0 145-6OO8/94/1805- 1242$3.Oo/O ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 18, No. 5 September/October 1994

Prenatal Alcohol Exposure Alters the Hypothalamic- Pituitary-Adrenal Axis Response of Immature Offspring

to Interleukin-1 : Is Nitric Oxide Involved? Soon Lee and Catherine Rivier

We have previously shown that following prenatal alcohol exposure, immature offspring showed blunted ACTH released in response to the peripheral administration of interleukin-16 (IL-16). The present studies were conducted to investigate the role of changes in corti- costeroii feedback (measured by altered adrenal responses to ACTH), corticotropin-releasing factor (CRF) content of the median eminence (ME), and the influence of endogenous nitric oxide (NO). The injection of several doses of ACTH failed to indicate measurable differences between the corticosterone responses of offspring born to dams fed ad libitum [control (C)], pair-fed (PF), or fed alcohol [ethanol (EtOH) = El. CRF content in the ME, taken as an index of the amount of releasable peptide, showed a small, but statistically significant, decrease following prenatal alcohol exposure. A com- parable change, however, was also noted in PF rats. As expected, the subcutaneous injection of IL-16 (0.5 pg/kg) induced smaller increases in plasma ACTH levels of E than C pups. The response of PF animals was intermediate between that of E and C rats. Finally, we observed that inhibition of NO formation by the administration of the arginine derivative Initro-L-arginine-methylester significantly augmented ACTH secretion in all three experimental groups, and reversed the decreased corticotrophs' response to IL-16 caused by prenatal alcohol.

Taken together, our results suggest that the ability of prenatal alcohol exposure to alter ACTH released by immature pups in re- sponse to blood-borne IL-16 is probably not mediated through changes in adrenal responsiveness. We propose, on the other hand, that endogenous NO levels may be increased by the prenatal treat- ment, thereby interfering with the release of peptides from nerve terminals in the ME.

Key Words: Alcohol, Interleukin-1, ACTH, Rats, Prenatal.

XPOSURE TO alcohol during prenatal development E influences the response of the offspring's hypothal- amic-pituitary-adrenal (HPA) axis, and in particular, alters the release of ACTH and/or corticosterone in response to various stimuli, such as physical stresses or the adminis- tration of alcohol, morphine, and interleukins. ' - I 2 The precise mechanisms that mediate these changes remain elusive and may depend on the type of signal Although alcohol-induced increases in maternal corticos-

From the the Clayton Foundation Laboratories for Peptide Biology,

Received for publication December 6, 1993; accepted April 6 , I994 This study was supported by the National Institute on Alcohol Abuse

and Alcoholism Grant 08924. Reprint requests: Soon Lee, Ph. D., the Clayton Foundation Labora-

tories for Peptide Biology, the Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037.

the Salk Institute, La Jolla, California.

Copyright 0 1994 by The Research Society on Alcoholism.

1242

terone levels,I3 or changes in hippocampal glucocorticoid receptors, l 4 are probably not involved, altered hypothal- amic corticotropin-releasing factor (CRF) content',' and/ or the response of CRF-dependent pathways, remain po- tential candidates.

We have recently reported that ACTH released in re- sponse to two stimuli, exposure to mild electroshocks, and administration of interleukin- 1 p (IL- 1 p), were differen- tially altered following prenatal alcohol exposure: indeed, whereas immature offspring born to dams exposed to alcohol showed an increased response to shocks, their response to the peripheral administration of the cytokine was markedly blunted.'3I2 Interestingly, the site of CRF- dependent pathways at which these two signals exert their effect is different: electroshocks primarily stimulate CRF neurons within the hypothalamus, 1 5 ~ 1 6 whereas the acute effect of peripherally injected IL- 1 p takes place on nerve terminals within the median eminence (ME)."," Our results thus suggest that prenatal alcohol exposure may exert a differential influence on CRF perikarya and ter- minals. Additionally, changes in adrenal responsiveness of immature rats administered alcohol during embryonic development may also play a role in the altered activation of the HPA axis. Indeed, as we reported earlier: prenatal alcohol exposure increases ACTH released in response to stress, but does not alter corticosterone secretion. Al- though ACTH levels may have been sufficient to cause maximum adrenal activation in all groups of rats, the possibility of alcohol-induced changes in adrenal function needs to be explored.

