BACKGROUND

Measurement of adrenocorticotropin levels in plasma from the inferior petrosal sinuses of patients with Cushing's syndrome can distinguish adrenocorticotropin-secreting pituitary tumors (Cushing's disease) from other causes of the syndrome, principally ectopic adrenocorticotropin secretion from an occult tumor. However, it is unknown whether such measurement consistently identifies patients with Cushing's disease and whether testing with corticotropin-releasing hormone (CRH) enhances the value of the procedure.

METHODS

We prospectively studied 281 patients with Cushing's syndrome to evaluate the diagnostic efficacy of the procedure. Bilateral sampling was successfully accomplished in 278 patients, with no major morbidity; 262 of these patients underwent sampling before and after administration of ovine CRH. The adrenocorticotropin levels in the samples were used to calculate the ratio of the concentration in plasma from the inferior petrosal sinuses to the concentration in peripheral-blood plasma (the IPS:P ratio).

RESULTS

The diagnosis of 246 patients was confirmed surgically as Cushing's disease in 215, as ectopic adrenocorticotropin syndrome in 20, and as primary adrenal disease in 11. An IPS:P ratio greater than or equal to 2.0 in basal samples identified 205 of the 215 patients with Cushing's disease (sensitivity, 95 percent), with no false positive results (specificity, 100 percent). A peak IPS:P ratio greater than or equal to 3.0 after CRH administration identified all 203 of the patients with Cushing's disease who received CRH (sensitivity, 100 percent), with no false positive results (specificity, 100 percent). The sensitivity was much lower when the adrenocorticotropin concentrations in the samples from one sinus were considered alone. In patients with Cushing's disease a difference of greater than or equal to 1.4-fold between the concentrations in the two sinuses (the adrenocorticotropin gradient) predicted the location of the microadenoma in 68 percent of 104 patients during basal sampling and in 71 percent of 105 patients after CRH administration.

CONCLUSIONS

Simultaneous bilateral sampling of plasma from the inferior petrosal sinuses, with the adjunctive use of CRH, distinguishes patients with Cushing's disease from those with ectopic adrenocorticotropin secretion with high diagnostic accuracy.

Previously, we demonstrated that transection of the fimbria/fornix blocked the excitatory effect of corticotropin-releasing hormone (CRH) on startle (CRH-enhanced startle), suggesting that the hippocampus and its efferent target areas that communicate via the fimbria may be critically involved in CRH-enhanced startle. The bed nucleus of the stria terminalis (BNST) receives direct projections from the ventral hippocampus via the fimbria/fornix. Therefore, the role of the ventral hippocampus, the BNST, and the amygdala in CRH-enhanced startle was investigated. NMDA lesions of the BNST completely blocked CRH-enhanced startle, whereas chemical lesions of the ventral hippocampus and the amygdala failed to block CRH-enhanced startle. However, the same amygdala-lesioned animals showed a complete blockade of fear-potentiated startle, a conditioned fear response sensitive to manipulations of the amygdala. In contrast, BNST-lesioned rats had normal fear-potentiated startle. This indicates a double dissociation between the BNST and the amygdala in two different paradigms that enhance startle amplitude. Microinfusions of CRH into the BNST, but not into the ventral hippocampus, mimicked intracerebroventricular CRH effects. Furthermore, infusion of a CRH antagonist into the BNST blocked CRH-enhanced startle in a dose-dependent manner. Control studies showed that this blockade did not result from either leakage of the antagonist into the ventricular system or a local anesthetic effect caused by infusion of the antagonist into the BNST. The present studies strongly suggest that CRH in the CSF can activate the BNST, which could lead to activation of brainstem and hypothalamic BNST target areas involved in anxiety and stress responses.

OBJECTIVE

Preliminary studies have suggested that low doses of corticosteroids might rapidly improve hemodynamics in late septic shock treated with catecholamines. We examined the effect of hydrocortisone on shock reversal, hemodynamics, and survival in this particular setting.

METHODS

Prospective, randomized, double-blind, placebo-controlled study.

METHODS

Two intensive care units of a University hospital.

METHODS

Forty-one patients with septic shock requiring catecholamine for >48 hrs.

METHODS

Patients were randomly assigned either hydrocortisone (100 mg i.v. three times daily for 5 days) or matching placebo.

