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Hypothalamic-pituitary-adrenal regulation in rats prenatally exposed to ethanol Glavas, Maria Matilda 2003

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HYPOTHALAMIC-PITUITARY-ADRENAL REGULATION IN RATS PRENATALLY EXPOSED TO ETHANOL by Maria Matilda Glavas B.Sc. (Physiology Honours), The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Neuroscience Program) We accept this thesis as conforming to the required standard THE UNFVERSITY OF BRITISH COLUMBIA April 2003 © Maria Matilda Glavas, 2003 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ArsccW The University of British Columbia Vancouver, Canada Date r ^ P R i * - " 2 3 , ^ L o o 3 > DE-6 (2/88) ABSTRACT Rats prenatally exposed to ethanol (E) exhibit a hyperresponsive hypothalamic-pituitary-adrenal (HPA) axis, demonstrated by increased and/or prolonged elevations of adrenocorticotropic hormone (ACTH) and/or corticosterone (CORT) following stress; however, basal levels are normal. The major objective of this thesis was to elucidate the underlying mechanisms of HPA hyperresponsiveness in E rats. We hypothesized that HPA hyperresponsiveness in E rats is mediated, in part, by enhanced HPA drive and/or by alterations in CORT feedback regulation. In all studies, male and female Sprague-Dawley offspring from E, pair-fed (PF) and control (C) groups were tested in adulthood at 90-120 d of age. The mechanisms mediating HPA hyperresponsiveness in E rats and the role of CORT in mediating any alterations were investigated using several manipulations: adrenalectomy (ADX) with or without CORT replacement, CORT receptor blockade and restraint stress. ADX revealed that E males exhibit enhanced biosynthesis of corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) and elevated plasma ACTH levels independent of CORT alterations, indicating that HPA hyperresponsiveness in E animals is not due solely to altered CORT feedback. Enhanced CRH synthesis also occurred in sham-operated E females. Measures of CRH Type 1 receptor and pro-opiomelanocortin mRNA suggested that hyperresponsiveness in E animals is likely not mediated by enhanced pituitary sensitivity to CRH. CORT receptor (mineralocorticoid receptors, MRs, and glucocorticoid receptors, GRs) mRNA levels in the hippocampus indicated that E males may have a decreased sensitivity to CORT feedback regulation of these genes in response to ADX with CORT replacement. As well, in response to MR or GR blockade, E females exhibited greater HPA activity compared to controls, indicative of enhanced HPA drive. Taken together, these studies indicate that prenatal ethanol exposure permanently reprograms the HPA axis of the rat such that HPA drive is enhanced into adulthood. Although some ii compensatory mechanisms appear to be in place to maintain normal basal ACTH and CORT levels, in response to challenge these mechanisms break down and alterations are revealed. HPA dysregulation in E rats appears to be mainly suprapituitary, mediated by enhanced drive to the hypothalamus as reflected by increased CRH biosynthesis. ( iii TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES x LIST OF TABLES xv ABBREVIATIONS USED IN TEXT xvii ACKNOWLEDGEMENTS xx CHAPTER I: GENERAL INTRODUCTION 1 A. Alcohol Consumption During Pregnancy: Effects on Human Offspring 1 1. History 1 2. Diagnosis 1 3. Rates of Occurrence 3 4. Effects of Timing, Dose, Pattern and Genetics 3 B. Prenatal Ethanol Exposure in Animals 4 1. Ethanol is a Teratogen 4 2. Animal Models of FAS 5 3. Ethanol-Related Effects in Animals 5 4. Mechanisms of Ethanol Teratogenesis 6 5. Behavioural Effects of Ethanol in Animals 7 C. Stress and the HPA Axis 8 1. Stress 8 2. The HPA Axis 9 3. The PVN: Site of Convergence 10 4. Neural Inputs to the PVN 11 iv 5. The Anterior Pituitary Gland * 11 6. The Adrenal Cortex 12 7. Corticosteroid Receptors 12 8. Glucocorticoid Actions 13 9. Glucocorticoid Negative Feedback 14 10. HPA Dysregulation 16 11. Allostatic Load 17 D. Prenatal Ethanol Exposure and the HPA Axis 18 1. Prenatal Ethanol and HPA Ontogeny 18 2. E Rats are Hyperresponsive to Stressors in Adulthood 18 3. Allostasis in the E Rat 20 4. FAS and the HPA Axis 21 E. Rationale and Thesis Objectives 21 CHAPTER II: GENERAL METHODS 24 A. Animals and Mating 24 B. Ethanol Administration 24 C. Diets and Feeding 26 D. Adrenalectomy 28 E. Corticosterone Replacement 29 F. Blood Sampling 30 1. Decapitation 30 2. Jugular Cannulation 31 G. Corticosterone and ACTH Radioimmunoassays 32 1. Corticosterone 32 2. ACTH 33 v CHAPTER III: EFFECTS OF PRENATAL ETHANOL EXPOSURE ON HYPOTHALAMIC-PITUITARY-ADRENAL RESPONSIVENESS FOLLOWING ADRENALECTOMY AND CORTICOSTERONE REPLACEMENT 34 A. Introduction 34 B. Methods 35 1. Breedings and Animals 35 2. Corticosterone Replacement 35 3. Experiment 1: Determination of CORT Dosage 36 4. ADX with or without CORT Replacement in E, PF and C Rats 37 a. Experiment 2 37 b. Experiment 3 38 5. Statistical Analyses 38 C. Results 38 1. Experiments la&b 38 2. Experiment 2 43 a. Developmental Data 43 b. Adult Body Weight 47 c. CORT Intake 47 d. Thymus Weight 47 e. Non-Stressed CORT Levels 47 f. Non-Stressed ACTH Levels 48 3. Experiment 3 54 a. Developmental Data 54 b. Adult Body Weight 54 c. CORT Intake 55 vi d. Stress CORT Levels e. Stress ACTH Levels * D. Discussion 1. Summary CHAPTER IV: GENE EXPRESSION IN THE LIMBIC-HYPOTHALAMIC-PITUITARY-ADRENAL AXIS OF RATS PRENATALLY EXPOSED TO ETHANOL A. Introduction B. Methods 1. Breeding and Animals 2. Testing 3. Probes and Labeling 4. Northern Hybridization 5. In Situ Hybridization a. Oligonucleotides: CRH, AVP and POMC b. cRNA Probes: MR, GR and CRH-R1 6. Densitometric Analysis 7. Statistical Analyses C. Results 1. Developmental Data 2. Adult Body Weight 3. CORT Intake 4. Plasma CORT Levels 5. Plasma ACTH Levels 6. Confirmation of Probe Specificity 7. CRH mRNA in the PVN vii 8. AVP mRNA in the PVN 77 9. CRH-R1 mRNA in the Anterior Pituitary 77 10. POMC mRNA in the Anterior Pituitary 78 11. Hippocampal MR mRNA 88 12. Hippocampal GR mRNA 88 D. Discussion 95 1. Paraventricular Nucleus: CRH and AVP mRNA 95 2. Anterior Pituitary: CRH-R1 and POMC 96 3. MR and GR mRNA 97 4. CORT Replacement: Effects on mRNA Expression 98 5. Sexual Dimorphism . 100 6. Summary 101 CHAPTER V: EFFECTS OF MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR BLOCKADE ON HYPOTHALAMIC-PITUITARY-ADRENAL FUNCTION IN RATS PRENATALLY EXPOSED TO ETHANOL 102 A. Introduction 102 1. Evidence of Feedback Deficits in E Rats 102 2. Effects of MR and GR Antagonists 103 3. Purpose 104 B. Methods 104 1. Surgery and Testing 104 2. Drug Injections 105 3. Blood Sampling and Novel Confinement 106 4. Statistical Analyses 107 C. Results 107 viii 1. Developmental Data 2. Plasma ACTH Following MR or GR Blockade 3: Plasma CORT Following MR or GR Blockade D. Discussion 1. Summary CHAPTER VI: GENERAL DISCUSSION A. Summary and Discussion B. Clinical Relevance C. Future Directions D. Conclusions REFERENCES APPENDIX ix LIST OF FIGURES Figure 1. Characteristic facial features of FAS Figure 2. The hypothalamic-pituitary-adrenal axis Figure 3. Plasma CORT levels in adrenalectomized control males and females and plasma ACTH in males and females at the diurnal peak following 7 d of CORT replacement Figure 4. Plasma CORT and ACTH levels at three different times of day: 0700h, 1600h and 2000h in adrenalectomized control males provided with 25 ug/ml CORT and females provided with 75 ug/ml CORT as drinking water Figure 5. Maternal body weights during gestation and lactation for Chapter III Expt. 2 breeding Figure 6. Mean pup body weights males and females during postnatal days 1 to 22 from Chapter III Expt. 2 breeding Figure 7. Mean thymus weight in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement Figure 8. Plasma CORT levels in male and female E, PF and C rats 7 d following sham surgery or ADX with or without CORT replacement Figure 9. Plasma ACTH levels in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement Figure 10. Plasma CORT levels in E, PF and C male and female rats immediately after a 15 min restraint stress 7 d following sham surgery or ADX with or without CORT replacement Figure 11. Plasma ACTH levels in E, PF and C male and female rats immediately after a 15 min restraint stress 7 d following sham surgery or ADX with or without CORT replacement 58 Figure 12. Plasma CORT levels in male and female E, PF and C rats 7 d following sham surgery or ADX with or without CORT replacement 74 Figure 13. Plasma ACTH levels in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement 75 Figure 14. Northern analysis of anterior pituitary gland total RNA from SHAM, ADX and ADX+CORT control males hybridized with digoxigenin-labeled POMC oligonucleotide 79 Figure 15. Location of pPVN within hypothalamus and representative sections demonstrating CRH expression pattern in SHAM, ADX and ADX+CORT C male rats 7 d following surgery 80 Figure 16. CRH mRNA in the dorsomedial pPVN in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement 81 Figure 17. Location of parvocellular and magnocellular PVN within hypothalamus and representative sections demonstrating AVP expression pattern in SHAM, ADX and ADX+CORT C male rats 7 d following surgery 82 Figure 18. AVP mRNA in the dorsomedial pPVN in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement 83 Figure 19. Representative autoradiography of anterior pituitary sections demonstrating CRH-R1 expression in SHAM, ADX and ADX+CORT C male rats 7 d following surgery 84 xi Figure 20. CRH-R1 mRNA in the anterior pituitary in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement Figure 21. Representative autoradiography of anterior pituitary sections demonstrating POMC expression in SHAM, ADX and ADX+CORT C male rats 7 d following surgery Figure 22. POMC mRNA in the anterior pituitary in E, PF and C male and female rats 7 d following sham surgery or ADX with or without CORT replacement Figure 23. Representative autoradiography of dorsal hippocampus sections demonstrating MR expression in SHAM, ADX and ADX+CORT C male rats 7 d following surgery Figure 24. MR mRNA in the dorsal hippocampus in E, PF and C males measured in hippocampus 7 d following sham surgery or ADX with or without CORT replacement Figure 25. MR mRNA in the dorsal hippocampus