Chronic daily ethanol and withdrawal: 2. Behavioral changes during prolonged abstinence.

BACKGROUND: Our previous investigations have suggested that alcohol abuse may induce persistently compromised hypothalamo-pituitary-adrenal (HPA) function, which could increase risk for subsequent alcohol abuse. The apparent similarity of chronic alcohol abuse-induced HPA defects to the compromised HPA functions associated with posttraumatic stress disorder and atypical depression also suggest potential common mechanisms shared with varied neurobehavioral disorders. Accordingly, we have investigated persistent behavioral effects of previous repetitive daily ethanol consumption and withdrawal. METHODS: Male Sprague-Dawley rats received either daily ad libitum chow, ad libitum liquid diet containing ethanol, or pair-fed isocaloric control liquid diet. The ethanol was gradually introduced over 4 weeks, maintained at 5% w/v for four subsequent weeks, and then gradually removed over 1 week. RESULTS: Four weeks after removal of ethanol from the diet, the previously ethanol-consuming rats exhibited greater (p < 0.05) evidence of anxiety in the elevated plus-maze test and in the novel cork-gnawing test, as well as greater (p < 0.05) locomotor response to a novel environment, compared with controls. CONCLUSIONS: These results suggest that repetitive daily alcohol consumption and withdrawal can induce not only persistent defects in HPA function, but also persistent increases in anxiety and behavioral responsiveness to novelty, all consistent with characteristics of abstinent alcoholics as well as with rats that self-administer increased amounts of alcohol and other drugs of abuse. This suggests that alcohol abuse can induce persistent common or interacting changes in neuroendocrine and behavioral responses that may increase risk for subsequent abuse.

Aging-dependent changes in the effect of daily melatonin supplementation on rat metabolic and behavioral responses.

Pineal melatonin secretion has been reported to commonly decrease with aging, whereas intra-abdominal adiposity, plasma insulin and plasma leptin levels tend to increase. We recently demonstrated that daily melatonin administration starting at middle age suppressed male rat intra-abdominal fat, plasma leptin and plasma insulin to youthful levels, suggesting that aging-related changes in pineal melatonin secretion and in energy regulation may be functionally related. Accordingly, we have now investigated the effects of daily melatonin treatment on energy regulation in young versus middle-aged male Sprague Dawley rats. Addition of melatonin to the drinking water (0.2 microg/mL) produced nocturnal and diurnal plasma melatonin concentrations in middle-aged rats (12 months) equivalent to those of young adult (5 months) rats. Administration of this melatonin dosage every day for 10 wk starting at 10 months of age suppressed (P < 0.01) relative intra-abdominal fat, non-fasted plasma insulin and plasma leptin by 27, 39, and 51%, respectively (vs. vehicle-treated controls). In contrast, administration of melatonin for 10 wk starting at 3 months of age did not significantly alter (P> 0.10) any of these parameters. The melatonin administration stimulated (102%, P < 0.001) behavioral responsiveness of the middle-aged rats in a test of response to novelty, restoring youthful levels, but did not significantly alter behavioral responsiveness of the young rats. These results suggest that suppression of intra-abdominal adiposity and plasma leptin and insulin levels and stimulation of behavioral responsiveness in response to daily exogenous melatonin begins at middle age, coincident with and likely dependent upon the aging-associated decline in endogenous pineal melatonin secretion. These results further suggest that appropriate melatonin supplementation may potentially provide therapy or prophylaxis not only for the insulin resistance, increased intra-abdominal fat and resulting pathologies that occur with aging, but also for some aging-associated behavioral changes.

Daily melatonin administration to middle-aged male rats suppresses body weight, intraabdominal adiposity, and plasma leptin and insulin independent of food intake and total body fat.

