Major Projects

Investigation of the mechanisms through which acute or long-term alcohol exposure alters the activity of the HPA axis.

Effect of alcohol on the HPA axis.
 Alcohol exerts a wide spectrum of effects that affect virtually every cell in the body. It is a unique drug because it appears to have multiple primary targets that include ligand-gated ion channels such as those associated with GABA, NMDA and serotonin, transporters, neurotransmitters, peptides and steroids. At present, it is not entirely clear whether alcohol exerts similar effects on most signaling pathways (for example, by similarly altering c-AMP-dependent processes, or gene transcription, or binding of ligands to their receptors), or whether these effects are system-specific. Indeed, the ability of the drug to indiscriminately distribute itself throughout the body (including the brain) renders studies of its specific influence within a particular system difficult. Also, as alcohol does not have a receptor, the mechanisms through which it alters cellular function are not easy to elucidate.

Study of the molecular and cellular aspects of the effects of alcohol, which is routinely done in isolated cell systems, presents the challenge of determining the molar concentrations of the drug within which results can be interpreted as being relevant for the whole organism. Study of the influence of alcohol in the intact animal, on the other hand, presents the challenge of determining whether results obtained with forced exposure to alcohol are relevant for conditions associated with spontaneous alcohol consumption, or whether one should dissociate between the two. Most of the results available in the published literature were obtained in laboratory animals exposed to alcohol through an experimenter-controlled procedure. This is primarily due to the fact that unselected animals do not spontaneously drink alcohol or, if they are forced to do so (for example when presented with a chocolate-based alcohol diet as the sole source of nutrients), only consume it in limited amounts. There is no doubt that results obtained with this experimental approach have been very interesting and useful.

Many studies have shown that alcohol administration to laboratory rodents causes a rapid and significant activation of the HPA axis. Present consensus is that increased levels of CRF and possibly VP in the brain are important in modulating the effect of the drug on this axis. There is some controversy regarding the acute effect of alcohol in humans, as some investigators claim that increased HPA axis function in human volunteers who consumed an alcoholic beverage is only present in the subjects that experienced gastrointestinal discomfort. Nevertheless, the overall consensus appears to be that humans who consume large amounts of alcohol exhibit increased HPA axis activity [though with a great deal of variability] as indicated by the fact that enhanced basal cortisol production is found in some alcoholics and can lead to a pseudo-Cushing’s syndrome. To state that the HPA axis of alcoholics is activated, however, is simplistic. Indeed, possibly as a consequence of increased corticosteroid feedback and/or downregulated pituitary CRF receptors, many alcoholics show a blunted response to exogenous CRF injection or exposure to a non-alcohol-related stress. Furthermore, the HPA axis remains pathologically altered in short-term abstinent alcoholics, who also show blunted responses to CRF or exposure to non-alcohol stresses. Therefore, while there appears to be reasonable evidence for alcohol-induced changes in the HPA axis of humans who abuse this drug, much remains to be investigated.