in E, PF and C females measured in hippocampus following sham surgery or ADX with or without CORT replacement Figure 26. Representative autoradiography of dorsal hippocampus sections demonstrating GR expression in SHAM, ADX and ADX+CORT C male rats 7 d following surgery Figure 27. GR mRNA in the dorsal hippocampus in E, PF and C males measured in hippocampus 7 d following sham surgery or ADX with or without CORT replacement xii Figure 28. GR mRNA in the dorsal hippocampus in E, PF and C females measured in hippocampus 7 d following sham surgery or ADX with or without CORT replacement Figure 29. Plasma ACTH in ethanol pair-fed and control males following sc injection of either propylene glycol vehicle, spironolactone or RU38486 Figure 30. Plasma ACTH in ethanol pair-fed and control females following sc injection of either propylene glycol vehicle, spironolactone or RU38486 Figure 31. Plasma CORT in ethanol, pair-fed and control males following sc injection of either propylene glycol vehicle, spironolactone or RU38486 Figure 32. Plasma CORT in ethanol, pair-fed and control females following sc injection of either propylene glycol vehicle, spironolactone or RU38486 Figure 33. Maternal body weights during gestation and lactation for Chapter III Expt. 3 breeding Figure 34. Mean pup body weights in males and females during postnatal days 1 to 22 from Chapter III Expt. 3 breeding Figure 35. Maternal body weights during gestation and lactation for Chapter IV breeding Figure 36. Mean pup body weights males and females during postnatal days 1 to 22 from Chapter IV breeding xiii Figure 37. Maternal body weights during gestation and lactation for Chapter V breeding Figure 38. Mean pup body weights in males and females during postnatal days 1 to 22 from Chapter V breeding xiv LIST OF TABLES Table 1. Caloric profile of ethanol and pair-fed diets Table 2. Twenty four hour fluid intake prior to and 60 d following ADX Table 3. Gestation length, # live pups and # dead pups of E, PF and C dams for Chapter III Expt. 2 breeding Table 4. Adult Body Weight one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females, Chapter III Expt. 2 Table 5. Mean CORT intake during 24 hr prior to testing after 7 days of CORT replacement in Chapter III Expt. 2 Table 6. Mean CORT intake during 24 hr prior to testing after 7 days of CORT replacement in Chapter III Expt. 3 Table 7. Mean CORT intake during 24 hr prior to testing after 7 days of CORT replacement, Chapter IV experiment Table 8. Body weight of adult E, PF and C males and females following jugular cannulation, 2 d prior to testing, Chapter V experiment Table 9. Gestation length, # live pups and # dead pups of E, PF and C dams for Chapter III Expt. 3 breeding Table 10. Adult Body Weight one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females, Chapter III Expt. 3 xv Table 11. Gestation length, # live pups and # dead pups of E, PF and C dams for Chapter IV breeding Table 12. Adult Body Weight one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females, Chapter IV experiment Table 13. Gestation length, # live pups and # dead pups of E, PF and C dams for Chapter V breeding xvi ABBREVIATIONS USED IN TEXT ACTH adrenocorticotropic hormone ADX adrenalectomy ANOVA analysis of variance ARBD alcohol-related birth defect ARND alcohol-related neurodevelopmental disorder AVP arginine vasopressin BAC blood alcohol concentration bw body weight C control CA • cornu ammonis CBG corticosteroid binding globulin CNS central nervous system CORT corticosterone CRH corticotropin-releasing hormone CRH-R1 corticotropin-releasing hormone type 1 receptor d day DEPC diethyl pyrocarbonate DEX dexamethasone DIG digoxigenin DTT dithiothreitol E prenatally exposed to ethanol EDTA ethylenediaminetetraacetic acid expt. experiment xvii FAS Fetal Alcohol Syndrome g gram G gestation day GC glucocorticoid GR glucocorticoid receptor GRE glucocorticoid responsive element HPA hypothalamic-pituitary-adrenal hr hours hsp heat shock protein icv intracerebroventricular KIU kallikrein-inhibiting units MC2 melanocortin 2 receptor min minutes mpPVN medial parvocellular paraventricular nucleus MR mineralocorticoid receptor mRNA messenger ribonucleic acid NE norepinephrine NMDA N-methyl-D-aspartate PBS phosphate-buffered saline PE polyethylene PF pair-fed PN postnatal day POMC pro-opiomelanocortin pPVN parvocellular paraventricular nucleus PR progesterone receptor xviii PVN paraventricular nucleus of the hypothalamus RIA radioimmunoassay s seconds sc subcutaneous SEM standard error of the mean SSC 300 mM NaCl/30 mM sodium citrate xix ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Joanne Weinberg, for taking me into her lab, for being so supportive and giving me a push when I needed it. I couldn't have done it without you. I would like to thank current and past members of my supervisory committee, including Dr. Christopher Mcintosh, Dr. Calvin Roskelley, Dr. Lakshimi Yatham and Dr. Athanasios Zis for their advice and guidance, and Dr. Victor Viau for the use of his imaging system. Wayne Yu and Linda Ellis deserve special thanks for providing technical assistance and always being there when I needed help and advice. Also, these studies would not have been logistically possible without the assistance of all the other graduate students, technicians, work study students and directed study students who willingly gave their time to assist me. I would like to thank my father for his patience throughout my seemingly endless education, my sisters Natalie, Suzana (for starting the Glavas family Ph.D. trend) and Katarina, and the rest of my relatives and friends for always keeping me entertained. I dedicate this dissertation to my late mother who will always be my inspiration. xx CHAPTER I: GENERAL INTRODUCTION Alcohol Consumption During Pregnancy: Effects on Human Offspring History In 1973, Jones et al. brought the issue of alcohol and pregnancy into the spotlight with the report of eight children from three different ethnic groups born to chronic alcoholic mothers, exhibiting a similar pattern of craniofacial, limb, and cardiovascular defects as well as growth deficiencies and developmental delay (Jones et al., 1973). This condition was subsequently termed "the fetal alcohol syndrome" (Jones & Smith, 1973). It was initially difficult to convince the public that alcohol, a widely consumed, legal substance which was even used clinically to delay premature labour (Patel & MacNaughton, 1969) was harmful to the developing fetus. It took animal studies to show that alcohol was indeed a teratogenic agent. Diagnosis The diagnostic criteria for Fetal Alcohol Syndrome, or FAS, are confirmed maternal alcohol exposure, a characteristic pattern of facial anomalies (Fig. 1) including short palpebral fissures (eye openings), flattened philtrum, and a flattened midface, growth retardation (low birth weight for gestational age, decelerating weight over time not due to nutrition, and/or disproportional low weight to height), and central nervous system (CNS) abnormalities (decreased cranial size at birth, structural brain abnormalities such as microcephaly, impaired fine motor skills) (Stratton et al., 1996). In addition, there is a complex pattern of behavioural or cognitive abnormalities, including learning difficulties, aggression and delinquent behaviours, memory, attention or judgement problems, and deficits in socialization and adaptive behaviours (Streissguth et al., 1989, 1991, 1994; Sood et al., 2001; Whaley et al., 2001; Thomas et al., 1998). However, FAS represents only the extreme end of a spectrum of alcohol-related effects. Levels of alcohol 1 exposure below those needed to produce FAS can also result in physical, behavioural and/or physiological abnormalities. Figure 1. Characteristic facial features of FAS (Warren & Foudin, 2001) As yet there is no biological marker that can reliably identify FAS or other alcohol-related effects. The diagnoses made by trained clinicians and dysmorphologists therefore vary from clinic to clinic, particularly when all the criteria of FAS are not met. The current diagnostic criteria for FAS and related conditions, established in 1996 by the Institute of Medicine of the National Academy of Sciences, can be divided into five categories dependent on the clinical signs present and whether a clear history of prenatal alcohol exposure exists (Stratton et al., 1996). Category 1, FAS with confirmed maternal alcohol exposure, requires the characteristic facial dysmorphology of FAS, evidence of growth retardation, as well as CNS abnormalities. Category 2, FAS without confirmed maternal alcohol exposure, is assigned to a case in which all of the criteria in category 1 are met but a history of maternal alcohol consumption cannot be confirmed. Category 3, partial FAS with confirmed maternal alcohol exposure, requires some facial features of FAS plus any one of the following: growth deficits, CNS abnormalities, or behavioural and cognitive abnormalities. Category 4, alcohol-related birth defects (ARBDs), 2 requires a congenital malformation or dysplasia which has been shown through animal studies to be related to prenatal alcohol exposure (e.g. cardiac, skeletal, renal, ocular, or auditory defects). Finally, Category 5, alcohol-related neurodevelopmental disorders (ARNDs), requires the presence of a CNS neurodevelopmental abnormality known to result from prenatal alcohol exposure (e.g. decreased cranial size, structural brain abnormalities, impaired fine motor skills). Since categories 4 and 5 are assigned when there are no facial features of FAS, diagnosis is difficult and therefore confirmed maternal alcohol exposure is a requirement. Rates of Occurrence FAS is the leading cause of mental retardation in the Western world (Abel & Sokol, 1987). Recent estimates of worldwide FAS incident rates are 0.97 cases per 1000 live births, with a rate of 1.95 cases per 1000 in the United States (Abel, 1995). However, these rates vary greatly from community to community, with the highest known occurrence in the Western Cape Province of South Africa, estimated at 39.2 to 46.4 cases per 1000 births (May et al., 2000). Estimates of other alcohol-related outcomes, including partial FAS, ARBDs and ARNDs, are more difficult to determine, but are thought to range from 10-30 