Pineal melatonin secretion declines with aging, whereas visceral fat, plasma insulin, and plasma leptin tend to increase. We have previously demonstrated that daily melatonin administration at middle age suppressed male rat intraabdominal visceral fat, plasma leptin, and plasma insulin to youthful levels; the current study was designed to begin investigating mechanisms that mediate these responses. Melatonin (0.4 microg/ml) or vehicle was administered in the drinking water of 10-month-old male Sprague Dawley rats (18/treatment) for 12 weeks. Half (9/treatment) were then killed, and the other half were submitted to cross-over treatment for an additional 12 weeks. Twelve weeks of melatonin treatment decreased (P<0.05) body weight (BW; by 7% relative to controls), relative intraabdominal adiposity (by 16%), plasma leptin (by 33%), and plasma insulin (by 25%) while increasing (P<0.05) locomotor activity (by 19%), core body temperature (by 0.5 C), and morning plasma corticosterone (by 154%), restoring each of these parameters toward more youthful levels. Food intake and total body fat were not changed by melatonin treatment. Melatonin-treated rats that were then crossed over to control treatment for a further 12 weeks gained BW, whereas control rats that were crossed to melatonin treatment lost BW, but food intake did not change in either group. Feed efficiency (grams of BW change per g cumulative food intake), a measure of metabolic function, was negative in melatonin-treated rats and positive in control rats before cross-over (P<0.001); this relationship was reversed after cross-over (P<0.001). Thus, melatonin treatment in middle age decreased BW, intraabdominal adiposity, plasma insulin, and plasma leptin, without altering food intake or total adiposity. These results suggest that the decrease in endogenous melatonin with aging may alter metabolism and physical activity, resulting in increased BW, visceral adiposity, and associated detrimental metabolic consequences.

Chronic daily ethanol and withdrawal: 1. Long-term changes in the hypothalamo-pituitary-adrenal axis.

BACKGROUND: Hypothalamo-pituitary-adrenal (HPA) function has been demonstrated to be compromised for weeks and even months after alcoholics cease ethanol consumption. Because nonalcoholic subjects with family history-associated increased risk for alcoholism also exhibit compromised HPA function, it is not clear whether defects in the HPA axis of abstinent alcoholics reflect a preexisting condition that may be responsible for increased risk for alcohol abuse versus a persisting adaptational change in response to prolonged alcohol abuse. Consequently, we investigated whether chronic daily ethanol consumption and withdrawal by male Sprague Dawley rats would induce persistent HPA changes consistent with those demonstrated in abstinent alcoholics. METHODS AND RESULTS: In an initial experiment in which ethanol (5%, w/v) was incrementally introduced to liquid diet over a 1 week period followed by 4 weeks of chronic ethanol consumption, not only ethanol-treated rats but also pair-fed control rats exhibited decreased (p < 0.05 vs. ad-libitum-fed controls) anterior pituitary pro-opiomelanocortin (POMC) mRNA concentrations and associated decreases in plasma corticosterone and adrenocorticotropin (ACTH) levels for at least 3 weeks after gradual withdrawal of ethanol from the diet. Pair-feeding-induced decreases (p < 0.05) in thymus and spleen weights suggested that the pair-fed controls were likely stressed in this model, probably in response to the marked and irregular suppression of liquid diet consumption immediately after introduction of ethanol. Consequently, a second model was developed in which ethanol was introduced to the liquid diet much more gradually (i.e., over 3 weeks). In contrast with the rapid ethanol-introduction model, this more prolonged ethanol introduction followed by 4 weeks of chronic daily ethanol consumption increased plasma corticosterone levels (p < 0.05), increased adrenal gland weight (p < 0.05), and decreased thymus and spleen weights (both p < 0.01) without altering any of these parameters in the pair-fed controls. Three weeks after gradual withdrawal of ethanol from the diet, anterior pituitary POMC mRNA concentrations were suppressed (p < 0.05) and thymus and spleen weights were increased (p < 0.05) versus both pair-fed and ad-libitum-fed controls, accompanied by trends for decreased basal plasma corticosterone and adrenal weights. CONCLUSIONS: Chronic daily ethanol treatment induced changes in the HPA axis that persisted for at least 3 weeks after complete cessation of ethanol consumption. These persistent alterations in the HPA axis are similar to the aberrant HPA regulation of abstinent alcoholics, sons of alcoholics, Lewis rats, and individuals who suffer from posttraumatic stress disorder and some types of depression, that is, categories of individuals who all exhibit increased risk for high ethanol consumption. Thus, these chronic daily ethanol-induced persistent changes in the HPA axis may have significant roles in alcohol abstinence syndrome and may increase vulnerability to relapse.

Reduced gene expression for dopamine biosynthesis and transport in midbrain neurons of adult male rats exposed prenatally to ethanol.

