Physiological and Psychological Stress Response in Youth at Clinical High-Risk for Psychosis.

Deficits in stress-response systems are a characteristic of schizophrenia and psychosis spectrum illnesses, and recent evidence suggests that this impairment may be evident in those at clinical high-risk (CHR) for the development of a psychotic disorder. However, there is limited research specifically investigating biological and subjective stress reactivity in CHR individuals. In the present study, 38 CHR individuals and group of 38 control individuals participated in the Trier Social Stress Test (TSST), an experimentally induced psychosocial stressor. Changes in salivary cortisol and alpha amylase, as well as self-reported units of distress (SUDS), were evaluated. Interestingly, the TSST did not induce a change in cortisol levels in either group, though the CHR group did show higher overall cortisol levels throughout the TSST (pre-anticipation period through recovery period). However, indicative of an effective task manipulation, the TSST did illicit an increase in alpha amylase in both groups. CHR participants exhibited higher levels of subjective stress prior to the stressor compared to the control group and CHR SUDs did not significantly increase in response to the stressor. In contrast, the control group showed an increase in SUDS in response to the stressor. Notably, SUDS for the control group post task mirrored the levels CHR youth endorsed prior to the stressor. Taken together, these findings suggest that there may be a functional relationship between persistently elevated cortisol and chronic high levels of subjective distress in CHR individuals.

Reflections on Bruce S. McEwen's contributions to stress neurobiology and so much more.

The authors highlight, from a firsthand perspective, Bruce S. McEwen's seminal influence on the field of stress neurobiology and beyond, and how these investigations have yielded important insights, principles and critical questions that continue to guide stress research today. Featured are discussion of: 1) the important inverted-U relationship between stress/glucocorticoids and optimal physiological function, 2) stress adaptation and the role of adaptive stress responses, 3) mechanisms by which the short-term stress response promotes heightened immune function and immunity, and 4) the far reaching impact of the theoretical framework of allostasis and allostatic load-concepts that have created new bridges between stress physiology, biomedical sciences, health psychology and sociology.

Memory and the circadian system: Identifying candidate mechanisms by which local clocks in the brain may regulate synaptic plasticity.

The circadian system is an endogenous biological network responsible for coordinating near-24-h cycles in behavior and physiology with daily timing cues from the external environment. In this review, we explore how the circadian system regulates memory formation, retention, and recall. Circadian rhythms in these memory processes may arise through several endogenous pathways, and recent work highlights the importance of genetic timekeepers found locally within tissues, called local clocks. We evaluate the circadian memory literature for evidence of local clock involvement in memory, identifying potential nodes for direct interactions between local clock components and mechanisms of synaptic plasticity. Our discussion illustrates how local clocks may pervasively modulate neuronal plastic capacity, a phenomenon that we designate here as circadian metaplasticity. We suggest that this function of local clocks supports the temporal optimization of memory processes, illuminating the potential for circadian therapeutic strategies in the prevention and treatment of memory impairment.

Circadian misalignment has differential effects on affective behavior following exposure to controllable or uncontrollable stress.

In modern 24 h society, circadian disruption is pervasive, arising from night shift work, air travel across multiple time zones, irregular sleep schedules, and exposure to artificial light at night. Disruption of the circadian system is associated with many adverse health consequences, including mood disorders. Here we investigate whether inducing circadian misalignment using a phase advance protocol interferes with the ability to cope with a stressor, thereby increasing susceptibility to the negative consequences of stress. Male rats were maintained on a standard 12:12 light: dark (LD) cycle or subjected to a chronic phase advance (CPA) protocol involving 4 weekly 6 h phase shifts (earlier light onset) of the LD cycle. Rats were then exposed to escapable stress (ES), inescapable stress (IS), or no stress (home cage control; HC) and performance on juvenile social exploration and active escape learning in the two-way shuttlebox test was assessed 24 h and 48 h following stress, respectively. CPA alone had no effect on pre-stress juvenile social exploration, and it also did not interfere with the protective effect of ES on the stress-induced reduction in juvenile social exploration. In contrast, CPA impaired escape learning in the two-way shuttlebox to the same extent as IS in all subjects, regardless of stress history. Additionally, CPA produced somatic alterations that included increased body mass, increased epididymal adiposity, and decreased adrenal mass. These data indicate that CPA differentially modulated the stress-protective effects of behavioral control depending on the type of affective behavior examined.