A variety of experimental approaches can be used to investigate the potential influence of prenatal alcohol ex- posure on the activity of the offspring's HPA axis. These include measurement of the secretory profiles of secreta- gogues (CRF, vasopressin, and catecholamines) by push- pull cannulation or microdialysis, assessment of steady- state mRNA levels of hypothalamic peptides, and respon- siveness of elements of the HPA axis to specific stimuli. Using in situ hybridization techniques, we have previously reported that treatment of pregnant dams with alcohol increased CRF mRNA levels of the hypothalamus of 3- week-old offspring' and proposed a possible role of this change in mediating the augmented ACTH secretion o b served following exposure of the pups to mild electro-

Alcohol CIin Exp Res, Vol 18, No 5, 1994 pp 1242-1247

PRENATAL ALCOHOL EXPOSURE 1243

shocks. Because, on the other hand, the response of the HPA axis to cytokines was blunted,I2 it is possible that the release of CRF (or other secretagogues) from the ME might be impaired by prenatal alcohol treatment. However, the assessment of peptide secretion in immature rats raises enormous technical difficulties. We therefore used an indirect approach and measured basal concentrations of CRF in extracts of the ME as an index of the amount of peptide releasable to the pituitary. We also examined possible changes in adrenal responsiveness caused by pre- natal alcohol exposure.

We have previously observed that nitric oxide (NO) appears to exert a restraining influence on ACTH released in response to the peripheral injection of IL-l&’9 but a stimulatory effect on ACTH secreted in rats exposed to mild electroshocks.20 The possible connection between alcohol and this gas has only started to receive attentioq2’ and at present very little is known of its putative role in modulating the action of alcohol on the HPA axis. Chronic alcohol treatment increases the number of glutamate bind- ing sites,22 and stimulation of N-methy1-D-aspartic acid (NMDA) receptors releases N0.23,24 Thus, it is not unrea- sonable to speculate that prenatal alcohol exposure might upregulate NO production in the brains of the fetuses. This change, if it takes place, might participate in the ability of prenatal alcohol to decrease pituitary response to IL-lp’z and to increase ACTH released by physical stress.8 If this hypothesis is correct, blockade of NO for- mation should reverse the inhibitory influence of prenatal alcohol exposure on IL- 1 -induced ACTH secretion. Therefore, the last set of experiments described herein pertains to the ability of arginine derivatives to reverse the effect of alcohol.

METHODS

Animals All animals were kept under standard lighting conditions (1 2-hr

light: 12-hr darkness; lights off at 1800). Virgin female Sprague-Dawley rats were housed individually with an adult male. The presence of a plug was checked every morning, and the morning that a plug was detected was designated as day 1 of gestation. Standard laboratory chow and water ad libitum were offered to all three groups until day 2 of gestation. At that time, the rats were randomly divided into three experimental groups: a group fed a standard laboratory chow diet [control (C)], a group fed an alcohol diet [ethanol (EtOH) = El, and a group fed an isocaloric diet, in which sucrose replaced ethanol [pair-fed (PF)]. Alcohol was introduced gradually over 3 days, until EtOHderived calories represented 35% of the total diet. In the PF group, the females were yoked to E animals so that pairs of females received the same amount of diet with or without ethanol on a ml/kg basis. The diets, which have been previously described in detail,12~2s were prepared freshly every day, and offered in late after- noon through day 20 of gestation. At that time, all rats were placed on the standard diet with free access to rat chow and water.