BACKGROUND: Prenatal ethanol exposure affects brain dopaminergic neuronal systems, and many of these alterations are permanent. METHODS: The primary objective of this study was to determine the effects of prenatal ethanol exposure on adult mRNA expression for two key regulatory proteins in the mesolimbic and nigrostriatal dopaminergic cell groups which mediate behavioral responses to alcohol and other drugs of abuse: tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). To also address the effects on noradrenergic regulation, we quantitated mRNA expression for TH and norepinephrine transporter (NET) in the noradrenergic loci of the locus coeruleus (LC). RESULTS: Daily dietary ethanol consumption by female Sprague-Dawley rats for 3 weeks before, and continuing throughout, pregnancy decreased both DAT (approximately 68%,p < 0.002) and TH (approximately 45%,p < 0.002) mRNA expression in the VTA of adult male offspring. This prenatal exposure also suppressed DAT mRNA expression in the SNpc (approximately 81 %;p < 0.03), although TH mRNA expression in this region was not significantly altered. Prenatal ethanol exposure did not alter significantly either TH or NET mRNA expression in the LC of adult male offspring, which suggests that this brain catecholaminergic response may be limited to DA neurons. CONCLUSION: These results demonstrated that prenatal maternal ethanol consumption suppresses mRNA expression for important regulatory proteins in the mesolimbic and nigrostriatal dopaminergic systems of adult male rat offspring. These persistent prenatal ethanol-induced changes in mRNA expression may thus contribute to the persistent effects of fetal ethanol exposure on the diverse behavioral and/or metabolic responses mediated by the mesolimbic and nigrostriatal dopaminergic systems in the adult.

Daily melatonin administration at middle age suppresses male rat visceral fat, plasma leptin, and plasma insulin to youthful levels.

Human and rat pineal melatonin secretion decline with aging, whereas visceral fat and plasma insulin levels increase. Melatonin modulates fat metabolism in some mammalian species, so these aging-associated melatonin, fat and insulin changes could be functionally related. Accordingly, we investigated the effects of daily melatonin supplementation to male Sprague-Dawley rats, starting at middle age (10 months) and continuing into old age (22 months). Melatonin was added to the drinking water (92% of which was consumed at night) at a dosage (4 microg/ml) previously reported to attenuate the aging-associated decrease in survival rate in male rats, as well as at a 10-fold lower dosage. The higher dosage produced nocturnal plasma melatonin levels in middle-aged rats which were 15-fold higher than in young (4 months) rats; nocturnal plasma melatonin levels in middle-aged rats receiving the lower dosage were not significantly different from young or middle-aged controls. Relative (% of body wt) retroperitoneal and epididymal fat, as well as plasma insulin and leptin levels, were all significantly increased at middle age when compared to young rats. All were restored within 10 weeks to youthful (4 month) levels in response to both dosages of melatonin. Continued treatment until old age maintained suppression of visceral (retroperitoneal + epididymal) fat levels. Plasma corticosterone and total thyroxine (T4) levels were not significantly altered by aging or melatonin treatment. Plasma testosterone, insulin-like growth factor I (IGF-I) and total triiodothyronine (T3) decreased by middle age; these aging-associated decreases were not significantly altered by melatonin treatment. Thus, visceral fat, insulin and leptin responses to melatonin administration may be independent of marked changes in gonadal, thyroid, adrenal or somatotropin regulation. Since increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest that appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with aging.

Effects of chronic nicotine treatment and withdrawal on hypothalamic proopiomelanocortin gene expression and neuroendocrine regulation.

