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Influence of prenatal ethanol exposure on hormonal responses to clonidine and naloxone in prepubescent male and female rats
Psychoneuroendocrinology,Vol.11, No. 1, pp. 105- 110,1986. Printedin GreatBritain.
INFLUENCE
0306-4530/86 $3.130+ 0.130 © 1986PergamonPressLtd.
OF PRENATAL
HORMONAL RESPONSES PREPUBESCENT
ETHANOL
EXPOSURE
ON
TO CLONIDINE AND NALOXONE MALE AND FEMALE RATS
IN
ROBERT F . McGIVERN,*'['~ RUSSELL E . POLAND,*'~ ERNEST P . NOBLE'l" a n d LESLIE A . L A N E *
*Department of Psychiatry, Bldg. F-5, Harbor-U.C.L.A. Medical Center, 1000 W. Carson Street, Torrance, CA 90509 U.S.A.; and 1"Department of Psychiatry and Behavioral Sciences, University of California at Los Angeles, Los Angeles, CA 90024, U.S.A.
(Received 2 October 1984; in final form 9 May 1985) SUMMARY The expression of sexually dimorphic behavior has been found to be altered in adult animals following prenatal alchohol exposure. The present study examined whether such exposure would alter the sexually dimorphic response of luteinizing hormone (LH) to cionidine 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. INTRODUCTION
(1982) reported that the rise in plasma LH following administration of naloxone and clonidine is sexually dimorphic in prepuberal male and female Sprague-Dawley rats. Females exhibited a rise in plasma LH levels in response to naloxone but not clonidine administration, whereas male levels of LH were elevated following clonidine but not naloxone. These results were of particular interest because of our finding of alterations in adult sexually dimorphic behavior in animals exposed to alcohol in utero (McGivern et al., 1984b) and our observation that LH levels following gonadectomy in fetal alcohol exposed (FAE) animals are approximately one-half those of controls (Handa et al., 1985). Therefore, we studied FAE animals at 16 days of age in the present experiments to determine whether prenatal ethanol exposure influences the normal expression of this sexually dimorphic LH response. Exposure of animals to alcohol prenatally has been reported to produce long-term alterations in both opioid and noradrenergic systems of the brain (Detering et aL, 1980, SCHULTZ e t al.
~To whom reprint requests and correspondence should be addressed. 105
106
ROBERTF. McGIVERN,RUSSELLE. POLAND,ERNESTP. NOBLEand LESLIEA. LANF
1981; M c G i v e r n et al., 1984a; S h o e m a k e r et al., 1983). Since b o t h o f these systems are i n v o l v e d in the m o d u l a t i o n o f p it u i ta r y h o r m o n e release ( H e r z & Millan, 1984; K al r a & Simpkins, 1981; Meites et al., 1979), we m e a s u r e d these h o r m o n e s in the p l a s m a o f F A E a n i m al s a n d c o n t r o l s f o l l o w i n g a d m i n i s t r a t i o n o f the a l p h a - a d r e n e r g i c agonist, cl o n i d i n e or the o p i o i d a n t a g o n i s t , n a l o x o n e . In the ad u l t rat, a l p h a - a d r e n e r g i c agonists depress p l a s m a CS ( J o n e s et a k , 1976; W e i n e r & G a n o n g , 1978) b u t elevate p l a s m a L H levels (Kalra & Simpkins, 1981), whereas opiate a n t a g o n i st s st i m u l at e b o t h CS (Eisenberg, 1984) and L H secretion (Meites et al., 1979).
METHODS Pregnant, experienced Sprague--Dawley dams were purchased from Charles River breeders (Wilmington, MA, U.S.A.) to arrive on day 12 of gestation. On day 14 of gestation, randomly selected dams were switched to a liquid diet containing 35% ethanol-derived calories or an isocaloric liquid control diet containing no ethanol (Bio-Serv, Frenchtown NJ, U.S.A.). The dams given the isocaloric control diet were pair-fed to weight-matched dams in the ethanol diet group. The remainder of the dams received the normal diet of Purina lab chow and tap water ad libitum. These diets were continued until parturition, whereupon the dams on the liquid diets were switched to Purina lab chow and tap water. The light cycle was 12 h on/off with lights on at 1000 h. All animals were raised with their biological dam. On postnatal day 16, male and female pups from each litter were weighed and injected i.p. with saline, naloxone (2 mg/kg), or clonidine (0.1 mg/kg) 30 min prior to decapitation. Trunk blood was collected, and CS and LH were measured by radioimmunoassay (Poland et al., 1980, 1981). All samples for CS and LH were analyzed in a single assay. The maximum intra-assay coefficient of variation for both assays was 15%. The values of LH are expressed in terms of the RP-1 standard. Animals were decapitated between 1500 and 1800 h. They were dervied from eight chow-fed litters, 12 ethanol-exposed litters and 14 pair-fed litters. Each litter was represented in the three drug treatments for both sexes as much as litter size allowed. No more than three animals of each sex were used from a litter, and no more than one animal of each sex from a litter was represented in a drug (saline, naloxone, or clonidine) condition. LH and CS data were analyzed by separate 3 (fetal alcohol exposed (FAE), pair-fed (PF) and chow fed (CF) × 3 (drug) × 2 (sex) analyses of variance. The LH data were log-transformed prior to analysis. Post-hoc analyses were conducted by the Newman - Keuls procedure to test for differences between means.