Coordination between Prefrontal Cortex Clock Gene Expression and Corticosterone Contributes to Enhanced Conditioned Fear Extinction Recall.

Post-traumatic stress disorder (PTSD) is associated with impaired conditioned fear extinction learning, a ventromedial prefrontal cortex (vmPFC)-dependent process. PTSD is also associated with dysregulation of vmPFC, circadian, and glucocorticoid hormone function. Rats have rhythmic clock gene expression in the vmPFC that requires appropriate diurnal circulatory patterns of corticosterone (CORT), suggesting the presence of CORT-entrained intrinsic circadian clock function within the PFC. We examined the role of vmPFC clock gene expression and its interaction with CORT profiles in regulation of auditory conditioned fear extinction learning. Extinction learning and recall were examined in male rats trained and tested either in the night (active phase) or in the day (inactive phase). Using a viral vector strategy, Per1 and Per2 clock gene expression were selectively knocked down within the vmPFC. Circulating CORT profiles were manipulated via adrenalectomy (ADX) ± diurnal and acute CORT replacement. Rats trained and tested during the night exhibited superior conditioned fear extinction recall that was absent in rats that had knock-down of vmPFC clock gene expression. Similarly, the superior nighttime extinction recall was absent in ADX rats, but restored in ADX rats given a combination of a diurnal pattern of CORT and acute elevation of CORT during the postextinction training consolidation period. Thus, conditioned fear extinction learning is regulated in a diurnal fashion that requires normal vmPFC clock gene expression and a combination of circadian and training-associated CORT. Strategic manipulation of these factors may enhance the therapeutic outcome of conditioned fear extinction related treatments in the clinical setting.

Adrenal-dependent and -independent stress-induced Per1 mRNA in hypothalamic paraventricular nucleus and prefrontal cortex of male and female rats.

Oscillating clock gene expression gives rise to a molecular clock that is present not only in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), but also in extra-SCN brain regions. These extra-SCN molecular clocks depend on the SCN for entrainment to a light:dark cycle. The SCN has limited neural efferents, so it may entrain extra-SCN molecular clocks through its well-established circadian control of glucocorticoid hormone secretion. Glucocorticoids can regulate the normal rhythmic expression of clock genes in some extra-SCN tissues. Untimely stress-induced glucocorticoid secretion may compromise extra-SCN molecular clock function. We examined whether acute restraint stress during the rat's inactive phase can rapidly (within 30 min) alter clock gene (Per1, Per2, Bmal1) and cFos mRNA (in situ hybridization) in the SCN, hypothalamic paraventricular nucleus (PVN), and prefrontal cortex (PFC) of male and female rats (6 rats per treatment group). Restraint stress increased Per1 and cFos mRNA in the PVN and PFC of both sexes. Stress also increased cFos mRNA in the SCN of male rats, but not when subsequently tested during their active phase. We also examined in male rats whether endogenous glucocorticoids are necessary for stress-induced Per1 mRNA (6-7 rats per treatment group). Adrenalectomy attenuated stress-induced Per1 mRNA in the PVN and ventral orbital cortex, but not in the medial PFC. These data indicate that increased Per1 mRNA may be a means by which extra-SCN molecular clocks adapt to environmental stimuli (e.g. stress), and in the PFC this effect is largely independent of glucocorticoids.

Glucocorticoid hormones are both a major circadian signal and major stress signal: How this shared signal contributes to a dynamic relationship between the circadian and stress systems.

Glucocorticoid hormones are a powerful mammalian systemic hormonal signal that exerts regulatory effects on almost every cell and system of the body. Glucocorticoids act in a circadian and stress-directed manner to aid in adaptation to an ever-changing environment. Circadian glucocorticoid secretion provides for a daily waxing and waning influence on target cell function. In addition, the daily circadian peak of glucocorticoid secretion serves as a timing signal that helps entrain intrinsic molecular clock phase in tissue cells distributed throughout the body. Stress-induced glucocorticoid secretion also modulates the state of these same cells in response to both physiological and psychological stressors. We review the strong functional interrelationships between glucocorticoids and the circadian system, and discuss how these interactions optimize the appropriate cellular and systems response to stress throughout the day. We also discuss clinical implications of this dual aspect of glucocorticoid signaling, especially for conditions of circadian and HPA axis dysregulation.

