Mediation of diurnal fluctuations in pain sensitivity in the rat by food intake patterns: reversal by naloxone.

Rats maintained on a 12-hour light-dark cycle were tested for pain sensitivity after being deprived of food during either the dark or the light phase of the cycle. Diurnal fluctuations in pain sensitivity were observed. The fluctuations followed food intake patterns rather than a natural circadian rhythm, with food deprivation producing a decrease in pain sensitivity. The analgesic response produced by this mild food deprivation was strongly attenuated by naloxone or feeding, suggesting that endogenous opioid systems may be related to patterns of food intake.

Prenatal alcohol exposure alters adult expression of sexually dimorphic behavior in the rat.

Saccharin preference and performance in a Lashley III maze were found to be altered in adult male and female rats that had been exposed to alcohol during gestation. Specifically, the sexual dimorphism normally observed in both behaviors was absent in fetal alcohol-exposed animals. The lack of sexual dimorphism appeared to result from a masculinization of the exposed females and a feminization of the exposed males.

Maternal adrenalectomy eliminates a surge of plasma dehydroepiandrosterone in the mother and attenuates the prenatal testosterone surge in the male fetus.

Previous work has established a number of sex-related deficits in immune function, behavior, and endocrine responses to stress in the offspring of dams exposed to ethanol. To examine the potential role of maternal glucocorticoids as a mediator of these sexually dimorphic effects in the fetus, we examined the influence of prenatal alcohol exposure in the presence or absence of maternal glucocorticoids on fetal plasma corticosterone (CORT) production. An additional question to be addressed by these studies was whether maternal adrenalectomy could eliminate the known inhibition by ethanol of the prenatal surge of plasma testosterone in male fetuses. Pregnant dams were adrenalectomized (ADX) or sham-adrenalectomized on gestational day (G) 7 and placed on a liquid diet containing 35% ethanol-derived calories or pair-fed an isocaloric control diet throughout the experiment. On G18, G19, and G21, plasma levels of CORT, testosterone, and dehydroepiandrosterone (DHEA) were measured in male and female fetuses and their mothers. Ethanol administration consistently increased maternal plasma CORT levels but did not significantly alter CORT levels in the fetus. Maternal ADX resulted in compensatory increases in fetal CORT levels that were lower in fetuses of ADX dams on alcohol, suggesting a direct effect of ethanol on fetal pituitary-adrenal activity. There were no significant sex differences in fetal plasma CORT levels in response to any of these manipulations. A novel surge of maternal plasma DHEA was found on G19 that was absent in plasma from ADX dams. In spite of the absence of a surge on G19, plasma DHEA levels of ADX dams rose from very low levels at G18 to levels on G21 that were significantly higher than in Sham dams. A normal testosterone surge was observed in male fetuses on G18 and G19 from sham-adrenalectomized dams administered the pair-fed diet. However, this surge was greatly attenuated in males administered ethanol and also in male fetuses from ADX dams. These results reveal a direct inhibitory influence of ethanol on fetal CORT secretion as well as on the prenatal testosterone surge in males. Furthermore, these studies demonstrate the presence of a surge of DHEA in the pregnant rat. Overall, these data suggest that there is a critical adrenal factor in the rat that regulates the maternal surge of DHEA on G19 and the prenatal testosterone surge of male fetuses on G18-19.

Plasma testosterone surge and luteinizing hormone beta (LH-beta) following parturition: lack of association in the male rat.