RESULTS Figure 1 shows the p l a s m a c o r t i c o s t e r o n e responses f o l l o w i n g drug t r e a t m e n t . T h e C F and P F an i m als o f b o t h sexes exhibited a significant rise in p l a s m a h o r m o n e levels f o l l o w i n g clonidine, whereas no significant h o r m o n e elevations o c c u r r e d in the F A E animals. T h e m e a n p l a s m a c o r t i c o s t e r o n e level in the F A E males was significantly lower f o l l o w i n g n a l o x o n e a d m i n i s t r a t i o n c o m p a r e d to saline. T h e r e were no o t h er effects p r o d u c e d by n a l o x o n e . T h e analysis o f CS levels indicated m a i n effects for t r e a t m e n t (F = 7.86; 2163 d . f ; p < 0.001) a n d drug (F = 20.38; 2163 d . f . ; p < 0.02), as well as f o r the drug sex i n t e r a c t i o n (F = 3.89; 2163 d . f . ; p < 0.03). T h e analysis o f L H levels i n d ic a t e d significant m a i n effects for t r e a t m e n t (F = 5.44; 2156 d.f.; p < 0.01), drug (F = 19.11; 2156 d . f . ; p < 0.001), an d sex (F = 11.19, 1156 d.f; p < 0.001). I n t e r a c t i o n s were significant b et w een d r u g an d sex (F -- 49.59; 2156 d.f.; p < 0.001) an d a m o n g t r e a t m e n t , d r u g a n d sex (F -- 5.43; 4156 d .f .; p < 0.001). F i g u r e 2 shows that b o t h P F a n d F A E males exhibited a m a r k e d rise in L H levels f o l l o w i n g
HORMONE RESPONSES TO PRENATAL ETHANOL IN RATS
107
administration of clonidine, whereas the CF rats did not respond to clonidine. The LH rise in the FAE males was significantly greater than that in the PF males. Naloxone produced a significant decrease in LH levels in the PF males but produced no effect in the males of the other two groups. A significant rise in LH levels following naloxone injection occurred in all three groups of females. Clonidine produced no LH effect in the CF or FAE females, whereas it significantly decreased LH levels in the PF females.
CORTICOSTERONE 16 DAYS OF AGE :525-
*
FEMALE
[ ] SALINE ~ ] NALOXONE • CLONIOINE ¢-
MALE
e-
v
Chow Fed
Pair Fed
FAE
FIG. I. Mean levels of plasma corticosterone in animals exposed to three maternal treatments during the last week of gestation, at 30 min after injection of saline, naloxone (2 mg/kg), or clonidine (0.1 mg/kg). Vertical lines represent S.E.M. Number of animals in each condition is shown at bottom of graph. Asterisk indicates a significant difference (p < 0.05) from each saline control group.
DISCUSSION
The results of the present study indicate that clonidine stimulates CS release in normal rats of both sexes at 16 days of age. This stimulatory influence is opposite to the inhibitory effect of clonidine on CS release by stress in the adult dog (Ganong, 1977) and in the adult rat following i.v. infusion of 0.1 mg (McGivern and Poland, unpublished results). This inhibitory effect of clonidine in the adult animal is consistent with other data showing that activation of the noradrenergic system by electrical stimulation or norepinephrine
108
ROBERT F. McGIVERN, RUSSELL E. POLAND, ERNEST P. NOBLE and LESI IE A. LANE
LUTEINIZING HORMONE !6 DAYS OF AGE 200180-
FEMALE
160-
[ ] SALINE ]
NALOXONE
140120""
I00806040-
6,O "_t ~ 20-
140
MALE
120
.
o, c
I00
80
60 4
4oq 20Chow Fed
Poir
Fed
FAE
FIG. 2. Mean levels of luteinizing hormone in plasma at 30 min after injection of saline, naloxone (2 mg/kg), or clonidine (0. l mg/kg). Vertical lines represent S.E.M. Number of animals in each condition is shown at bottom of graph. Asterisk indicates a significant difference (p < 0.05) from each saline control group.