RESULTS

Reversal of shock was defined by a stable systolic arterial pressure (>90 mm Hg) for>> or =24 hrs without catecholamine or fluid infusion. Of the 22 hydrocortisone-treated patients and 19 placebo-treated patients, 15 (68%) and 4 (21%) achieved 7-day shock reversal, respectively, a difference of 47% (95% confidence interval 17% to 77%; p = .007). Serial invasive hemodynamic measurements for 5 days did not show significant differences between both groups. At 28-day follow-up, reversal of shock was higher in the hydrocortisone group (p = .005). Crude 28-day mortality was 7 (32%) of 22 treated patients and 12 (63%) of 19 placebo patients, a difference of 31% (95% confidence interval 1% to 61%; p = .091). Shock reversal within 7 days after the onset of corticosteroid therapy was a very strong predictor of survival. There were no significant differences in outcome in responders and nonresponders to a short corticotropin test. The respective rates of gastrointestinal bleeding and secondary infections did not differ between both groups.

CONCLUSIONS

Administration of modest doses of hydrocortisone in the setting of pressor-dependent septic shock for a mean of >96 hrs resulted in a significant improvement in hemodynamics and a beneficial effect on survival. These beneficial effects do not appear related to adrenocortical insufficiency.

Studies in humans suggest that exposure to life stressors is correlated with compulsive drug abuse and relapse to drugs during periods of abstinence. The behavioral and neurobiological mechanisms involved in the effect of stress on drug abuse, however, are not known. Here, we review data from studies using preclinical models in rats on the effect of environmental stressors on opiate and psychostimulant reinforcement, as measured by the intravenous drug self-administration and conditioned place preference procedures, on relapse to these drugs, as measured by the reinstatement procedure, and on the subjective effects of these drugs, as measured by the drug discrimination procedure. The results of the studies reviewed here suggest that while stressors are important modulators of the behavioral effects of opiate and psychostimulant drugs, the effect of stress on behavior in these animal models is stressor-specific, and to some degree, procedure- and drug-class-specific. The review of studies on the neurobiological mechanisms underlying stress-drug interactions in these animal models indicate that central noradrenaline and extrahypothalamic corticotropin-releasing factor mediate the effect of one form of stress (intermittent footshock) on reinstatement of opiate and psychostimulant seeking after prolonged drug-free periods. At present, however, little is known about the neuronal events that mediate the effect of environmental stressors on opiate and psychostimulant reinforcement or discrimination. The broader implications of the data reviewed here for future research and for the treatment of opiate and psychostimulant addiction are briefly discussed.

The molecular and cellular mechanisms by which plasticity is induced in the mature CNS (and, specifically, in the hippocampus) by environmental input are progressively being elucidated. However, the mechanisms - and even the existence - of functional and structural effects of environmental input (and, particularly, stress) early in life are incompletely understood. Here, we discuss recent evidence that stressful stimuli have a significant impact on neonatal (rat) and prenatal (human) hippocampal function and integrity. Stressful signals provoke expression and release of neuromodulators, including the peptide corticotropin-releasing hormone (CRH), leading to activation of CRH receptors on principal hippocampal neurons. Although physiological activation of these receptors promotes synaptic efficacy, pathological levels of CRH at hippocampal synapses contribute to neuronal death. Thus, early-life stress could constitute a 'double-edged sword': mild stress might promote hippocampal-dependent cognitive function, whereas severe stress might impair neuronal function and survival, both immediately and in the long-term. Importantly, these CRH-mediated processes could be targets of preventive and interventional strategies.

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic-pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and alpha melanocyte stimulating hormone (alpha-MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic-pituitary-adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ-cultured human scalp hair follicles respond to CRH stimulation by up-regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and alpha-MSH, which must have been processed from POMC. CRH, alpha-MSH, and ACTH also modulate expression of their cognate receptors (CRH-R1, MC1-R, MC2-R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up-regulates cortisol-IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up-regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis-like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down-regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

The CRH receptor (CRH-R) is a member of the secretin family of G protein-coupled receptors. Wide expression of CRH-Rs in the central nervous system and periphery ensures that their cognate agonists, the family of CRH-like peptides, are capable of exerting a wide spectrum of actions that underpin their critical role in integrating the stress response and coordinating the activity of fundamental physiological functions, such as the regulation of the cardiovascular system, energy balance, and homeostasis. Two types of mammal CRH-R exist, CRH-R1 and CRH-R2, each with unique splicing patterns and remarkably distinct pharmacological properties, but similar signaling properties, probably reflecting their distinct and sometimes contrasting biological functions. The regulation of CRH-R expression and activity is not fully elucidated, and we only now begin to fully understand the impact on mammalian pathophysiology. The focus of this review is the current and evolving understanding of the molecular mechanisms controlling CRH-R biological activity and functional flexibility. This shows notable tissue-specific characteristics, highlighted by their ability to couple to distinct G proteins and activate tissue-specific signaling cascades. The type of activating agonist, receptor, and target cell appears to play a major role in determining the overall signaling and biological responses in health and disease.

