Effects of prenatal ethanol exposure on basal limbic-hypothalamic-pituitary-adrenal regulation: role of corticosterone

BACKGROUND: Rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness and changes in central HPA regulation following exposure to stressors. Whether ethanol-induced alterations in basal HPA regulation play a role in mediating HPA hyperresponsiveness remains unclear. We utilized adrenalectomy (ADX), with or without corticosterone (CORT) replacement, to investigate basal HPA function and the role of CORT in mediating ethanol-induced alterations. METHODS: Adult males and females from prenatal E, pair-fed (PF), and ad lib-fed control (C) groups were terminated at the circadian peak, 7 days following sham surgery or ADX, with or without CORT replacement. Plasma levels of CORT and adrenocorticotropin (ACTH), and mRNA levels of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the paraventricular nucleus, CRH Type 1 receptor (CRH-R1) and pro-opiomelanocortin (POMC) in the anterior pituitary, and mineralocorticoid (MR) and glucocorticoid (GR) receptors in the dorsal hippocampus were determined. RESULTS: Adrenalectomy resulted in significantly greater plasma ACTH elevations in E and PF males, and parallel CRH mRNA elevations in both E and PF males and females compared with their C counterparts. In contrast, pituitary CRH-R1 mRNA levels were lower in E compared with C males, with no differences in POMC. In addition, in response to ADX, E females showed a greater MR mRNA response, and E males showed a greater GR mRNA response compared with their C counterparts, and CORT replacement was ineffective in normalizing ADX-induced alterations in ACTH levels in E and PF females, hippocampal MR mRNA levels in E males, and AVP mRNA levels in PF males and females. CONCLUSIONS: Together, these data indicate that the prenatal ethanol exposure induces HPA dysregulation under basal conditions at multiple levels of the axis, resulting in alterations in both HPA drive and feedback regulation and/or in the balance between drive and feedback. While some effects may be nutritionally mediated, it appears that the mechanisms underlying basal HPA dysregulation may differ between E and PF animals rather than occurring along a continuum of effects on the same pathway. Altered basal HPA tone may play a role in mediating the HPA hyperresponsiveness to stressors observed in E offspring.     

Onset of glucocorticoid responsiveness of anterior pituitary corticotrophs during development is scheduled by corticotropin-releasing factor

Expression of the CRF gene in the hypothalamus and that of the POMC gene in the anterior pituitary are reduced during the first week of life in the rat. During this so-called stress nonresponsive period (SNRP), stimuli such as ether vapors, electroshocks, and hypoxia do not elicit ACTH secretion from the pituitary, as occurs later in development. The current hypothesis to explain the SNRP is an increased negative glucocorticoid feedback on POMC and CRF synthesis and/or release during this time. To test this hypothesis we studied the effects of adrenalectomy (ADX) on anterior pituitary POMC mRNA expression. In 7-day-old rats POMC mRNA levels were increased only 3-fold 48 h post-ADX, compared to a 7-fold increase in 14-day-old animals. This blunted effect of endogenous glucocorticoid removal on pituitary POMC mRNA could be due to decreased up-regulation of CRF after removal of glucocorticoids or normal up-regulation of CRF but decreased pituitary responsiveness to CRF relative to those in 14-day-old animals. Therefore, we studied in vitro beta-endorphin release from pituitaries obtained from 7- and 14-day-old rats. CRF stimulated basal beta-endorphin release to the same extent in pituitaries from both groups. The inhibition by corticosterone of CRF-stimulated beta-endorphin secretion was also indistinguishable in pituitaries obtained from 7- or 14-day-old rats. Since the responsiveness of the 7-day-old pituitary was normal, the blunted enhancement of POMC biosynthesis after ADX must be mediated at the level of the hypothalamus. Indeed, in situ hybridization showed that while in 14-day-old rats ADX induced a significant increase [190 +/- 10% (+/- SE) of control; n = 5; P less than 0.0005] in hypothalamic mRNA levels, ADX did not change the expression of the CRF gene in the paraventricular nucleus of 7-day-old rats, indicating a lack of glucocorticoid modulation of hypothalamic CRF synthesis. Finally, we studied the effects of 48 h CRF treatment on the post-ADX increase in POMC mRNA levels in 7-day-old rats. Daily injections of 200 ng CRF/rat induced an increase in anterior pituitary POMC mRNA concentrations [669 +/- 139% (+/- SE) of control; n = 6; P less than 0.02 vs. adrenalectomized vehicle-treated rats] comparable to that in adrenalectomized untreated 14-day-old rats. In conclusion, our data indicate that the glucocorticoid regulation of hypothalamic CRF gene expression is not mature during the first week of life, i.e. within the so-called SNRP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased Postnatal Testosterone Surge in Male Rats Exposed to Ethanol During the Last Week of Gestation

