Over twenty five years of research by our laboratories as well as others resulted in a number of landmark publications providing evidence implicating alterations in specific neuroendocrine networks in FAE-related abnormalities. The HPA axis is particularly vulnerable to the effects of prenatal alcohol exposure, which manifests as augmented responsiveness to a variety of stimuli in the mature offspring. Indeed, this hyperactivity of the HPA axis represents a hallmark of the FAE model. In theory, changes in ACTH release could stem from altered function at any level along the HPA axis: (a) the PVN of the hypothalamus, which integrates afferent signals and prompts the axis by manufacturing CRF; (b) the hypothalamus-median eminence pathway, through which CRF produced in the PVN perikarya is transported to nerve terminals in the median eminence for release into the portal circulation; (c) the anterior pituitary, where CRF binds to its receptors and regulated expression of the gene for proopiomelanocortin, the precursor polypeptide of ACTH; and (d) the adrenal glands, where ACTH binds to its receptors to stimulate the release of glucocorticoids which, in turn, regulate general metabolism as well as immune and reproductive functions.
At present, the most probable mechanisms include impaired steroid feedback and upregulated activity of PVN CRF neurons. Indeed, in a study evaluating the time course of action of in utero alcohol exposure, we observed increases in PVN CRF mRNA levels under basal conditions in 21-d-old male and female rats exposed to alcohol during the second week of gestation. More recently, we found that following alcohol treatment during embryonic development, 55-60-d-old offspring of both genders have enhanced activity of the HPA axis in general, and the PVN in particular (measured by CRF hnRNA and immediate-early gene transcript levels) in response to both neurogenic and systemic stressors, and that this upregulation occurs concomitant with augmented stress-induced ACTH secretion. Taken together, our laboratory's findings and those of others therefore indicate that prenatal alcohol-induced effects are primarily mediated at the level of the hypothalamus, not the pituitary or adrenal glands. However, we still do not know whether or not prenatal alcohol alters the activity of PVN cell bodies themselves, versus that of their afferents; and if it exerts this influence through specific neurotransmitters. Studies of the influence of maternal alcohol on the rat fetus’s developping brain have uncovered a vast array of possible mechanisms, including changes in overall CNS development, cell proliferation or survival, synaptic plasticity, levels of retinoids and neurotransmitters, electrophysiological, behavioral or memory-linked events, and poor nutrition, to quote only a few. We based our own studies on what we knew about some of the critical signals that influence PVN CRF neuronal activity, and will illustrate two such mechanisms here. The first pertains to nitric oxide (NO), an unstable gas that acts as a transmitter in many parts of the brain, including the hypothalamus. As discussed above, we had shown that the injection of NO donors into the brain lateral ventricles rapidly increased plasma ACTH levels, and that this response depended on endogenous CRF. As there was some evidence that prenatal alcohol might alter brain NO levels in the offspring, we tested the hypothesis that our FAE model was accompanied by upregulated hypothalamic levels of this gas or by an altered the HPA axis response to NO. We did not measure significant changes in gene expression levels of NO synthase, the enzyme responsible for NO formation, in the PVN of FAE rats. On the other hand, we observed that these animals displayed an altered HPA axis response to NO. We then showed that acute blockade of NO abolished the difference between the HPA axis activity of control rats and rats exposed to alcohol during embryonic development, thereby suggesting that dysregulated NO formation participated in the mechanisms modulating the effect of prenatal alcohol. In order to test the hypothesis that the HPA axis of rats exposed to alcohol prenatally responds abnormally to NO, we measured the ACTH responses of mature offspring of dams exposed to alcohol during gestation, to the intracerebroventricular (icv) injection of 3-morpholinosydnonimine (SIN-1), a drug that is readily converted to NO without requiring enzymatic bioactivation. We found that indeed, prenatal alcohol exposure exerted a significant, sexually dimorphic influence on the ACTH secretory response to icv NO. Compared to like-gender controls, female offspring exposed to alcohol prenatally were hyperresponsive while males were hyporesponsive to the lowest dose of SIN-1. Blunted ACTH release in E males when compared to controls was also observed after injection of the highest dose of SIN-1. These data support the hypothesis that alterations in HPA axis activity in adult offspring of alcohol-exposed dams may be related to changes in hypothalamic responsiveness to NO, and that this influence is gender-specific.