Animals were weighed every 3-5 days. Blood alcohol levels were measured in tail blood samples obtained between 2000 and 2 100 on day 18 of gestation. PF and C dams were also subjected to tail vein bleeding. Starting on day 20, the colony was checked morning and evening for births. The day of birth was designated postnatal day 1. Following parturition, litters were weighed, sexed, pooled within each of the three

experimental groups, randomized, and culled to 5-6 males and 5-6 females whenever possible. Pups born to dams from PF and E rats were fostered to control nursing dams to avoid possible effects of restricted calorie intake on the nursing process. Pups were kept with their litter mates and dams until day 22. To minimize handling, the pups were only weighed at birth and on the day of the experiment. Pups were weaned on day 22, then kept six/box. Studies were conducted between postnatal days 22 and 27. Pilot studies were first conducted in naive rats (no prenatal treatment) to establish dose-response curves, time courses of hormone responses, and optimum conditions for preparation of CRF extracts. Following prenatal treatments, each experiment was done at least twice with different batches of prenatally treated rats.

Treatments I G l & a generous gift of Dr. S. Gillis (Immunex, Seattle, WA), was

diluted in 0.04 M phosphate buffer (pH 7.4) containing 0.1% bovine serum albumin and 0.01 % ascorbic acid, and injected subcutaneously. This vehicle was used for treatment of control rats. Cortrosyn (ACTH 1-24) was purchased from Sigma Chemical Co. (St. Louis, MO) and injected subcutaneously. The arginine derivative L,nitro-L-arginine- methylester (L-NAME) was also purchased from Sigma, diluted in Satine following adjustment of the pH to 7.35, and injected subcutaneously.

ME Extract Following rapid decapitation of the rats, the brains were gently sepa-

rated from the skull, taking care not to disturb the ME. This tissue was removed from the rest of the brain under a dissecting scope, and placed in 0.5 ml of 1.0 N HOAc:0.5 N H a (1: 1 v:v) containing pepstatin (4.5 pg/ml) and 0.5% @-mercaptoethanol. The extracts were boiled for 5 min, then cooled, and lyophilized as previously described.26 Despite reported alcohol-induced changes in brain weights? our preliminary experiments indicated no significant differences in the weight or protein content of tissues obtained from the three experimental groups (data not shown). We therefore present results in ng CRF/ME.

Radioimmunoassay

Plasma ACTH levels were measured in duplicate samples, using a commercially available kit (Allegro kit, Nichols Institute, San Juan Capistrano, CA) that we have validated for rat ACTH.I9 Plasma corti- costerone and CRF levels were measured by radi~irnrnunoassay.’~~’~

Statistical Analysis Data were analyzed by one- or two-way ANOVA, followed by Dun-

nett’s one-sided Duncan’s multiple range test for individual differences, Bonferroni/Dunn test, and/or Fisher’s protected least significant differ- ence test.

RESULTS AND DISCUSSION

The work presented herein was conducted to examine some of the mechanisms that mediate the ability of pre- natal alcohol exposure to blunt ACTH released in response to the peripheral injection of IL-lp. We had previously reported that changes in pituitary responsiveness to CRF did not play a major roleI2 and show herein that differ- ences in adrenal sensitivity to ACTH do not seem impor- tant either. Indeed, as illustrated in Fig. 1 , administration of Cortrosyn induced comparable ACTH levels in C, PF, and E rats. Two possible mechanisms, on the other hand, emerge from our results. First, we observed decreased CRF

1244 LEE AND RlVlER

400

300

200

100

0 0 0.05 0.2 0.8

-Yn (u)

Fig. 1. Effect of the subcutaneous injection of the vehicle or Cortrosyn (ex- pressed in units) on plasma wrtiisterone levels of pups born to C, PF, or E dams. Blood samples were obtained in different groups of rats decapitated under basal conditions, or 15, 30, and 60 min after the subcutaneous injection. Each point represents the integrated means (over the 60-min period) -e SEM of 6-8 male rats, aged 24-25 days. There were no statistical ( p > 0.05) dflerences between basal plasma wrtiwsterone levels of C, PF, and E rats. The subcutaneous injection of 0.05-0.8 units Cortrosyn induced comparable increases in plasma cOrtiCosterone levels, integrated over a 6 h i n period, in all three experimental groups.