Studies examining the role of luteinizing hormone (LH) in the initiation of the postnatal surge of testosterone in the male rat have produced ambiguous results. We examined the pattern of postnatal LH secretion in the newborn male rat, coincident with plasma testosterone levels, using a specific monoclonal antibody for LH-beta. In some males, we attempted to block LH secretion and the postnatal testosterone surge by injecting males with a gonadotropin-releasing hormone (GnRH) antagonist, an LH antibody or progesterone immediately after delivery by cesarean section on day 22. Following injection, animals were immediately sacrificed (time 0) or housed in a humidified incubator maintained at 30 degrees C until sacrifice at 60, 120, 240, 360 or 480 min after delivery. Plasma from individual animals was measured subsequently for LH-beta and testosterone by radioimmunoassay. Results revealed a postnatal surge of testosterone which peaked at 2 h after delivery in males from all treatment groups. This testosterone surge was not accompanied by a postnatal rise in plasma LH-beta in any group. Administration of the GnRH antagonist or the ethanol vehicle produced a transient drop of approximately 25% in LH-beta levels at 60 min but did not decrease the postnatal testosterone surge in the same animals. Additional studies in untreated males and females born by cesarean section or natural birth also failed to reveal a postnatal rise in plasma LH-beta during the first 3 h after birth. Plasma levels in both sexes were significantly lower in animals delivered by cesarean section compared to natural birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of prenatal ethanol exposure on hormonal responses to clonidine and naloxone in prepubescent male and female rats.

The expression of sexually dimorphic behavior has been found to be altered in adult animals following prenatal alcohol exposure. The present study examined whether such exposure would alter the sexually dimorphic response of luteinizing hormone (LH) to clonidine and naloxone observed in normal prepubescent animals. Both LH and corticosterone (CS) were measured in 16 day old male and female rats 30 min after injection of naloxone (2 mg/kg) or clonidine (0.1 mg/kg). Prenatal alcohol exposure did not influence the LH response to either drug in females. An LH response to clonidine in normal males did not occur, but it was present in the males exposed to alcohol in utero and in the pair-fed controls. Prenatal alcohol exposure influenced the CS response to both drugs. CS levels were depressed in the naloxone-treated males prenatally exposed to alcohol compared to their saline-injected counterparts. The CS levels of other groups following naloxone administration were unchanged compared to saline injection. Normal animals of both sexes exhibited an elevation in CS levels following clonidine. However, this stimulatory effect of clonidine on CS release was absent in both female and male animals prenatally exposed to alcohol. The results of this study indicate that prenatal alcohol exposure may alter noradrenergic and opioid modulation of corticosterone and possibly of LH in young animals.

Alterations in the estrogen sensitivity of hypothalamic proenkephalin mRNA expression with age and prenatal exposure to alcohol.

Studies suggest that exposure to alcohol in utero causes reproductive and neuroendocrine deficits in adult female rats. The ventromedial nucleus of the hypothalamus (VMN) is an estrogen-sensitive brain region which is regarded as a primary locus for modulating female reproduction. Proenkephalin (PE) mRNA expression in the VMN is dramatically increased by estrogen and this elevation is thought to be involved in modulating female reproductive behavior and neuroendocrine function. To examine whether prenatal alcohol exposure has long-term effects on the ability of estrogen to influence hypothalamic PE mRNA levels, female rats at 2-3, 6-7 or 15-18 months of age, derived from alcohol- or control-fed dams, were studied. 7 days following ovariectomy, animals received either estrogen or sham treatment for 2 days prior to sacrifice. PE mRNA levels in the VMN and striatum were determined by in situ hybridization histochemistry. Film autoradiogram density, numbers of PE mRNA-expressing cells and exposed silver grains/cell were analyzed. Estrogen treatment increased hybridization density, the number of PE mRNA-expressing cells and PE mRNA (grains) level/cell in the VMN of normal adult female rats. In old rats, estrogen increased the number of PE mRNA-expressing cells without up-regulating PE mRNA grain density/cell. In fetal alcohol-exposed (FAE) female rats, the number of cells that expressed PE mRNA did not increase following estrogen treatment at any age. Elevation of grain density/cell following estrogen was observed in FAE animals but only at 7-8 months of age. Overall, these data indicate that the estrogen responsiveness of PE mRNA expression in the VMN declines with age and, furthermore, prenatal exposure to alcohol blunts estrogen's effects on PE mRNA expression in the adult VMN. These finding may help to explain the mechanisms underlying the loss of reproductive function observed in FAE females.

Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat.