A variety of neurotransmitters are considered to exert key roles in regulating CRF PVN neurons, among which catecholamines (adrenaline and noradrenaline) occupy a central position to play a role in stress-induced activation of the HPA axis. Of the catecholaminergic neurons (adrenergic and noradrenergic) that innervate the PVN and respond to stress, most reside in the brain stem. Indeed, one of the main innervating pathways to the PVN is the brainstem noradrenergic system, in particular, the locus coeruleus, which is activated by stressors and modulates the HPA axis response to various stimuli. Likewise, adrenergic neurons of the brain stem (C1, C2, and C3) also innervate the PVN, where the most prominent projections (70%) are from the C1 region. They primarily innervate the parvocellular region of the PVN, where CRF perikarya reside. These neurons respond to stress and can influence ACTH in circulation via arterial pressure. Blockade of adrenergic receptors are known to interfere with this neuroendocrine function, and stress enhances NA release while lesions of catecholamineregic neurons impair HPA axis activity. Lastly, CRF-containing neurons in the bed nucleus of the stria terminalis are surrounded by cells that express tyrosine hydroxylase (TH), the rate-limiting step in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline that is used as a general marker of this synthesis. Likewise, neurons of the PVN are surrounded by projections from neurons that express phenylethanolamine N-methyltransferase (PNMT), an enzyme that converts noradrenaline to adrenaline. In view of these findings, we hypothesized that exposure to alcohol during embryonic development might alter HPA axis activity via changes in catecholamine-dependent mechanisms. There were no significant differences in response to adrenergic receptor agonists or in shock-induced CRF/TH immunoreactivity (ir) and neuronal activity, as determined by c-fos colocalization. In contrast, FAE female offspring exposed to footshocks showed a significant increase in the activity of adrenergic neurons in the C1 region of the brain stem. This is a very novel finding which suggests that catecholamine fibers originating from the brain stem play an hitherto unsuspected role in modulating the HPA axis hyperactivity that is the hallmark of the FAE model.
As indicated above, the PVN of rodents exposed to alcohol prenatally has been shown to exhibit upregulated CRF synthesis. An important and interesting observation is that in humans, many of the pathologies linked to prenatal alcohol exposure, including hyperactivity, increased susceptibility to infections and vulnerability to drug abuse, resemble conditions that can be induced by increased CRF levels in the brains of laboratory animals. Experiments designed to determine the consequences of prenatal alcohol treatment in rodents lacking the gene for CRF or its receptors will help unravel the putative role of increased brain CRF levels in these conditions. These studies will need to be done in mutant mice, and until very recently, prenatal alcohol delivery models in mice were cumbersome and difficult to implement. We therefore developed a system of individual chambers through which controlled delivery of alcohol vapors allows us to target specific blood alcohol levels (BALs) in mice without requiring the administration of an alcohol dehydrogenase inhibitor. As a proof of concept, we demonstrated that this new system could be used to expose pregnant BALB/c or C57BL/6 mice to alcohol, and that the hypothalamic-pituitary-adrenal (HPA) axis of their mature offspring exhibited the well known hyperactivity that has been previously documented in rats. A first series of experiments was designed to establish the parameters that resulted in specific BALs in non-pregnant adult male and female BALB/c as well as C57BL/6 mice exposed to various alcohol flow rates. Using information gathered from these experiments, we then chose a regimen of 6 h of daily vapor exposure in pregnant mice in order to determine whether this regimen would alter their mature offspring' HPA axis activity. Control dams were maintained in similar chambers but without alcohol. We first used control mice to assess plasma ACTH levels as a function of shock intensity as well as total duration of the shock session. The most suitable protocol was then used to measure shock-induced ACTH release in two-month old male and female offspring exposed to alcohol prenatally or not. We found that BALs increased as a function of the alcohol flow rates and remained within an acceptable range of homogeneity, consistency and reproducibility over the desired periods of time. There were no gender differences in BALs while vapors were delivered. On the other hand, there was a strain difference in that BALB/c mice displayed slightly higher BALs than C57BL/6. Females also exhibited a slightly more pronounced decrease in BALs, compared to males, once removed from the drug. Measurement of plasma ACTH levels as a function of the intensity and duration of the shock sessions indicated that 0.3 mA intensity, 1 sec duration shocks at the rate of 2 shocks/min for 20 min provided the most reliable protocol. We then used this protocol in pregnant mice. Alcohol exposure did not interfere with maternal weights during gestation. When offspring were tested at 8-9 weeks of age, male and female BALB/c, as well as female C57BL/6 mice exposed to alcohol vapors prenatally, exhibited significantly higher shock-induced plasma ACTH levels, compared to controls of the same strain. Collectively, our results indicate that the individual alcohol chamber system that we have developed offers a reliable means of exposing mice to alcohol so that they reach predetermined BALs in the absence of the pharmacological manipulations often used to influence alcohol metabolism in this species. This system, which is compatible with normal weight gains, was used to provide evidence that as previously demonstrated in rats, adult murine offspring of alcohol-treated dams exhibit an hyperactive HPA axis. The development of protocols for use in mice offers the possibility of investigating the influence of alcohol in mutant animals with manipulations of specific genes of interest.