Prenatal alcohol exposure in the rat is known to interfere with the neurobehavioral sexual differentiation of the male brain. Because normal sexual differentiation of the male brain requires adequate levels of perinatal testosterone, we examined the effect of prenatal ethanol exposure on (1) the postnatal surge of testosterone and (2) the in vitro secretion of testosterone in response to luteinizing hormone (LH) stimulation of testes from fetal alcohol exposed (FAE) animals and controls. Sprague-Dawley dams were administered a fortified liquid diet containing 35% ethanol-derived calories, a pair-fed (PF) isocaloric liquid diet, or given ad libitum access to dry lab chow (CF). Dams were administered the liquid diets from days 7 or 14 through parturition. The postnatal surge of testosterone in FAE males was studied only in animals exposed to ethanol from day 14 through parturition. In the first experiment, FAE and PF males and females were delivered by cesarian section on day 22 of gestation (E22) and trunk blood collected at 0, 60, 120, and 240 min after parturition. Experiment 2 measured plasma testosterone in male pups that were killed at 0, 60, 120, 240, 360, and 480 min after delivery. Results showed that the postnatal testosterone surge of FAE males in both experiments was significantly attenuated compared with PF controls. No effect of prenatal ethanol was observed in female offspring. Female testosterone levels were several fold lower than male littermates, and no evidence of a postnatal testosterone surge was observed. Production of testosterone from testes was studied using an automated perfusion system. The secretory testosterone response to LH stimulation of testes on day 18 (E18) of gestation and at birth (E22) was similar in testes from FAE animals to that observed in testes from PF and CF controls. The response of all testes to a pulse of LH (5 nM) delivered over a 5-min period (flow rate = 200 μl/min) from animals at E18 was significantly greater than the response observed at day 22. No morphological abnormalities were detected by visual inspection of electron photomicrographs of testes from FAE animals at E18 or E22. The in vitro response to LH of testes from FAE animals indicates that the reduction in the postnatal testosterone surge in FAE animals is not due to a decrease in testicular sensitivity to LH.     

Reduced hypothalamic POMC and anterior pituitary CRF1 receptor mRNA levels after acute, but not chronic, daily "binge" intragastric alcohol administration

BACKGROUND: Endogenous corticotropin-releasing factor (CRF), its pituitary CRF1 receptor, and proopiomelanocortin (POMC) may be involved in the hypothalamic-pituitary-adrenal (HPA) responses to alcohol. METHODS: Alcohol (1.5 g/kg) or water was administered intragastrically to male Fischer rats after the "binge" pattern regimen, that is, three times daily at 1 hr intervals at the beginning of the light cycle. The levels of CRF, CRF1 receptor, and POMC mRNAs in the hypothalamic-pituitary axis were measured after acute (1 day) or chronic (14 days) binge pattern alcohol administration. Plasma levels of ACTH and corticosterone were measured to examine time-dependent alterations of HPA responses. RESULTS: Plasma ACTH and corticosterone levels were elevated dramatically after 1 day of acute binge pattern alcohol administration. After 14 days of chronic alcohol, however, no elevation in plasma ACTH levels and an attenuated elevation in plasma corticosterone levels were found. CRF mRNA levels in the hypothalamus were not altered after either acute or chronic alcohol administration. CRF1 receptor mRNA levels in the anterior pituitary were decreased significantly after acute administration, with no change after chronic alcohol administration. POMC mRNA levels in the anterior pituitary were not altered by either acute or chronic alcohol administration. In the hypothalamus, POMC mRNA levels were decreased significantly after acute but not chronic binge alcohol administration. CONCLUSIONS: These results suggest that (1) rats exposed to chronic binge alcohol develop tolerance in HPA activity, as shown by no elevation of ACTH and an attenuated corticosterone response to chronic alcohol after initial dramatic elevations by acute alcohol administration; (2) a concurrent acute decrease in CRF1 receptor mRNA levels in the anterior pituitary is associated with increased HPA activity, and (3) alterations of POMC gene expression in the hypothalamic region may have implications for a molecular understanding of the neuroendocrine response to alcohol.     