In the adult male rat, androgen and estrogen synergize in the regulation of male reproductive behaviors. To explore some of the molecular mechanisms underlying this synergism we examined the distribution and hormonal regulation of androgen receptor (AR) and estrogen receptor (ER) mRNAs in the medial preoptic area (MPOA) and bed nucleus of the stria terminalis (BST) of the adult male rat. Using in situ hybridization, AR and ER mRNAs were found to be distributed in overlapping but unique patterns. The highest density of AR mRNA was found in the central part of the medial preoptic n. and the principal n. of the BST. Gonadectomy (GDX) of adult male rats caused an increase in hybridization density in both brain areas after 4 days followed by a decrease after 2 months. In contrast, ER mRNA was increased following GDX and remained high regardless of length of time. Treatment of adult GDX'd males with dihydrotestosterone (DHT) reversed the effects of GDX on AR mRNA at both the short and long-term castrate but had no effect on ER mRNA in both the MPOA and BST. Estrogen treatment increased AR mRNA in the long-term castrate only and decreased ER mRNA in both long- and short-term castrates. Immunocytochemical detection of AR revealed a similar distribution to AR mRNA; however, AR immunoreactivity was reduced in the MPOA and BST after both short- and long-term GDX. In vitro [3H]DHT binding in cytosols of the preoptic area showed appreciable binding but there was no effect of length of time following GDX. These data show that the pattern of regulation of AR mRNA is unique to this receptor type and does not follow the pattern of regulation of the ER mRNA. Furthermore, although the distribution of AR mRNA and AR protein coincide within the MPOA, changes in mRNA levels as a result of castration or hormone treatment do not result in corresponding changes in binding. This mismatch between mRNA and binding suggests a complex regulation of AR beyond simply changes in transcription.

A novel endogenous corticotropin release inhibiting factor.

ACTH is the major regulator of the body's adaptive response to stress and the physiological stimulus for glucocorticoid secretion. A hypothalamic corticotropin release inhibiting factor (CRIF) that inhibits ACTH synthesis and secretion has long been postulated but was not characterized until recently. We have recently identified a 22 amino acid peptide, prepro-thyrotropin releasing hormone (TRH) 178-199 that inhibits basal and stimulated ACTH synthesis and secretion in vitro and stress-induced ACTH secretion in vivo. Prepro-TRH 178-199 is abundant in several brain regions, including the external zone of the median eminence, where its concentration changes in response to stress. We propose that this peptide is a physiological regulator of ACTH production: an endogenous CRIF. Because prepro-TRH 178-199 is encoded within the same precursor as TRH, its expression is likely to be negatively regulated by thyroid hormones leading to changes in endogenous glucocorticoid levels. Streptococcal cell wall (SCW)-induced inflammation, a model of rheumatoid arthritis (RA), was alleviated after long-term thyroxine treatment. Inversely, a hypothyroid milieu led to decreased basal hypothalamic-pituitary-adrenal activity, but increased expression of IL-1 beta and MIP-1 alpha, specific markers for RA in humans. These results suggest that this putative CRIF may be an important component in the development of RA and that regulation of prepro TRH may be highly relevant to the development of other autoimmune diseases that are also exacerbated by low endogenous glucocorticoid levels.

Prenatal inhibition of hypothalamic sex steroid uptake by cocaine: effects on neurobehavioral sexual differentiation in male rats.

Several adrenergically active drugs have been shown to prevent the masculinizing and/or defeminizing effects of testosterone on brain sexual differentiation. We examined the ability of the neuronal norepinephrine uptake blocker, cocaine, to produce similar effects. The ability of cocaine to inhibit sex steroid incorporation into the hypothalamus during a critical period for sexual differentiation of the brain was examined in females treated at birth with testosterone. Sixty minutes after administration, cocaine was observed to inhibit both testosterone and estradiol incorporation into the hypothalamus by approximately 50%. Long-term consequences of prenatal cocaine exposure were studied by injecting Sprague-Dawley dams twice daily with 3, 10 or 30 mg/kg of cocaine hydrochloride on days 15 through 20 of gestation and examining the offspring. In adulthood, cocaine-exposed males, but not females were found to exhibit significantly less marking behavior than controls. Cocaine-exposed males in the 10 mg/kg group tested for sex behavior exhibited demasculinization in some aspects of the behaviors tested. Measurement of plasma hormone levels in this group revealed elevated levels of plasma LH, but normal levels of FSH and testosterone. No differences were observed in cocaine-exposed males with respect to sex organ or adrenal weights, but thymus was approximately 25% smaller compared to control males at 80 days of age. In a separate experiment, dams were treated with 3 mg/kg of cocaine twice daily from days 15 through 21 of gestation and half of the male pups received additional injections twice a day for the first 5 days postnatally.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of postnatal testosterone fails to demasculinize the male rat's corpus callosum.