cases per 1000 live births in the United States (Hannigan & Armant, 2000). Effects of Timing, Dose, Pattern and Genetics Alcohol effects on the fetus manifest as a continuum, with FAS and fetal death at one extreme. The range of effects relates to the timing, dose, pattern and duration of alcohol exposure, as well as genetic factors. Timing of alcohol exposure is a major contributor to outcome, since the organs undergoing most rapid development and differentiation at a given time will be most vulnerable to alcohol's effects (Coles, 1994). For instance, first trimester alcohol exposure, during the period of organogenesis, can affect maturation of craniofacial areas 3 resulting in facial dysmorphology (Ernhart et al., 1987), whereas exposure through the third trimester, a time of rapid brain development, may result in microcephaly, cognitive deficits and motor problems (Coles et al. 1985, 1991; Smith et al, 1986; Autti-Ramo & Granstrom, 1991; Day et al., 1994). Sensitivity to alcohol's effects is dependent not only upon the developmental period of exposure, but also the level of alcohol consumption. Animal studies have shown that the critical factor determining outcome magnitude is the peak blood alcohol concentration (BAC) achieved at a given time (West & Goodlett, 1990). Peak BAC is a factor of both the dose and the pattern of alcohol consumption, such that a smaller dose of alcohol may actually be more harmful than a larger dose if it is consumed within a short period of time, thereby resulting in higher BACs (Bonthius & West, 1990). Binge drinking is thus particularly harmful because of the high BACs achieved. Not every child born of a chronic alcoholic mother presents with FAS. This may partly be due to genetic factors which make some children more vulnerable than others. Twin studies do suggest a genetic component, with monozygotic twins showing higher concordance rates for FAS than dizygotic twins (Streissguth & Dehaene, 1993). This may be due in part to genetic diversity in the enzymes responsible for alcohol metabolism in both the mother and fetus, both of which affect fetal BACs (McCarver et al., 1997; Viljoen et al., 2001). Other possible mechanisms may involve genetic variability in the sensitivity of target organs to alcohol. Prenatal Ethanol Exposure in Animals Ethanol is a Teratogen Following the recognition of FAS in 1973, there was initial skepticism that alcohol itself was the culprit, and not nutritional and/or environmental effects associated with chronic alcohol abuse. This debate was ended by animal studies which demonstrated that ethanol was indeed a 4 teratogen, and characteristics similar to FAS could be produced in the absence of malnutrition, smoking, disease, or other drug use (Randall et al. 1977; Kronick, 1976; Abel & Dintcheff, 1978). In one particularly striking study, administration of just two small doses of ethanol to pregnant mice on gestational day 7 resulted in distinctive craniofacial anomalies similar to those seen in children with FAS (Sulik et al., 1981). Since these early studies, animal models have been instrumental in predicting outcomes in humans with FAS. In addition, animal models allow us to investigate the mechanisms of ethanol teratogenesis and the influence of environmental and genetic factors on fetal outcome. Animal Models of FAS Numerous animal models of prenatal ethanol exposure now exist, including monkey (Clarren et al., 1987), sheep (Cudd et al., 2002), rat (Abel & Dintcheff, 1978; Weinberg, 1989), and mouse (Randall et al., 1977) models. Although animal models can mimic effects seen in humans with FAS, species differences often exist in the magnitude of specific ethanol effects. For instance, whereas mice are more sensitive to craniofacial anomalies and organ malformations resulting from ethanol exposure, rats better model the neurodevelopmental and neurobehavioural abnormalities (Sulik et al., 1981; Riley & Meyer, 1984; Diaz & Samson, 1980). Although craniofacial anomalies are not clearly evident in rats, radiographic skull measurements do reveal facial dysmorphology characteristic of FAS (Edwards & Dow-Edwards, 1991). The choice of animal model is therefore typically based on sensitivity to the effect under investigation, in addition to practical considerations. Ethanol-Related Effects in Animals Animal studies have shown that prenatal ethanol exposure affects a wide range of systems, resulting in growth deficiencies (Abel & Dintcheff, 1978; Gallo & Weinberg, 1986), altered 5 neuronal proliferation and migration (Miller, 1986, 1993), alterations in several neurotransmitter systems including glutamate, serotonin, dopamine, norepinephrine and GABA (Maier et al., 1996; Rudeen & Weinberg, 1993; Spuhler-Phillips et al., 1997; Druse et al., 1991), behavioural abnormalities (Royalty, 1990; Anandam et al., 1980; Riley et al., 1979a,b; Bond & Di Giusto, 1976), and neuroendocrine abnormalities (Breese et al., 1993; Cudd et al., 2002; McGivern et al., 1993; Portoles et al., 1988; Taylor et al., 1982; Weinberg, 1989; Wilson et al., 1995). Although similar investigations in humans are not always possible, animal studies have reproduced most of the deficits observed in humans, and most of the effects studied in animals also appear in humans with FAS. Mechanisms of Ethanol Teratogenesis The wide range of deficits resulting from prenatal ethanol exposure is not likely mediated by a single mechanism. However, two or more primary mechanisms may in turn cause numerous secondary effects. Ethanol is a small, lipophilic molecule that readily crosses the placenta, resulting in fetal BACs similar to those of the mother (Guerri & Sanchis, 1985). Ethanol can pass through the blood-brain barrier, diffuse across the plasma membrane, and can directly interact with fetal tissue. In addition, ethanol may indirectly affect the fetus through effects on the mother's physiology. Ethanol may alter placental blood flow and impair placental transfer of nutrients including glucose (Falconer, 1990; Jones et al., 1981; Singh et al., 1989). As well, ethanol can alter the utilization of nutrients in both the mother and fetus and affect maternal intestinal absorption (Lieber, 2000). In addition, ethanol-induced hormonal alterations may disrupt the normal maternal-fetal endocrine balance, thereby altering fetal endocrine development (Cudd et al., 2001, 2002). While the teratogenic effects of ethanol are well established, some effects on the fetus may be due to ethanol metabolites, particularly acetaldehyde which is also a known teratogen (Webster et al., 1983; Ali & Persaud, 1988). 6 Ethanol may have direct effects on the fetus through a number of possible mechanisms. For instance, various neurotransmitter systems are affected by ethanol. In particular, NMDA receptor-mediated glutamate transmission may be inhibited by ethanol but is enhanced during ethanol withdrawal, which may trigger cell excitotoxicity (Spuhler-Phillips et al., 1997; Thomas et al., 1997). Ethanol has also been shown to inhibit cell-cell adhesion (Charness et al., 1994; Chen et al., 2001), induce oxidative stress (Henderson et al., 1995; Chen & Sulik, 1996), and increase apoptosis (Dunty et al., 2001; Ikonomidou et al., 2000), all of which could impair development. In addition, ethanol alters, the activity of numerous growth factors required for normal development, including nerve growth factor and insulin-like growth factors (Heaton et al., 2000; Mauceri et al., 1993). Although there are a number of candidate mechanisms for ethanol teratogenesis, it remains difficult to identify the primary mechanisms and subsequent pathways. Behavioural Effects of Ethanol in Animals Direct comparisons between human and animal behaviours are difficult to make. However, some general similarities can be seen between humans with FAS and animals exposed to ethanol in utero. Rats prenatally exposed to ethanol, or E rats, exhibit increased activity, a lack of response inhibition, aggression and hyperreactivity (Royalty, 1990; Anandam et al., 1980; Riley et al., 1979a,b; Bond & Di Giusto, 1976). Some behavioural effects in E rats, such as increased activity, are transient in that they are pronounced in young animals, typically not evident in adulthood, and reappear in old age (Bond & Di Giusto, 1976; Abel & Dintcheff, 1986). Interestingly, these behaviours may re-emerge in adults when challenged pharmacologically (Means et al., 1984). One explanation is that adult E animals develop compensatory mechanisms to overcome behavioural abnormalities, but that these mechanisms break down under challenge and with age (Riley, 1990). Another challenge that may unmask behavioural abnormalities is 7 exposure to stressors. E rats exhibit increased stress-induced ethanol consumption (Nelson et al., 1983), increased stress-induced analgesia (Nelson et al., 1985), and inability to adapt to a stressful swimming paradigm (Taylor et al., 1983). E rats not only exhibit abnormal behaviours in response to stressors, but they may also mount an abnormally heightened hormonal stress response, mediated by the hypothalamic-pituitary-adrenal (HPA) axis (Taylor et al., 1982; Weinberg, 1989; Lee et al., 2000). Stress and the HPA Axis Stress The use of the term stress in the biological sense was popularized by Hans Selye, who described a triad of symptoms he called the "general adaptation syndrome" caused by a variety of noxious stimuli, all resulting in adrenal cortical enlargement, thymic atrophy and ulcers (Selye, 1936). He later theorized that certain "diseases of adaptation" were the result of abnormal reactions to stress (Selye, 1946; Mason, 1975). Stress may be defined as a state of threatened homeostasis, whereas the internal or external challenges which cause this threat are termed stressors (Miller & O'Callaghan, 2002). Stressors can range from real threats to survival, including immune challenges or physical stressors, such as cold or pain, to perceived threats such as psychological or social stressors. The hormonal response to a stressor is mediated by two systems, namely the sympathoadrenal medullary system and the HPA axis. The sympathoadrenal medullary system responds rapidly to a stressor with the secretion of norepinephrine from sympathetic nerves and epinephrine from the adrenal medulla. This system is involved in the "fight or flight" response and enables the organism to rapidly react to and deal with the threat. The HPA axis acts over a longer time frame than the sympathetic response and helps orchestrate the body's response and adaptation to the stressor to maintain homeostasis. 