Catherine Rivier - US grants

Corticotropin-releasing factor (CRF) is recognized as the principal regulator of the activity of the hypothalamic-pituitary-adrenal (HPA) axis under both basal and stressful circumstances. In doing so, CRF acts in concert with established secretagogues such as vasopressin, catecholamines and corticosteroids. Recent evidence is accumulating that additionally, a number of other factors [such as melanocyte-concentrating hormone (MCH), activin and interleukin-1(Il-1)] may play a role in modulating the HPA axis. At present, the mechanisms through which stimulation of the HPA axis regulates CRF expression, and the specific role of some of these newly described factors in modulating the HPA axis' response to stimuli, are far from being entirely understood. The purpose of this proposal is, first, to investigate the effect of stress on CRF expression in the brain, using Northern blot analysis and in situ hybridization techniques. In particular, we will examine the possibility that CRF expression is regulated differentially according to the type and/or regimen of administration of the stress, and the age of the animal exposed to the stress. Second, we will explore the pharmacological effects and putative physiological role of newly described factors (MCH, activin and Il-1) reported to alter ACTH secretion, and investigate their interaction with CRF, vasopressin and/or catecholamines in mediated stress-induced ACTH, corticosterone and Beta-endorphin secretion. Third, we will explore some of the mechanisms (including the role of endogenous CRF and Il-1) through which immune activation or inflammation, two powerful stimuli of ACTH secretion, modify the activity of the corticotrophs. Finally, this proposal will evaluate the in vivo activity of selected CRF and GRF analogs, which represent indispensable tools for the studies described above.

Preliminary results recently obtained in our laboratory have indicated that cocaine stimulates ACTH and corticosterone through a CRF-dependent mechanism. Consequently, this application requests funding for the investigation of the effects of cocaine on the hypothalamic-pituitary- adrenal (HPA) axis, and the mechanisms which mediate these effects in the rat. Under SPECIFIC AIM #1, we will investigate the effects of various doses of cocaine on ACTH and corticosterone secretion, as well as on corticotropin- releasing factor (CRF) biosynthesis. These hormonal measurements will be obtained during continuous (Study 1) or intermittent (Study 2) administration of cocaine, as well as following cessation of treatment with the drug (Study 3). Because CRF interacts with factors such as vasopressin, catecholamines, and corticoids to modulate ACTH secretion, SPECIFIC AIM #2 will explore the role of such interactions in mediating the effect of cocaine on pituitary function. We will investigate the possibility that cocaine modifies the pituitary responsiveness to CRF, vasopressin and/or catecholamines during continuous (Study 4) or intermittent (Study 5) treatment with cocaine, as well as following cessation of treatment (Study 6). In order to explore the possibility that steroid feedback participates in cocaine-induced changes in pituitary responsiveness, interactions between cocaine, CRF, vasopressin and catecholamines will also be investigated in adrenalectomized rats (Study 7). SPECIFIC AIM #3 will test the hypothesis, supported by our preliminary results, that cocaine will modify the HPA axis's response to stress. The ability of cocaine to alter stress-induced ACTH secretion will be investigated during continuous (Study 8) or intermittent Study 9 treatment with the drug, as well as following cessation of treatment (Study) 10. We believe that the results obtained will provide presently unavailable information concerning the effect of cocaine on pituitary function, the mechanisms which modulate these effects, and possible interactions with cocaine and the pituitary's response to stress.

Human alcoholics and FAS children often have an increased incidence of infectious diseases, which suggests that their immune functions are impaired. Abnormally elevated levels of glucocorticoids exert profound inhibitory effects on immune cell number and function, while abnormally decreased levels can induce inflammatory reactions. Our working hypothesis is that alcohol-induced activation of the hypothalamic-pituitary-adrenal (HPA axis alters the ability of this axis to respond to circulating interleukins with an appropriate secretion of glucocorticoid, corticotrophin-releasing factor (CRF), and/or ACTH, and that these pathological levels will compromise immune functions. The proposed work makes use of the novel finding in a rodent model that prior exposure to alcohol alters the ability of immune mediators such as interleukins to stimulate ACTH and glucocorticoid secretion. The purpose of this proposal is therefore to verify this finding under a variety of alcohol regimens,then extend it to address the question of the stage of development during which alcohol can influence the HPA axis' responsiveness to interleukins, and to investigate the mechanisms responsible for these changes. Exposure to an antigen represents a threat to homeostasis. In order to survive, mammalian organisms must make appropriate endocrine, metabolic and immune changes to this challenge. Essential to this response is the increased secretion of immune mediators produced by stimulated macrophages, which are called cytokines or interleukins. It is presently believed that one of the functions of these interleukins is to convey the occurrence of immune activation to the brain, and in particular to the hypothalamus where they stimulate the release of endogenous CRF and activate the hypothalamic- pituitary-adrenal (HPA) axis. While the release of interleukins is essential for orchestrating the early part of the immune response, equally important for the health of the organism is the presence of a mechanisms which, following completion of this initial phase, terminates interleukin release to prevent an "overshooting" reaction. Increased secretion of glucocorticoids, which is caused by the stimulatory effect of interleukins on the HPA axis, represents such a mechanism. Prior stimulation of the HPA axis, occurs following exposure to post- or prenatal alcohol, and can result in either the inhibition or the enhancement of the HPA axis' responsiveness to another stimulus. We presently do not know why such opposite effects can occur, but believe that hyper- or hyporesponsiveness of the HPA axis depends on the mode of alcohol exposure. Our hypothesis is that in rats treated with alcohol, the ability of the HPA axis to be activated by exogenously administered cytokines will be modified. Specific Aims #1 and 2 will investigate the pattern of cytokine-induced release of corticosterone, ACTH and/or CRF in rats exposed to alcohol post- or prenatally, respectively. The possible mechanisms mediating the effects of pre-or postnatal exposure to alcohol on HPA axis activity, such as alteration in pituitary responsiveness to CRF, in glucocorticoid feedback, and/or in CRF biosynthesis, will be investigated under Specific Aim #3.