We had previously shown that elimination of testosterone from embryonic day 17 through adulthood reduced the midsagittal area of the male rat corpus callosum (CC). However, day 1 castration, performed after the 2-h post-birth testosterone surge, was without effect. To elucidate the contribution of this surge on the CC, male rats were delivered by cesarean section and castrated within 20 min. This procedure eliminated the 2-h postnatal rise in testosterone levels. The prenatal surge in testosterone, which occurs on embryonic day 18, remained intact. In adulthood, callosal area was examined in castrate males, sham males, and intact females. Castrate males and sham males had significantly larger CCs as compared to females. The two male groups did not differ from each other. Body weight was significantly higher in sham versus castrate males, establishing the effectiveness of the castration. These results show that hormonal organization of the CC in the male is the result of the independent action of prenatal testicular androgens, and suggest that the end of this period marks the end of callosal sensitivity to testicular hormone influence. In addition, this report documents sexual dimorphism of the CC in a third rat strain.

Testosterone dose-dependency of sexual and nonsexual behaviors in the gonadotropin-releasing hormone antagonist-treated male rat.

The testosterone dose-dependency of several mating and nonmating behaviors was examined in the male rat, chemically castrated with a GnRH antagonist analog. Graded doses of testosterone enanthate (TE) were given to male rats to reinstate behaviors abolished by GnRH antagonist treatment. GnRH antagonist treatment alone markedly lowered serum LH, FSH and T concentrations and ventral prostate and testis weights. Open field behaviors were not significantly affected by GnRH antagonist treatment or castration. Scent-marking behavior was markedly suppressed by both castration and GnRH antagonist and restored by the lowest dose of TE (0.05 mg). All measures of male sexual behavior were impaired by GnRH antagonist treatment and castration and restored by the lowest dose of TE (0.05 mg). The doses of TE required to restore normal ventral prostate weights and testis weights were higher than those required to maintain scent marking and mating behaviors. No direct behavioral effects of the GnRH antagonist, other than those that can be explained by GnRH antagonist-induced suppression of testosterone were observed. The finding that sexual and nonsexual behaviors in the male rat have different testosterone requirements from those maintaining spermatogenesis and fertility may have significant implications for contraception.

Prolonged intermittent footshock stress decreases Met and Leu enkephalin levels in brain with concomitant decreases in pain threshold.

The influence of a 21 day intermittent footshock regimen upon enkephalin levels in brain and adrenals was examined in the rat. Changes in pain sensitivity as well as analgesic and hyperthermic responsiveness to morphine (7.5 mg/kg) were also monitored. Following the stress regimen, Met and Leu enkephalin levels were decreased by 40 to 50% in brain, but were unchanged in adrenals. Post-stress pain thresholds were markedly decreased in stressed animals while the analgesic properties of morphine were enhanced. Core body temperature of stressed animals was significantly raised, but the hyperthermic response to morphine was unchanged.

Exposure to alcohol in utero alters the adult patterns of luteinizing hormone secretion in male and female rats.