infusion also inhibits CS release in adult animals (see Weiner and Ganong, 1978 for review), but is in contrast to the work of Eisenberg (1983) indicating that clonidine infusion, in the same dose range which we have employed, stimulates CS release in the rat. Further work will be needed to understand the effect of clonidine on CS release in the rat. This apparent differential influence of clonidine in the 16 day old animal vs the adult may reflect the relative morphological immaturity of the noradrenergic system at 16 days. The density of NE terminals in the mesencephalon and hypothalamus of animals of this age is roughly half that observed in adult animals (Loizou, 1972). Amphetamine, which stimulates both noradrenergic and dopamine release, also has been reported to have an opposite effect in the prepubescent animal than it has in the adult. Spear (1979) found that amphetamine inhibited locomotor activity in animals under 35 days of age, in contrast to the stimulatory effect of the drug on adult animals. Interestingly, the stimulatory effect of clonidine on CS release in the young animal is similar to the stimulatory effect of the betaadrenergic agonist, isoproterenol, in the adult rat (Tilders et al., 1982). This suggests the possibility of differential development of noradrenergic receptor subtypes during ontogeny. In contrast to the CF and PF controls, the FAE female and male animals exhibited no increase in CS levels 30 min after clonidine administration. This apparent noradrenergic insensitivity at this age is consistent with the work of Detering et al. (1980, 1981) showing depressed NE levels in FAE animals at 3 weeks of age, which persisted into adulthood. Other Work has shown adult FAE animals to exhibit a hypersensitive locomotor response
HORMONE RESPONSESTO PRENATAL ETHANOLIN RATS
109
to methylphenidate compared to controls (Means et aL, 1984), suggesting a long-term functional influence of prenatal alcohol exposure on noradrenergic systems. Adult animals have been reported to exhibit an elevation in CS levels following naloxone administration (see Eisenberg, 1984 for review). Most of the animals in the present study failed to show any CS response to naloxone, suggesting that the role of the opioid system in the modulation of CS is not yet developed at this age. The FAE males appear to be an exception, however, since their CS levels were significantly depressed following naloxone administration compared to their saline-injected counterparts. Since normal CS levels in unstressed males at 16 days of age are in the 40 ng/ml range (Poland et al., 1981), the elevated CS levels following saline treatment in all groups in the present study appear to be the result of injection-related stress. Therefore, naloxone apparently partially antagonized this stress-induced elevation of CS levels in the FAE males. It should be noted that elevations in CS levels in this study were not due to time of day, since the diurnal rhythm of CS does not appear until postnatal day 18 or 19 (Poland et aL, 1981). The effects on LH plasma levels which we observed following naloxone and clonidine administration are in partial agreement with the findings of Schultz et ai. (1982). These investigators reported that naloxone progressively increased plasma LH concentrations in normal females from day 1 through day 16 postnatally, reaching a maximal stimulatory effect between day 10 and day 16. Clonidine was relatively ineffective in inducing a rise in plasma LH levels in females during this period. The opposite effect of these drugs on LH secretion was reported for males; a rise in LH was observed following clonidine administration but not after naloxone administration. The stimulatory effect of clonidine on LH was maximal on day 10 and decreased progressively until it was absent on day 30. Our findings on postnatal day 16 differ from those of Schultz et al. (1982) only in regard to the lack of a clonidine-induced LH rise in CF males. This may reflect an accelerated maturation rate of the noradrenergic pathways in the CNS in our control animals compared to those employed by Schultz et al., or, conversely, may be indicative of a delayed CNS maturation in the PF and FAE animals. The present results indicate that the pair-feeding diet also may produce a selective effect on the LH response to naloxone and clonidine, which is sex-dependent. The PF males exhibited a decreased LH response to naloxone, whereas the PF females exhibited a decreased LH response to clonidine. A pair-feeding-induced effect on the development of the circadian rhythm of plasma CS in rats has been reported previously (Taylor et al., 1982). In light of the widespread use of pair-feeding diets in fetal alcohol research, these hormonal findings appear to warrant further study. In. summary, the present results reveal a notable insensitivity of CS in prepubescent FAE male and female rats to the stimulatory influence of clonidine. CS levels in FAE males were depressed following naloxone administration compared to saline injection, but were unchanged in the other groups. Overall, these results appear to provide functional support for reported alterations in the CNS noradrenergic and opioid systems following prenatal exposure to ethanol (Detering et al., 1980, 1981; McGivern et al., 1984a; Shoemaker et al., 1983). This study was supported by NIAAA grants AA 06466 and AA 06478. The authors wish to thank Mrs Debra Hanaya for her editorial assistance.