While of interest in themselves, the importance of studies of the effect of alcohol on the HPA axis of any mammalian species extends beyond the mechanisms that they will uncover. Indeed because of its pivotal influence as a general regulator and coordinator of the stress responses, and because its hormones exert such a wide range of effects, changes in the activity of this axis are likely to contribute to many of the effects of this drug. We mentioned above that CRF is the primary regulator of the HPA axis, but this peptide also exerts many other effects. Thanks to studies of the distribution of CRF through the brain, of its pharmacological effects on a wide array of parameters, and of the consequence of immunoneutralizing it or blocking its receptors, we now know that this peptide also controls or participates in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis, growth hormone release, gastrointestinal functions and natural killer cell activity, which it inhibits; opioids and catecholamines, which it stimulates; depression, which it appears to induce, and anxiety, which it promotes. Any alterations in CRF production and the activity of CRF-dependent circuitries (such as those seen after alcohol, for example) will therefore have profound consequences for the organism, both under basal conditions and during attempts to restore homeostasis. A full and complete knowledge of what alcohol does and how, is therefore crucial. Another important point is that in experimental animals, CRF has been shown to induce or participate in many responses that are very similar to those associated with fetal alcohol exposure in humans, such as hyperactivity, decreased attentiveness, aggressiveness, increased incidence of infections, augmented activity of the HPA axis, abnormal sexual behavior and premature aging. Many of these pathologies might therefore be directly or indirectly caused by elevated CRF levels. However, while our understanding of the consequences of prenatal alcohol on CRF-dependent circuitries is increasing, to our knowledge there is a great paucity of studies testing the hypothesis that this peptide participates in fetal alcohol syndrome (FAS)/fetal alcohol effects (FAE)-related disorders. Finally, there is a very interesting fact that has only recently been uncovered, namely that animals displaying increased voluntary alcohol consumption have elevated corticosterone levels under basal conditions. If true, this finding suggests the intriguing possibility, discussed below, that increased brain CRF levels may be associated with increased drinking.

CRF also exerts direct effects in many systems outside of the brain. In view of the ability of alcohol to upregulate the CRF gene, it seems reasonable to propose that probing the hypothesis that cardiovascular and immune effects of the drug, to mention only a few, might be modulated through this peptide, is of great interest. While it is outside the scope of this document to review this field, it may be useful to remember that CRF is reported to be manufactured and has receptors in macrophages and other immune cells, and to be present in arthritic tissue and inflamed tissues, where it is believed to participate in the inflammatory process. Indeed, the concept of a “tissue CRF” that is released in response to immune challenges and plays a local regulatory role has long been recognized. However, the effect of alcohol on this CRF has not been extensively studied. CRF is also present in steroid-producing cells, where it is reported to play a (mostly inhibitory) physiological role in regulating sex steroid production. Finally, CRF and/or its receptors are found in the gastrointestinal (GI) tract and in the heart.

In addition to exerting effects by itself, CRF alters homeostasis by stimulating the release of glucocorticoids (GC). As is well known, these steroids influence immune functions – if their levels are too high, infection can develop because the activity of immune cells is inhibited. If GC levels are too low, inflammation can take place because of increased reactivity. GC also plays a critical role in the general metabolism by regulating carbohydrate levels, and by influencing the tone of blood vessels. Within the brain, GC maintain the integrity of neuronal networks and chronic elevations of its levels can enhance susceptibility to neurodegeneration and premature aging. Collectively, these few examples show that alcohol can disrupt numerous pathways that are essential for health.

The phenomenon of tolerance.
Acute exposure to alcohol stimulates HPA axis activity, including the release of ACTH and the synthesis of CRF, CRF receptors and VP in the PVN. This endocrine response can be observed regardless of the route through which alcohol is delivered. Activation of pituitary CRF receptors type 1 is essential for increased ACTH secretion, as shown by the inability of mice lacking the gene for this receptor to mount an appropriate response when exposed to alcohol. This ACTH response also depends on upregulated CRF and VP synthesis and release because CRF/VP antibodies or receptor antagonists, or lesions of CRF neurons in the PVN, markedly decrease the ability of alcohol to release ACTH. Stimulation of the HPA axis is retained during prolonged, continuous exposure to alcohol, and in humans this can lead to a pseudo-Cushing's syndrome. However, the response of the HPA axis to alcohol often decreases with time, as does its ability to respond to other homeostatic challenges. Changes in PVN neuronal activation (i.e., the inability of these neurons to respond to alcohol or other stressors), rather than in levels of pituitary CRF and/or VP receptors, appear to be the primary mediators of this neuroendocrine habituation phenomenon. It is possible that dysregulated production of NO, a gas that stimulates PVN CRF synthesis, may participate in the blunted HPA axis activity. An interesting and recent discovery is that the HPA axis continues to exhibit a blunted HPA axis response to alcohol for days, if not weeks, after the drug is discontinued. While CRF and VP stores are decreased in the external zone of the median eminence of rats previously treated with alcohol and allowed to recover for at least 7 days, these changes are unlikely to represent an important mechanism because the HPA axis retains its ability to respond adequately to other homeostatic challenges. In contrast, we found that the PVN neuronal response to alcohol was significantly decreased. We proposed that this phenomenon might play a role in human alcohol abuse: If we accept the concept that some of the individuals who abuse alcohol do so, in part, because of the changes that the drug induces in their CNS, it is possible that if such changes cannot be achieved with a given dose of alcohol, these individuals may consume more drug in an attempt to regain the wanted changes. This might provide the basis for a testable, neuroendocrine-based hypothesis of vulnerability to alcohol abuse. Research done in rodent lines specifically bred for high spontaneous alcohol consumption may help us determine whether or not this hypothesis is tenable, at least in laboratory models.

