Pair-fed Females Research Articles

Prenatal alcohol exposure alters methyl metabolism and programs serotonin transporter and glucocorticoid receptor expression in brain.

Prenatal alcohol exposure (PAE) programs the fetal hypothalamic-pituitary-adrenal (HPA) axis, resulting in HPA dysregulation and hyperresponsiveness to stressors in adulthood. Molecular mechanisms mediating these alterations are not fully understood. Disturbances in one-carbon metabolism, a source of methyl donors for epigenetic processes, contributes to alcoholic liver disease. We assessed whether PAE affects one-carbon metabolism (including Mtr, Mat2a, Mthfr, and Cbs mRNA) and programming of HPA function genes (Nr3c1, Nr3c2, and Slc6a4) in offspring from ethanol-fed (E), pair-fed (PF), and ad libitum-fed control (C) dams. At gestation day 21, plasma total homocysteine and methionine concentrations were higher in E compared with C dams, and E fetuses had higher plasma methionine concentrations and lower whole brain Mtr and Mat2a mRNA compared with C fetuses. In adulthood (55 days), hippocampal Mtr and Cbs mRNA was lower in E compared with C males, whereas Mtr, Mat2a, Mthfr, and Cbs mRNA were higher in E compared with C females. We found lower Nr3c1 mRNA and lower nerve growth factor inducible protein A (NGFI-A) protein in the hippocampus of E compared with PF females, whereas hippocampal Slc6a4 mRNA was higher in E than C males. By contrast, hypothalamic Slc6a4 mRNA was lower in E males and females compared with C offspring. This was accompanied by higher hypothalamic Slc6a4 mean promoter methylation in E compared with PF females. These findings demonstrate that PAE is associated with alterations in one-carbon metabolism and has long-term and region-specific effects on gene expression in the brain. These findings advance our understanding of mechanisms of HPA dysregulation associated with PAE.

Effects of Prenatal Ethanol Exposure on Hypothalamic‐Pituitary‐Adrenal Function Across the Estrous Cycle

Rats prenatally exposed to ethanol (E) typically show increased hypothalamic-pituitary-adrenal (HPA) responses to stressors in adulthood. Importantly, prenatal ethanol may differentially alter stress responsiveness in male and female offspring, suggesting a role for the gonadal hormones in mediating the effects of ethanol on HPA activity. We investigated the role of ethanol-induced changes in hypothalamic-pituitary-gonadal (HPG) activity in the differential HPA regulation observed in E compared to control females across the estrous cycle. Peripheral hormones and changes in central neuropeptide mRNA levels were measured across the estrous cycle in adult female offspring from E, pair-fed (PF) and ad libitum-fed control (C) dams. Ethanol females showed normal estrous cyclicity (vaginal smears) but delayed sexual maturation (vaginal opening). Both HPG and HPA activity were differentially altered in E (and in some cases, PF) compared to control females as a function of estrous cycle stage. In relation to HPG activity, E and PF females had higher basal and stress estradiol (E(2)) levels in proestrus compared to other phases of the cycle, and decreased GnRH mRNA levels compared to C females in diestrus. Further, E females had greater variation in LH than PF and C females across the cycle, and in proestrus, only E females showed a significant LH increase following stress. In relation to HPA activity, both basal and stress CORT levels and overall ACTH levels were greater in E than in C females in proestrus. Furthermore, AVP mRNA levels were increased overall in E compared to PF and C females. These data demonstrate ethanol-induced changes in both HPG and HPA activity that are estrous phase-specific, and support the possibility that changes in HPA activity in E females may reflect differential sensitivity to ovarian steroids. E females appear to have an increased HPA sensitivity to E(2), and a possible shift toward AVP regulation of HPA activity. That PF were similar to E females on some measures suggests that nutritional effects of diet or food restriction played a role in mediating at least some of the changes observed.

Effects of Prenatal Ethanol Exposure on Basal Limbic–Hypothalamic–Pituitary–Adrenal Regulation: Role of Corticosterone

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. 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. 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. 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.