BACKGROUND

Interferon (IFN)-alpha has been used to study the effects of innate immune cytokines on the brain and behavior in humans. The degree to which peripheral administration of IFN-alpha accesses the brain and is associated with a central nervous system (CNS) inflammatory response is unknown. Moreover, the relationship among IFN-alpha-associated CNS inflammatory responses, neurotransmitter metabolism, and behavior has yet to be established.

METHODS

Twenty-four patients with hepatitis C underwent lumbar puncture and blood sampling after approximately 12 weeks of either no treatment (n = 12) or treatment with pegylated IFN-alpha 2b (n = 12). Cerebrospinal fluid (CSF) and blood samples were analyzed for proinflammatory cytokines and their receptors as well as the chemokine, monocyte chemoattractant protein-1 (MCP-1), and IFN-alpha. Cerebrospinal fluid samples were additionally analyzed for monoamine metabolites and corticotropin releasing hormone. Depressive symptoms were assessed using the Montgomery Asberg Depression Rating Scale.

RESULTS

Interferon-alpha was detected in the CSF of all IFN-alpha-treated patients and only one control subject. Despite no increases in plasma IL-6, IFN-alpha-treated patients exhibited significant elevations in CSF IL-6 and MCP-1, both of which were highly correlated with CSF IFN-alpha concentrations. Of the immunologic and neurotransmitter variables, log-transformed CSF concentrations of the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were the strongest predictor of depressive symptoms. Log-transformed CSF concentrations of IL-6, but not IFN-alpha or MCP-1, were negatively correlated with log-transformed CSF 5-HIAA (r(2) = -.25, p < .05).

CONCLUSIONS

These data indicate that a peripherally administered cytokine can activate a CNS inflammatory response in humans that interacts with monoamine (serotonin) metabolism, which is associated with depression.

Alcoholism can be defined by a compulsion to seek and take drug, loss of control in limiting intake, and the emergence of a negative emotional state when access to the drug is prevented. Alcoholism impacts multiple motivational mechanisms and can be conceptualized as a disorder that includes a progression from impulsivity (positive reinforcement) to compulsivity (negative reinforcement). The compulsive drug seeking associated with alcoholism can be derived from multiple neuroadaptations, but the thesis argued here is that a key component involves the construct of negative reinforcement. Negative reinforcement is defined as drug taking that alleviates a negative emotional state. The negative emotional state that drives such negative reinforcement is hypothesized to derive from dysregulation of specific neurochemical elements involved in reward and stress within the basal forebrain structures involving the ventral striatum and extended amygdala, respectively. Specific neurochemical elements in these structures include not only decreases in reward neurotransmission, such as decreased dopamine and γ-aminobutyric acid function in the ventral striatum, but also recruitment of brain stress systems, such as corticotropin-releasing factor (CRF), in the extended amygdala. Acute withdrawal from chronic alcohol, sufficient to produce dependence, increases reward thresholds, increases anxiety-like responses, decreases dopamine system function, and increases extracellular levels of CRF in the central nucleus of the amygdala. CRF receptor antagonists also block excessive drug intake produced by dependence. A brain stress response system is hypothesized to be activated by acute excessive drug intake, to be sensitized during repeated withdrawal, to persist into protracted abstinence, and to contribute to the compulsivity of alcoholism. Other components of brain stress systems in the extended amygdala that interact with CRF and that may contribute to the negative motivational state of withdrawal include norepinephrine, dynorphin, and neuropeptide Y. The combination of loss of reward function and recruitment of brain stress systems provides a powerful neurochemical basis for a negative emotional state that is responsible for the negative reinforcement driving, at least partially, the compulsivity of alcoholism.

The CRF receptors belong to the VIP/GRF/PTH family of G-protein coupled receptors whose actions are mediated through activation of adenylate cyclase. Two CRF receptors, encoded by distinct genes, CRF-R1 and CRF-R2, and that can exist in two alternatively spliced forms, have been cloned. The type-1 receptor is expressed in many areas of the rodent brain, as well as in the pituitary, gonads, and skin. In the rodent, one splice variant of the type-2 receptor, CRF-R2 alpha, is expressed mainly in the brain, whereas the other variant, CRF-R2 beta, is found not only in the CNS, but also in cardiac and skeletal muscle, epididymis, and the gastrointestinal tract. The poor correlation between the sites of expression of CRF-R2 and CRF, as well as the relatively low affinity of CRF for CRF-R2, suggested the presence of another ligand, whose existence was confirmed in our cloning of urocortin. This CRF-like peptide is found not only in brain, but also in peripheral sites, such as lymphocytes. The broad tissue distribution of CRF receptors and their ligands underscores the important role of this system in maintenance of homeostasis. Functional studies of the two receptor types reveal differences in the specificity for CRF and related ligands. On the basis of its greater affinity for urocortin, in comparison with CRF, as well as its brain distribution, CRF-R2 may be the cognate receptor for urocortin. Mutagenesis studies of CRF receptors directed toward understanding the basis for their specificity, provide insight into the structural determinants for hormone-receptor recognition and signal transduction.