Site of of action of alcohol in stimulating the HPA axis.

In vitro studies.

To determine the cellular and molecular mechanisms through which ethanol regulates CRF gene expression, we compared the effect of ethanol and forskolin on CRF peptide secretion and messenger RNA levels in hypothalamic primary cell cultures, and on CRF promoter activity in the NG108-15 cell line. CRF secretion, mRNA levels and gene transcription significantly increased in response to ethanol or forskolin. Mutation of the cAMP-response element (CRE) reduced luciferase activity under basal conditions as well as in response to forskolin or ethanol. On the other hand, plasmid with five CRE repeats yielded dramatically elevated basal luciferase activity and significantly increased up-regulation by ethanol. Inclusion of adenosine deaminase reduced the promoter response to ethanol. Finally a PKA inhibitor and a cAMP antagonist both decreased ethanol-induced CRF peptide secretion, gene expression and transcription. These results suggest that ethanol up-regulates CRF expression through cAMP/PKA-dependent pathways.

In vivo studies.

As discussed above, the peripheral injection of alcohol stimulates the activity of the HPA axis, but the ready penetration of this drug in most bodily compartments has made it difficult to identify its specific sites of action. We therefore determined whether alcohol acts directly on the corticotrophs. We first determined whether alcohol stimulated neurons in the PVN) of the hypothalamus, that synthesize CRF and VP. To test this hypothesis, we injected alcohol intracerebroventricularly (icv) and compared these results with those obtained following its intraperitoneal (ip) administration. While not causing neuronal damage and not leading to detectable levels of the drug in the general circulation, icv alcohol significantly upregulated PVN CRF heteronuclear RNA levels and increased plasma ACTH levels, a change comparable to the one observed in the ip model. To determine whether alcohol stimulated the corticotrophs independently of CRF and/or VP, we injected the drug ip or icv and measured changes in anterior pituitary POMC transcripts and ACTH release in the presence or absence of endogenous CRF and/or VP. Icv and ip alcohol significantly increased POMC primary transcript levels, measured by RNase protection assay, over a time-course that corresponded to ACTH release. Both the POMC and the ACTH responses were completely abolished by removal of CRF and VP. Collectively, these results indicate that alcohol-induced activation of the corticotrophs does not represent a direct influence of the drug on the pituitary, but requires CRF and VP.

Role of neurotransmitters. Nitric oxide.