110
ROBERT F. McGIVERN, RUSSELL E. POLAND, ERNEST P. NOBLE and LESLIE A. LANE REFERENCES
Detering N, Collins R M Jr, Hawkins R L, Ozand P, Karahasan A (1980) Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: changes in neurotransmitter levels. J Neurochem 34" 1587 - 1593. Detering N, Collins R M Jr, Hawkins R L, Ozand P T, Karahasan A (1981) Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: a long-lasting effect in the hypothalamus. J Neurochem 36" 2 0 9 4 - 2096. Eisenberg R M (1983) Influence of clonidine on the acute dependence response elicited in naive rats by naloxone. Life Sci 32: 1 5 4 7 - 1552. Eisenberg R M (1984) Effects of naltrexone on plasma corticosterone in opiate-naive rats: a central action. Life Sci 34: 1 1 8 5 - 1191. Ganong W F (1977) Neurotransmitters involved in A C T H secretion: catecholamines. Ann N Y Acad Sci 297: 509 - 517. H a n d a R J, McGivern R F, Noble E P, Gorski R A (1985) Exposure to alcohol in utero alters the adult pattern of luteinizing hormone secretion in male and female rats. Life Sci 37:1683 - 1690. Herz A, Millan M J (1984) Opioid peptides in the hypothalamic-pituitary axis. In: Hughes J, Collier H O J, Rance M J, Tyres M B (Eds) Opioids: Past, Present and Future. Taylor and Francis, London, pp 7 9 - 102. Jones M T, Hitlhouse E W, Burden J (1976) Effect of various putative neurotransmitters on the secretion of corticotrophin-releasing hormone from the rat hypothalamus in vitro; a model of the neurotransmitter involved. J Endocrinol 69:1 - 10. Kalra S P, Simpkins J W (1981) Evidence for noradrenergic mediation of opioid effects on luteinizing hormone secretion. Endocrinology 109: 7 7 6 - 7 8 2 . Loizou L A (1972) The postnatal ontogeny of monoamine-containing neurons in the central nervous system of the albino rat. Brain Res 40: 3 9 5 - 4 1 8 . McGivern R F, Clancy A N, Mousa S, Couri D, Noble E P (1984a) Prenatal alcohol exposure alters enkephalin levels without affecting ethanol preference. Life Sci 34:585 - 589. McGivern R F, Clancy A N, Hill M A, Noble E P (1984b) Prenatal alcohol exposure alters adult expression of sexually dimorphic behavior in the rat. Science 224: 8 9 6 - 898. Means L W, Medlin C W, Hughes V D, Gray S L (1984) Hyperresponsiveness to methylphenidate in rats following prenatal ethanol exposure. Neurobehav Toxicol Teratol 6 : 1 8 7 - 192. Meites J, Bruni J F, VanVugt D A, Smith A F (1979) Relation of endogenous opioid peptides and morphine to neuroendocrine functions. Life Sci 2 4 : 1 1 8 5 - 1191. Poland R E, Rubin R T, Weischel M E Jr (1980) Circadian patterns of rat anterior pituitary and target gland hormones in serum: determination of the appropriate sample size by statistical power analysis. Psychoneuroendocrinology 5 : 2 0 9 - 224. Poland R E, Weischel M R Jr, Rubin R T (1981) Neonatal dexamethasone administration. 1. Temporary delay of development of the circadian serum corticosterone rhythm in rats. Endocrinology 108: 1 0 4 9 - 1054. Schultz R, Wilhelm A, Pirke K M, Herz A (1982) Regulation of luteinizing h o r m o n e secretion in prepuberal male and female rats. Life Sci 31: 2 1 6 7 - 2170. Shoemaker W J, Baetge G, Azad R, Sapin V, Bloom F E (1983) Effect of prenatal alcohol exposure on amine and peptide neurotransmitter systems. Monogr Neural Sci 9: 1 3 0 - 139. Spear L P (1979) The use of psychopharmacological procedures to analyze the ontogeny of learning and retention: issues and concerns. In: Spear N E, Campbell B A (Eds) Ontogeny o f Learning and Memory. Wiley and Sons, New York, pp 1 3 5 - 156. Taylor A N, Branch B J, Cooley-Matthews B, Poland R E (1982) Effects of maternal ethanol consumption in rats on basal and rhythmic pituitary-adrenal function in neonatal offspring. Psychoneuroendocrinology 7: 49 - 58. Tilders F J H, Berkenbosch F, Smelik P G (1982) Adrenergic mechanisms involved in the control of pituitary adrenal activity in the rat: a beta-adrenergic stimulatory mechanism. Endocrinology 1 1 0 : 1 1 4 - 120. Weiner R I, Ganong W F (1978) Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev 58:905 - 976.