OBJECTIVE

The authors sought to carefully test, by using a technique of continuous CSF sampling, the hypothesis that basal elevations in CSF corticotropin-releasing hormone (CRH) concentrations exist in patients with posttraumatic stress disorder (PTSD). They also sought to assess the relationship among PTSD symptoms, adrenocortical activity, and CSF CRH levels.

METHODS

CSF was withdrawn by means of a flexible, indwelling subarachnoid catheter over a 6-hour period, and hourly CSF concentrations of CRH were determined for 11 well-characterized combat veterans with PTSD and 12 matched normal volunteers. Twenty-four-hour urinary-free cortisol excretion was also determined. PTSD and depressive symptoms were correlated with the neuroendocrine data.

RESULTS

Mean CSF CRH levels were significantly greater in PTSD patients than in normal subjects (55.2 [SD = 16.4] versus 42.3 pg/ml [SD = 15.6]). No correlation was found between CSF CRH concentrations and PTSD symptoms. While there was no significant difference between groups in 24-hour urinary-free cortisol excretion, the correlation between 24-hour urinary-free cortisol excretion and PTSD symptoms was negative and significant.

CONCLUSIONS

By using a serial CSF sampling technique, the authors found high basal CSF CRH concentrations and normal 24-hour urinary-free cortisol excretion in combat veterans with PTSD, a combination that appears to be unique among psychiatric conditions studied to date.

Initially the hypothalamic factor responsible for the release of corticotropin (CRF), was thought to be a simple peptide. More recent work has led to the conclusion that CRF is a multifactorial complex. In 1979 we proposed that vasopressin, much disputed as a CRF candidate, was a major constituent of the complex, interacting with a potentiating the CRF activity of the other component(s). The recent characterization of a 41 residue ovine hypothalamic peptide capable of releasing adrenocorticotropic hormone (ACTH) in a dose-related manner has allowed us to compare its CRF bioactivity with that of vasopressin and simple extracts of the hypothalamus, and to investigate any interaction it may have with vasopressin and other hypothalamic factors in the release of ACTH. We report here that the new CRF is more potent than vasopressin in releasing ACTH. When given simultaneously with vasopressin a fourfold potentiation of CRF activity with steep dose-response characteristics were observed. It also potentiated vasopressin-free hypothalamic extracts, suggesting that a new CRF does not account for all the nonvasopressin portion of the CRF complex.

Stress represents a complex stimulus to neuroendocrine systems regulating homeostasis. By and large, stress effects are mediated by stress-integrative corticotropin-releasing hormone (CRH) neurons present in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN). These neurons summate a large variety of neuronal and hormonal signals to eventually yield a physiologically meaningful level of circulating glucocorticoids. In the present experiments, we examined the effects of a chronic variable-stressor paradigm on indices of adrenocorticotropic hormone (ACTH) secretagogue biosynthesis in the PVN and adrenocorticosteroid receptor mRNA expression in the hippocampal formation, PVN and cortex. The variable-stressor paradigm produces a syndrome consistent with chronic stress, including baseline hypersecretion of corticosterone, ACTH and prolactin, and adrenal hypertrophy. CRH mRNA levels in the PVN are increased some 61%, consistent with the observed hypothalamo-pituitary-adrenal (HPA) up-regulation. There was a small but significant increase in arginine vasopressin (AVP) mRNA expression in individual parvocellular PVN neurons (16%), and no demonstrable increase in the number of AVP mRNA-containing neurons. No change in AVP expression was seen in the magnocellular PVN, supraoptic or suprachiasmatic nuclei. In all, these data highlight the importance of CRH in maintaining HPA up-regulation in the face of prolonged challenge. To investigate effects of chronic stress on the regulation of glucocorticoid receptivity, mineralocorticoid receptor (MR) and glucocorticoid receptor mRNA expression was assessed in the hippocampus, frontoparietal cortex and PVN. Chronic stress significantly down-regulated MR mRNA expression in subfields CA1, CA3 and the dentate gyrus (DG), and GR mRNA expression in subfields CA1, the DG and frontoparietal cortex. The reduction in receptor biosynthesis suggests the capacity for stress to modulate the impact of glucocorticoid on hippocampal cell physiology at the genomic level, potentially influencing processes ranging from cognition to feedback regulation of the HPA axis. At the level of the parvocellular PVN, GR mRNA expression was decreased to 60% of control values. GR mRNA expression was negatively correlated with PVN CRH mRNA expression, suggesting a relationship between elevated CRH gene expression and down-regulation of GR at the level of the PVN.