NO is an unstable gas presently considered to be an important neurotransmitter in the brain where it modulates many central nervous system processes, including neuroendocrine functions. We have shown that NO stimulated the activity of the PVN of the hypothalamus, specifically of these cells that express the gene for VP and CRF, and that blockade of NO formation through the activity of its constitutive (c) enzyme blunted the HPA axis response to physical stresses. These results indicated that within structures protected by the blood-brain barrier, NO exerted a stimulatory influence on the HPA axis. In view of the importance of this axis in allowing the organism to successfully respond to homeostatic threats, studies of the ability of NO to modulate its activity during exposure to alcohol appear to be of interest. Information regarding functional interactions between alcohol and NO remains somewhat scant. Much of the research has focused on NO production through the inducible (i) isoform of the enzyme responsible for its formation, NO synthase (NOS), an isoform that appears inhibited by alcohol. The emergent literature pertaining to NO formation through neuronal (n) NOS, the isoform that is constitutively present in the brain, remains controversial. For example, prolonged alcohol exposure has been reported to both increase or decrease NOS activity in the frontal cortex and striatum. Results obtained in other parts of the brain appear equally contradictory, with reports of decreased NADPH-diaphorase staining (an index of nNOS activity) in the superior colliculus but increased NOS activity in the hypothalamus. We chose to study putative functional relationships between NO and acute alcohol exposure by using a three-pronged approach: First, we tested the hypothesis that endogenous NO participated in the ability of alcohol to release ACTH. Indeed, it seemed logical to us that if NO played no role, additional studies designed to investigate potential influences of alcohol on the release of this gas would not necessarily be meaningful. In the past, we successfully used a variety of NOS inhibitors to probe the role of NO in HPA activity, and the present work focused on the arginine derivative Nω−nitro-L-arginine-methylester (L-NAME), which non-specifically inhibited the activity of all cNOS (7-NI), a specific nNOS antagonist. The acute ig injection of alcohol significantly (P<0.01) increased NO levels in the paraventricular nucleus of the hypothalamus, the anterior pituitary and peripheral blood. The ACTH response to acute ig alcohol injection was significantly (P<0.01) decreased by the arginine derivative Nω−nitro-L-arginine-methylester (L-NAME), which non-specifically blocks NOS activity, as well as by the specific neuronal NOS antagonist 7-nitroindazole (7-NI).

We then determined whether alcohol would alter the stimulatory influence of increased hypothalamic NO levels on ACTH release. As we had shown that the icv injection of the NO donor SIN-1, a compound readily converted to NO without requiring enzymatic bioactivation, induced dose-related increases in plasma ACTH levels and upregulated PVN CRF and VP transcripts, we determined whether exposing rats to alcohol vapors for 1-5 days would alter their ACTH response to SIN-1 injection. Our previous studies had shown that while ACTH levels increased during vapor treatment, they had returned to baseline levels by the time of SIN-1 injection, which was done 24 h after the last alcohol vapor exposure. In addition, plasma corticosterone levels were also comparable in all groups (ng corticosterone/ml: controls, 22.1 + 2.4; 1 d vapors, 25.8 + 3.5; 3 d vapors, 19.7 + 3.8; 5 d vapors, 23.4 + 2.9; P>0.05). Exposure to alcohol vapors for 5, but not 1 or 3 days, significantly (P<0.01) blunted the increase in ACTH levels elicited by the icv injection of SIN-1. In conclusion, we have shown that NO, which is acutely released by alcohol in the anterior pituitary and the PVN, facilitates the stimulatory effect of alcohol on ACTH release. In turn, prolonged exposure to this drug decreases NO-induced ACTH release. These results provide novel information regarding reciprocal relationships between the NO and alcohol in modulating HPA axis activity in the rat.

Catecholamines. While as discussed above in isolated systems alcohol acts directly on the CRF promoter through cAMP-PKA-dependent pathways, its effect in intact animals likely involves intermediates. There is evidence that alcohol releases dopamine and stimulates fos signals in catecholamine-rich brain regions. However, with the exception of nitric oxide (whose role is discussed above), the physiological importance of specific modulators is unknown. In recent work we focused on norepinephrine, a bioamine that stimulates the PVN through activation of α1-adenergic receptors, and whose release within this nucleus is critical for many HPA axis responses to stressors. Aminergic pathways to the PVN originate from the lower brain stem in the ventrolateral medulla, the nucleus of the solitary tract (NTS) and the locus coeruleus (LC). As this latter only provides sparse direct innervation to the PVN, its influence on the HPA axis response to stressors is thought to involve innervation of the prefrontal cortex, while the NTS and the related A1-A2/C1-C3 cell groups represent the most important direct catecholaminergic input. However when we embarqued on these studies, the importance of adrenergic circuits in mediating the HPA axis response to alcohol remains largely untested. Importantly, because categorically distinct stressors stimulate the HPA axis through different circuits and mechanisms, results obtained with other stimuli cannot automatically be considered relevant during alcohol exposure. We therefore first evaluated the effect of this drug on medulla noradrenergic cells and on afferent circuits to the PVN, using freely-moving rats bearing intragastric (ig) cannulae. This protocol has the advantage over many previous studies, that alcohol is administered without the stress of handling the animals and is delivered to the stomach rather than the abdominal cavity, which can release pro-inflammatory cytokines whose effect on the HPA axis interferes with the interpretation of the results.