Brain CRF on one hand, and circulating GC on the other, exert important influences on key responses of the body to homeostatic challenges. Consequently, deregulation of their release is likely to induce significant pathogeneses. Children born to mothers who abused alcohol during pregnancy can display, for example, increased occurrence of infections, disorders in which abnormally elevated GC levels often play a role. These children also often show increased activity levels, attention deficits, higher incidence of drug abuse and/or depression. While none of these disorders have been unambiguously demonstrated to result from elevated CRF levels in humans, the influence of this peptide on behavior, drug consumption and mood disorders is well documented in pre-clinical studies (see above). In view of the effects of GC on the mobilization of sugars and fat reserves, as well as the peptides that regulate food intake, it is also likely that individuals who consistently release too much cortisol in response to various stimuli may develop metabolic disorders. Experiments carried out in mice lacking CRF or its receptors, or the use of CRF antagonists in human medicine, may allow us to determine the role played by CRF in disorders caused by prenatal alcohol exposure, and to develop therapies that are palliative or restorative.
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Lee S and Rivier C. 1994 Prenatal alcohol exposure alters the hypothalamic-pituitary-adrenal axis response of immature offspring to interleukin-1: is nitric oxide involved? Alcoholism: Clin Exp Res 18:1242-1247.
Ogilvie K and Rivier C. 1997 Prenatal alcohol exposure results in hyperactivity of the hypothalamic-pituitary-adrenal axis of the offspring: Modulation by fostering at birth and postnatal handling. Alcoholism: Clin Exp Res 21:424-429.
Kim CK, Giberson PK, Yui W, Zoeller RT and Weinberg J. 1999 Effects of prenatal ethanol exposure on hypothalamic-pituitary-adrenal responses to chronic cold stress in rats. Alcoholism: Clin Exp Res 23:301-310.
Lee SY, Schmidt D, Tilders F and Rivier CL. 2000 Increased activity of the hypothalamic-pituitary-adrenal axis of rats exposed to alcohol in utero: Role of altered pituitary and hypothalamic function. Mol Cell Neurosci 16:515-528.
Lee S, Blanton CA and Rivier C. 2003 Prenatal ethanol exposure alters the responsiveness of the rat hypothalamic-pituitary-adrenal axis to nitric oxide. Alcoholism: Clin Exp Res 27:962-969.
Kang S, Cole M, Lee S and Rivier C. 2004 Development of individual inhalation chambers for mice: Validation in a model of prenatal alcohol. Alcoholism: Clin Exp Res 28:1549-1556.
Choi IY, Lee SO and Rivier CR. 2008 Novel role of adrenergic neurons in the brain stem in mediating the hypothalamic-pituitary axis hyperactivity caused by prenatal alcohol exposure. Neuroscience 155:888-901.
Lee S, Choi I, Kang S, and Rivier C. 2008 Role of various neurotransmitters in mediating the long-term endocrine consequences of prenatal alcohol exposure. Ann NY Acad Sci 1144:176-188
Allen CD, Rivier CL, Koob GF and Lee S. 2011 Adolescent alcohol exposure alters the central brain circuits known to regulate the stress response. Neuroscience 182:162-168. PMCID: PMC3085552.
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