INFLUENCE
0306-4530/86 $3.130+ 0.130 © 1986PergamonPressLtd.
OF PRENATAL
HORMONAL RESPONSES PREPUBESCENT
ETHANOL
EXPOSURE
ON
TO CLONIDINE AND NALOXONE MALE AND FEMALE RATS
IN
ROBERT F . McGIVERN,*'['~ RUSSELL E . POLAND,*'~ ERNEST P . NOBLE'l" a n d LESLIE A . L A N E *
*Department of Psychiatry, Bldg. F-5, Harbor-U.C.L.A. Medical Center, 1000 W. Carson Street, Torrance, CA 90509 U.S.A.; and 1"Department of Psychiatry and Behavioral Sciences, University of California at Los Angeles, Los Angeles, CA 90024, U.S.A.
(Received 2 October 1984; in final form 9 May 1985) SUMMARY The expression of sexually dimorphic behavior has been found to be altered in adult animals following prenatal alchohol exposure. The present study examined whether such exposure would alter the sexually dimorphic response of luteinizing hormone (LH) to cionidine 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. INTRODUCTION
(1982) reported that the rise in plasma LH following administration of naloxone and clonidine is sexually dimorphic in prepuberal male and female Sprague-Dawley rats. Females exhibited a rise in plasma LH levels in response to naloxone but not clonidine administration, whereas male levels of LH were elevated following clonidine but not naloxone. These results were of particular interest because of our finding of alterations in adult sexually dimorphic behavior in animals exposed to alcohol in utero (McGivern et al., 1984b) and our observation that LH levels following gonadectomy in fetal alcohol exposed (FAE) animals are approximately one-half those of controls (Handa et al., 1985). Therefore, we studied FAE animals at 16 days of age in the present experiments to determine whether prenatal ethanol exposure influences the normal expression of this sexually dimorphic LH response. Exposure of animals to alcohol prenatally has been reported to produce long-term alterations in both opioid and noradrenergic systems of the brain (Detering et aL, 1980, SCHULTZ e t al.
~To whom reprint requests and correspondence should be addressed. 105
106
ROBERTF. McGIVERN,RUSSELLE. POLAND,ERNESTP. NOBLEand LESLIEA. LANF
1981; M c G i v e r n et al., 1984a; S h o e m a k e r et al., 1983). Since b o t h o f these systems are i n v o l v e d in the m o d u l a t i o n o f p it u i ta r y h o r m o n e release ( H e r z & Millan, 1984; K al r a & Simpkins, 1981; Meites et al., 1979), we m e a s u r e d these h o r m o n e s in the p l a s m a o f F A E a n i m al s a n d c o n t r o l s f o l l o w i n g a d m i n i s t r a t i o n o f the a l p h a - a d r e n e r g i c agonist, cl o n i d i n e or the o p i o i d a n t a g o n i s t , n a l o x o n e . In the ad u l t rat, a l p h a - a d r e n e r g i c agonists depress p l a s m a CS ( J o n e s et a k , 1976; W e i n e r & G a n o n g , 1978) b u t elevate p l a s m a L H levels (Kalra & Simpkins, 1981), whereas opiate a n t a g o n i st s st i m u l at e b o t h CS (Eisenberg, 1984) and L H secretion (Meites et al., 1979).
METHODS Pregnant, experienced Sprague--Dawley dams were purchased from Charles River breeders (Wilmington, MA, U.S.A.) to arrive on day 12 of gestation. On day 14 of gestation, randomly selected dams were switched to a liquid diet containing 35% ethanol-derived calories or an isocaloric liquid control diet containing no ethanol (Bio-Serv, Frenchtown NJ, U.S.A.). The dams given the isocaloric control diet were pair-fed to weight-matched dams in the ethanol diet group. The remainder of the dams received the normal diet of Purina lab chow and tap water ad libitum. These diets were continued until parturition, whereupon the dams on the liquid diets were switched to Purina lab chow and tap water. The light cycle was 12 h on/off with lights on at 1000 h. All animals were raised with their biological dam. On postnatal day 16, male and female pups from each litter were weighed and injected i.p. with saline, naloxone (2 mg/kg), or clonidine (0.1 mg/kg) 30 min prior to decapitation. Trunk blood was collected, and CS and LH were measured by radioimmunoassay (Poland et al., 1980, 1981). All samples for CS and LH were analyzed in a single assay. The maximum intra-assay coefficient of variation for both assays was 15%. The values of LH are expressed in terms of the RP-1 standard. Animals were decapitated between 1500 and 1800 h. They were dervied from eight chow-fed litters, 12 ethanol-exposed litters and 14 pair-fed litters. Each litter was represented in the three drug treatments for both sexes as much as litter size allowed. No more than three animals of each sex were used from a litter, and no more than one animal of each sex from a litter was represented in a drug (saline, naloxone, or clonidine) condition. LH and CS data were analyzed by separate 3 (fetal alcohol exposed (FAE), pair-fed (PF) and chow fed (CF) × 3 (drug) × 2 (sex) analyses of variance. The LH data were log-transformed prior to analysis. Post-hoc analyses were conducted by the Newman - Keuls procedure to test for differences between means.