In vehicle-injected rats, CRF-ir neurons were found exclusively in the parvocellular part of the paraventricular hypothalamic nucleus (pPVN). Alcohol (EtOH) induced a significant (P<0.01) increase in c-fos signals in pPVN CRF, measured 2 h later. In addition, and in agreement with previous reports using other alcohol protocols, we found that ig alcohol increased c-fos signals in the central nucleus of the amygdala. When we examined catecholaminergic-rich regions, we first focused on DBH, the enzyme that catalyzes the conversion of dopamine to noradrenaline and is expressed selectively in noradrenaline (A1-A2) and adrenaline (C1-C3) neurons. In these regions, alcohol significantly (P<0.01) increased c-fos levels in DBH-ir cells, measured 2 hr later. We then used tyroxine hydroxylase (TH), the rate limiting enzymes in biosynthesis of dopamine and norepinephrine, to investigate the influence of acute alcohol delivery on the LC, and reported that stress-free injection of this drug into the stomach significantly (P<0.01) upregulated c-fos signals in TH-immunoreactive cells in the LC (A6), measured 2 h later.

(Nor)adrenergic medullary cell groups convey interoceptive information in a stress-specific manner and their lesion, through knife cuts or catecholamine depletion induced by toxins, interferes with the ACTH response to a variety, but not all stressors. While we therefore hypothesized that this area might also mediate the HPA axis' response to alcohol, it was important to demonstrate it. Six brain stem cell groups are currently considered to provide the catecholaminergic input required for normal PVN CRF responses to many stressors, i.e., the noradrenergic A1, A2 and A6 (LC) regions on one hand, and the adrenergic C1, C2 and C3 (including the NTS) regions on the other hand. As 6-OHDA lesions are considered to interrupt (nor)adrenergic inputs from the brain stem to the PVN, results of this type of experiments give us general information regarding the functional importance of these terminals on CRF neurons. We found that the loss of noradrenergic terminals to the PVN (6-OHDA treatment) significantly, but not completely reduced this response. We also measured corticosterone levels even though the rapid saturation of this curve at relatively low ACTH values makes this steroid a less informative parameter of HPA axis activity because even relatively modest increases in ACTH release prompt large elevations in corticosterone levels. While basal corticosterone levels remained <40 ng/ml in all animals, peak values in alcohol-injected rats, measured 30 min post-drug, were 312 + 43 ng/ml in the vehicle-pretreated group, and 157 + 23 ng/ml in animals with 6-OHDA lesions. Finally, we used the axonally transported immunotoxin anti-dopamine-β-hydroxylase-saporin (anti-DBH-saporin), which is an antibody to DBH (the enzyme responsible for NE synthesis) coupled to saporin. This toxin, which arrests protein synthesis in the neuronal cell body, specifically eliminates noradrenergic as well as adrenergic terminals and their neurons while preserving dopaminergic and cholinergic cell bodies. The toxin was microinfused directly in the A1/C1 and A2-C2/C3 region of the brain stem in order to provide more discrete lesions that those caused by icv treatment, which also destroys noradrenergic neurons of the LC. Compared to results obtained in rats that did not receive anti-DBH-saporin, the toxin induced the following changes in brain tissues obtained 2 hr after alcohol injection: they significantly decreased the density of DBH-ir fibers in the PVN, indicating the loss of catecholaminergic input to this hypothalamic region; and they also lowered the number of positive immunoreactive cells for PNMT in the C1-C3 region of the brain stem.