Table 1. CRF Content in the ME of Pups Born to C, PF, or E Dams

Treatments Males Females

C 6.88 f 0.49 7.24 f 0.39 PF 6.06 f 0.35' 5.86 f 0.40' E 6.10 f 0.35't 6.03 f 0.30't

Results were obtained in 24- to 27day-old, rapidly decapitated rats and are

' p < 0.05 from C rats. t p > 0.05 from PF rats.

expressed as ng CRF/ME f SEM (n = 8 for each group).

concentrations in the ME of E animals (Table 1). As blood-borne cytokines act in this region to stimulate CRF release (for references, see ref. 30), a smaller pool of releasable CRF might account at least in part for the blunted ACTH response of E rats. Indeed, CRF concen- trations in the ME represent the readily releasable pool of this hormone present in axon terminals, and ME peptide contents are reportedly correlated with ACTH responses to ~tresses.~' The mechanism leading to this decrease, however, remains to be determined. We have previously shown that, in contrast to dams exposed to alcohol vapors during gestatioq8 pregnant rats fed alcohol gave birth to pups that did not exhibit significantly different steady- state CRF mRNA levels in their hypothalamus,12 an ob- servation also reported by other investigator^.^' Whether the lower CRF concentrations we observed were thus caused by an alcohol-induced decrease in translational efficiency of the CFW message and/or enhanced CRF protein degradation, remains to be determined. Another, not mutually exclusive possibility, concerns the role played by catecholamines. Indeed 3-week-old offspring of dams fed alcohol during pregnancy are reported to have significantly decreased norepinephrine content of the hy-

pot ha la mu^.^^ Because catecholamines influence CRF synthesis and release,34 this might represent one possible mechanism of the influence of alcohol.

Another mechanism we considered involves NO. As previously discussed, the role played by NO at the level of the brain and the potential effects of various stimuli on the levels of this gas remain both controversial and still largely unexplored. Nevertheless, it does not seem unrea- sonable to consider the possibility that rats exposed to alcohol during embryonic development might exhibit en- hanced NO formation. We have recently proposed that NO restrains the corticotrophs' response to blood-borne cytokines by modulating the release of ACTH secreta- gogues from nerve terminals." Because IL-I@ increases plasma ACTH levels in part by stimulating the secretion of peptides and amines from the ME,17,30,35,36 it is possible that alcohol blunts the effect of IL-1 by altering peptide concentrations in, and/or release from, this tissue. Whether this plays a role in the decreased CRF levels we measured in the ME of alcohol-exposed pups remains to be determined. We have, on the other hand, recently reported that, in contrast to its effect at the level of the ME, NO facilitates the release of ACTH measured in rats exposed to stress.'' A proposed role of NO could thus also explain our earlier finding that prenatal alcohol exposure augments ACTH released by mild electroshocks.8

If our hypothesis is correct, blockade of NO formation should reverse the inhibitory influence of prenatal alcohol on IL- 1 -induced pituitary stimulation. This was indeed the case. As illustrated in Fig. 2, E rats showed the expected blunted ACTH response to IL- I @ whereas administration of the arginine derivative L-NAME restored this response to levels at least comparable, if not slightly higher, than those of C animals. Furthermore, comparison of the per- centage increase in ACTH values before and after L- NAME injection indicated a significantly higher value in pups exposed to alcohol during embryonic development than in C animals. Whether this corresponds to a com- pensatory mechanism remains to be determined. Indeed, the remarkable ability of arginine derivatives to markedly increase ACTH released by peripherally injected cytokines has only recently been described," and much remains to be learned concerning the consequences of NO formation blockade on the activity of the HPA axis. Additionally, whereas our results suggest that alcohol may alter NO formation in immature offspring of dams exposed to the drug, it must be noted that NO is an ubiquitous gas whose effects and synthesis are linked to several neurotransmit- ters modulating the activity of the HPA axis, such as NMDA.23,374' Thus, whereas our results suggest the pos- sibility that NO participates in the decreased ACTH re- sponse of alcohol-treated offspring injected with IL-16, it will be necessary to identify the mechanisms involved in this effect.