RESULTS Figure 1 shows the p l a s m a c o r t i c o s t e r o n e responses f o l l o w i n g drug t r e a t m e n t . T h e C F and P F an i m als o f b o t h sexes exhibited a significant rise in p l a s m a h o r m o n e levels f o l l o w i n g clonidine, whereas no significant h o r m o n e elevations o c c u r r e d in the F A E animals. T h e m e a n p l a s m a c o r t i c o s t e r o n e level in the F A E males was significantly lower f o l l o w i n g n a l o x o n e a d m i n i s t r a t i o n c o m p a r e d to saline. T h e r e were no o t h er effects p r o d u c e d by n a l o x o n e . T h e analysis o f CS levels indicated m a i n effects for t r e a t m e n t (F = 7.86; 2163 d . f ; p < 0.001) a n d drug (F = 20.38; 2163 d . f . ; p < 0.02), as well as f o r the drug sex i n t e r a c t i o n (F = 3.89; 2163 d . f . ; p < 0.03). T h e analysis o f L H levels i n d ic a t e d significant m a i n effects for t r e a t m e n t (F = 5.44; 2156 d.f.; p < 0.01), drug (F = 19.11; 2156 d . f . ; p < 0.001), an d sex (F = 11.19, 1156 d.f; p < 0.001). I n t e r a c t i o n s were significant b et w een d r u g an d sex (F -- 49.59; 2156 d.f.; p < 0.001) an d a m o n g t r e a t m e n t , d r u g a n d sex (F -- 5.43; 4156 d .f .; p < 0.001). F i g u r e 2 shows that b o t h P F a n d F A E males exhibited a m a r k e d rise in L H levels f o l l o w i n g
HORMONE RESPONSES TO PRENATAL ETHANOL IN RATS
107
administration of clonidine, whereas the CF rats did not respond to clonidine. The LH rise in the FAE males was significantly greater than that in the PF males. Naloxone produced a significant decrease in LH levels in the PF males but produced no effect in the males of the other two groups. A significant rise in LH levels following naloxone injection occurred in all three groups of females. Clonidine produced no LH effect in the CF or FAE females, whereas it significantly decreased LH levels in the PF females.
CORTICOSTERONE 16 DAYS OF AGE :525-
*
FEMALE
[ ] SALINE ~ ] NALOXONE • CLONIOINE ¢-
MALE
e-
v
Chow Fed
Pair Fed
FAE
FIG. I. Mean levels of plasma corticosterone in animals exposed to three maternal treatments during the last week of gestation, at 30 min after injection of saline, naloxone (2 mg/kg), or clonidine (0.1 mg/kg). Vertical lines represent S.E.M. Number of animals in each condition is shown at bottom of graph. Asterisk indicates a significant difference (p < 0.05) from each saline control group.
DISCUSSION
The results of the present study indicate that clonidine stimulates CS release in normal rats of both sexes at 16 days of age. This stimulatory influence is opposite to the inhibitory effect of clonidine on CS release by stress in the adult dog (Ganong, 1977) and in the adult rat following i.v. infusion of 0.1 mg (McGivern and Poland, unpublished results). This inhibitory effect of clonidine in the adult animal is consistent with other data showing that activation of the noradrenergic system by electrical stimulation or norepinephrine
108
ROBERT F. McGIVERN, RUSSELL E. POLAND, ERNEST P. NOBLE and LESI IE A. LANE
LUTEINIZING HORMONE !6 DAYS OF AGE 200180-
FEMALE
160-
[ ] SALINE ]
NALOXONE
140120""
I00806040-
6,O "_t ~ 20-
140
MALE
120
.
o, c
I00
80
60 4
4oq 20Chow Fed
Poir
Fed
FAE
FIG. 2. Mean levels of luteinizing hormone in plasma at 30 min after injection of saline, naloxone (2 mg/kg), or clonidine (0. l mg/kg). Vertical lines represent S.E.M. Number of animals in each condition is shown at bottom of graph. Asterisk indicates a significant difference (p < 0.05) from each saline control group.