Although there is a close correspondence between fear and anxiety, and the study of fear in animals has been extremely valuable for understanding brain systems that are important for anxiety, it is equally clear that a richer animal model of human anxiety disorders would include measures of both stimulus-specific fear and something less stimulus specific, more akin to anxiety. Studies in patients with posttraumatic stress syndrome indicate these individuals seem to show normal fear reactions but abnormal anxiety measured with the acoustic startle reflex. Studies in rats, also using the startle reflex, indicate that highly processed explicit cue information (lights, tones, touch) activates the central nucleus of the amygdala, which in turn activates hypothalamic and brain stem target areas involved in specific signs of fear. Somewhat less explicit information, such as that produced by exposure to a threating environment for several minutes or by intraventricular administration of the peptide corticotropin-releasing hormone may activate a brain area closely related to the amygdala, called the bed nucleus of the stria terminalis, which in turn activates hypothalamic and brain stem target areas involved in specific signs of fear or anxiety. Because the nature of this information may be less specific than that produced by an explicit cue, and of much longer duration, activation of the bed nucleus of the stria terminalis may be more akin to anxiety than to fear.

Under normal conditions, the adrenal glucocorticoids, the endproduct of the hypothalamic-pituitary-adrenal (HPA) axis, provide a frontline of defence against threats to homeostasis (i.e. stress). On the other hand, chronic HPA drive and glucocorticoid hypersecretion have been implicated in the pathogenesis of several forms of systemic, neurodegenerative and affective disorders. The HPA axis is subject to gonadal influence, indicated by sex differences in basal and stress HPA function and neuropathologies associated with HPA dysfunction. Functional cross-talk between the gonadal and adrenal axes is due in large part to the interactive effects of sex steroids and glucocorticoids, explaining perhaps why several disease states linked to stress are sex-dependent. Realizing the interactive nature by which the hypothalamic-pituitary-gonadal and HPA systems operate, however, has made it difficult to model how these hormones act in the brain. Manipulation of one endocrine system is not without effects on the other. Simultaneous manipulation and assessment of both endocrine systems can overcome this problem. This dual approach in the male rat reveals that testosterone can act and interact on different aspects of basal and stress HPA function. Basal adrenocorticotropic hormone (ACTH) release is regulated by testosterone-dependent effects on arginine vasopressin synthesis, and corticosterone-dependent effects on corticotropin-releasing hormone (CRH) synthesis in the paraventricular nucleus (PVN) of the hypothalamus. In contrast, testosterone and corticosterone interact on stress-induced ACTH release and drive to the PVN motor neurones. Candidate structures mediating this interaction include several testosterone-sensitive afferents to the HPA axis, including the medial preoptic area, central and medial amygdala and bed nuclei of the stria terminalis. All of these relay homeostatic information and integrate reproductive and social behaviour. Because these modalities are affected by stress in humans, a dual systems approach holds great promise in establishing further links between the neuroendocrinology of stress and the central bases of sex-dependent disorders, including psychiatric, cardiovascular and metabolic disease.

The primary structure of a precursor protein that contains beta-neo-endorphin, dynorphin and a third leucine-enkephalin sequence with a carboxyl extension has been deduced from the nucleotide sequence of cloned DNA complementary to the porcine hypothalamic mRNA encoding it. The three peptides are each bounded by Lys-Arg. This precursor protein, like adrenal preproenkephalin and the corticotropin/beta-lipotropin precursor, comprises multiple repetitive units and a cysteine-containing amino-terminal sequence preceded by a signal peptide.

We showed previously that brief footshock stress and priming injections of heroin reinstate heroin-seeking after prolonged drug-free periods. Here, we examined whether the adrenal hormone, corticosterone, and brain corticotropin-releasing factor (CRF) were involved in such reinstatement. We tested the effects of adrenalectomy, chronic exposure to the corticosterone synthesis inhibitor metyrapone (100 mg/kg, s.c., twice daily), acute exposure to metyrapone, acute intracerebroventricular injections of CRF (0.3 and 1.0 microgram), and intracerebroventricular injections of the CRF antagonist alpha-helical CRF (3 and 10 micrograms). Rats were trained to self-administer heroin (100 micrograms/kg/infusion, i.v.) for 12-14 d. Extinction sessions were given for 4-8 d (saline substituted for heroin). Tests for reinstatement were given after priming injections of saline and of heroin (0.25 mg/kg, s.c.), and after intermittent footshock (15 or 30 min, 0.5 mA). Adrenalectomy (performed after training) did not affect reinstatement by heroin but appeared to potentiate the reinstatement by footshock. Chronic exposure to metyrapone (from the beginning of extinction) or an acute injection of metyrapone (3 hr before testing) did not alter the reinstatement of heroin-seeking induced by footshock or heroin. Acute exposure to metyrapone alone potently reinstated heroin-seeking. In addition, acute exposure to CRF reinstated heroin-seeking, and the CRF antagonist alpha-helical CRF attenuated stress-induced relapse. The effect of the CRF antagonist on reinstatement by heroin was less consistent. These results suggest that CRF, a major brain peptide involved in stress, contributes to relapse to heroin-seeking induced by stressors.