Corticotrope response to removal of releasing factors and corticosteroids in vivo

In the intact rat, adrenalectomy (ADX) is known to result in increased ACTH synthesis, content, and secretion from the anterior pituitary compared with those in the sham-adrenalectomized control. Treatment of adrenalectomized, rats with corticosterone prevents or reverses these changes in ACTH. Because corticosterone is known to act both at the corticotrope and at the level of CRF secretion, it is not clear to what extent the ACTH response to ADX is a result of removal of glucocorticoids from the pituitary per se. To test the role of brain input as well as the role of glucocorticoids on the corticotrope response to ADX, we performed the following experiment. Rats were prepared with anterolateral hypothalamic deafferentations (lesion) which severed CRF and arginine vasopressin cell bodies in the hypothalamus from their axonal endings in the median eminence and posterior pituitary. Control rats were subjected to sham lesions. Two days later, half of the rats in each group were subjected to either ADX or sham ADX; a subgroup of the lesioned rats was provided at the time of adrenal surgery with a constant infusion of rat CRF. Five days later, all rats were killed, and anterior pituitary levels of proopiomelanocortin (POMC) mRNA, ACTH, and protein; plasma ACTH and corticosterone, and adrenal and thymus weights were measured. In sham-lesioned rats, ADX resulted in increases in POMC mRNA, and plasma ACTH of 2.5- and 12-fold, respectively, compared to sham-adrenalectomized controls. In the absence of hypothalamic drive (lesion only), there were no responses of any of these variables to ADX. In lesioned rats driven with CRF, ADX resulted in increases in POMC mRNA and plasma ACTH of 2.2- and 2.6-fold, respectively, compared to sham ADX. After consideration of the three variables indicating ACTH synthesis, storage, and secretion and comparison of the results of ADX vs. sham ADX within and across the sets of animals, we conclude that 1) there is no autonomous response of the corticotrope to ADX; 2) the removal of corticosterone from the anterior pituitary may account for the majority of the effects of ADX on ACTH synthesis; and 3) the normal response to ADX requires secretion of CRF and increased secretion of another ACTH-releasing factor (possibly arginine vasopressin) that causes increased secretion but little synthesis of ACTH.     

Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat.

Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH. In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression. These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.     

Effects of prenatal ethanol exposure on hypothalamic-pituitary-adrenal responses to chronic cold stress in rats

Animals prenatally exposed to ethanol typically exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors. In contrast to previous studies that have investigated effects of prenatal ethanol exposure on HPA responses to acute or intermittent stressors, our study investigated HPA responses to a chronic continuous stressor, cold stress (4 degrees C for 0, 1, or 3 days). We tested the hypothesis that prenatal ethanol exposure would result in increased plasma corticosterone (CORT) and adrenocorticotropin (ACTH) responses and increased peptide [corticotropin-releasing factor and vasopressin] mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus compared to that in control animals. In addition, CORT and ACTH responses were measured after exposure to an acute stressor (i.p. isotonic saline injection), superimposed during chronic cold exposure, to examine possible sensitization of the HPA response to the acute stress. Thus, blood samples were collected at the end of each of the three periods of cold exposure, either before (0 min) or 15 min after acute stress. The subjects were adult male and female Sprague-Dawley rat offspring from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) treatment groups. Exposure to cold stress resulted in significant body weight loss in E males at 1 day and in both males and females of all prenatal treatment groups by 3 days of cold stress. Males in all prenatal groups also exhibited significant increases in adrenal weight:body weight ratios. Cold stress alone (0 min condition) increased CORT levels in E males and overall ACTH levels in E males and females compared to controls. ACTH levels were also higher overall in E compared to control males after acute stress (15 min condition). Sensitization of the CORT response to acute stress was observed in males but not females across all prenatal treatment groups. Corticotropin-releasing factor and vasopressin mRNA levels in the PVN were not significantly affected by prenatal treatment or chronic cold stress in either males or females. In contrast, both males and females displayed increases in PVN thyrotropin-releasing hormone (TRH) mRNA levels after cold stress. These data support and extend previous work demonstrating differential effects of prenatal ethanol exposure on HPA responsiveness of male and female offspring, and suggest that E males may be more vulnerable to the effects of chronic cold stress than E females.     