Analysis of c-Fos induction in response to social interaction in male and female Fisher 344 rats.

Sex differences in the expression of social behavior are typically apparent in adolescent and adult rats. While the neurobiology underlying juvenile social play behavior has been well characterized, less is known about discrete brain regions involved in adult responsiveness to a same sex peer. Furthermore, whether adult males and females differ in their responsiveness to a social interaction in terms of neuronal activation indexed via immediate early gene (IEG) expression remains to be determined. Thus, the present study was designed to identify key sites relevant to the processing of sensory stimuli (generally) or social stimuli (specifically) after brief exposure to a same-sex social partner by assessing IEG expression. Four-month-old male and female Fisher (F) 344 rats (N=38; n=5-8/group) were either left undisturbed in their home cage as controls (HCC), exposed to a testing context alone for 30min (CXT), or were placed in the context for 20min and then allowed to socially interact (SI) with a sex-matched conspecific for 10min. Females demonstrated greater levels of social behavior, relative to males. Analysis of c-Fos induction revealed that females exhibited greater c-Fos expression in the prefrontal cortex, regardless of condition. In many brain regions, induction was similar in the CXT and SI groups. However, in the bed nucleus of the stria terminalis (BNST), females exhibited greater c-Fos induction in response to the social interaction relative to their male counterparts, indicating a sex difference in responsivity to social stimuli. Taken together, these data suggest that the BNST is a sexually dimorphic region in terms of activation in response to social stimuli.

The relationship between cannabis use and cortisol levels in youth at ultra high-risk for psychosis.

Recent studies have posited a relationship between cannabis use and the biological stress system, but this critical relationship has not been evaluated during the ultra high-risk (UHR) period immediately preceding the onset of psychotic disorders. Salivary cortisol samples were collected on 46 UHR and 29 control adolescents; these individuals were assessed for current cannabis use with a urine panel and self-report. UHR participants where separated into two groups: Current Cannabis Use (UHR-CU) and No Current Cannabis Use (UHR-NC). Healthy Control participants (HC) were free of cannabis use. Consistent with the literature, results indicate UHR individuals showed elevated cortisol levels when compared to HC participants. Further, we also observed that UHR-CU participants exhibited elevated levels when compared to both the non-using UHR and HC groups. Findings suggest that cannabis use may interact with underlying biological vulnerability associated with the hypothalamic-pituitary-adrenal (HPA) axis system.

Dynamic glucocorticoid-dependent regulation of Sgk1 expression in oligodendrocytes of adult male rat brain by acute stress and time of day.

Recent studies support plasticity in adult brain white matter structure and myelination in response to various experiential factors. One possible contributor to this plasticity may be activity-dependent modulation of serum- and glucocorticoid-inducible kinase 1 (Sgk1) expression in oligodendrocytes. We examined whether Sgk1 expression in adult rat brain white matter is increased by acute stress-induced elevations in endogenous corticosterone and whether it fluctuates with diurnal variations in corticosterone. We observed rapid increases (within 30 min) in Sgk1 mRNA in the corpus callosum in response to acute stress, as well as large increases at the beginning of the rat's active period (the time of peak corticosterone secretion). These increases were absent in adrenalectomized rats. Corticosterone treatment of adrenalectomized rats also rapidly increased corpus callosum Sgk1 mRNA. The majority of Sgk1 mRNA in corpus callosum was co-localized with myelin basic protein mRNA, suggesting that mature oligodendrocytes respond dynamically to acute stress and circadian rhythms. The regulation of Sgk1 expression by acute stress and time of day was selective for white matter, with limited alteration of Sgk1 expression by these factors in hippocampus and somatosensory cortex. These results indicate a unique sensitivity of oligodendrocyte Sgk1 expression to activity-dependent fluctuations in corticosterone hormone secretion, and raises the prospect that hypothalamic-pituitary-adrenal axis dysregulation or glucocorticoid pharmacotherapy may compromise the normal activity-dependent interactions between oligodendrocytes and neurons.

A users guide to HPA axis research.