Effects of Mineralocorticoid and Glucocorticoid Receptor Blockade on Hypothalamic–Pituitary–Adrenal Function in Female Rats Prenatally Exposed to Ethanol

Rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness, demonstrated by increased and/or prolonged elevations of adrenocorticotropic hormone (ACTH) and/or corticosterone (CORT) in response to stressors. The present study examined the possible role of CORT feedback deficits in mediating this hyperresponsiveness by examining HPA function following mineralocorticoid (MR) and glucocorticoid (GR) receptor blockade. Adult female Sprague-Dawley offspring from E, pair-fed (PF), and control (C) groups were injected subcutaneously with the MR antagonist spironolactone (SPIRO; 30 mg/kg bw), the GR antagonist RU38486 (120 mg/kg bw), or vehicle. One hour postinjection, blood samples (0 minutes) were taken via jugular cannulae to obtain a measure of prestress ACTH and CORT levels. Rats were then loosely restrained for 1 hour, and samples were taken during (15, 30, and 60 minutes) and then 1 hour following stress, for determination of plasma ACTH and CORT levels. Both SPIRO and RU38486 significantly increased prestress ACTH levels in E compared with both PF and C females. In contrast, RU38486 significantly increased ACTH levels in C compared with PF females during stress and in C compared with E females during recovery. CORT levels were increased during stress in E females in response to SPIRO, and RU38486 increased the CORT response during stress in PF and during recovery in E and PF females compared with vehicle. E females showed enhanced HPA responses to both MR and GR blockade compared with PF and C before restraint as well as a different pattern of responsivity during and following restraint. While receptor blockade had some effect on CORT responses in PF females, changes in ACTH appear specific to ethanol. These findings suggest that the balance between HPA drive and feedback may be altered in E compared with C females.

Prenatal ethanol exposure alters sensitivity to the effects of corticotropin-releasing factor (CRF) on behavior in the elevated plus-maze

Animals prenatally exposed to ethanol (E) exhibit behavioral alterations in a wide variety of stressful or challenging tasks. The hypothalamic peptide corticotropin-releasing factor (CRF) is known to play a crucial role in integrating an organism's behavioral responses to environmental stressors or challenges. Previous research indicates that E animals exhibit increased hypothalamic-pituitary-adrenal (HPA) reactivity, including increased hypothalamic CRF expression under both basal and stress conditions. However, the possible role of CRF in mediating the behavioral changes observed in E animals remains to be determined. The current study investigated the hypothesis that E animals may be differentially sensitive to the effects of CRF on behavior in the elevated plus-maze, a task widely used to assess anxiety-like behavior in rodents. Sprague-Dawley offspring from prenatal E, pair-fed (PF), and ad lib-fed control (C) groups were tested at 60-90 days of age. Thirty minutes prior to a 5 min test on the elevated plus-maze, animals received an icv infusion of vehicle (VEH) or CRF (males: 0.75 microg or 1.5 microg ; females: 1.0 microg or 2.0 microg ). Under VEH conditions, E males showed greater activity (more total arm entries) than PF and C males and both E males and E and PF females showed less anxiety-like behavior (more open arm entries) than their PF and/or C counterparts. As expected, CRF treatment resulted in fewer open arm, closed arm and total arm entries, and total rears in both males and females in all prenatal groups, and increased time in the closed arms in males compared to that in their VEH-treated counterparts. Importantly, the effects of CRF were most pronounced in E animals. That is, when normalized for prenatal group differences following VEH treatment, CRF-treated E males showed fewer total arm entries and total rears than PF and C males, and CRF-treated E and PF females showed fewer open arm entries than C females. These results support and extend previous findings demonstrating that E animals show altered behavior in aversive or stressful situations. While some effects of CRF in females may be mediated partially by nutritional effects of ethanol, the data overall suggest that the behavioral alterations observed in E animals may be due, at least in part, to increased sensitivity to CRF.