Luteinizing hormone (LH) secretory patterns were characterized in adult male and female rats exposed to ethanol during the last week of fetal life. Gonadectomized fetal alcohol exposed (FAE) males and females had significantly reduced plasma LH titers as compared to those of pair-fed (PF) controls. The phasic afternoon LH secretory response to estrogen and progesterone priming was also significantly reduced in FAE females. These differences do not appear to be a result of altered pituitary sensitivity to luteinizing hormone releasing hormone (LHRH), since the infusion of LHRH resulted in an equal response in PF and FAE females. Subsequent characterization of the episodic pattern of LH secretion in FAE males revealed significantly reduced mean LH level as well as a decreased pulse amplitude and frequency when compared to PF males. Taken together, these data indicate that some of the central mechanisms controlling pituitary LH secretion are altered by prenatal exposure to alcohol.

Prenatal alcohol exposure alters enkephalin levels, without affecting ethanol preference.

Pregnant dams were pair-fed a liquid diet containing 35% ethanol derived calories or isocaloric sucrose during the last two trimesters of pregnancy. No differences were observed in adult ethanol preference between fetal alcohol exposed (FAE) animals and pair-fed controls. However, Met- and Leu-enkephalin levels were significantly elevated in globus pallidus of adult FAE animals. Pituitary levels were unaffected.

Adrenalectomy reverses stress-induced suppression of luteinizing hormone secretion in long-term ovariectomized rats.

An inhibitory effect of stress on reproductive function is well established. This inhibition involves activation of the hypothalamic-pituitary-adrenal (HPA) axis, which leads to a suppression of LH secretion. It has been proposed that this suppression is mediated by a direct effect of CRF that is independent of glucocorticoid actions. We tested this proposition by examining plasma LH levels in adult rats that were both ovariectomized (OVX) and adrenalectomized (ADX). Each animal was surgically implanted with an indwelling atrial cannula and exposed to intermittent foot shock for 100 min. Blood samples were drawn just prior to putting the animals into the test cage and then at 20-min intervals. Results revealed normal castrate levels of plasma LH in both ADX and ADX/OVX animals prior to shock. A significant shock-induced suppression of LH was observed in OVX animals within 20 min after the onset of shock and remained throughout the duration of the session. In contrast, no evidence was obtained for a suppression of LH in OVX/ADX animals at any time point. Additional studies demonstrated a marked suppression of LH in experimentally naive OVX/ADX females implanted with corticosterone capsules for 2 weeks prior to blood sampling. Overall, these results support a primary role for glucocorticoid actions in the stress-induced inhibition of reproductive function.

Effects of prenatal testosterone propionate treatment on saccharin preference of adult rats exposed to ethanol in utero.

Prenatal exposure to alcohol feminizes saccharin consumption patterns in adult male rats. To study the involvement of testosterone in this effect, testosterone propionate (TP) was administered to pregnant dams in an attempt to reverse that feminized saccharin consumption pattern in the male offspring. Female offspring were also studied to determine the effect of TP on saccharin preference in normal males and females. During the last week of gestation, dams were administered a liquid diet containing 35% ethanol derived calories, an isocaloric liquid diet containing no ethanol, or Purina Lab Chow. Half of the dams in each group received twice daily injections of TP, the other half were injected with the oil vehicle. Saccharin consumption of adult fetal alcohol exposed (FAE) males from dams administered oil or TP was significantly greater than controls, indicating that the feminized pattern of saccharin consumption of FAE males cannot be overcome with TP administration during the prenatal period. In controls, prenatal TP exposure alone was found to increase adult saccharin consumption in both sexes. Prenatal administration of TP was also found to markedly depress body weight of offspring of dams receiving the liquid diets compared to offspring from dams receiving the same diets plus oil injections. Body weights of offspring from TP or oil injected dams receiving the chow fed diets during pregnancy did not differ.

Sex difference in daily water consumption of rats: effect of housing and hormones.

Females rats consume more water than males per day when consumption is indexed to body weight. We examined the developmental expression of this sex difference as well as the organizational and activational influences of testosterone (T). The amount of water consumed from weaning to adulthood exhibited a linear decrease with age in both sexes. The development of a sex difference in water consumption was evident immediately after weaning in singly housed animals, but did not emerge until about Day 42-45 in group-housed animals, when females began to consume greater amounts of water than males. Castration at weaning had minimal effects on the sex difference. Treating dams with testosterone propionate (TP; 0.3 mg/kg; E15-E20) resulted in a significant increase in adult water consumption in offspring of both sexes, but the sex difference remained. Overall, these data indicate that gonadal steroids are not the primary organizational influence on this sex difference. The greater water consumption in females is consistent with other studies demonstrating sex differences in plasma vasopressin levels, as well as differences in vasopressin sensitivity.