Lastly, we examined the type of receptors involved in mediating the influence of (nor)adrenergic pathways in our alcohol model. In general, the bulk of the stimulatory catecholaminergic drive to the central limb of the HPA axis is provided by adrenergic receptors type α, and subtype α1 receptors have been localized in the endocrine PVN. However, their central blockade interferes with the effect of only selective stressors. Whether this represents poor penetration into the brain of systemically-injected drugs, inadequate reach by these drugs of the brain areas that are important in the model studied, or a differential reliance of various stressors on catecholaminergic inputs into the PVN remains unclear, though the issue of the blood-brain barrier was circumvented in some studies by the icv administration of the antagonists. Alpha1 adrenergic receptors are also reported to mediate CRF release from the median eminence. We found that blockade of β receptors did not significantly alter the ability of alcohol to release ACTH, and that on the other hand this drug was not capable of inducing ACTH secretion in the absence of functional α1-adrenergic receptors. We had previously shown that alcohol acts on PVN CRF neurons and not directly on the pituitary. As corynanthine does not significantly interfere with ACTH release induced by exogenous CRF, we propose that the primary site of action of this antagonist is exerted in brain regions protected by the blood-brain barrier. Finally, as alcohol increases circulating levels of epinephrine, which may be able to penetrate the brain, one might argue that blood-borne amines might play a role in our model. However, activation of the HPA axis by epinephrine is mediated by β-adrenergic receptors. As blockade of these receptors did not alter the effect of alcohol, it seems unlikely that changes in peripheral catecholamines represent an important mechanism in the stimulatory effect of this drug on the HPA axis. Collectively, our work extends our knowledge of the ability of this drug to upregulate catecholamine circuitry in the rat brain. It also shows that medullary catecholamine innervation of the hypothalamus plays an important role in modulating the stimulatory effect of alcohol on the HPA axis, an effect exerted through activation of α1-adrenergic receptors.

References

Rivier C. 1995 Adult male rats exposed to an alcohol diet exhibit a blunted ACTH response to immune or physical stress: possible role of nitric oxide. Alcoholism: Clin Exp Res 19:1474-1479.

Rivier C. 1996 Alcohol stimulates ACTH secretion in the rat: mechanisms of action and interactions with other stimuli. Alcoholism: Clin Exp Res 20:240-254.

Rivier C and Lee S. 1996 Acute alcohol administration stimulates the activity of hypothalamic neurons that express corticotropin-releasing factor and vasopressin. Brain Res 726:1-10.

Rivier C, Rivier J and Lee S. 1996 Importance of pituitary and brain receptors for corticotrophin-releasing factor in modulating alcohol-induced ACTH secretion in the rat. Brain Res 721:83-90.

Lee S and Rivier C. 1997 Alcohol increases the expression of type 1, but not type 2a corticotropin-releasing factor (CRF) receptor messenger ribonucleic acid in the rat hypothalamus. Mol Brain Res 52:78-89.

Ogilvie K, Lee S and Rivier C. 1997 Role of arginine vasopressin and corticotropin-releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei. Brain Res 744:83-95.

Ogilvie K and Rivier C. 1997 Effect of three different modes of alcohol administration on the activity of the rat hypothalamic-pituitary-adrenal axis. Alcoholism: Clin Exp Res 21:467-476.

Rivier C. 1997 Effect of pretreatment with alcohol on subsequent endocrine and immune responses in the adult male rat. Alcoholism: Clin Exp Res 21:1690-1694.

Lee S and Rivier C. 1997 An initial, three-day long treatment with alcohol, induces a long-lasting phenomenon of selective tolerance in the activity of the rat hypothalamic-pituitary-adrenal axis. J Neurosci 17:8856-8866.