infusion also inhibits CS release in adult animals (see Weiner and Ganong, 1978 for review), but is in contrast to the work of Eisenberg (1983) indicating that clonidine infusion, in the same dose range which we have employed, stimulates CS release in the rat. Further work will be needed to understand the effect of clonidine on CS release in the rat. This apparent differential influence of clonidine in the 16 day old animal vs the adult may reflect the relative morphological immaturity of the noradrenergic system at 16 days. The density of NE terminals in the mesencephalon and hypothalamus of animals of this age is roughly half that observed in adult animals (Loizou, 1972). Amphetamine, which stimulates both noradrenergic and dopamine release, also has been reported to have an opposite effect in the prepubescent animal than it has in the adult. Spear (1979) found that amphetamine inhibited locomotor activity in animals under 35 days of age, in contrast to the stimulatory effect of the drug on adult animals. Interestingly, the stimulatory effect of clonidine on CS release in the young animal is similar to the stimulatory effect of the betaadrenergic agonist, isoproterenol, in the adult rat (Tilders et al., 1982). This suggests the possibility of differential development of noradrenergic receptor subtypes during ontogeny. In contrast to the CF and PF controls, the FAE female and male animals exhibited no increase in CS levels 30 min after clonidine administration. This apparent noradrenergic insensitivity at this age is consistent with the work of Detering et al. (1980, 1981) showing depressed NE levels in FAE animals at 3 weeks of age, which persisted into adulthood. Other Work has shown adult FAE animals to exhibit a hypersensitive locomotor response
HORMONE RESPONSESTO PRENATAL ETHANOLIN RATS
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to methylphenidate compared to controls (Means et aL, 1984), suggesting a long-term functional influence of prenatal alcohol exposure on noradrenergic systems. Adult animals have been reported to exhibit an elevation in CS levels following naloxone administration (see Eisenberg, 1984 for review). Most of the animals in the present study failed to show any CS response to naloxone, suggesting that the role of the opioid system in the modulation of CS is not yet developed at this age. The FAE males appear to be an exception, however, since their CS levels were significantly depressed following naloxone administration compared to their saline-injected counterparts. Since normal CS levels in unstressed males at 16 days of age are in the 40 ng/ml range (Poland et al., 1981), the elevated CS levels following saline treatment in all groups in the present study appear to be the result of injection-related stress. Therefore, naloxone apparently partially antagonized this stress-induced elevation of CS levels in the FAE males. It should be noted that elevations in CS levels in this study were not due to time of day, since the diurnal rhythm of CS does not appear until postnatal day 18 or 19 (Poland et aL, 1981). The effects on LH plasma levels which we observed following naloxone and clonidine administration are in partial agreement with the findings of Schultz et ai. (1982). These investigators reported that naloxone progressively increased plasma LH concentrations in normal females from day 1 through day 16 postnatally, reaching a maximal stimulatory effect between day 10 and day 16. Clonidine was relatively ineffective in inducing a rise in plasma LH levels in females during this period. The opposite effect of these drugs on LH secretion was reported for males; a rise in LH was observed following clonidine administration but not after naloxone administration. The stimulatory effect of clonidine on LH was maximal on day 10 and decreased progressively until it was absent on day 30. Our findings on postnatal day 16 differ from those of Schultz et al. (1982) only in regard to the lack of a clonidine-induced LH rise in CF males. This may reflect an accelerated maturation rate of the noradrenergic pathways in the CNS in our control animals compared to those employed by Schultz et al., or, conversely, may be indicative of a delayed CNS maturation in the PF and FAE animals. The present results indicate that the pair-feeding diet also may produce a selective effect on the LH response to naloxone and clonidine, which is sex-dependent. The PF males exhibited a decreased LH response to naloxone, whereas the PF females exhibited a decreased LH response to clonidine. A pair-feeding-induced effect on the development of the circadian rhythm of plasma CS in rats has been reported previously (Taylor et al., 1982). In light of the widespread use of pair-feeding diets in fetal alcohol research, these hormonal findings appear to warrant further study. In. summary, the present results reveal a notable insensitivity of CS in prepubescent FAE male and female rats to the stimulatory influence of clonidine. CS levels in FAE males were depressed following naloxone administration compared to saline injection, but were unchanged in the other groups. Overall, these results appear to provide functional support for reported alterations in the CNS noradrenergic and opioid systems following prenatal exposure to ethanol (Detering et al., 1980, 1981; McGivern et al., 1984a; Shoemaker et al., 1983). This study was supported by NIAAA grants AA 06466 and AA 06478. The authors wish to thank Mrs Debra Hanaya for her editorial assistance.