Human alcoholics and FAS children often have an increased incidence of infectious diseases, which suggests that their immune functions are impaired. Abnormally elevated levels of glucocorticoids exert profound inhibitory effects on immune cell number and function, while abnormally decreased levels can induce inflammatory reactions. Our working hypothesis is that alcohol-induced activation of the hypothalamic-pituitary-adrenal (HPA axis alters the ability of this axis to respond to circulating interleukins with an appropriate secretion of glucocorticoid, corticotrophin-releasing factor (CRF), and/or ACTH, and that these pathological levels will compromise immune functions. The proposed work makes use of the novel finding in a rodent model that prior exposure to alcohol alters the ability of immune mediators such as interleukins to stimulate ACTH and glucocorticoid secretion. The purpose of this proposal is therefore to verify this finding under a variety of alcohol regimens,then extend it to address the question of the stage of development during which alcohol can influence the HPA axis' responsiveness to interleukins, and to investigate the mechanisms responsible for these changes. Exposure to an antigen represents a threat to homeostasis. In order to survive, mammalian organisms must make appropriate endocrine, metabolic and immune changes to this challenge. Essential to this response is the increased secretion of immune mediators produced by stimulated macrophages, which are called cytokines or interleukins. It is presently believed that one of the functions of these interleukins is to convey the occurrence of immune activation to the brain, and in particular to the hypothalamus where they stimulate the release of endogenous CRF and activate the hypothalamic- pituitary-adrenal (HPA) axis. While the release of interleukins is essential for orchestrating the early part of the immune response, equally important for the health of the organism is the presence of a mechanisms which, following completion of this initial phase, terminates interleukin release to prevent an "overshooting" reaction. Increased secretion of glucocorticoids, which is caused by the stimulatory effect of interleukins on the HPA axis, represents such a mechanism. Prior stimulation of the HPA axis, occurs following exposure to post- or prenatal alcohol, and can result in either the inhibition or the enhancement of the HPA axis' responsiveness to another stimulus. We presently do not know why such opposite effects can occur, but believe that hyper- or hyporesponsiveness of the HPA axis depends on the mode of alcohol exposure. Our hypothesis is that in rats treated with alcohol, the ability of the HPA axis to be activated by exogenously administered cytokines will be modified. Specific Aims #1 and 2 will investigate the pattern of cytokine-induced release of corticosterone, ACTH and/or CRF in rats exposed to alcohol post- or prenatally, respectively. The possible mechanisms mediating the effects of pre-or postnatal exposure to alcohol on HPA axis activity, such as alteration in pituitary responsiveness to CRF, in glucocorticoid feedback, and/or in CRF biosynthesis, will be investigated under Specific Aim #3.

antiserum; radioimmunoassay; peptide hormone; biomaterial development /preparation; biomedical facility; progesterone; prolactin; pituitary hormones; gonadotropin releasing factor; follicle stimulating hormone; luteinizing hormone; testosterone; estradiol; immunoaffinity chromatography; western blottings; enzyme linked immunosorbent assay; laboratory rabbit; laboratory rat; antibody titering; antibody; synthetic peptide;

Activin and inhibin are gonadal proteins that alter both pituitary and ovarian function. The work presented in the present proposal represents an extension of our previous studies of the effects of, and the role played by these proteins. We have observed that the peripheral administration of activin increased GnRH mRNA levels in the hypothalamus. Under Specific Aim 1, we will determine whether inhibin also alters GnRH gene expression; whether the effect of activin (and possibly inhibin) on GnRH transcripts are mediated by sex steroids; whether changes in brain GnRH levels are accompanied by altered release of the peptide from the median eminence; and whether endogenous opiates play a role in these effects. We will also investigate the role of activin during the estrous cycle and during the early increase in gonadotropin levels caused by gonadectomy, by blocking activin with specific neutralizing antibodies of the ligand binding domain. These experiments will address as yet unexplored aspects of the role and effects of inhibin/activin.