Another important question to discuss concerns the ability of pair-feeding itself to produce effects which, al-

PRENATAL ALCOHOL EXPOSURE

1200-

1245

a T

IL-1 p I IL-1 p + L-NAME

1000

400

200

a T

4 i C PF E

Fig. 2. Effect of maternal exposure to an ad libitum diet (C), PF diet, or E diet on plasma ACTH levels of offspring injected with IL-1,9 in the presence or absence of L-NAME. Blood samples were obtained 2 hr after injection of IL-1s (0.5 pg/kg, sc) or IL-ls + L-NAME (40 mg/kg, SC). Each bar represents the mean f SEM of eight 24- to 26day-old females. *, p < 0.05 from plasma ACTH levels of C rats injected with the cytokine. a, p < 0.05 from plasma ACTH levels of rats not injected with L-NAME. To facilitate the reader’s analysis of these results, we also present calculations of the percentage increases in plasma ACTH levels (levels in the presence of L-NAME/levels in the absence of L-NAME, pg/ml f SEM): C rats, 330% (1009 & 70/306 f 46); PF rats, 396% (910 f 1151230 f 36); and E rats, 594% (1446 f 71/193 31). Analysis by Bonferroni/Dunn test indicated that following L- NAME treatment, significant differences were found between C and E rats (p = 0.0019) and PF and E rats (p = 0.0035), whereas the difference between C and PF rats was not significant (p = 0.3741). Administration of L-NAME alone did not measurably alter plasma ACTH values over those of vehicle-administered rats in any group (data not shown).

though smaller than those of alcohol and often not signif- icantly different from those of control animals, are never- theless observable. The fact that pair-feeding represents an experimental treatment has long been r e c o g n i ~ e d . ~ * ~ ~ ’ ~ ~ , ~ ~ Although nutritional factors have been implicated in these effects, our PF dams showed weight gains that were not markedly different from those of control animals (Fig. 3). Additionally, whereas offspring of PF dams were slightly smaller than those of C rats, they were obviously larger than pups born to E dams (Table 2). Other mechanisms are therefore probably important. The stress of being hungry, for example, activates the HPA axis of PF dams3 and possibly that of their fetuses. It is also feasible that changes in the basal circadian rhythm of the dams’ HPA axes caused by restricted feeding4346 might play a role, although Weinberg and Gallo3 have argued that no such shift occurs when the diet is presented in late afternoon (the method used in our laboratory). Our observations thus lead to the following questions: Are the neuroendo- crine changes observed in PF and E offspring linked to changes in maternal HPA axis? And, are the mechanisms

total weight gains (grn)

(OC OPF H E )

b

0 3 6 9 12 15 10 21

days of pregnancy Fig. 3. Cumulative weight gains of pregnant dams fed ad libitum (C), PF, or E

diet. Each point represents the means f SEM of 12-15 animals. *, p < 0.05. Statistical analysis of maternal weight gains indicated a significant (group x time) interaction. Although increases in body weights, expressed as cumulative gains over day 1 of pregnancy, showed some differences between PF and C dams, the overall changes were comparable. In contrast, the alcohol diet measurably inter- fered (p < 0.05) with normal weight gains throughout the treatment, a finding also reported by others? At birth, there was a significant (group x gender x treatment) interaction rF(2.21) = 7.6111 with males and females born to PF or E dams significantly smaller ( p < 0.05) than those born to C dams (Table 2). These differences, which represent common findings with these types of per- sisted in 23- to 24day-dd females [F(2,20) = 30.9; p < 0.051 and 22- to 23day d d males tF(2.45) = 9.98, p < 0.051.