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ROBERT F. McGIVERN, RUSSELL E. POLAND, ERNEST P. NOBLE and LESLIE A. LANE REFERENCES
Detering N, Collins R M Jr, Hawkins R L, Ozand P, Karahasan A (1980) Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: changes in neurotransmitter levels. J Neurochem 34" 1587 - 1593. Detering N, Collins R M Jr, Hawkins R L, Ozand P T, Karahasan A (1981) Comparative effects of ethanol and malnutrition on the development of catecholamine neurons: a long-lasting effect in the hypothalamus. J Neurochem 36" 2 0 9 4 - 2096. Eisenberg R M (1983) Influence of clonidine on the acute dependence response elicited in naive rats by naloxone. Life Sci 32: 1 5 4 7 - 1552. Eisenberg R M (1984) Effects of naltrexone on plasma corticosterone in opiate-naive rats: a central action. Life Sci 34: 1 1 8 5 - 1191. Ganong W F (1977) Neurotransmitters involved in A C T H secretion: catecholamines. Ann N Y Acad Sci 297: 509 - 517. H a n d a R J, McGivern R F, Noble E P, Gorski R A (1985) Exposure to alcohol in utero alters the adult pattern of luteinizing hormone secretion in male and female rats. Life Sci 37:1683 - 1690. Herz A, Millan M J (1984) Opioid peptides in the hypothalamic-pituitary axis. In: Hughes J, Collier H O J, Rance M J, Tyres M B (Eds) Opioids: Past, Present and Future. Taylor and Francis, London, pp 7 9 - 102. Jones M T, Hitlhouse E W, Burden J (1976) Effect of various putative neurotransmitters on the secretion of corticotrophin-releasing hormone from the rat hypothalamus in vitro; a model of the neurotransmitter involved. J Endocrinol 69:1 - 10. Kalra S P, Simpkins J W (1981) Evidence for noradrenergic mediation of opioid effects on luteinizing hormone secretion. Endocrinology 109: 7 7 6 - 7 8 2 . Loizou L A (1972) The postnatal ontogeny of monoamine-containing neurons in the central nervous system of the albino rat. Brain Res 40: 3 9 5 - 4 1 8 . McGivern R F, Clancy A N, Mousa S, Couri D, Noble E P (1984a) Prenatal alcohol exposure alters enkephalin levels without affecting ethanol preference. Life Sci 34:585 - 589. McGivern R F, Clancy A N, Hill M A, Noble E P (1984b) Prenatal alcohol exposure alters adult expression of sexually dimorphic behavior in the rat. Science 224: 8 9 6 - 898. Means L W, Medlin C W, Hughes V D, Gray S L (1984) Hyperresponsiveness to methylphenidate in rats following prenatal ethanol exposure. Neurobehav Toxicol Teratol 6 : 1 8 7 - 192. Meites J, Bruni J F, VanVugt D A, Smith A F (1979) Relation of endogenous opioid peptides and morphine to neuroendocrine functions. Life Sci 2 4 : 1 1 8 5 - 1191. Poland R E, Rubin R T, Weischel M E Jr (1980) Circadian patterns of rat anterior pituitary and target gland hormones in serum: determination of the appropriate sample size by statistical power analysis. Psychoneuroendocrinology 5 : 2 0 9 - 224. Poland R E, Weischel M R Jr, Rubin R T (1981) Neonatal dexamethasone administration. 1. Temporary delay of development of the circadian serum corticosterone rhythm in rats. Endocrinology 108: 1 0 4 9 - 1054. Schultz R, Wilhelm A, Pirke K M, Herz A (1982) Regulation of luteinizing h o r m o n e secretion in prepuberal male and female rats. Life Sci 31: 2 1 6 7 - 2170. Shoemaker W J, Baetge G, Azad R, Sapin V, Bloom F E (1983) Effect of prenatal alcohol exposure on amine and peptide neurotransmitter systems. Monogr Neural Sci 9: 1 3 0 - 139. Spear L P (1979) The use of psychopharmacological procedures to analyze the ontogeny of learning and retention: issues and concerns. In: Spear N E, Campbell B A (Eds) Ontogeny o f Learning and Memory. Wiley and Sons, New York, pp 1 3 5 - 156. Taylor A N, Branch B J, Cooley-Matthews B, Poland R E (1982) Effects of maternal ethanol consumption in rats on basal and rhythmic pituitary-adrenal function in neonatal offspring. Psychoneuroendocrinology 7: 49 - 58. Tilders F J H, Berkenbosch F, Smelik P G (1982) Adrenergic mechanisms involved in the control of pituitary adrenal activity in the rat: a beta-adrenergic stimulatory mechanism. Endocrinology 1 1 0 : 1 1 4 - 120. Weiner R I, Ganong W F (1978) Role of brain monoamines and histamine in regulation of anterior pituitary secretion. Physiol Rev 58:905 - 976.