The stress system coordinates the adaptive response of the organism to real or perceived stressors. The main components of the stress system are the corticotropin-releasing hormone (CRH) and locus ceruleus-norepinephrine/ autonomic (LC/NE) systems and their peripheral effectors, the hypothalamic-pituitary-adrenal (HPA) axis, and the limbs of the autonomic system. Activation of the stress system leads to behavioral and peripheral changes that improve the ability of the organism to adjust homeostasis and increase its chances for survival. Thus, CRH and the LC/NE system stimulate arousal and attention, as well as the mesocorticolimbic dopaminergic system, which is involved in anticipatory and reward phenomena, and the amygdala, which are responsible for the generation of fear. Hypothalamic CRH plays an important role in inhibiting gonadotropin-releasing hormone secretion during stress, while via somatostatin it also inhibits growth hormone, thyrotropin-releasing hormone and thyrotropin secretion, suppressing thus reproduction, growth and thyroid function. Glucocorticoids directly inhibit pituitary gonadotropin, growth hormone and thyrotropin secretion and make the target tissues of sex steroids and growth factors resistant to these substances. In addition, glucocorticoids stimulate hepatic gluconeogenesis, and inhibit or potentiate insulin actions on skeletal muscle and adipose tissue respectively, ultimately promoting visceral adiposity and the metabolic syndrome. Glucocorticoids also have direct effects on the bone, inhibiting osteoblastic activity and causing osteoporosis. Obese subjects with psychiatric manifestations ranging from those of melancholic depression to anxiety with perception of 'uncontrollable' stress, frequently have mild hypercortisolism, while carefully screened obese subjects with no such manifestations are eucortisolemic. The former may have stress-induced glucocorticoid-mediated visceral obesity and metabolic syndrome manifestations, which in the extreme may be called a pseudo-Cushing state that needs to be differentiated from frank Cushing syndrome. Stress-induced hypercortisolism and visceral obesity and their cardiovascular and other sequelae increase the all-cause mortality risk of affected subjects by 2-3-fold and curtail their life expectancy by several years.

Drug addiction has been conceptualized as a chronically relapsing disorder of compulsive drug seeking and taking that progresses through three stages: binge/intoxication, withdrawal/negative affect, and preoccupation/anticipation. Drug addiction impacts multiple motivational mechanisms and can be conceptualized as a disorder that progresses from positive reinforcement (binge/intoxication stage) to negative reinforcement (withdrawal/negative affect stage). The construct of negative reinforcement is defined as drug taking that alleviates a negative emotional state. Our hypothesis is that the negative emotional state that drives such negative reinforcement is derived from dysregulation of key neurochemical elements involved in the brain stress systems within the frontal cortex, ventral striatum, and extended amygdala. Specific neurochemical elements in these structures include not only recruitment of the classic stress axis mediated by corticotropin-releasing factor (CRF) in the extended amygdala as previously hypothesized but also recruitment of dynorphin-κ opioid aversive systems in the ventral striatum and extended amygdala. Additionally, we hypothesized that these brain stress systems may be engaged in the frontal cortex early in the addiction process. Excessive drug taking engages activation of CRF not only in the extended amygdala, accompanied by anxiety-like states, but also in the medial prefrontal cortex, accompanied by deficits in executive function that may facilitate the transition to compulsive-like responding. Excessive activation of the nucleus accumbens via the release of mesocorticolimbic dopamine or activation of opioid receptors has long been hypothesized to subsequently activate the dynorphin-κ opioid system, which in turn can decrease dopaminergic activity in the mesocorticolimbic dopamine system. Blockade of the κ opioid system can also block anxiety-like and reward deficits associated with withdrawal from drugs of abuse and block the development of compulsive-like responding during extended access to drugs of abuse, suggesting another powerful brain stress/anti-reward system that contributes to compulsive drug seeking. Thus, brain stress response systems are hypothesized to be activated by acute excessive drug intake, to be sensitized during repeated withdrawal, to persist into protracted abstinence, and to contribute to the development and persistence of addiction. The recruitment of anti-reward systems provides a powerful neurochemical basis for the negative emotional states that are responsible for the dark side of addiction. This article is part of a Special Issue entitled 'NIDA 40th Anniversary Issue'.