Considerable evidence suggest that some responses to smoking and nicotine are mediated by forebrain beta-endorphinergic opioid mechanisms. It has also been demonstrated that nicotine stimulates rat tuberoinfundibular dopaminergic activity. Since we have proposed that interactions between mediobasohypothalamic (MBH) dopaminergic and beta-endorphinergic mechanisms have a key role in neuroendocrine integration, we investigated the effects of chronic nicotine treatment and withdrawal on: (1) MBH concentrations of proopiomelanocortin (POMC, precursor for beta-endorphin biosynthesis) mRNA; (2) MBH concentrations of tyrosine hydroxylase (TH, rate limiting enzyme in catecholamine biosynthesis) mRNA; (3) corresponding serum prolacin, corticosterone, luteinizing hormone (LH), and testosterone concentrations. POMC and TH mRNA levels were measured by RNase protection/solution hybridization assay; serum hormone levels were measured by radioimmunoassay. Adult male rats received subcutaneous injections of either nicotine or saline during the dark period of each day on an increasing frequency (1-3 injections/day) and dosage (0.4-0.5 mg nicotine/kg body weight) schedule over 4 weeks. The rats were sacrificed after 4 weeks treatment and at 1, 3, 7, 14 and 21 days withdrawal. Chronic daily nicotine administration induced significant changes in serum corticosterone, serum prolactin, MBH TH mRNA, and MBH POMC mRNA concentrations that tended to persist through day 3 of withdrawal; serum prolactin and MBH POMC mRNA concentrations were suppressed whereas serum corticosterone and MBH TH mRNA concentrations were stimulated. None of the parameters were significantly different from control levels following 7 or more days of withdrawal from nicotine, except for a significant decrease of MBH POMC mRNA concentrations on day 21. Chronic daily nicotine or withdrawal did not significantly alter serum LH or testosterone concentrations. These results suggest that chronic nicotine inhibited POMC gene expression and thus, probably, biosynthesis of beta-endorphin and other opiomelanocortins. We hypothesize that suppression of forebrain beta-endorphin synthesis in response to long-term nicotine exposure produces a chronically opioid deficient condition which may play an important role in maintaining nicotine self-administration and in mediating some changes during the nicotine withdrawal syndrome.

Acute alcohol effects on opiomelanocortinergic regulation.

To assess acute effects of alcohol on forebrain and pituitary opiomelanocortinergic regulation, a model was developed in which "experienced" (previously introduced to ethanol administration, so the subjective response was not a novel stimulus) male Sprague-Dawley rats received pulsatile intragastric ethanol infusions during the dark (active) photophase to produce and sustain (for 3 hr) behaviorally relevant (0, 40 to 70, 80 to 110, or 120 to 150 mg/dl) plasma ethanol levels. The effects of alcohol on hypothalamo-pituitary-adrenal (H-P-A) axis function were biphasic with respect to dosage (inhibition with low dosage and stimulation with higher dosages) and time (initial stimulation with higher dosages was followed by rapid return to control levels even though elevated plasma ethanol levels were maintained). The effects of alcohol on H-P-A activation were also inconsistent; some of the animals did not appear to respond even though elevated (i.e., >100 mg/dl) plasma ethanol levels were produced. Induction of moderate (80 to 110 mg/dl) plasma ethanol levels acutely (within 30 min) increased immunoreactive (i) beta-endorphin concentrations in the ventral tegmental area of the brain; higher (120 to 150 mg/dl) plasma ethanol levels increased i beta-endorphin concentrations in both the ventral tegmental area and the nucleus accumbens, whereas i beta3-endorphin concentrations were not significantly altered in other brain areas. High (120 to 150 mg/dl) plasma ethanol levels also increased mediobasohypothalamic pro-opiomelanocortin (biosynthetic precursor of forebrain beta-endorphin) mRNA concentrations at 3 and 6 hr after initiation of ethanol infusions. Results demonstrate that atraumatic induction of physiologically meaningful plasma alcohol levels by gastric ethanol infusion activates the forebrain opiomelanocortinergic opioid system and exerts complex effects on the interrelated H-P-A system, consistent with evidence that these systems may interact to mediate or modulate some responses to alcohol ingestion.

Episodic gonadotropin-releasing hormone release from the rat isolated median eminence in vitro.

Several studies have demonstrated that the rat isolated mediobasohypothalamus can release gonadotropin-releasing hormone (GnRH) in a pulsatile manner in vitro. We have now used an in vitro perfusion system to characterize GnRH release from the adult male Sprague-Dawley rat isolated median eminence (ME) alone. After 1 h stabilization periods, eight MEs released GnRH in an episodic pattern during 4-hour experiments (samples collected every 4 min, RIA in triplicate) with mean (+/- SE) release rates of 2.2 +/- 0.2 pg GnRH/4 min and peak amplitudes of 1.3 +/- 0.2 pg/4 min. The intervals between significant peaks were variable, although 70% were in the range of 12-24 min, i.e. similar to the non-gonadal steroid-modulated frequency of pulsatile luteinizing hormone secretion in gonadectomized rats in vivo. During nine additional experiments, MEs were perifused with control medium (containing 2.5 mM calcium) through the first hour of sample collection, then either calcium-free medium containing 0.5 mM of the calcium chelator EGTA and 100 microM of the intracellular calcium antagonist TMB-8 or calcium-free medium containing just 100 microM TMB-8 for 2 h, followed by a final hour with control medium. Removal of calcium activity decreased the frequency of GnRH peaks from 3.2 +/- 0.3 to 1.3 +/- 0.2/h, which then increased to 2.3 +/- 0.3/h with subsequent replacement of control medium. Removal of available calcium also decreased mean GnRH release by 51 +/- 11%, which returned to basal levels with subsequent calcium replacement.(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal modulation of rat hypothalamic gonadotropin-releasing hormone release by melatonin in vitro.