Prenatal Ethanol Exposure Induces a Sexually Dimorphic Effect on Daily Water Consumption in Prepubertal and Adult Rats

Previous studies have shown that female rats consume significantly more water than males on a weight basis. Because exposure to alcohol during the last week of gestation is associated with incomplete behavioral defeminization in male rats, we examined daily water intake in fetal alcohol-exposed (FAE) males and females. Time-pregnant multiparous Sprague-Dawley dams were administered an ethanol liquid diet containing 35% ethanol-derived calories from day 14 through parturition. At 80 days of age, daily water consumption of FAE males and female litter representatives was measured for 7 days. FAE males, but not females, consumed significantly more water than their pair-fed counterparts. Subsequent experiments determined that the increased water consumption in FAE males is present prepubertally, persists into mature adulthood, and is not influenced by prenatal or postnatal castration. Chronic estrogen treatment induced large increases in water consumption, but consumption of FAE males remained elevated over elevated pair-fed male consumption, indicating that pituitary sensitivity to estrogen was not increased in FAE males. Morphometric studies of hypothalamic nuclei containing vasopressin cells revealed no long-term effects of prenatal ethanol exposure on the volume of the supraoptic nucleus or paraventricular nucleus in males, nor was an effect observed in the ventromedial nucleus measured as a control. In FAE females, the volume of the paraventricular nucleus was significantly smaller than chow-fed controls. Whereas baseline plasma and pituitary arginine vasopressin (AVP) levels of FAE animals and pair-fed controls were not significantly different, AVP content was significantly reduced in the septal/bed nucleus region in brains of FAE animals of both sexes. Overall, these data indicate that prenatal ethanol exposure increases male water consumption in the absence of alterations in basal plasma AVP.     

Changes in the hypothalamo-corticotrope axis after bilateral adrenalectomy: evidence for a median eminence site of glucocorticoid action.

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of fetal adrenalectomy on messenger ribonucleic acid for proopiomelanocortin in the anterior pituitary and for corticotropin-releasing hormone in the paraventricular nucleus of the ovine fetus