Alcohol Affects the Skeletal Muscle Proteins, Titin and Nebulin in Male and Female Rats

Alcoholic myopathy is characterized by decreased protein synthesis and contents resulting in atrophy of muscle fibers. We investigated the effect of alcohol on the cytoskeletal muscle proteins, nebulin and titin. Because women are more susceptible than men to the toxic effects of alcohol, male and female rats were included. Four groups were investigated: alcoholic males, pair-fed males, alcoholic females, pair-fed females. Alcohol consumption per unit body weight was 12.9 g/kg.d, with no difference between males and females. After 10 wk, male and female rats fed alcohol had lower gastrocnemius and plantaris protein and RNA contents (P < 0.001), with no effect on soleus, indicating myopathy of type II fibers. The gastrocnemius was fractionated to measure myofibrillary protein contents. Low percentage SDS-gel electrophoresis was performed to determine myosin heavy chain (MHC), nebulin and titin contents. Alcohol reduced gastrocnemius myofibrillary protein and MHC contents, and the plantaris RNA/protein ratio (P < 0.01). The titin/MHC and nebulin/MHC ratios were unaffected, suggesting a concomitant reduction in titin and nebulin. The decreases in titin and nebulin contents may affect muscle function. An interaction between gender and alcohol was noted for the plantaris RNA/protein ratio (P < 0.025), suggesting a reduced capacity for muscle protein synthesis in females.

Glucocorticoid Fast Feedback Is Not Altered in Rats Prenatally Exposed to Ethanol

Animals exposed in utero to ethanol exhibit hormonal hyperresponsiveness to stressors in adulthood. One possible mechanism for this hyperresponsiveness is a deficit in negative feedback regulation of the hypothalamic-pituitary-adrenal axis. The present study tested the hypothesis that a deficit in the fast feedback time domain may play a role in the hormonal hyperresponsiveness in ethanol-exposed rats. Sprague-Dawley offspring from prenatal ethanol (E), pair-fed (PF), and ad lib-fed control (C) groups were tested in two experiments. Experiment 1 used a swim stress paradigm and tested animals at the trough of the corticosterone (CORT) circadian rhythm. Experiment 2 used ether stress and tested animals at the peak of the circadian rhythm. Animals were injected subcutaneously with CORT or saline and were immediately subjected to either a 5-min swim stress or a 1-min ether stress. Half the animals were terminated immediately after stress (5-min postinjection), and the rest were terminated 25 min later. Plasma levels of CORT and ACTH were assayed to determine whether E animals differed from control animals in showing a CORT-induced blunting of the ACTH response to the stressor, indicating alterations in fast feedback regulation. Injection of CORT significantly blunted the ACTH response to swim stress (experiment 1) in E, PF, and C females and males, compared with their saline-injected counterparts. There were no significant differences among groups. Similarly, CORT-injected males in E, PF, and C groups all exhibited a significantly blunted ACTH response to ether stress (experiment 2). CORT-injected C females also exhibited a significantly blunted ACTH response to ether stress, whereas E females showed an obvious CORT decrease that approached significance. However, PF females showed a clear deficit in fast feedback regulation. Together, these data suggest that: (1) CORT injection can serve as a fast feedback signal that can blunt the ACTH response to a stressor; and (2) prenatal ethanol exposure does not produce a deficit in hypothalamic-pituitary-adrenal feedback regulation in the fast feedback time domain.