8 The HPA Axis The HPA axis (Fig. 2) consists of a number of structures, including the paraventricular nucleus (PVN) of the hypothalamus, the anterior pituitary gland and the adrenal cortex, that produce a hormonal cascade in response to stressors. Corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are synthesized in the parvocellular PVN (pPVN) and released into the median eminence, reaching the anterior pituitary via the hypophysial portal system. CRH and AVP act synergistically at the anterior pituitary to stimulate the release of adrenocorticotropic hormone (ACTH, the precursor of which is pro-opiomelanocortin, or POMC) (Gillies et al., 1982). ACTH released from the anterior pituitary travels through the systemic circulation and acts at the adrenal cortex to stimulate the synthesis and release of glucocorticoids, namely Cortisol in humans and corticosterone (CORT) in rats. In addition to a variety of actions throughout the body, CORT has a negative feedback function which inhibits further HPA activity by acting at the anterior pituitary, the PVN and other brain regions, particularly the hippocampus. Like other neuroendocrine systems, the HPA axis exhibits a circadian rhythm, with peak activity occurring near the beginning of the active cycle (morning for humans, evening for rats). This circadian rhythm is more exaggerated in female rats, who achieve higher blood glucocorticoid levels at the circadian peak than males; however, the magnitude of this difference is dependent on estrous cycle stage (Atkinson & Waddell, 1997). Although much research has been conducted on basic HPA function, differences in. the age and strain of animals as well as experimental paradigms utilized have limited our understanding of the system. As well, the majority of studies are conducted in males leaving limited information on HPA function in females. 9 stressors other brain reg { ions PVN of the hypothalamus anterior pituitary adrenal cortex Figure 2. The hypothalamic-pituitary-adrenal axis. (PVN: paraventricular nucleus; CRH: corticotropin-releasing hormone; A V P : arginine vasopressin; POMC: pro-opiomelanocortin; A C T H : adrenocorticotropic hormone; GCs: glucocorticoids. Dashed lines indicate glucocorticoid negative feedback inhibition) The PVN: Site of Convergence Different types of stressors activate the HPA axis through distinct neural circuits and neurotransmitter pathways, but all converge on the medial parvocellular PVN (mpPVN) (Pacak 10 & Palkovits, 2001). For instance, life-threatening physical stressors, such as hemorrhage, stimulate the PVN through a rapid, direct pathway from the brainstem, whereas psychological stressors, such as an air-puff startle, take a more indirect route to the PVN including forebrain and limbic regions, suggesting higher cognitive processing and comparison to previous experiences (Thrivikraman et al., 2000); Regardless of the pathway, if the stimulus is interpreted as a threat to homeostasis the PVN is ultimately activated and an HPA response is mounted. Neural Inputs to the PVN The PVN receives numerous neural inputs, both excitatory and inhibitory (Herman et al., 1996; Cole & Sawchenko, 2002). The pattern of these inputs, which is dependent not only on the type of stressor but the interpretation of that stressor, determines the magnitude of the resulting HPA response. Neural inputs to the PVN include serotonergic (Korte et al., 1991), glutamatergic (van den Pol et al., 1990; Feldman & Weidenfeld, 1997), and catecholaminergic pathways (Liu et al., 1991; Radant et al., 1992), with GABA mediating the main inhibitory inputs (Roland & Sawchenko, 1993). In addition to these neurotransmitters, numerous neuropeptides modulate HPA activity at the PVN (Herman et al., 1996). Stimulation of the mpPVN results in the release of CRH and AVP into the portal circulation and also stimulates further synthesis of these releasing hormones in order to replenish protein stores. Stimulus-induced release occurs prior to and independently of gene activation, suggesting that distinct mechanisms mediate these two effects (Tanimura et al., 1998). The Anterior Pituitary Gland CRH and AVP released from the median eminence act on the anterior pituitary to stimulate ACTH release and POMC synthesis. POMC is a large precursor protein which is differentially cleaved into smaller peptides in different cell populations. Whereas the intermediate pituitary 11 processes POMC primarily into a-melanocyte stimulating hormone (a-MSH) and P-endorphin, the major products in anterior pituitary corticotrophs are ACTH and p-lipotropin (Eipper & Mains, 1980). At the anterior pituitary, CRH activates an adenylate cyclase/cAMP-dependent pathway, via the CRH type 1 receptor (CRH-R1), to stimulate ACTH release (Giguere et al., 1982; Castro et al., 1989). AVP potentiates CRH-stimulated ACTH release via Vlb receptors which act through a phosphatidylinositol pathway (Jard et al., 1986; Castro et al., 1989). CRH but not AVP also stimulates POMC synthesis, whereas AVP may instead mobilize a rapid turnover pool of ACTH (Antoni, 1993). Thus, in response to enhanced PVN stimulation, CRH and AVP stimulate pituitary ACTH release and consequently blood ACTH levels are elevated. The Adrenal Cortex The primary peripheral target for ACTH is the adrenal cortex, where it acts on melanocortin-2 (MC2) receptors to stimulate the synthesis and release of CORT (Raikhinstein et al., 1994; Liakos et al., 2000). Circulating CORT is bound to the plasma protein corticosterone-binding globulin (CBG), which is synthesized in the liver (Weiser et al., 1979). CORT is biologically inactive when bound to CBG, thus only the unbound fraction of CORT is active and able to freely diffuse into cells. Corticosteroid Receptors CORT actions are mainly mediated by two receptor subtypes, namely the mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). MRs have a high affinity for both CORT (Kj ~0.5 nM) and aldosterone, are substantially occupied even at low basal CORT levels, and mainly mediate the tonic effects of CORT (Reul & De Kloet, 1985; Reul et al., 1987; Ratka et al., 1989). GRs have a lower affinity for CORT (Kd -2.5-5 nM), a high affinity for the synthetic glucocorticoid dexamethasone (DEX), are only significantly occupied during stress and at the 12 peak of the circadian rhythm, and mainly mediate the stress-induced effects of CORT (Reul & De Kloet, 1985; Reul et al., 1987; Ratka et al., 1989). However, recent studies suggest that MRs may play a more dynamic role in HPA regulation than previously thought by facilitating GR-mediated effects, particularly at the circadian peak and with low intensity stressors (Ratka et al., 1989; Spencer et al., 1998; Pace et al., 2001). MRs and GRs are intracellular receptors which, when unbound, are associated with a protein complex that includes the heat shock proteins hsp90, hsp70, and hsp56, and are loosely associated with the cell nucleus (Hutchison et al., 1993; Adcock, 2000). CORT is a steroid hormone which readily diffuses across the plasma membrane. Upon ligand binding, the protein complex dissociates from the receptor and the receptor-ligand complex translocates into the nucleus. This complex can then bind via zinc fingers to glucocorticoid response elements (GREs) and negative GREs in the upstream promoter region of CORT-responsive genes, thereby acting as transcription factors to promote or repress gene transcription, respectively (Adcock, 2000). The ligand-receptor complex can also enhance the activity of or interfere with other transcription factors via protein-protein interactions, thereby indirectly affecting gene transcription (Adcock, 2000). In addition to intracellular receptors, there is mounting evidence of rapid, non-genomic membrane-related CORT effects; however, a membrane-bound receptor has not yet been characterized in rats (Makara & Haller, 2001). Glucocorticoid Actions Following Selye's description of the general adaptation syndrome, early studies focused on the role of glucocorticoids in mediating adaptation to stressors. However, with the discovery in 1949 of the anti-inflammatory actions of glucocorticoids (Hench et al., 1949), much research switched towards pharmacological applications and glucocorticoids came to be viewed as mainly inhibitory. This led to some confusion, since glucocorticoids should facilitate the response to 13 stressors yet appear to inhibit the response to immune challenges. This paradox was resolved by Munck, who hypothesized that glucocorticoids serve to prevent damage due to overactivation of defense mechanisms (Munck et al., 1984). However, such a suppressive role for glucocorticoids is just one of many actions. Glucocorticoids not only inhibit, but they also modulate the stress response and alter the response to subsequent stressors. Basal glucocorticoid levels have an important permissive role in priming defense mechanisms in order to respond appropriately to future stressors, and help maintain normal neuronal activity and survival (Sapolsky et al., 2000). These permissive effects of CORT are thought to be mediated mainly by MRs, which are nearly fully occupied at all times (Reul & De Kloet, 1985; Sapolsky et al., 2000). In addition, glucocorticoids have numerous catabolic functions which mobilize energy, including stimulation of hepatic gluconeogenesis, inhibition of glucose uptake, and stimulation of fat depletion and muscle breakdown (Sapolsky et al., 2000). As well, numerous systems which are not essential to immediate survival, including reproduction and growth, are inhibited in order to redirect energy to coping with the stressor and reinstating homeostasis (Ferin, 1999; Plourde, 1999; Habib et al., 2001). Such suppressive effects of CORT are thought to be mediated mainly by GRs, which only become significantly occupied and thereby activated following stress and at the circadian peak (Reul & De Kloet, 1985; Sapolsky et al., 2000). Glucocorticoid Negative Feedback In addition to the numerous CORT effects which mediate the stress