Nicotine, the main psychoactive ingredient of tobacco, induces negative emotional symptoms during abstinence that contribute to a profound craving for nicotine. However, the neurobiological mechanisms underlying how nicotine produces dependence remains poorly understood. We demonstrate one mechanism for both the anxiety-like symptoms of withdrawal and excessive nicotine intake observed after abstinence, through recruitment of the extrahypothalamic stress peptide corticotropin-releasing factor (CRF) system and activation of CRF(1) receptors. Overactivation of the CRF-CRF(1) system may contribute to nicotine dependence and may represent a prominent target for investigating the vulnerability to tobacco addiction.

Early-life stimulation (e.g., brief handling) attenuates the behavioral and neuroendocrine responses to stressors encountered in adulthood, particularly with respect to activation of hypothalamic-pituitary-adrenal (HPA) activity. In contrast, if neonates were subjected to a more severe stressor, such as protracted separation from the dam or exposure to an endotoxin, then the adult response to a stressor was exaggerated. These early-life experiences program HPA functioning, including negative feedback derived from stimulation of hippocampal glucocorticoid receptors, and corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) coexpression in PVN neurons, to modify the response to subsequent stressor experiences. The persistent variations of HPA activity observed in handled/stimulated animals may stem from alterations in dam-pup interactions (e.g. increased arched-back feeding, licking, grooming). In addition genetic makeup is critical in determining stress reactivity. For instance, BALB/cByJ mice are more reactive to stressors than C57BL/6ByJ mice, exhibiting greater HPA hormonal alterations and behavioral disturbances. BALB/cByJ also fail to acquire a spatial learning response in a Morris water-maze paradigm, which has been shown to be correlated with hippocampal cell loss associated with aging. Early-life handling of BALB/cByJ mice prevented these performance deficits and attenuated the hypersecretion of ACTH and corticosterone elicited by stressors. The stressor reactivity may have been related to maternal and genetic factors. When BALB/cByJ mice were raised by a C57BL/6ByJ dam, the excessive stress-elicited HPA activity was reduced, as were the behavioral impairments. However, cross-fostering the more resilient C57BL/6ByJ mice to a BALB/cByJ dam failed to elicit the behavioral disturbances. It is suggested that genetic factors may influence dam-pup interactive styles and may thus proactively influence the response to subsequent stressors among vulnerable animals. In contrast, in relatively hardy animals the early-life manipulations may have less obvious effects.

Relapse is a major characteristic of drug addiction, and remains the primary problem in treating drug abuse. Without an understanding of the factors that determine renewed drug-seeking, the urge to use drugs, and the persistent craving for them, it is unlikely that health care professionals can provide effective treatment. Using an animal model of relapse, the author and her team are studying factors that induce reinstatement of drug-taking behaviour after short and long periods of abstinence, and they are exploring the neurobiological basis of these effects. In their experiments, rats are trained to self-administer drugs intravenously by pressing 1 of 2 levers. During a subsequent period, the drug is no longer available, but the rats are free to try to obtain the drug (a period of "extinction training"). After extinction of responding, the investigators test for the ability of various events to reinitiate drug-seeking. On this background of renewed drug-seeking or relapse, the investigators search for pharmacological and neurochemical manipulations that might block or attenuate such behaviour. They have found that the 2 most effective events for reinstating responding after both short and long drug-free periods are re-exposure to the drug itself and exposure to a brief period of stress. The critical neurochemical pathways mediating drug-induced relapse are not identical to those mediating stress-induced relapse. Relapse induced by "priming" injections of heroin or cocaine involves activation of the mesolimbic dopaminergic pathways, whereas relapse induced by stress involves actions of corticotropin-releasing factor (CRF) in the brain, and of brain noradrenergic (NE) systems. In addition, evidence shows that CRF and NE may interact at the level of the bed nucleus of the stria terminalis in stress-induced relapse. By contrast, relapse induced by "priming" injections of drugs is relatively unaffected by manipulation of CRF and NE systems of the brain.

BACKGROUND

Studies in rodents have determined that intermittent exposure to alcohol vapor can increase subsequent ethanol self-administration, measured with operant and 2-bottle choice procedures. Two key procedural factors in demonstrating increased alcohol intake are the establishment of stable alcohol self-administration before alcohol vapor exposure and the number of bouts of intermittent vapor exposure. The present studies provide additional behavioral validation and initial pharmacological validation of this withdrawal-associated drinking procedure.

METHODS

Studies at 2 different sites (Portland and Scripps) examined the effect of intermittent ethanol vapor exposure (3 cycles of 16 hours of ethanol vapor+8 hours air) on 2-hour limited access ethanol preference drinking in male C57BL/6 mice. Separate studies tested 10 or 15% (v/v) ethanol concentrations, and measured intake during the circadian dark. In one study, before measuring ethanol intake after the second bout of intermittent vapor exposure, mice were tested for handling-induced convulsions (HICs) indicative of physical dependence on ethanol. In a second study, the effect of bilateral infusions of the corticotropin-releasing factor (CRF) receptor antagonist D-Phe-CRF(12-41) (0.25 microg/0.5 microL) into the central nucleus of the amygdala (CeA) on ethanol intake was compared in vapor-exposed animals and air controls.