Androgen regulation of adrenocorticotropin and corticosterone secretion in the male rat following novelty and foot shock stressors.

To examine mechanisms responsible for sex differences in hypothalamo-pituitary-adrenal (HPA) axis responsiveness to stress, we studied the role of androgens in the regulation of the adrenocorticotropin (ACTH) and corticosterone (CORT) responses to foot shock and novelty stressors in gonadectomized (GDX) or intact male F344 rats. Foot shock or exposure to a novel open field increased plasma ACTH and CORT, which was significantly greater in GDX vs. intacts. Testosterone (T) or dihydrotestosterone propionate (DHT) treatment of GDX animals returned poststress levels of ACTH and CORT to intact levels. Estrogen treatment of GDX males further increased poststress CORT secretion above GDX levels. There was no difference in the ACTH response of anterior pituitaries from intact, GDX, and GDX+DHT animals to CRF using an in vitro perifusion system. There were no differences in beta max or binding affinity of type I or II CORT receptors in the hypothalamus or hippocampus of intact, GDX, or GDX+DHT groups. These data demonstrate an effect of GDX on hormonal indices of stress. The increased response in GDX rats appears to be due to the release from androgen receptor mediated inhibition of the HPA axis. This inhibition by androgen is not due to changes in anterior pituitary sensitivity to CRH, nor to changes in type I or type II corticosteroid receptor concentrations.

Decreased tolerance to ethanol-induced hypothermia in long-term castrate male rats.

A potential role for central stores of vasopressin in the development of tolerance was studied in the long-term castrate rat. Vasopressin stores in the septal region are known to be dramatically depressed following long-term castration. Sprague-Dawley male rat littermates were castrated at 26 days of age or given a sham surgery. Experiments began when animals reached 130 days of age. Tolerance to the hypothermic effects of ethanol occurred in intact but not castrate animals over the course of six daily IP injections of 3.0 g/kg ethanol. Both groups exhibited tolerance to the length of time needed to return to baseline temperature over the 6 days of ethanol injections. Tolerance to this effect of ethanol was still evident in intact animals but not castrates following another injection of ethanol 1 week later. No tolerance developed to the rebound hyperthermia that occurred in both groups. Blood ethanol levels did not differ significantly between castrate and intact littermates administered a single dose of ethanol. Overall, these results support the hypothesis that endogenous vasopressin is involved in the development of some aspects of tolerance to ethanol.

Androstenedione synergizes with stress or prenatal drug exposure to retard fetal growth: role of IGF.

Pregnant Sprague-Dawley dams were implanted with a Silastic capsule (3 or 10 mm) containing androstenedione (AN) or cholesterol prior to being administered one of several treatments that create an endocrine profile of stress: maternal exposure to alcohol, pair feeding (PF), cocaine (COC), or restraint stress (RS). Controls (chow fed, CF) were left undisturbed during pregnancy. Treatments were administered from day 14 to day 22 of gestation. Fetuses were delivered by cesarean section on day 22. Results revealed that administration of AN to pregnant dams at a dose that does not influence fetal growth by itself can retard fetal growth in the presence of alcohol, PF, COC or RS. Data indicate that these effects are not directly attributable to changes in adrenocorticotropin (ACTH) or corticosterone levels. Preliminary results suggest a role for insulinlike growth factor (IGF) binding proteins (IGFBPs). Overall, these data demonstrate that AN can synergize with drugs and/or stress to enhance intrauterine growth retardation (IUGR). One underlying cause of this synergism between stress-related environmental events and androgenic actions on fetal growth may be increased expression of IGFBPs, which can sequester IGFs, thereby inhibiting their trophic actions on fetal and/or placental tissue.