Table 2. Body Weights of Offspring Born to C, PF, or E Dams

Age (days)

Treatments 1 22-23

Males C 6.50 f 0.1 1 57.8 i 0.8 PF 6.22 f 0.13’ 53.3 f 0.8t E 5.79 f 0.08tS 49.6 f 0.7t*

Age (days)

Treatments 1 23-24

Females C 6.18 f 0.1 1 51.9 f 0.6 PF 5.98 f 0.09’ 50.9 f 0.5’ E 5.45 f 0.13tS 48.6 f 0.5tt

Weights given in g f SEM (n = 20-25). ‘ p > 0.05 from C rats. t p < 0.05 from C rats. $ p < 0.05 from PF rats.

mediating the consequences of pair-feeding similar to those operative during prenatal alcohol exposure? Wein- berg and B e ~ i o ~ ~ have shown that both PF and E diets augmented the activity of the maternal HPA axes, and the qualitative (if not quantitative) similarities between the neuroendocrine results of the two diets suggest that indeed comparable mechanisms may be operative. We investi- gated the role of increased maternal corticosterone levels in mediating the changes observed in the offspring and concluded that, at least in the paradigm we used (admin- istration of alcohol through vapors), this steroid did not appear to be of primary importance.13 Our results, how- ever, do not allow us to rule out the putative contribution

1246 LEE AND RlVlER

of other factors, such as altered release of peptides, cate- cholamines, and/or excitatory amino acids. Examination of the pattern of secretion of these secretagogues may yield information regarding the similarities or differences be- tween the effects of pair-feeding versus the alcohol diet in the dams.

In conclusion, we show herein that the ability of prenatal alcohol exposure to blunt ACTH released by pups injected with IL- 1 p does not appear to be mediated by increased adrenal responsiveness to ACTH. Although these studies do not give complete information regarding possible alter- ations in corticosteroid feedback, the lack of changes in receptors for these steroids in the hippocampus of rats administered alcohol during embryonic development l4 is not congruent with the potential importance of this mech- anism. Our results are consistent, on the other hand, with the hypothesis that following alcohol administration dur- ing embryonic development, increased endogenous NO levels may alter the response of the HPA axis to stimuli. This phenomenon would explain both the decreased ac- tivity of the HPA axis to blood-borne cytokines'2 and the increased stimulatory effect of stress on this axis.*

REFERENCES 1. Taylor AN, Branch BJ, Liu SH, Wiechmann AF, Hill MA, Kokka

N: Fetal exposure to ethanol enhances pituitary-adrenal and temperature responses to ethanol in adult rats. Alcohol Clin Exp Res 5:237-246,198 1

2. Taylor AN, Branch BJ, Liu SH, Kokka N Long-term effects of fetal ethanol exposure on pituitary-adrenal response to stress. Pharmacol Biochem Behav 16585-589, 1982

3. Weinberg J, Gallo P V Prenatal ethanol exposure: Pituitary-ad- renal activity in pregnant dams and offspring. Neurobehav Toxicol Teratol45 15-520, 1982

4. Weinberg J: Hyperresponsiveness to stress: Differential effects of prenatal ethanol on males and females. Alcohol Clin Exp Res 12:647- 652, 1988

5 . Weinberg J: Prenatal ethanol exposure alters adrenocortical de- velopment of offspring. Alcohol Clin Exp Res 13:73-83, 1989

6. Nelson LR, Taylor AN, Lewis JW, Poland RE, Redei E, Branch BJ: Pituitary-adrenal responses to morphine and footshock stress are enhanced following prenatal alcohol exposure. Alcohol Clin Exp Res 10:397-402, 1986

7. Angelogianni P, Gianoulakis C: Ontogeny of the &endorphin response to stress in the rat: Role of the pituitary and the hypothalamus. Neuroendocrinology 50:372-38 1, 1989

8. Lee SY, Imaki T, Vale W, Rivier CL: Effect of prenatal exposure to ethanol on the activity of the hypothalamic-pituitary-adrenal axis of the offspring: Importance of the time of exposure to ethanol and possible modulating mechanisms. Mol Cell Neurosci 1 : 168- 177, 1990