RESULTS

Intermittent ethanol vapor exposure significantly increased ethanol intake by 30 to 40%, and the mice had higher blood ethanol concentrations than controls. Intra-amygdala infusions of D-Phe-CRF(12-41) significantly decreased the withdrawal-associated increase in ethanol intake without altering ethanol consumption in controls. Following the second bout of intermittent vapor exposure, mice exhibited an increase in HICs, when compared with their own baseline scores or the air controls.

CONCLUSIONS

Intermittent alcohol vapor exposure significantly increased alcohol intake and produced signs of physical dependence. Initial pharmacological studies suggest that manipulation of the CRF system in the CeA can block this increased alcohol intake.

The principal modulators of the hypothalamic-pituitary-adrenal (HPA) axis are corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP). Corticotropin-releasing hormone is not exclusively produced in the hypothalamus. Its presence has been demonstrated at peripheral inflammatory sites. Ovulation and luteolysis bear characteristics of an aseptic inflammation. CRH was found in the theca and stromal cells as well as in cells of the corpora lutea of human and rat ovaries. The cytoplasm of the glandular epithelial cells of the endometrium has been shown to contain CRH and the myometrium contains specific CRH receptors. It has been suggested that CRH of fetal and maternal origin regulates FasL production, thus affecting the invasion (implantation) process through a local auto-paracrine regulatory loop involving the cytotrophoblast cells. Thus, the latter may regulate their own apoptosis. During pregnancy, the plasma level of circulating maternal immunoreactive CRH increases exponentially from the first trimester of gestation due to the CRH production in the placenta, decidua, and fetal membranes. The presence in plasma and amniotic fluid of a CRH-binding protein (CRHbp) that reduces the bioactivity of circulating CRH by binding is unique to humans. Maternal pituitary ACTH secretion and plasma ACTH levels rise during pregnancy-though remaining within normal limits-paralleling the rise of plasma cortisol levels. The maternal adrenal glands during pregnancy gradually become hypertrophic. Pregnancy is a transient, but physiologic, period of hypercortisolism. The diurnal variation of plasma cortisol levels is maintained in pregnancy, probably due to the secretion of AVP from the parvicellular paraventricular nuclei. CRH is detected in the fetal hypothalamus as early as the 12th week of gestation. CRH levels in fetal plasma are 50% less than in maternal plasma. The circulating fetal CRH is almost exclusively of placental origin. The placenta secretes CRH at a slower rate in the fetal compartment. AVP is detected in some neurons of the fetal hypothalamus together with CRH. AVP is usually detectable in the human fetal neurohypophysis at 11 to 12 weeks gestation and increases over 1000-fold over the next 12 to 16 weeks. The role of fetal AVP is unclear. Labor appears to be a stimulus for AVP release by the fetus. The processing of POMC differs in the anterior and intermediate lobes of the fetal pituitary gland. Corticotropin (ACTH) is detectable by radioimmunoassay in fetal plasma at 12 weeks gestation. Concentrations are higher before 34 weeks gestation, with a significant fall in late gestation. The human fetal adrenal is enormous relative to that of the adult organ. Adrenal steroid synthesis is increased in the fetus. The major steroid produced by the fetal adrenal zone is sulfoconjugated dehydroepiandrosterone (DHEAS). The majority of cortisol present in the fetal circulation appears to be of maternal origin, at least in the nonhuman primate. The fetal adrenal uses the large amounts of progesterone supplied by the placenta to make cortisol. Another source of cortisol for the fetus is the amniotic fluid where cortisol converted from cortisone by the choriodecidua, is found. In humans, maternal plasma CRH, ACTH, and cortisol levels increase during normal labor and drop at about four days postpartum; however, maternal ACTH and cortisol levels at this stage are not correlated. In sheep, placental CRH stimulates the fetal production of ACTH, which in turn leads to a surge of fetal cortisol secretion that precipitates parturition. The 10-day-long intravenous administration of antalarmin, a CRH receptor antagonist, significantly prolonged gestation compared to the control group of animals. Thus, CRH receptor antagonism in the fetus can also delay parturition. The HPA axis during the postpartum period gradually recovers from its activated state during pregnancy. The adrenals are mildly suppressed in a way analogous to postcure Cushing's syndrome. Provocation testing has shown that hypothalamic CRH secretion is transiently suppriently suppressed at three and six weeks postpartum, normalizing at 12 weeks.