In the ovine fetus, adrenalectomy at 90-120 days gestational age (dGA) results in a gradual increase in basal concentrations of fetal plasma ACTH beginning at approximately 122 dGA. Bilateral adrenalectomy at 116-119 dGA also results in an increase in POMC mRNA in the fetal pituitary. It is not known whether both the paraventricular nuclei (PVN) of the hypothalamus and the anterior pituitary of the ovine fetus are responsive in late gestation to the removal of cortisol negative feedback. The purpose of this study was to determine the subsequent effect of fetal adrenalectomy at 118-121 dGA on the CRH mRNA content in fetal PVN and on POMC mRNA in the fetal anterior pituitary at 134 dGA. Mature Rambouellet-Columbia cross-bred ewes (n = 10), bred on a single occasion only and carrying fetuses of known gestational ages, were used. Both fetal adrenal glands were exposed via a retroperitoneal approach and removed [adrenalectomized (ADX); n = 5]. In control fetuses (CONT; n = 5) adrenal glands were exposed and isolated, but not removed. At 134 dGA, fetal plasma cortisol concentrations were significantly greater in CONT fetuses (7.2 +/- 2.5 ng/ml) than in ADX fetuses (mean +/- SD, 1.97 +/- 0.9 ng/ml; P less than 0.025). At 134 dGA the fetal PVN was removed by micropunching, and the anterior pituitary was separated from neurointermediate and posterior lobes after necropsy. Total RNA was prepared by the guanidium isothiocyanate-cesium chloride method and subjected to Northern analysis using specific cDNA probes to CRH and POMC. After autoradiography, quantification of mRNA was performed by scanning densitometry. Quantities of specific hybridization signal for POMC and CRH were normalized to the content of actin mRNA in each individual sample. RNA prepared from PVN exhibited a single specifically hybridizing band for CRH of approximately 1300 nucleotides. RNA prepared from anterior pituitary exhibited a single specifically hybridizing band for POMC at approximately 1300 nucleotides. Anterior pituitary POMC mRNA was significantly increased (P less than 0.025) in ADX fetuses (236 +/- 32% of CONT). CRH mRNA in PVN was greater in ADX fetuses than in CONT fetuses (P less than 0.05; mean +/- SEM, 179 +/- 21% of CONT). Adrenalectomy in fetal sheep significantly increased expression of CRH and POMC. We conclude that the increased levels of mRNA for CRH and POMC indicate that both the fetal PVN (CRH) and the anterior pituitary (POMC) are responsive to removal of the primary source of circulating glucocorticoid at this gestational age.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sex Differences in Ethanol-Induced Hypothermia in Ethanol-Naïve and Ethanol-Dependent/Withdrawn Rats

Background: Human and animal findings indicate that males and females display major differences in risk for and consequences of alcohol abuse and alcoholism. These differences are in large part mediated by sex‐specific hormonal environments. Gonadal and adrenal secretory products are known to modulate the neurobehavioral responses of ethanol (EtOH) dependence and withdrawal. However, the effects of these steroids on physiological adaptations, such as thermoregulation, are less well established. To study the role of sex‐related hormones in mediating sex differences in the hypothermic response to acute challenge with EtOH, we compared the EtOH‐induced hypothermic responses of EtOH‐naïve male and female rats and EtOH‐dependent (on the third day of withdrawal) male and female rats before (intact) and after depletion of all gonadal and adrenal steroids by gonadectomy (GDX) with or without adrenalectomy (ADX). Methods: Intact and GDX male and female rats, with or without ADX, were fed an EtOH‐containing liquid diet for 15 days while control (EtOH‐naïve) rats were pairfed the isocaloric liquid diet without EtOH or fed normal rat chow and water. On the third day of withdrawal from the EtOH diet we tested the hypothermic response to EtOH challenge (1.5 g/kg BWt, ip). Blood alcohol content (BAC) and corticosterone (CORT) content were analyzed in a separate series of intact and GDX males and females with and without ADX in response to the EtOH challenge. Results: Ethanol‐induced hypothermia was significantly greater and its duration significantly longer in intact males than females when subjects were EtOH‐naïve. EtOH‐induced hypothermia was significantly greater in intact females than males by the third day of withdrawal from EtOH dependence. GDX in males significantly shortened the duration of the hypothermic response and tended to blunt EtOH‐induced hypothermia while response duration was significantly extended by GDX in females that tended to enhance EtOH‐hypothermia. EtOH‐induced hypothermia was significantly enhanced and its duration significantly lengthened by combined GDX and ADX in EtOH‐naïve and ‐withdrawn males and by combined GDX and ADX in EtOH‐naïve but not EtOH‐withdrawn females. These differential EtOH‐induced hypothermic responses did not appear to be caused by differences in EtOH handling among the groups. The absence of adrenal activation by EtOH in the GDX–ADX males and females contributes to their enhanced EtOH‐induced hypothermic responses. Conclusions: These results implicate the direct and indirect effects of removal of gonadal and adrenal secretory products as mediators of the thermoregulatory actions of EtOH.     