The mechanism(s) by which melatonin (MEL) can regulate gonadotropin secretion remains unresolved. Accordingly, we used acute in vitro incubations of male rat hypothalamic tissues to investigate effects of MEL on hypothalamic gonadotropin-releasing hormone (GnRH) secretion. At 10:00 h (3.5 after lights on), addition of 1 nM MEL to the medium inhibited (p = 0.028) GnRH release from the median eminence (ME) by 24%, 100 nM MEL had no significant effect on GnRH release, and 10 microM MEL tended to inhibit (p = 0.056, 21%) GnRH release. At 15:00 h, none of these MEL dosages significantly altered GnRH release from the ME. When the effect of a lower range of MEL dosages was evaluated, 1 nM MEL again inhibited (24%, p = 0.032) GnRH release from the ME at 10:00 h, and a linear dose-response relationship between initially increasing dosages (0, 0.01, 0.1, 1 nM) of MEL treatment and decreasing GnRH release was evident (r = 0.88), although a further 10 fold increase in MEL dosage (10 nM) resulted in a loss of suppression by MEL. Treatment with these dosages of MEL did not significantly alter ME GnRH release at 15:00 h. In contrast to the results with the ME alone, treatment with 0.1 microM MEL increased (p = 0.018) GnRH release from the arcuate-median eminence region (ARC-ME) by 60.2% at 10:00 h, whereas treatment with either 0.001 or 10 microM MEL did not significantly affect GnRH release. Treatment with these dosages of MEL did not significantly alter ARC-ME GnRH release at 15:00 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive correlation between proopiomelanocortin and tyrosine hydroxylase mRNA levels in the mediobasohypothalamus of ovariectomized rats: response to estradiol replacement and withdrawal.

We have investigated putative dopaminergic regulation of opiomelanotropinergic activity in the arcuate/periarcuate mediobasohypothalamus (MBH) by assessing the changes in MBH tyrosine hydroxylase (TH; rate-limiting enzyme in catecholamine synthesis) and proopiomelanocortin (POMC; opiomelanotropin precursor) mRNA levels under conditions in which endogenous tuberinfundibular dopaminergic activity exhibits marked changes. Adult Sprague-Dawley rats were sacrificed at 09.00 and 15.00 h, and individual MBH POMC and TH cytoplasmic mRNA levels were simultaneously quantified by multiplex solution hybridization-RNase protection assay with protected fragments separated by polyacrylamide gel electrophoresis. In ovariectomized (OVX) rats treated for 3 days with low-dose estradiol (E2) implants (resulting in 18 +/- 4 pg E2/ml serum), the MBH levels of POMC and TH mRNAs were approximately 17 and 31% lower than those measured in OVX controls, respectively. In OVX rats implanted for 20 days with larger E2 implants (99 +/- 9 pg E2/ml serum), POMC and TH mRNA levels were approximately 29 and 41% lower than in OVX controls, respectively. Additional groups were exposed to the higher E2 dose for 20 days and then killed 10 or 20 days after removal of the E2 implant. In these rats, POMC mRNA levels rebounded to the same level seen in OVX controls, while TH mRNA levels even exceeded control values by 22-27%. TH and POMC mRNA levels did not change significantly between 09.00 and 15.00 h, except 10 days after removal of the E2 implants, when 09.00 h POMC mRNA levels were higher than the 15.00 h levels. MBH POMC and TH mRNA levels were positively correlated with each other within individual animals. This correlation is maintained when both POMC and TH mRNA levels are suppressed in response to both 3-day low-dose and 20-day high-dose E2 treatment. However, although rat MBH opiomelanotropinergic and tuberoinfundibular dopaminergic mRNA biosynthesis thus appear to be positively correlated, the coregulation or functional interactions of these two neuronal systems remain to be determined.