neuropeptides; radioimmunoassay; biomedical facility; vasopressins; somatotropin; corticotropin releasing factor; interleukin 6; interleukin 1; corticosterone; tumor necrosis factor alpha; adrenocorticotropic hormone; radiotracer; enzyme linked immunosorbent assay; laboratory rat; laboratory mouse;

As part of the functional relationship which exists between the immune and the neuroendocrine systems, proteins released by activated immune cells, called interleukins (ILs) or cytokines, convey the occurrence of immune stimulation to the hypothalamic-pituitary-adrenal (HPA) axis. The resulting increased activity of the HPA axis induced by ILs is essential to allow the organism to mount proper immune, endocrine and metabolic responses to an antigenic challenge. Consequently, pathological secretory rates of corticotropin-releasing factor (CRF), ACTH and/or corticosteroids during antigenic challenges can result in increased susceptibility immunologic disorders. Alcoholics and children of alcoholic mothers suffer from an increased occurrence of both infectious and inflammatory diseases, conditions that are associated, respectively, with abnormally elevated or blunted activity of the HPA axis. The distribution of these diseases in predisposed individuals indicates a distribution that is skewed by gender. Based on our animal studies, we hypothesize that alcohol-induced changes in the normal response of the HPA axis to immune signals participate in these pathologies, and that the sex steroid milieu influences both the stimulatory effect of cytokines on neuroendocrine functions, and the ability of alcohol to alter these responses. Our studies are therefore aimed at gaining a better understanding of the mechanisms responsible for the ability of alcohol alters the normal functional link between the immune system and the HPA axis. We have shown that exposure to alcohol during embryonic development or postnatally alters the stimulatory effect of ILs on ACTH and corticosterone release. Studies described in the present proposal will extend these results and explore the mechanisms responsible for the influence of alcohol. We will use IL-1beta, endotoxins or a small volume of turpentine to investigate the effect of a single cytokine, of a cascade of cytokines, or of a true inflammatory response, to probe the mechanisms through which alcohol disrupts the response of the HPA axis to immune signals. Under Specific Aim 1, we will study the influence of gender and sex steroids, the role of CRF, vasopressin and their receptors, and of nitric oxide, in mediating the influence of prenatal alcohol exposure on the response of the HPA axis to immune challenges. Under Specific Aim 2, a similar approach will be used to investigate the ability of post-natal alcohol administration to alter the response of the HPA axis of peripubertal and mature rats to cytokines. In both sets of experiments, sophisticated surgical approaches will be combined with techniques of molecular biology to provide a comprehensive investigation of the hypotheses we propose to test. We believe that the concept we present is original, and that the information gained from our studies will form the basis for a novel approach to the treatment of alcohol-related immune dysfunction.

We recently uncovered the existence of a neural brain-testicular pathway that interferes with Leydig cell function independently of the pituitary. Specifically, we showed that within 5 min of their intracerebroventricular (icv) injection, corticotropin- releasing factor (CRF) or isoproterenol (ISO) blocked the stimulatory effect of human chorionic gonadotropin (hCG) on testosterone (T) release. Systemic alcohol injected 15 min prior to hCG exerted a similar effect. The ability of icv CRF or ISO, or of alcohol, to block the T response was neither mimicked nor reversed by iv pretreatment with a GnRH antagonist, indicating that their inhibitory effect was not due to low LH levels. In contrast, the icv injection of an adrenergic antagonist partially reversed the effect of icv ISO, icv CRF or systemic alcohol. These results support the existence of a neural pathway that rapidly inhibits Leydig cell function through an adrenergic mechanism. At present, neuromorphological evidence for this pathway is missing. Under Specific Aim 1, we will use a viral transneuronal labeling method to identify sites in the central nervous system (CNS) that are involved in this pathway. This powerful neuroanatomical tool consists of the injection of pseudorabies virus (PRV) into the testis. Following replication, PRV is transported in a retrograde fashion to the perikarya of first-order neurons innervating the gonad, then to second-, third- and fourth-order neurons as the infection proceeds to further synaptically linked cell bodies. Virus-labeled neurons are identified by means of immunocytochemistry using polyclonal antibodies. Specificity of labeling will be determined by the ability of spermatic denervation and/or spinal cord T1 section, to eliminate PRV labeling in higher structures. After we have identified brain areas that belong to the proposed pathway, we will conduct functional experiments to determine its physiological importance. Under Specific Aim 2, we will microinfuse CRF or ISO in selected brain areas. We anticipate that by activating the proposed inhibitory pathway, these treatments will decrease hCG-induced T secretion, compared to results obtained in animals infused with the vehicle. Under Specific Aim 3, we will sever the spinal cord at the T1 level or lesion specific hypothalamic areas thought to be involved in the proposed pathway, to determine whether these procedures block the inhibitory effect of icv CRF or systemic alcohol on hCG-induced T secretion. These experiments will provide the first morphological and functional evidence of the existence of a multisynaptic neural pathway between the testes and the CNS, that is influenced by alcohol.