Ontogeny of expression of the corticotropin-releasing factor gene in the hypothalamic paraventricular nucleus and of the proopiomelanocortin gene in rat pituitary

The ontogeny of expression of the CRF gene in the paraventricular hypothalamic nucleus and POMC gene in the pituitary was studied in rats using in situ hybridization histochemistry and Northern blotting techniques, respectively. CRF mRNA was first detected on day 17 of gestation (E17) in the paraventricular nucleus of the hypothalamus. The levels of hypothalamic CRF mRNA increased progressively from E17 to E19-E20, decreased during the perinatal period, and increased thereafter. The levels of POMC mRNA in the pituitary paralleled the variations in hypothalamic CRF mRNA, showing a peak on E20-E21. POMC mRNA levels in the anterior pituitary were decreased on days 4-7 after birth (P4-P7) and increased steadily thereafter. In contrast to levels in the anterior pituitary, POMC mRNA levels increased steadily from P1 to P21 in the neurointermediate lobe of the pituitary. These data indicate that the expression of both the CRF and POMC genes in the paraventricular nucleus and anterior pituitary, respectively, are reduced during the first week of life, i.e. within the so-called stress nonresponsive period. Our observations suggest that an impaired regulation of ACTH and CRF synthesis due to an immature neuronal pathway within the brain or increased glucocorticoid feedback may account for the stress nonresponsive period.     

The Effect of Cold Stress on Lymphocyte Proliferation in Fetal Ethanol-Exposed Rats

Prenatal ethanol exposure and stress have each been shown to have significant effects on the immune system. This study examined the possible interactive effects of prenatal ethanol exposure and exposure to stress later in life on the immune system. Differential vulnerability to these challenges in female and male offspring was assessed. At 5 to 6 months of age, female and male offspring from prenatal ethanol-exposed (E), pair-fed (PF), and ad libitum-fed control (C) conditions were exposed to 0, 1, or 3 days of cold (4°C). At the end of the cold period, the proliferative response of splenic lymphocytes to the mitogens concanavalin A (Con A) and pokeweed mitogen (PWM) was assessed. The data demonstrate a significant interactive effect between prenatal ethanol exposure and cold stress in female offspring. After 1 day of cold stress, E females had significantly increased PWM-induced lymphocyte proliferation compared with PF and C females, and significantly increased Con A-induced lymphocyte proliferation compared with PF females. There were no differences in PWM or Con A-induced lymphocyte proliferation among E, PF, and C females after 0 or 3 days of cold stress, nor among E, PF, and C males on any test day. Regardless of prenatal treatment, females exposed to 1 or 3 days of cold had significantly greater basal plasma corticosterone levels than females not exposed to cold. In contrast, only E males exposed to 1 or 3 days of cold had significantly increased basal plasma corticosterone levels, compared with E males not exposed to cold; PF and C males showed no significant change in basal corticosterone after cold stress. These data demonstrate that, in response to the challenge of cold stress, changes in lymphocyte proliferation to PWM and Con A may occur selectively in E females. Moreover, the interactive effects of prenatal ethanol and cold stress may result in enhanced rather than suppressed immune responsiveness.     

Effects of Prenatal Ethanol Exposure on Hypothalamic-Pituitary-Adrenal Regulation After Adrenalectomy and Corticosterone Replacement

BACKGROUND: Previous studies have demonstrated that rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness, demonstrated by increased and/or prolonged elevations of adrenocorticotropin (ACTH) and/or corticosterone (CORT) after stress. This study investigated possible mechanisms of HPA hyperresponsiveness in E rats by manipulating CORT feedback regulation of HPA activity via adrenalectomy (ADX) with or without CORT replacement. METHODS: Male Sprague-Dawley rat offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) groups were tested at 90 to 120 days of age. Rats were either sham-operated or underwent ADX, with or without CORT replacement. CORT (25 microg/ml) was replaced via the drinking water to achieve basal plasma CORT levels and maintain a phasic CORT signal. Seven days after surgery, animals were decapitated at the diurnal peak either under basal conditions or after a 15-min restraint stress, and trunk blood was collected. RESULTS: After ADX, loss of the CORT feedback signal resulted in increased plasma ACTH in all groups compared with those in sham animals. In addition, under basal conditions, ADX E rats had significantly greater plasma ACTH levels than both PF and C rats. However, no differences were seen in ADX rats after stress. CORT replacement after ADX was partially effective in normalizing ACTH levels under both basal and stress conditions, with no differences among E, PF, and C animals. CONCLUSIONS: These results suggest that E males may exhibit enhanced stimulatory inputs to the hypothalamus, increased pituitary sensitivity to secretagogues, or both, which may be revealed after ADX. In contrast, E animals seem similar to controls in their ability to use an exogenous CORT signal to regulate HPA activity.     