response, CORT maintains basal HPA tone (mainly MR-mediated) and inhibits the HPA axis in order to return activity to basal levels following a stressor (mainly GR-mediated). This negative feedback acts at the level of the anterior pituitary, the PVN of the hypothalamus as well as other brain regions (Keller-Wood & Dallman, 1984; Young & Vazquez, 1996). The extent and pattern of this 14 negative feedback is stressor-specific and may target those areas of the brain initially activated by the stressor (Tanimura et al., 1998; Thrivikraman et al., 2000). Glucocorticoid negative feedback can be divided into three time domains: fast, intermediate and slow feedback (Keller-Wood & Dallman, 1984). Fast feedback, occurring within seconds to minutes of stressor onset, is dependent on the rate of change of blood glucocorticoid levels, inhibits release of CRH and thereby ACTH release, and regulates the extent and duration of an HPA response (Kaneko & Hiroshige, 1978; De Souza & Van Loon, 1989). Intermediate feedback, which begins 30-90 min post-stressor and peaks at about 5 hr, is dependent on the level of blood glucocorticoids achieved and inhibits mainly CRH and ACTH release, as well as CRH synthesis (Young & Vazquez, 1996; Tanimura et al., 1998; Keller-Wood & Dallman, 1984). Slow feedback requires prolonged elevations in glucocorticoids, only occurs with chronic stress or various pathological conditions, and inhibits POMC synthesis in the anterior pituitary as well as ACTH release (Keller-Wood & Dallman, 1984). The rapidity of fast feedback suggests non-genomic, membrane effects, and numerous brain regions have been implicated including the hippocampus, hypothalamus and cerebral cortex (Hinz & Hirschelmann, 2000; Makara & Haller, 2001). Intermediate feedback requires de novo protein synthesis and may be mediated in part by glucocorticoid-mediated inhibition of pituitary CRH binding, direct inhibition of CRH neurons and/or by altering afferent inputs to the PVN from extra-hypothalamic CORT-responsive brain regions (Ochedalski et al., 1998; Childs et al., 1986; Tanimura & Watts, 2001). Although the mechanisms and neuronal circuitry of glucocorticoid negative feedback have not been clearly elucidated, lesion studies, localized glucocorticoid implants and location of MRs and GRs offer some insight. Although the anterior pituitary does contain GRs and ACTH release can be blocked by localized DEX injection, the anterior pituitary is not considered to be a major site for direct glucocorticoid feedback inhibition (Keller-Wood & Dallman, 1984). The PVN also 15 contains GRs, suggesting direct inhibition of HPA activity (Lipostis et al., 1987; Alexis et al., 1982). As well, implantation of DEX near the PVN suppresses adrenalectomy-induced increases in CRH and AVP immunoreactivity (Kovacs et al., 1986). However, tonic regulation of CRH and AVP mRNA in the PVN appears to be largely mediated by indirect glucococorticoid-mediated projections from the limbic system and not through direct inhibition at the PVN (Whitnall, 1993). Numerous extra-hypothalamic brain regions express GRs and MRs, and may mediate HPA feedback inhibition through various stressor-specific pathways (Chao et al., 1989; Thrivikraman et al., 2000). The hippocampus has been a major focus as a putative feedback target, since it shows the highest level of glucocorticoid binding in the brain (McEwen et al., 1968). As well, hippocampal lesions result in increased HPA activity (Herman et al., 1989a; Magarinos et al., 1987). CRH and AVP appear to show differential responsivity to extrahypothalamic glucocorticoid-mediated feedback and recent studies suggest that AVP is the major target of glucocorticoid feedback (Kovacs et al., 2000). As well, AVP appears to be more sensitive to changes in the glucocorticoid environment and may therefore play a more dynamic role in HPA regulation, with CRH mainly providing stimulatory tone (Antoni, 1993; Ma & Aguilera, 1999). HPA Dysregulation Normal HPA function is of vital importance to the organism in order to respond appropriately to stressors and thereby maintain homeostasis. While an adequate HPA response is essential to enable the body to adapt to a stressor, it is as important for the organism to rapidly and appropriately return to baseline, thereby preventing any harmful effects of prolonged glucocorticoid elevations. Chronically elevated glucocorticoid levels, resulting from chronic stress or a pathological condition, can have numerous detrimental consequences. These include immunosuppression, alteration of other neuroendocrine systems, affective disorders and 16 neuronal degeneration (Habib et al., 2001; Magarinos et al., 1996; De Kloet et al., 1998). A proper balance between MRs and GRs is also vital to maintain neuronal excitability and appropriate stress responses (De Kloet, 1995). HPA dysregulation may result in an imbalance, in excitatory inputs and subsequent inhibitory feedback. This may result from genetic factors (Gomez et al., 1996; King & Edwards, 1999), early life experiences (Plotsky & Meaney, 1993; Sutanto et al., 1996) or various pathological conditions. Conditions which may present with increased HPA activity include depression (Checkley, 1996), Alzheimer's disease (O'Brien et al., 1996) and old age (Ferrari et al., 2001), whereas decreased HPA activity occurs with post-traumatic stress disorder (Kanter et al., 2001) and chronic fatigue syndrome (Cleare et al., 2001). Allostatic Load An emerging model in the understanding of HPA dysregulation is the concept of allostatic load. Allostasis is a process of adaptation which allows an organism to maintain stability, or homeostasis, through change (Sterling & Eyer, 1988). In contrast to homeostasis, which is a steady-state condition with defined set points, allostasis is a dynamic condition without set points which allows the organism to adapt to challenge, and the HPA axis is one system which mediates such adaptation (Sterling & Eyer, 1988). Allostatic load is a measure of the cumulative burden of these challenges on the organism, or the price of adaptation (McEwen, 1998; Seeman et al., 2001; McEwen & Stellar, 1993). Over time, prolonged, repeated, or inadequate allostatic responses can result in allostatic load, leading to maladaptation and impaired responses to further challenges (Pacak & Palkovits, 2001). Allostatic load may thus result in HPA dysregulation or, alternatively, HPA dysregulation due to genetic factors or disease conditions may increase susceptibility to excessive allostatic load and lead to further dysfunction. 17 Prenatal Ethanol Exposure and the HPA Axis Prenatal Ethanol and HPA Ontogeny In addition to genetic factors and early environmental effects, certain prenatal insults may also result in HPA dysregulation. One such insult is prenatal exposure to ethanol, which has been shown to alter the development of the HPA axis as well as HPA responsiveness in adulthood. Rodent neonates prenatally exposed to ethanol exhibit significantly greater brain, plasma and adrenal CORT levels and decreased CBG binding capacity compared to controls (Kakihana et al., 1980; Taylor et al., 1983; Weinberg, 1989). By 3-5 days of age, basal CORT levels are normalized. However, the HPA response to stress is suppressed compared to controls and this suppression continues throughout the preweaning period (Taylor et al., 1986; Weinberg, 1989). Ontogeny of CRH and POMC gene expression is also affected by prenatal ethanol exposure during the preweaning period, but effects differ between males and females. E females demonstrate significantly lower CRH mRNA in the PVN at 7 d of age and significantly greater CRH mRNA at 14 d of age compared to control females, whereas levels in E males do not differ from controls during this period (Aird et al., 1997). Conversely, POMC mRNA in the anterior pituitary is significantly reduced in E males compared to controls at 7 d of age whereas POMC mRNA in E females does not differ from controls during the preweaning period (Aird et al., 1997). Thus, there appears to be a sexual dimorphism in the effects of prenatal ethanol on development of the HPA axis. E Rats are Hyperresponsive to Stressors in Adulthood By the time of weaning (approximately 22 d of age), CRH mRNA levels have been shown to be either similar or increased in E compared to controls, whereas POMC mRNA does not appear to differ between E and control males.or females (Aird et al., 1997; Lee et al., 1990). By adulthood, both E males and females exhibit hormonal hyperresponsiveness to a number of 18 stressors (Taylor et al., 1982; Weinberg, 1988; Lee et al., 2000). In adult E rats, basal non-stressed plasma CORT, CBG and ACTH levels are typically normal (Taylor et al., 1983; Weinberg & Gallo, 1982; Kim et al., 1999b). However, in response to several stressors including foot shock (Nelson et al., 1986; Lee et al., 2000), novel environments (Weinberg, 1988), restraint (Weinberg, 1988, 1992), ether (Angelogianni & Gianoulakis, 1989; Weinberg & Gallo, 1982), cold (Angelogianni & Gianoulakis, 1989; Kim et al., 1999a), and immune challenges (Lee & Rivier, 1996; Lee et al., 2000), CORT and/or ACTH levels may show increased and/or prolonged elevations compared to controls. The degree of hyperresponsiveness is dependent upon the nature and intensity of the stressor, the timepoint as well as the hormonal endpoint measured. In addition, although both E males and females exhibit this HPA hyperresponsiveness, the pattern may differ between the sexes (Weinberg, 1992). The mechanisms underlying HPA hyperresponsiveness in E rats are unclear at present. One possibility is that prolonged HPA responsiveness following stress in E rats may be, at least partially, due to deficits in CORT negative feedback regulation. Previous studies in our laboratory suggest that E animals may exhibit deficits in the intermediate but not the fast feedback time domain. Our data indicate that both E males and females demonstrate significantly greater CORT and/or ACTH responses to an ether stress 3-6 hr (intermediate feedback time domain) following HPA suppression by DEX, compared to control animals (Osborn et al., 1996), but do not differ significantly from controls in response to swim or ether stress at 5 or 30 min (fast feedback time domain) after a CORT injection (Hofmann