High postovariectomy LH levels are not due to decreased opioid inhibition of GnRH.

It has been proposed that endogenous opioid peptide (EOP) inhibition of hypothalamic GnRH secretion mediates and is dependent upon gonadal steroid feedback of LH secretion, although considerable conflicting data have been reported. Accordingly, a well-characterized replacement regimen was used to approximate physiological stimulation by estradiol (E2) and progesterone (P4) in adult rats 10 days after ovariectomy (OVX), followed by in vitro incubation of the isolated median eminence to evaluate the role of E2 and P4 in modifying GnRH release in response to the opiate receptor antagonist, naloxone (NAL). Basal (control) GnRH release from median eminences of OVX, OVX + E2, and OVX + E2 + P4 rats was similar, and NAL treatment elicited a comparable increase in GnRH release under all three gonadal steroid conditions. Thus, EOP suppression of median eminence GnRH secretion does not appear to mediate or be dependent upon negative feedback regulation of LH secretion by physiological concentrations of gonadal steroids.

Pulsatile GnRH-stimulated LH release from the human fetal pituitary in vitro: sex-associated differences.

An in-vitro perfusion system was utilized to examine LH release from human fetal (19-24 weeks gestation) anterior pituitaries during repetitive GnRH stimulations. Pituitaries (five male, four female) were dissected into halves, and one hemipituitary of each pair was stimulated with 10-min pulses of 1 nM GnRH administered at 60-min intervals over 24h, whereas the matching hemipituitary received pulses of medium alone. Basal (no GnRH stimulation) LH release from female hemipituitaries was significantly (P less than 0.01) greater than from male hemipituitaries, and the amplitude of LH release associated with GnRH pulses was sixfold greater (P less than 0.001) with female hemipituitaries. Furthermore, the magnitude of LH release associated with individual GnRH pulses was significantly (P less than 0.001) enhanced during the course of female hemipituitary perfusions, but not during perifusion of male hemipituitaries. These studies demonstrate that LH secretion by the female, but not male, mid-gestational human fetal pituitary is increased in response to a physiological pattern and interval of repeated pulsatile GnRH stimulation in vitro.

Synchronous Pulsatile Release of Luteinizing Hormone and Immunoreactive Beta-Endorphin from the Human Pituitary in vitro.

Abstract An in vitro perifusion system employing very frequent (30 s) perifusate collectionswas utilized to investigate the relationship between pulsatile release of luteinizing hormone (LH) and immunoreactive beta-endorphin (ibeta-END) during individual perifusions of adult human anterior hemipituitaries. Each of six hemipituitaries released LH and ibeta-END in a distinctlypulsatile fashion, with pulses occurring approximately every 3.2 min for each. Power spectral analysis revealed that pulsatile release of both LH and ibeta-END occurred in a rhythmic pattern, with a periodicity of 3.1 and 3.2 min, respectively, and that the periodicity of pulsatile LH and ibeta-END release was correlated within individual perifusions. Moreover, the relative amplitudes (% change) of the synchronous LH and ibeta-END pulses were correlated. The effluent fractions from two of the perifusions were also assessed for thyrotropin, and it was determinedthat thyrotropin pulses were synchronized to both LH and ibeta-END pulses. These studies confirm that LH and ibeta-END are released from human anterior pituitaries in vitro in an intrinsically pulsatile fashion, and demonstrate that the LH and ibeta-END pulses tend to occur rhythmically and in synchrony and proportion with each other. Furthermore, correlation of thyrotropin pulses to both the LH and ibeta-END pulses suggests a common fundamental intrapituitary pulse generating mechanism.

Alterations of the adrenocorticotropin-cortisol axis in normal weight bulimic women: evidence for a central mechanism.