Lee S, Kim CK and Rivier C. 1999 Nitric oxide stimulates ACTH secretion, as well as the transcription of the genes encoding for corticotropin-releasing factor, corticotropin-releasing factor receptors type 1 and vasopressin in the hypothalamus of the intact rat. J Neurosci 19:7640-7647.

Rivier C. 1999 Effect of acute alcohol treatment on the release of ACTH, corticosterone and pro-inflammatory cytokines in response to endotoxin. Alcoholism: Clin Exp Res 23:673-82.

Rivier C. 1999 Gender, sex steroids, corticotropin-releasing factor, nitric oxide and the HPA response to stress. Pharmacol Biochem Behav 64:739-51.

Lee S, Schmidt D, Tilders F, Cole M, Smith A and Rivier C. 2000 Prolonged exposure to intermittent alcohol vapors blunts hypothalamic responsiveness to immune and non-immune signals. Alcoholism: Clin Exp Res 24:110-122.

Lee S, Smith G, Vale W, Lee K-F and Rivier C. 2001 Mice that lack corticotropin-releasing factor (CRF) receptors type 1 show a blunted ACTH response to acute alcohol despite up-regulated constitutive hypothalamic CRF gene expression. Alcoholism: Clin Exp Res 25:427-433.

Rivier C and Lee S. 2001 Effect of repeated exposure to alcohol on the response of the hypothalamic-pituitary adrenal axis of the rat: II. Role of the length and regimen of alcohol treatment. Alcoholism: Clin Exp Res 25:106-111.

Lee S, Schmidt ED, Tilders FJH and Rivier C. 2001 Effect of repeated exposure to alcohol on the response of the hypothalamic-pituitary adrenal axis of the rat: I. Role of changes in hypothalamic neuronal activity. Alcoholism: Clin Exp Res 25:98-105.

Ryabinin AE, Bachtell RK, Heinrichs SC, Lee S, Rivier C, Olive MF, Mehmert KK, Camarini R, Kim JA, Koenig HN, Nannini MA, Hodge CW, Roberts AJ and Koob GF. 2002 The CRF/urocortin system and alcohol. Alcoholism: Clin Exp Res 26:714-722.

Rivier C, Grigoriadis D and Rivier J. 2002 Role of corticotropin-releasing factor receptors type 1 and 2 in modulating the rat ACTH response to stressors. Endocrinology 144:2396-2403.

Seo DO, Rivier C. 2003 Interaction between alcohol and nitric oxide on ACTH release in the rat. Alchoolism: Clin Exp Res 17:989-996.

Lee S, Selvage D and Rivier C. 2004 Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: Comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses. Endocrinology 145:4470-4479.

Seo DO, Lee S and Rivier C. 2004 Prolonged exposure to intermittent alcohol vapors decreases the ACTH as well as hypothalamic nitric oxide and cytokine responses to endotoxemia. Alcoholism: Clin Exp Res 28(6):848-854.

Li Z, Kang SS, Lee S and Rivier C. 2005 Effect of ethanol on the regulation of corticotropin-releasing factor (CRF) gene expression. Mol Cell Neurosci 29:345-354.

Lee S and Rivier C. 2005 Role played by hypothalamic nuclear factor-κB in alcohol-mediated activation of the rat hypothalamic-pituiatry-adrenal axis. Endocrinology 146:2006-2014.

Richardson HN, Zorrilla EP, Mandyam CD and Rivier CL. 2006 Exposure to repetitive versus varied stress during prenatal development generates two distinct anxiogenic and neuroendocrine profiles in adulthood. Endocrinology 147:2506-2517.

Lee S, Craddock Z and Rivier C. 2011 Brain stem catecholamines circuitry: Activation by alcohol and role in the hypothalamic-pituitary-adrenal response to this drug. J Neuroendocrinol 23(6):531-541