Moderate caloric restriction during gestation results in lower arcuate nucleus NPY‐ and αMSH‐neurons and impairs hypothalamic response to fed/fasting conditions in weaned rats

Aim: We aimed to characterize the developmental programming effects of moderate caloric restriction during early pregnancy on factors involved in hypothalamic control of energy balance. Methods: Twenty‐five‐days‐old offspring Wistar rats from 20% caloric restricted dams (from 1 to 12 days of pregnancy) (CR) and from control dams were studied under fed and 12 h fasting conditions. Morphometric studies on arcuate nucleus (ARC) and determinations of circulating parameters and hypothalamic levels of neuropeptide Y (NPY), proopiomelanocortin (POMC), long‐form leptin receptor (ObRb), insulin receptor (InsR) and suppressor of cytokine signalling‐3 (SOCS‐3) mRNA were performed. Results: CR animals did not show different body weight with respect to their controls, but presented higher food intake. They exhibited lower neuropeptide Y‐ and α‐melanocyte‐stimulating hormone‐neurons (decreases of 18 and 13% in males, and 10 and 18% in females respectively) and lower total cells (decrease of 3% in males and 18% in females) in ARC. Under fed conditions, CR animals presented lower circulating leptin and ghrelin levels (decreases of 37 and 43% in males, and 15 and 34% in females respectively); furthermore, hypothalamic POMC, NPY (only in females), ObRb and InsR mRNA levels were reduced (39, 16 and 26% in males, and 112, 33, 61 and 56% in females), and those of SOCS‐3 were increased (86% in males and 74% in females). Unlike control animals, under fasting conditions, ObRb, InsR and POMC mRNA levels did not decrease in CR females, and NPY mRNA decreased instead of increase in CR males. Conclusions: Moderate caloric restriction during gestation affects offspring hypothalamic structure and function, impairing its response to fed/fasting conditions, which suggests a predisposition to insulin and leptin resistance.     

Induction of fos-like immunoreactivity in hypothalamic corticotropin-releasing factor neurons after adrenalectomy in the rat

To identify brain sites responding to the removal of corticosterone feedback by adrenalectomy (ADX), rat brains were processed for fos immunocytochemistry 1, 3, and 7 days after ADX, sham-ADX, or no surgery using a polyclonal antiserum to fos residues 132-154. Compared to SHAM, ADX rats exhibited strong fos-like immunoreactivity (FLI) only in the parvocellular neurons of the paraventricular hypothalamic nuclei (PVN) 1, 3, and 7 days after surgery. Replacement with a corticosterone pellet at the time of adrenalectomy (ADX + B) prevented this increase in PVN FLI in three of four rats at 1 day, all rats at 3 days, and two of seven rats 7 days after surgery; 100 micrograms/ml corticosterone in the drinking water for 2 days before perfusion reversed ADX-induced increases in PVN FLI in 7-day ADX rats. Providing 25 micrograms/ml corticosterone in the drinking water to ADX rats for 5 days after surgery did not prevent expression of PVN FLI, even though this dose has been shown to normalize morning basal ACTH levels in ADX rats. Virtually all parvocellular PVN neurons expressing FLI after ADX costained for CRF. Some parvocellular neurons also expressed both fos and vasopressin. In all rats, many brain regions expressed FLI that was not related to adrenalectomy. We conclude that the changes in neuronal FLI correlate with demonstrated changes in neuroendocrine activity after ADX; however, suppression of ADX-induced FLI may require higher replacement levels of corticosterone than inhibition of ADX-induced ACTH secretion.