Glucocorticoid hormones (cortisol and corticosterone - CORT) are the effector hormones of the hypothalamic-pituitary-adrenal (HPA) axis neuroendocrine system. CORT is a systemic intercellular signal whose level predictably varies with time of day and dynamically increases with environmental and psychological stressors. This hormonal signal is utilized by virtually every cell and physiological system of the body to optimize performance according to circadian, environmental and physiological demands. Disturbances in normal HPA axis activity profiles are associated with a wide variety of physiological and mental health disorders. Despite numerous studies to date that have identified molecular, cellular and systems-level glucocorticoid actions, new glucocorticoid actions and clinical status associations continue to be revealed at a brisk pace in the scientific literature. However, the breadth of investigators working in this area poses distinct challenges in ensuring common practices across investigators, and a full appreciation for the complexity of a system that is often reduced to a single dependent measure. This Users Guide is intended to provide a fundamental overview of conceptual, technical and practical knowledge that will assist individuals who engage in and evaluate HPA axis research. We begin with examination of the anatomical and hormonal components of the HPA axis and their physiological range of operation. We then examine strategies and best practices for systematic manipulation and accurate measurement of HPA axis activity. We feature use of experimental methods that will assist with better understanding of CORT's physiological actions, especially as those actions impact subsequent brain function. This research approach is instrumental for determining the mechanisms by which alterations of HPA axis function may contribute to pathophysiology.

Sex differences in morning cortisol in youth at ultra-high-risk for psychosis.

Research suggests abnormalities in hypothalamic-pituitary-adrenal (HPA) axis function play an important role in the pathophysiology of psychosis. However, there is limited research on the biological stress system in young people at ultra high risk (UHR) for psychosis. Morning cortisol levels are particularly relevant to study in this context, as these markers reflect HPA regulation. This is the first examination of sex differences in morning cortisol levels in UHR individuals. Twenty-eight UHR and 22 matched healthy control participants were assessed in respect to symptoms and had home-based collection of salivary cortisol over three time points in the morning. It was predicted that the UHR participants would exhibit lower morning cortisol levels and lower cortisol would be associated with greater symptomatology (i.e. higher positive, negative, and depressive symptoms). Additionally, sex differences in morning cortisol levels were explored based on recent evidence suggesting that sex differences may play an important role in the exacerbation of psychosis. While there were no group differences in morning salivary cortisol secretion, there was a sex by time interaction among UHR individuals, such that only UHR males exhibited flat cortisol levels across two hours after awakening, whereas UHR females had a pattern of cortisol secretion similar to healthy controls, even among medication-free individuals (F=6.34, p=0.004). Cortisol AUC (area under the curve) across the three time points had a trend association (medium effect size; r=0.34, p=0.08) with depressive, but not positive or negative, symptom severity. These results stress the importance of considering sex differences in the psychosis-risk period, as they improve understanding of pathogenic processes.

Glucocorticoid Fast Feedback Inhibition of Stress-Induced ACTH Secretion in the Male Rat: Rate Independence and Stress-State Resistance.

Normal glucocorticoid secretion is critical for physiological and mental health. Glucocorticoid secretion is dynamically regulated by glucocorticoid-negative feedback; however, the mechanisms of that feedback process are poorly understood. We assessed the temporal characteristics of glucocorticoid-negative feedback in vivo using a procedure for drug infusions and serial blood collection in unanesthetized rats that produced a minimal disruption of basal ACTH plasma levels. We compared the negative feedback effectiveness present when stress onset coincides with corticosterone's (CORT) rapidly rising phase (30 sec pretreatment), high plateau phase (15 min pretreatment), or restored basal phase (60 min pretreatment) as well as effectiveness when CORT infusion occurs after the onset of stress (5 min poststress onset). CORT treatment prior to stress onset acted remarkably fast (within 30 sec) to suppress stress-induced ACTH secretion. Furthermore, fast feedback induction did not require rapid increases in CORT at the time of stress onset (hormone rate independent), and those feedback actions were relatively long lasting (≥15 min). In contrast, CORT elevation after stress onset produced limited and delayed ACTH suppression (stress state resistance). There was a parallel stress-state resistance for CORT inhibition of stress-induced Crh heteronuclear RNA in the paraventricular nucleus but not Pomc heteronuclear RNA in the anterior pituitary. CORT treatment did not suppress stress-induced prolactin secretion, suggesting that CORT feedback is restricted to the control of hypothalamic-pituitary-adrenal axis elements of a stress response. These temporal, stress-state, and system-level features of in vivo CORT feedback provide an important physiological context for ex vivo studies of molecular and cellular mechanisms of CORT-negative feedback.