9. Weinberg J: Prenatal ethanol effects-Sex differences in offspring stress responsiveness. Alcohol 8:2 19-223, 1992

10. Weinberg J: Prenatal ethanol exposure alters adrenocortical re- sponse to predictable and unpredictable stressors. Alcohol 9:427-432, 1992

1 1. Taylor AN, Branch BJ, Nelson LR, Lane LA, Poland RE: Prenatal ethanol and ontogeny of pituitary-adrenal responses to ethanol and morphine. Alcohol 3:255-259, 1986

12. Lee S, Rivier C: Prenatal alcohol exposure blunts interleukin- 18- induced ACTH and @-endorphin secretion by immature rats. Alcohol Clin Exp Res 17:940-945, 1993

13. Lee S, Rivier C: Administration of corticosterone to pregnant adrenalectomized dams does not alter the hypothalamic-pituitary-adre- nal axis' activity of the offspring. Mol Cell Neurosci 3: 1 18- 123, 1992

14. Weinberg J, Petersen T D Effects of prenatal ethanol exposure on glucocorticoid receptors in rat hippocampus. Alcohol Clin Exp Res

15. Imaki T, Nahon J-L, Rivier C, Sawchenko PE, Vale W Differ- ential regulation of corticotropin-releasing factor mRNA in rat brain cell types by glucocorticoids and stress. J Neurosci 1 1585-599, 1991

16. Imaki T, Shibasaki T, Hotta M, Demura H: Early induction of c-fos precedes increased expression of corticotropin-releasing factor mes- senger ribonucleic acid in the paraventricular nucleus after immobiliza- tion stress. Endocrinology 13 1 :240-246, 1992

17. Watanobe H, Sasaki S, Takebe K Evidence that intravenous administration of interleukin- 1 stimulates corticotropin releasing hor- mone secretion in the median eminence of freely moving rats: Estimation by push-pull perfusion. Neurosci Lett 133:7-10, 1991

18. Rivest S , Torres G, Rivier C Differential effects of central and peripheral injection of interleukin-l@ on brain cyos expression and neuroendocrine functions. Brain Res 587: 13-23, 1992

19. Rivier C, Shen G In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin- 18, vasopressin and oxytocin. J Neurosci 14:1985-1993, 1994

20. Rivier C: Endogenous nitric oxide participates in the activation of the hypothalamic-pituitary-adrenal axis by noxious stimuli. Endocr J

2 1. Lancaster FE: Alcohol, nitric oxide, and neurotoxicity: Is there a connection?-A review. Alcohol Clin Exp Res 16539-541, 1992

22. Brennan C, Crabbe J, Littleton J: Genetic regulation of dihydro- pyridine-sensitive calcium channels in brain may determine susceptibility to physical dependence on alcohol. Neuropharmacology 29:429-432, 1990

23. Moncada S, Palmer RMJ, Higgs EA: Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991

24. Snyder SH, Bredt DS: Biological roles of nitric oxide. Sci Am

25. Driscoll CD, Chen JS, Riley E: Operant DRL performance in rats following prenatal alcohol exposure. Neurobehav Toxicol 2:207-2 1 I , 1980

26. Plotsky P, Meaney M: Early, postnatal experience alters hypothal- amic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Mol Br Res 18: 195- 200, 1993

27. Ahmed 11, Shryne JE, Gorski RA, Branch BJ, Taylor AN: Prenatal ethanol and the prepubertal sexually dimorphic nucleus of the preoptic area. Physiol Behav 49:427-432, 1991

28. Rivier C Female rats release more corticosterone than males in response to alcohol: Influence of circulating sex steroids and possible consequences for blood alcohol levels. Alcohol Clin Exp Res 172354- 859, 1993

29. Vale W, Vaughan J, Yamamoto G, Bruhn T, Douglas C, Dalton D, Rivier C, Rivier J: Assay of corticotropin releasing factor, in Conn PM (ed): Methods in Enzymology: Neuroendocrine Peptides. New York, Academic Press, 1983, pp 565-577