MATERNAL ETHANOL CONSUMPTION INDUCES TRANSIENT COMPENSATORY HYPERPLASIA OF DEVELOPING CARDIAC TISSUE IN THE NEONATAL RAT

The effect of continuous exposure to ethanol in utero and postpartum on growth and cell division in developing cardiac tissue was studied in neonatal Fischer rats. Pregnant and lactating females were maintained on three dietary regimens; a control group fed rat chow ad libitum, an experimental group receiving an ethanol-containing (6% by volume) liquid diet, and a pair-fed control group, which received an isocaloric amount of control liquid diet. At days 1, 5, and 10 postpartum, five litters of pups from each control and experimental group were sacrificed and the body weights, heart weights, heart-to-body weight ratios, and mitotic frequency of the ventricular myocardium were measured. When compared to either group of controls, pups continuously exposed to dietary ethanol expressed significantly (P < 0.01) lower body weights. Pups maintained by the pair-fed females had significantly (P < 0.01) lower body weights at days 5 and 10 than pups maintained by the chow-fed females, indicating a pair-fed effect of suboptimal nutrition of the model. As the pups developed, the heart weights of pups maintained by the chow-fed females became progressively greater (P < 0.01) than the heart weights of pups maintained by the pair-fed and ethanol-fed females, which expressed no weight difference. The reduction of heart weight present in the ethanol-fed and pair-fed pups represents a pair-fed effect of suboptimal nutrition and not an obvious effect of exposure to dietary ethanol. The ratio of heart weight to body weight and mitotic frequency were significantly greater (P < 0.01) in 1- to 5-day-old pups exposed to ethanol. Following day 5, these parameters decreased and approached the control values. This indicates that growth of cardiac tissue is not suppressed in the 1- to 5-day-old rat pups exposed continuously to dietary ethanol. These observations further suggest the presence of a mechanism intrinsic to the heart which can provide stage-dependent protection from the adverse effects of ethanol during early development. The decline in heart weight to body weight ratios and mitotic frequency in pups of ethanol-fed females also suggests that ethanol may initiate suppression of the growth of cardiac tissue or may incur stage-dependent injury during the later stages of development. The possible mechanism of this stage-dependent protection during early neonatal development is an increased mitotic activity of the cardiac myocytes.

Prenatal ethanol exposure alters adrenocortical response to predictable and unpredictable stressors

In the present study, effects of psychological stressors and stressor predictability on the adrenocortical responses of animals exposed to ethanol in utero were examined. Male and female offspring from ethanol-exposed (E), pair-fed (PF), and ad lib-fed control (C) conditions were tested in adulthood. Separate sets of animals were exposed once daily for 5 consecutive days to either unpredictable (UNPRED) or predictable (PRED) restraint stress or to UNPRED novelty stress. On the fifth test day, blood samples for plasma corticosterone determination were taken either prior to stress (basal levels) or following stress. Males in all groups and C females had higher basal corticosterone levels if previously subjected to PRED restraint than if previously subjected to UNPRED restraint; E and PF females did not exhibit this differential responsiveness. Following restraint stress on the fifth test day, there were no significant differences in response to PRED and UNPRED restraint among E, PF, and C males. In contrast, C but not E or PF females exhibited a greater plasma corticosterone response to UNPRED than to PRED restraint. Following novelty stress, E males exhibited a significantly reduced plasma corticosterone response compared to PF and C males. For females, there was a trend for an increased corticoid response in E compared to PF females following novelty stress as well as a different pattern of response among groups from days 1 to 5 of testing. These data support and extend previous studies suggesting that, compared to PF and C animals, E animals may exhibit altered responses to stressors as well as deficits in the ability to use or respond to environmental cues.

The role of maternal alcohol damage on ethanol teratogenicity in the rat.

To study the severity and degree of in utero alcohol effects in relation to the rate of maternal alcohol damage, multiparous 1-year alcohol-fed rats were used, with an appropriate pair-fed control group. During pregnancy, alcoholic dams showed relatively high acetaldehyde levels (41 +/- 19 mumol/l) and blood alcohol levels of 22.8 +/- 14 mmol/l. They also showed marked histological alterations in liver as well as high serum aspartate-aminotransferase, alanine-aminotransferase, alkaline phosphatase, glutamate dehydrogenase, and gamma-glutamyltransferase activities. The increase in serum enzyme levels did not correlate with an increase in hepatic enzyme levels since only glutamate dehydrogenase was enhanced in liver after 1 year of alcohol intake. In addition, except for an increase in low Km aldehyde dehydrogenase activity, there were no changes in liver alcohol metabolizing enzymes in chronic alcohol vs. pair-fed females. Alcoholic rats showed a high incidence of damage in their progeny (resorptions, immature fetuses, decrease in fetal weight, etc.), and rats with the highest serum levels of the above enzymes (especially glutamate dehydrogenase and gamma-glutamyl transferase) had severely affected progeny. Rats with minimal histological liver damage, in contrast, did not show resorptions. Thus, the results presented suggest that the stage of maternal alcohol illness, as indicated mainly by the extent of liver damage, plays an important role in the frequency and severity of in utero alcohol effects in the rat.