Diurnal Corticosterone Presence and Phase Modulate Clock Gene Expression in the Male Rat Prefrontal Cortex.

Mood disorders are associated with dysregulation of prefrontal cortex (PFC) function, circadian rhythms, and diurnal glucocorticoid (corticosterone [CORT]) circulation. Entrainment of clock gene expression in some peripheral tissues depends on CORT. In this study, we characterized over the course of the day the mRNA expression pattern of the core clock genes Per1, Per2, and Bmal1 in the male rat PFC and suprachiasmatic nucleus (SCN) under different diurnal CORT conditions. In experiment 1, rats were left adrenal-intact (sham) or were adrenalectomized (ADX) followed by 10 daily antiphasic (opposite time of day of the endogenous CORT peak) ip injections of either vehicle or 2.5 mg/kg CORT. In experiment 2, all rats received ADX surgery followed by 13 daily injections of vehicle or CORT either antiphasic or in-phase with the endogenous CORT peak. In sham rats clock gene mRNA levels displayed a diurnal pattern of expression in the PFC and the SCN, but the phase differed between the 2 structures. ADX substantially altered clock gene expression patterns in the PFC. This alteration was normalized by in-phase CORT treatment, whereas antiphasic CORT treatment appears to have eliminated a diurnal pattern (Per1 and Bmal1) or dampened/inverted its phase (Per2). There was very little effect of CORT condition on clock gene expression in the SCN. These experiments suggest that an important component of glucocorticoid circadian physiology entails CORT regulation of the molecular clock in the PFC. Consequently, they also point to a possible mechanism that contributes to PFC disrupted function in disorders associated with abnormal CORT circulation.

Adolescent caffeine consumption increases adulthood anxiety-related behavior and modifies neuroendocrine signaling.

Caffeine is a commonly used psychoactive substance and consumption by children and adolescents continues to rise. Here, we examine the lasting effects of adolescent caffeine consumption on anxiety-related behaviors and several neuroendocrine measures in adulthood. Adolescent male Sprague-Dawley rats consumed caffeine (0.3g/L) for 28 consecutive days from postnatal day 28 (P28) to P55. Age-matched control rats consumed water. Behavioral testing for anxiety-related behavior began in adulthood (P62) 7 days after removal of caffeine. Adolescent caffeine consumption enhanced anxiety-related behavior in an open field, social interaction test, and elevated plus maze. Similar caffeine consumption in adult rats did not alter anxiety-related behavior after caffeine removal. Characterization of neuroendocrine measures was next assessed to determine whether the changes in anxiety were associated with modifications in the HPA axis. Blood plasma levels of corticosterone (CORT) were assessed throughout the caffeine consumption procedure in adolescent rats. Adolescent caffeine consumption elevated plasma CORT 24h after initiation of caffeine consumption that normalized over the course of the 28-day consumption procedure. CORT levels were also elevated 24h after caffeine removal and remained elevated for 7 days. Despite elevated basal CORT in adult rats that consumed caffeine during adolescence, the adrenocorticotropic hormone (ACTH) and CORT response to placement on an elevated pedestal (a mild stressor) was significantly blunted. Lastly, we assessed changes in basal and stress-induced c-fos and corticotropin-releasing factor (Crf) mRNA expression in brain tissue collected at 7 days withdrawal from adolescent caffeine. Adolescent caffeine consumption increased basal c-fos mRNA in the paraventricular nucleus of the hypothalamus. Adolescent caffeine consumption had no other effects on the basal or stress-induced c-fos mRNA changes. Caffeine consumption during adolescence increased basal Crf mRNA in the central nucleus of the amygdala, but no additional effects of stress or caffeine consumption were observed in other brain regions. Together these findings suggest that adolescent caffeine consumption may increase vulnerability to psychiatric disorders including anxiety-related disorders, and this vulnerability may result from dysregulation of the neuroendocrine stress response system.