DESCRIPTION (applicant's abstract): The gases NO and CO are produced within the rat hypothalamus, where they exert a physiological role in modulating the hypothalamic-pituitary-adrenal (HPA) axis. Specifically, we have shown that removal of either gas blunts the response of this axis to a neurogenic stress (mild electrofootshocks), which suggests that NO and CO stimulate the activity of neurons of the paraventricular nucleus (PVN) that are important for ACTH release. In the case of NO, but not yet CO, we have provided additional evidence for this stimulatory effect by showing that the intracerebroventricular injection of NO donors significantly activated the HPA axis. At present, the neural circuitry responsible for the influence of NO and CO is not known. In order to provide novel information in this regard, we have developed a number of testable hypotheses that are grouped into three Specific Aims. Under Specific Aim 1, we will identify the pathways, both within the PVN and among the afferents to this nucleus, that modulate the HPA axis response to footshocks via an NO- or CO-dependent mechanism. Specifically using double- or triple-labeling, we will determine whether the PVN neurons that express corticotropin releasing factor (CRF) and/or vasopressin (VP), and that respond to footshocks by expressing the immediate early gene Fos, also contain the enzymes responsible for NO or CO formation [called NO synthase (NO) or hemeoxygenase (HO)], or are in close proximity to cells that express NOS or HO. We will also determine whether the retrogradely NO- or CO- producing cells that are activated by footshocks send their projections to the vicinity of CRF or VP perikarya in the PVN. Under Specific Aim 2, we will use this information to test the hypothesis that blockade of NO or CO formation within the identified circuitry decreases the HPA axis response to footshocks. Changes in plasma ACTH levels and in PVN transcripts for Fos, CRF and VP will be used to monitor HPA axis activity. Conversely under Specific Aim 3, we will test the hypothesis that increasing levels of these gases in the identified circuitry stimulates the activity of the HPA axis measured as indicated above. Collectively, these results will provide novel and comprehensive information regarding the neurocircuitries through which NO or CO modulate the response of the rat HPA to a neurogenic stress.

Exposure to alcohol alters the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Work carried out in our laboratory as well as other's indicates that prenatal alcohol exposure augments HPA axis activity in mature offspring, while animals chronically exposed to the drug for the first time in adulthood display blunted HPA axis responses. The purpose of this proposal is to investigate some of the mechanisms responsible for these effects. First, we will focus on nitric oxide (NO), a gaseous neurotransmitter that, as we recently reported, significantly stimulates levels of corticotropin releasing factor (CRF) and vasopressin (VP) in the hypothalamus. Specifically, we will test the hypothesis that prenatal alcohol alters (a) hypothalamic levels of NO and NO synthase (NOS), the enzyme responsible for NO formation, (b) the CRF and/or VP neuronal response to NO donors, and (c) the physiological role played by NO, which will be studied with NOS antagonists (Specific Aim 1). Second, we will test the hypothesis that alcohol-induced increases in hypothalamic CRF levels, through activation of a hypothalamic feed forward mechanism previously reported by our laboratory, participates in the hyperactive HPA axis that is the hallmark of prenatal alcohol (Specific Aim 2). At present, there is no information regarding the effect of prenatal alcohol exposure on the HPA axis of murine offspring. As a first step, we will therefore develop a murine model of prenatal alcohol treatment and investigate HPA axis responses in wild-type mice born to dams exposed to alcohol during gestation. These experiments will rely on an automated system that was recently developed in our laboratory, and that allows us to deliver intermittent amounts of alcohol vapors that are customized for each animal. Once the parameters of this new model are established, we will investigate the role of alcohol-induced changes in CRF levels, by exposing to alcohol vapors pregnant wild-type mice and mice lacking the gene for CRF receptors type 1 or 2, and comparing the HPA axis of their offspring. The final part of our proposal will focus on the hypothesis that long-term alcohol vapor treatment of adult rats decreases hypothalamic NO production, thereby contributing to the blunted HPA axis activity that characterizes these animal (Specific Aim 3). All the proposed experiments rely on cutting-edge methodology that was developed in our laboratory, as well as on recent concepts of brain regulation by NO. These studies will provide important information regarding effects of alcohol that are well documented in animal models as well as in humans, but for which mechanisms remain poorly understood.