30. Rivier C Neuroendocrine effects of cytokines in the rat. Rev Neurosci 4:223-238, 1993

31. Murakami K, Akana S, Dallman MF, Ganong WF: Correlation between the stress-induced transient increase in corticotropin-releasing hormone content of the median eminence of the hypothalamus and adrenocorticotropic hormone secretion. Neuroendocrinology 49:233- 241, 1989

32. Angelogianni P, Gianoulakis C: Prenatal exposure to ethanol alters the ontogeny of the @-endorphin response to stress. Alcohol Clin Exp Res 13564-571, 1989

33. Detering N, Collins RM Jr, Hawkins RL, Ozand PT, Karahasan A Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: A long-lasting effect in the hypothalamus. J Neurochem 36:2094-2096, 1981

34. Plotsky P, Cunningham ETJ, Widmaier EP: Catecholaminergic

15:711-716, 1991

2~367-373, 1994

266:68-77, 1992

PRENATAL ALCOHOL EXPOSURE 1247

modulation of corticotropin-releasing factor and adrenocorticotropin secretion. Endocrine Rev 10:437-458, 1989

35. Matta SG, Singh J, Newton R, Sharp BM: The adrenocorticotro- pin response to interleukin-lg instilled into the rat median eminence

depends on the local release of catecholamines. Endocrinology 127:2 175- 2182, 1990

36, Barbanel B, G d e t s, Mekaouche M, ~ i ~ d ~ i ~ L, ~~m G, Siaud P, Szafarczyk s, Malaval F, Assenmacher I: Complex catecholaminergic modulation of the stimulatory effect of interleukin-10 on the cortico- tropic axis. Brain Res 626:31-36, 1993

37. Garthwaite J, Charles sL, Chess-wil~iams R: Endoaelium-de- rived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 336:385-388, 1988

38. Farah JM, Tadimedi SR, Mick SJ, Come KE, Iyengar S: N- methyl-Baspartate treatment increases circulating adrenocorticotropin and luteinizing hormone in the rat. Endocrinology 128: 1875-1880, 1991

merit increases Plasma ACTH in the neonatal female and male rat. Dev Brain Res 68:289-293, 1992

40. Chautard T, Boudouresque F, Guillaume V, Oliver C: Effect of excitatory amino acid on the hypothalamo-pituitary-adrenal axis in the

rat during the stress-hyporesponsive period. Neuroendocrinology 57:70- 78, 1993

41. Montague P, Gancayco C, Winn M, Marchase R, Friedlander M: Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex. Science 263:973-977, 1994

42. Taylor AN, Branch BJ, Cooley-Matthews B, Poland RE. Effects of maternal ethanol consumption in rats on basal and rhythmic pitui- --Adrenal function in neonatal offspring. Psychoneuroendocrinol- Ogy 7:49-589 1982

43. Krieger D Food and water restriction shifts corticosterone, tem- perature activity and amine periodicity. Endocrinology 95:1195-1201, 1974

44. Coover G, Murison R, Sundberg H, Jellestad F, Ursin H: Plasma corticosterone and meal expectancy in rats: Effects of low probability cues. physiol &hav 33:179-184, 1984

expres- sion of hypothalamic neurowptide m ~ ~ ~ s in fod-restricted and food-

46. Sander LD: Circadian influences on feeding-induced changes in ACTH and corticosterone secretion in rats. Regul Pept 4 1 : 109- 1 17, 1992

47. Weinberg J, Bezio S Alcohol-induced changes in pituitary-adre- nal activity during pregnancy. Alcohol Clin Exp Res 1 1:274-280, 1987

45. Brady Ls, Smith MA, Gold pw, Herkenham M:

39. Bardgett ME, Taylor GT, F m h JM Jr: Systemic NMDA treat- deprived rats. Neurmndoc-nology 52:441-447, 1990