et al., 1999). In addition to feedback deficits, an alternate but not incompatible hypothesis is that HPA hyperresponsiveness in E rats may be mediated by enhanced stimulatory inputs to the PVN and/or increased sensitivity of the anterior pituitary to secretagogues, such as CRH. In support of the latter, we have shown that following DEX suppression at the trough of the diurnal rhythm, E females had a greater ACTH response to a bolus iv infusion of CRH compared to controls 19 (Osborn et al., 2000). However, other studies in which DEX was not utilized showed no differential pituitary responsiveness to either CRH or AVP in E compared to controls (Lee et al., 1990, 2000). These differences may reflect decreased sensitivity to DEX blockade in our study and/or differential effects of endogenous CORT or CRH in E animals. Although pituitary POMC mRNA has been shown to be increased in E males under non-stressed conditions it is decreased with chronic ethanol consumption compared to controls (Redei et al., 1993; Halasz et al., 1993). Thus, the role of the anterior pituitary in mediating HPA hyperresponsiveness remains unclear. Increased activity has been more consistently observed at the level of the PVN, due possibly to increased basal tone or increased stimulatory inputs in response to stress. At the level of the PVN, CRH mRNA has been shown to be higher in 60 d old E males compared to controls (Redei et al., 1993). However, we and others have found no differences in basal CRH or AVP mRNA in E animals (Kim et al., 1999b; Lee et al., 2000). As well, Lee et al. (2000) found no differences in basal CRH heteronuclear RNA (hnRNA) and lower AVP hnRNA in E compared to controls, along with no differences in basal CRH and AVP levels in the median eminence. These findings suggest that E animals likely do not exhibit enhanced basal HPA tone under non-stressed conditions. However, in response to stressors, including footshock and endotoxemia, immediate early gene (c-fos and NGFI-B) and CRH hnRNA responses are greater in the pPVN of E compared to control animals (Lee et al., 2000), suggesting that HPA hyperresponsiveness may be mediated by enhanced stimulatory inputs to the PVN. Since E animals may exhibit greater elevations in stress CORT levels compared to controls and since there are suggestions of CORT feedback deficits in E animals, it remains to be determined whether this increased PVN activity is due to enhanced stimulatory inputs to the PVN and/or CORT feedback deficits. Allostasis in the E Rat HPA responses in the E rat are clearly dysregulated and allostatic responses are inadequate. If 20 we apply the concept of allostatic load, E rats would likely achieve a higher level of allostatic load over time than a normal rat. However, a typical laboratory rat which spends its life in a predictable environment with no shortage of food or water likely has little accumulation of allostatic load. Therefore, studying allostatic responses including HPA function in such an animal is likely an underestimate of the extent of maladaptation that may occur with this type of insult. On the other hand, a human with similar HPA dysfunction dealing with the numerous stressors of daily life may be more prone to excessive allostatic load, leading to further detrimental effects. FAS and the HPA Axis Although HPA hyperresponsiveness resulting from prenatal ethanol exposure was first discovered in rats (Taylor et al., 1981) and has now also been demonstrated in primates (Schneider et al., 2002), recent studies suggest similar abnormalities in humans. In response to a routine blood draw, which served as the stressor, infants with a history of prenatal alcohol exposure were found to exhibit higher salivary Cortisol levels compared to non-exposed infants (Jacobson et al., 1999). As of yet, HPA function has not been examined in older children or adults with FAS. However, if hyperresponsiveness does persist into adulthood as it does in animal models, it may exacerbate the numerous problems already present with this disorder. Rationale and Thesis Objectives Adult E rats exhibit HPA hyperresponsiveness in response to different types of stressors, ranging from systemic to psychological. This suggests a problem either within the HPA axis resulting in increased HPA responses to any input and/or extensive damage in numerous pathways mediated by multiple neurotransmitters and brain regions. Although the former seems more likely, the latter cannot be discounted considering the wide range of ethanol effects on 21 neuronal development. In addition, since E animals may exhibit differential CORT responses to stress and possibly decreased sensitivity to CORT regulation of HPA activity, it is difficult to determine whether any alterations observed in E animals are due to increased stimulatory inputs and/or to CORT feedback alterations. The present studies therefore aimed to examine the extent to which prenatal ethanol effects on HPA function are mediated by alterations in CORT feedback mechanisms. The present studies tested the hypotheses that 1) HPA hyperresponsiveness in E rats is mediated, in part, by enhanced stimulatory drive to the PVN and/or anterior pituitary and 2) HPA hyperresponsiveness in E rats is mediated, in part, by altered CORT feedback regulation. These hypotheses were tested by manipulating the CORT feedback signal, allowing the role of CORT to be examined and these two possible mechanisms to be investigated somewhat independently. Since both E males and females exhibit HPA hyperresponsiveness but often with a different pattern, both sexes were examined in all studies. In Chapter III, a series of studies is described in which removal of CORT by adrenalectomy (ADX) was used to assess hypothalamic-pituitary activity in E rats under basal and stress conditions without the possible confounding effects of variability in endogenous plasma CORT levels. CORT replacement via the drinking water (to similar levels in all animals) was used to assess CORT feedback function and to determine whether E rats can effectively use this CORT signal to regulate HPA activity as measured by plasma ACTH levels. Chapter IV extended this investigation by further examining HPA function under basal conditions by measuring mRNA levels of key HPA components. Again, ADX (with or without CORT replacement) was used as a probe to investigate the role of CORT in mediating any alterations. In order to determine whether enhanced HPA drive in E rats occurs at the hypothalamic and/or anterior pituitary level, we measured CRH and AVP mRNA in the PVN and CRH-R1 and POMC mRNA in the anterior pituitary. In addition, MR and GR mRNA was measured in the hippocampus as an indication of CORT feedback. If alterations in HPA function in E animals are mediated by differences in 22 CORT feedback, these alterations would be absent in ADX animals and restored by CORT replacement. Conversely, alterations present only with ADX would not be mediated by CORT feedback deficits and would perhaps be indicative of enhanced stimulatory inputs. Finally, the experiment in Chapter V was undertaken to investigate the role of MRs and GRs in mediating altered HPA responsivity in E animals. Function of MRs and GRs was examined independently using selective receptor antagonists to investigate HPA activity under non-stressed conditions as well as during and following restraint stress. Deficits in CORT feedback in E animals would be expected to present as a decreased response to either MR or GR blockade compared to controls, allowing us to determine whether any CORT feedback alterations are mediated via MR- or GR-mediated pathways, or by both receptors. 23 CHAPTER II: GENERAL METHODS Animals and Mating For all studies, Sprague-Dawley male (250 - 275 g) and female (225 - 250 g) rats were obtained from the Animal Care Centre, University of British Columbia, Vancouver, BC, Canada. Males and females were group housed by sex for a 1 - 2 week period prior to, mating, to allow for adaptation to the colony room and recovery from transportation. Males were then singly housed in stainless steel hanging cages (25 x 18 x 18 cm) with mesh front and floor, and maintained on standard laboratory chow (Jamieson's Pet Food Distributors Ltd., Delta, BC, Canada) and water. The colony room had controlled temperature (21 °C) and lighting, with lights on from 0600h to 1800h. Females were placed singly with males and cage papers were checked daily for vaginal plugs. The day the plug was found was considered day 1 of gestation (GI). Al l animal use procedures were in accordance with NIH guidelines and were approved by the University of British Columbia Animal Care Committee. Ethanol Administration Numerous methods of ethanol administration exist in animal studies, each having its advantages and drawbacks (Abel, 1980). Intragastric intubation allows the same dose to be given to all animals, and a high blood alcohol concentration (BAC) can be achieved. However, this method is rather stressful to the animal, and high BACs result in high mortality rates. Although administering ethanol in the drinking water may seem like the easiest and most logical solution, animals are generally aversed to the taste of ethanol and will not drink solutions with a high ethanol concentration. Therefore, only low BACs can be achieved. As well, rodents tend to decrease their food consumption when fluid intake decreases, leading to malnutrition. This 24 problem is overcome by using a liquid ethanol diet as the sole source of nutrition and fluids. Although consumption does decrease just before parturition and very high BACs are difficult to achieve, this method is nonstressful and provides adequate nutrition to the animal. Due to the appetite suppressing effects of ethanol, animals on the liquid ethanol diet decrease their food consumption compared to ad libitum-fed control animals. Thus, a pair-fed (PF) nutritional control group is necessary. Although dams consuming the PF control liquid diet are matched for body weight and consume the same amount of calories per gestation day as their ethanol-consuming counterparts, this diet does not control for possible differences in metabolic need or possible effects of the ethanol diet on nutrient absorption and/or utilization. As well, whereas ethanol-consuming dams have ad