We studied pituitary-adrenal function in eight women with normal weight bulimia and seven normal women by measuring plasma ACTH and serum cortisol levels at 20-min intervals for 24 h and the responses to human CRH (hCRH) and to a noon meal. The bulimic women had increased 24-h transverse mean plasma ACTH [1.09 +/- 0.06 (+/- SE) vs. 0.75 +/- 0.14 pmol/L; P less than 0.05] and serum cortisol (235 +/- 21 vs. 152 +/- 9 nmol/L; P less than 0.005) concentrations. While the 24-h ACTH and cortisol pulse frequencies were unaltered, the bulimic women had higher (P less than 0.05) mean peak ACTH (1.46 +/- 0.09 vs. 1.03 +/- 0.15 pmol/L) and cortisol values (331 +/- 33 vs. 239 +/- 17 nmol/L). Despite having higher mean and peak plasma ACTH and serum cortisol values, the bulimic women had a blunted response of both ACTH (P less than 0.001) and cortisol (P less than 0.005) to hCRH, which included a lower mean maximal plasma ACTH response, decreased (P less than 0.05) integrated area under the ACTH response curve (161 +/- 12 vs. 231 +/- 23 pmol/min.L), a lower (P less than 0.05) maximum cortisol response (284 +/- 35 vs. 413 +/- 19 nmol/L), and decreased (P less than 0.05) area under the cortisol curve (11.1 +/- 1.9 vs. 15.9 +/- 1.3 X 10(3) nmol/min.L). The circadian variations of both ACTH and cortisol were maintained in the bulimic women; the nadir and acrophase times were similar to those of the normal women. However, the rise in serum cortisol that occurred within 1 h after the lunch meal in the normal women (104 +/- 35 nmol) did not occur in the bulimic women (P less than 0.05). These data demonstrate that marked changes in hypothalamic-pituitary-adrenal function occur in bulimia in the absence of weight disturbance and suggest central activation of CRH and/or synergistic factors as well as alterations in signals from gut to brain in this syndrome.

Pulsatile gonadotropin-releasing hormone release from the human mediobasal hypothalamus in vitro: opiate receptor-mediated suppression.

An in vitro perifusion system was used to investigate pulsatile gonadotropin-releasing hormone (GnRH) release from the fetal (20-23 weeks of gestation) and adult human mediobasal hypothalamus (MBH). Fetal human MBHs released GnRH in discrete pulses, with a periodicity of approximately 1 h. Adult human MBHs also released GnRH in a pulsatile manner, with a periodicity of 60-100 min. The calcium-dependent pulsatile GnRH release from fetal human MBHs was suppressed by addition of morphine (10 microM) to the perifusion medium, and this suppression was reversed by addition of the opiate receptor antagonist naloxone (10 microM). These results indicate that the human hypothalamic GnRH pulse-generating mechanism is located entirely within the MBH, and that this pulse generator can maintain intrinsically pulsatile GnRH release independent of all innervation from outside this site. Our data also demonstrate that human hypothalamic pulsatile GnRH release can be suppressed by an opiate receptor-mediated mechanism located within the MBH.

Endogenous opioid inhibition and facilitation of gonadotropin-releasing hormone release from the median eminence in vitro: potential role of catecholamines.

The intrahypothalamic site(s) of endogenous opioid regulation of GnRH secretion remains to be resolved. Accordingly, we used an in vitro acute incubation system to evaluate GnRH, dopamine (DA), and norepinephrine (NE) release from adult male rat median eminences (MEs) in response to the opiate receptor agonist morphine (MOR) and the opiate receptor antagonist naloxone (NAL). MOR (2 mM) stimulated basal and K+-induced GnRH release from isolated MEs, but 0.25, 5, or 100 microM MOR was without significant effect. NAL (1 mg/ml; 2.8 mM) increased basal GnRH release, but 0.01 mg NAL/ml suppressed basal GnRH release, and neither 0.001 nor 0.1 mg NAL/ml had an appreciable effect. NAL did not significantly alter K+-induced GnRH release. In a separate experiment, 1 mg NAL/ml stimulated but 0.01 mg NAL/ml inhibited basal release of DA and NE from the ME. NAL (1 ng/ml) also decreased K+-induced DA and NE release. The rates of basal and K+-induced DA and NE release were highly correlated with GnRH release during corresponding 0, 0.01, and 1.0 mg/ml NAL treatments in the preceding experiment (r = 0.98 and 0.93, respectively). Thus, 2 mM MOR stimulated but different NAL dosages either stimulated or inhibited GnRH release from isolated MEs, suggesting complex opioid regulation at the level of the GnRH neurosecretory terminals. The precise correlation between GnRH and DA/NE release suggests that the catecholamine terminals close to both GnRH- and endorphin-containing terminals in the ME may mediate this opioid regulation.