libitum access to food, PF dams do not and typically receive less food than they would desire. As a result, PF dams often consume most of their food within a short period of time and are hungry by the time food is presented again the following day. PF dams therefore tend to be more agitated than their ethanol-consuming counterparts. This form of stress occurring during pregnancy may impact the developing fetus and could potentially alter HPA activity. Therefore, it is possible that not all the effects seen in PF offspring relate to nutritional effects, but may also be a consequence of prenatal stress. Although the PF group is not an ideal control, it is important to control for the nutritional effects of prenatal ethanol exposure as reflected by the decreased body weight in both E and PF offspring compared to controls in early life. An alternate method of ethanol exposure is the ethanol inhalation vapor, which eliminates the nutritional issue by bypassing the digestive system. Pregnant animals are placed into chambers into which ethanol vapors are delivered and remain in these chambers for many hours. This method allows for continuous ethanol exposure and fine control of BACs, is relatively non-stressful, and allows high BACs to be achieved. Another advantage is that once the animals are removed from the ethanol chamber, their food consumption does not appear to be affected. As a 25 result, body weights of ethanol-exposed offspring do not differ from controls. Thus, a PF group is not necessary and ethanol effects can be examined independently of nutritional effects. However, growth deficiencies are a consistent and significant effect of prenatal alcohol exposure in humans and a method that eliminates this mechanism may not mimic the human situation as accurately. Since humans ingest alcohol, some of the mechanisms of alcohol teratogenicity may be due to effects on food intake and/or nutrient absorption which would not be modeled by the ethanol inhalation method. As well, the ethanol inhalation method results in sustained high level BACs which again do not mimic typical human alcohol consumption in which, even with high levels of chronic alcohol consumption, result in varying BACs throughout the day. Since peak BACs are the key determinant of alcohol's teratogenic effects (West & Goodlett, 1990), the high and sustained BACs in the ethanol inhalation model may exaggerate some of the ethanol effects observed. Thus, although the ethanol inhalation chamber allows us to eliminate the nutritional control group, is relatively non-stressful and allows for high and controlled BACs, the fact that this method does not mimic human alcohol consumption as effectively as other methods has limited its popularity. The liquid diet method of ethanol administration is the model chosen in our laboratory due to the ease of administration and because it is non-stressful, which is important when studying HPA function. Since rats do not display the characteristic facial features of FAS, and due to the moderate BACs achieved with this method, ours is a model of ARND and not specifically of FAS. Diets and Feeding On GI, females were singly housed in polycarbonate cages (24 x 16 x 46 cm) and randomly assigned to one of three treatment groups: 1) ethanol (E) females received ad libitum access to a liquid diet containing 36.8% ethanol-derived calories, 2) pair-fed (PF) females received a liquid 26 diet with maltose-dextrin isocalorically substituted for ethanol, with each animal pair-fed the amount consumed by a female in the E group (g/kg body weight) on the same day of gestation, and 3) control (C) females received ad libitum access to standard laboratory chow and water. The liquid diets (see Table 1 for composition) were previously developed by our laboratory and prepared by Bio-Serv Inc. (Frenchtown, NJ, USA) to provide adequate nutrition to pregnant dams regardless of ethanol intake (Weinberg, 1985). Fresh diet was provided daily at approximately 1600h, near the diurnal peak, to prevent a shift in the corticosterone rhythm that may occur in animals on a restricted feeding schedule, such as the PF dams (Gallo & Weinberg, 1981). Diets from the previous day were removed at this time and weighed to determine daily consumption. E females were gradually introduced to the ethanol diet by providing a 1/3 ethanol:2/3 PF diet on G l , 2/3 ethanol:l/3 PF diet on G2, and ethanol diet alone by G3. Experimental diets were continued until G22, at which time they were replaced with standard laboratory chow and water, ad libitum. Pregnant females were undisturbed except for weighing and cage changing on G l , 7, 14 and 21. At birth, designated postnatal day 1 (PN1), litters were randomly culled to 5 males and 5 females. Dams and pups were weighed and cages changed on PN1, 8, 15 and 22. On PN22, pups were weaned and housed by litter and sex. Testing was done on males and females at 90 - 120 d of age, unless otherwise stated. 27 Table 1. Caloric profile of ethanol and pair-fed diets obtained from Bio-Serv Inc. (Frenchtown, NJ, USA). Actual values fall within 10% of these values due to analytical variability, sampling variability, and moisture levels during assay. Dietary Component Ethanol Diet (kcal/litre) Pair-Fed Diet (kcal/litre) Protein 258 258 Fat 255 255 Carbohydrates 118 486 Ethanol 368 0 Total 999 999 Adrenalectomy In the studies outlined in Chapters III and IV, ADX with or without CORT replacement, was utilized to examine the role of CORT in mediating any HPA alterations. One day prior to surgery, rats were singly housed in clean cages and assigned to one of three surgical conditions: 1) SHAM, 2) ADX, or 3) ADX with CORT replacement via the drinking water (ADX + CORT). In order to control for litter effects, no more than one animal from any one litter was used per test condition. Bilateral ADX was carried out via the dorsal approach under halothane anesthesia. The incision areas were shaved then disinfected with 70% ethanol. Two small bilateral incisions were made and the muscle wall was entered using blunt dissection. The adrenal glands were removed by grasping the surrounding adipose tissue with blunt forceps allowing the intact adrenal gland 28 to be lifted out. The muscle wall was then closed with two small sutures and the exterior incisions were closed using two wound clips per side. Sham surgery involved the same procedure except adrenal glands were not removed. ADX not only removes circulating CORT but other hormones as well, particularly aldosterone which is important in maintaining plasma osmolarity. To compensate for the loss of aldosterone, all ADX animals were provided with 0.9% NaCl as drinking water beginning immediately after surgery and continuing until the time of testing. Corticosterone Replacement A subset of ADX animals was given CORT in their drinking water in order to restore plasma CORT to low basal levels (ADX+CORT condition). Corticosterone (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 95% ethanol heated to 50°C, then dissolved in water containing 0.9% NaCl, to give a final ethanol concentration of 0.2% and a final CORT concentration of 25 ug/ml CORT for males and 75 ug/ml for females (unless otherwise stated). Animals in the ADX condition were provided with 0.2% ethanol and 0.9% NaCl as drinking water. CORT replacement in the drinking water produces a diurnal pattern of circulating CORT. In the evening, during the active period, food and fluid consumption increase and thus CORT intake increases, resulting in a rise in circulating CORT levels. During the day, when the animal is asleep and not consuming fluid, CORT levels drop. This mimics the normal diurnal pattern seen in adrenal intact rats. An alternative method of CORT replacement is a subcutaneous CORT pellet, which produces a tonic level of plasma CORT. With this method, the dose is typically chosen to produce plasma CORT levels similar to median levels in an intact rat. As a result, CORT levels are much higher than normal at the trough, resulting in suppression of ACTH, and lower than normal at the peak of the diurnal rhythm, resulting in higher than normal ACTH 29 levels (Akana et al., 1985; Jacobson et al., 1988). Since regulation of ACTH secretion requires very low CORT levels during the diurnal trough and high levels during the diurnal peak, CORT replacement in the drinking water is more effective in regulating basal HPA activity in ADX animals (Bradbury et al., 1991). As a result, this was the method we chose for CORT replacement. Blood Sampling For all experiments, blood was collected for measurement of plasma CORT and ACTH levels. Since ACTH readily binds to glass, all blood was collected in polystyrene or polypropylene tubes. EDTA disodium salt was added as an anticoagulant and aprotinin (ICN Pharmaceuticals, Inc., Costa Mesa, CA, USA), a protease inhibitor, was used to prevent ACTH degradation. Blood was collected on ice then centrifuged at 2200 x g for 10 min at 4°C. Plasma was then transferred to individual polypropylene tubes for CORT and ACTH determination and stored at -70°C. ACTH responds rapidly to stressors, with increases in plasma ACTH levels occurring within 2 minutes. Thus, in order to obtain an accurate plasma ACTH measurement the sampling method must be rapid enough that the stress of the sampling method is not reflected in the measurement. Decapitation One method of obtaining basal plasma ACTH levels in rodents is by rapid decapitation of an unanesthetized animal. Animals are taken in their home cage to an immediately adjacent room, removed from their home cage and decapitated. Trunk blood is then collected on ice into 12 x 75 mm polystyrene tubes containing 0.2 ml of EDTA (0.2M) and aprotinin (1000 KIU). The length of time from touching the cage to decapitating the animal is typically about 30 s, which is shorter than the timeframe for an ACTH response to be mounted. Thus, the plasma ACTH levels 30 measured are not affected by the sampling method. Decapitation was the method used for the experiments outlined in Chapters III and IV. Jugular Cannulation When serial blood samples are required within a brief period of time from one animal, a sampling method is required which causes minimal stress and therefore does not affect subsequent samples. This is typically achieved by withdrawing blood via an intravenous cannula. This method was utilized in the study detailed in Chapter V. The cannula consisted of 25 cm PE50 tubing with a short piece of silastic tubing attached to one end (3.5 cm for males, 3.3 cm for females) with a 45° bevel on the end inserted into the jugular vein. Animals were anesthetized with halothane, the skin was disinfected using 70% ethanol, then a small skin incision was made over the right jugular vein. Connective tissue was cleared away by blunt dissection to expose approximately 0.5 cm of the vessel. The vein was then ligated with silk thread at the cranial end and two additional ligatures were placed loosely at the caudal and cranial ends of the exposed vein. A small opening was made in the vein using a 22G needle and the cannula was inserted into the opening with the aid of a catheter introducer. The cannula was secured in place by tying the loose ligature around the vessel then tunneled subcutaneously and externalized dorsally between the scapulae. The patency of the cannula was verified then the external incision was closed using two sutures. The cannula was flushed with saline and the free end was folded and capped with a short piece of PE240 tubing until testing. Rats were singly housed immediately after surgery and weighed one day following surgery. Two days after surgery, rats were removed from the animal room to an adjacent testing room at lights on (0600h). At this time a 75 cm piece of PE50 tubing filled with 1% heparin/saline was attached to the externalized cannula by way of a blunted 22G needle attached to the end. The opposite end of this tubing was connected to an intermittent infusion plug, which allowed for 31 attachment of syringes for blood collection. The patency of the cannula was checked and the rat was placed into an opaque cylindrical bucket (height: 43 cm; diameter: 29 cm) with bedding on the bottom. The lid contained a hole in the middle (5 cm diameter) through which the sampling cannula was threaded and hung over the side of the bucket, allowing for blood sampling without disturbing the animal. Animals were allowed 3 hr to adapt to the new environment prior to blood sampling. Each blood sample taken during testing was a maximum of 0.4 ml in volume. The heparin/saline syringe was used to draw blood until it just reached the syringe barrel. An ice-cold EDTA/aprotinin-coated syringe was then used to withdraw 0.4 ml of blood, which was immediately transferred to a polypropylene tube on ice. The volume of blood removed was then replaced with an equal volume of saline (i.e., 0.4 ml), then 0.3 ml of heparin/saline was injected in order to fill the cannula and maintain patency, with care taken to ensure that the heparin solution was not injected into the animal. Corticosterone and ACTH Radioimmunoassays Corticosterone Total CORT (bound + free) was measured by radioimmunoassay (RIA) using our adaptation (Weinberg & Bezio, 1987) of the methods of Kaneko et al. (1981). CORT antiserum was obtained from Immunocorp (Montreal, PQ, Canada) and tracer was obtained from Mandel Scientific (Guelph, ON, Canada). Dextran-coated charcoal was used to absorb and precipitate free steroids after incubation. Antiserum cross-reactivity was 100% for corticosterone, 2.3% for desoxycorticosterone, 0.47% for testosterone, 0.17% for progesterone and 0.05% for aldosterone. The minimum detectable concentration for corticosterone was 2.5 ng/ml, and the intra- and interassay coefficients of variation were 1.55% and 4.26%, respectively. 32 ACTH Plasma ACTH was assayed using a modification of the DiaSorin ACTH 1 2 5I RIA Kit (DiaSorin Inc., Stillwater, MN, USA). Antiserum cross-reactivity was 100% for ACTH and less than 0.1% for all other peptides assessed (including a-MSH, (3-endorphin and P-lipotropin). Al l reagent volumes were halved and 50 ul of plasma per sample were used. Since repeated freeze-thaw may result in ACTH degradation, individual samples were analyzed only once. The minimum detectable dose for ACTH was 20 pg/ml, and the mid-range intra- and interassay coefficients of variation were 3.9% and 6.5%, respectively. 33 CHAPTER III: EFFECTS OF PRENATAL ETHANOL EXPOSURE ON HYPOTHALAMIC-PITUITARY-AD RENAL RESPONSIVENESS FOLLOWING ADRENALECTOMY AND CORTICOSTERONE REPLACEMENT INTRODUCTION As discussed in the General Introduction, our previous studies suggest that HPA hyperresponsiveness in E rats may be mediated by enhanced HPA drive and/or CORT feedback alterations. In order to investigate the role CORT may play in mediating any alterations, the present studies utilized ADX in order to assess hypothalamic-pituitary function in the absence of CORT. As well, CORT replacement following ADX allowed hypothalamic-pituitary function to be investigated in the presence of a controlled exogenous CORT feedback signal. In control rats, ADX typically results in elevated CRH mRNA in the PVN (Young et al., 1986a), increased POMC mRNA in the anterior pituitary, and elevated plasma ACTH levels (Akana et al., 1985; Dallman et al., 1985) due to loss of CORT negative feedback. CORT replacement, via subcutaneous pellets or in the drinking water, can return these elevations toward basal levels (Akana et al., 1985; Paull & Gibbs, 1983). CORT replacement using pellets produces a constant plasma CORT signal, whereas putting CORT in the drinking water results in a phasic plasma CORT signal which more closely mimics the normal CORT diurnal rhythm and thus may be more effective in normalizing basal HPA activity (Akana et al., 1985). In the present studies, manipulation of the CORT feedback, signal via ADX with or without CORT replacement, was used to assess the role of CORT in mediating any HPA alterations in E rats. Hypothalamic-pituitary activity was examined under basal and stress conditions without the possible confounding effects of variability in endogenous plasma CORT levels. CORT replacement via the drinking water (to similar levels in all animals) was used to assess CORT feedback function and to determine whether E rats can effectively use this exogenous CORT 34 signal to regulate HPA activity. Plasma ACTH levels were determined as an end-point measurement of hypothalamic-pituitary activity. Al l testing was done at the circadian peak, since we have previously shown greater HPA hyperresponsiveness in E animals at this time (Osborn et al., 1996) and since hypothalamic drive is increased toward the evening, allowing hypothalamic function to be better assessed. We hypothesized that any CORT-mediated alterations in HPA activity would be present in sham and/or ADX+CORT E animals, indicating decreased sensitivity to the CORT feedback signal, but would be absent with ADX, i.e., in the absence of CORT. Conversely, alterations following ADX, in the absence of CORT, would indicate enhanced HPA drive. METHODS Breeding and Animals For Experiment 1, only control animals (N=10) were bred and animals were approximately 60 d of age at the time of surgery. For Expt. la, CORT replacement was initiated 30 d later (at approximately 90 d of age) and blood samples obtained after 7 d of CORT replacement. For Expt. lb, CORT replacement was initiated 60 d after surgery (at approximately 120 d of age). Replicate breedings were done for Experiments 2 and 3 with E, PF and C prenatal treatment groups. For these studies, all surgeries were conducted on animals ranging from 90 to 110 d in age and testing was conducted 7 d after surgery. Both male and female rats were tested in all experiments. Corticosterone Replacement Corticosterone was dissolved in 0.2% ethanol/0.9% NaCl to give a final CORT concentration of 25, 50 or 75 ug/ml. Initially the highest CORT dose made was 100 ug/ml CORT. However, at this concentration small amounts of CORT appeared to precipitate out of solution within 2 d; 35. therefore, this dose was not used. CORT solution was provided to a subset of ADX rats as the sole source of drinking fluid immediately following ADX until termination. Fluid intake was measured daily at 1600h-1700h for determination of daily CORT intake. Each bottle was also shaken vigorously at this time to ensure that CORT would not precipitate out of solution (CORT visibly precipitates out of solution after approximately 3-4 days). CORT solutions were replaced every second day. Experiment 1: Determination of CORT Dosage Expt. 1 was a pilot study conducted in control males and females in order to determine appropriate CORT replacement doses. At 60 d of age, all rats underwent ADX surgery under halothane anesthesia and were provided with 0.9% NaCl as drinking water immediately after. In Expt. la, thirty days following surgery (i.e., at 90 d of age), saline drinking water was replaced in a subset of males and females with 25, 50 or 75 ug/ml CORT dissolved in 0.2% ethanol/0.9% NaCl. After 7 d of CORT replacement, animals were decapitated at the diurnal peak (2 hr after lights off) under red light conditions. Blood was Collected for plasma CORT and ACTH measurement to determine the optimal dose of CORT replacement. In Expt. lb, CORT concentrations of 25 ug/ml for males and 75 ug/ml for females (chosen based on ACTH and CORT results from Expt. la; see Results section below) were then given to another subset of control animals 60 d after surgery (i.e., 120 d of age) to assess ACTH and CORT levels at different times of day. After 7 d of CORT replacement, rats were decapitated at either 0700h (diurnal trough) or 1600h. Trunk blood was collected for plasma CORT and ACTH measurement in order to determine whether this dose of CORT replacement produced a diurnal pattern of both CORT and ACTH. 36 In order to asses effects of ADX and saline intake on fluid consumption, 24 hr water intake was measured in animals 2 d prior to ADX and saline intake was measured in a subset of animals 50 d following ADX. ADX with or without CORT Replacement in E, PF and C Rats Experiments 2 and 3 were carried out as separate replications. In each study, E, PF and C males and females underwent surgery at 90-120 d of age. One day prior to surgery, rats were singly housed in clean cages and assigned to one of three surgical conditions: 1) SHAM, 2) ADX, or 3) ADX + CORT. Immediately following surgery and continuing for 7 d until testing, ADX+CORT rats were provided with CORT in the drinking water at a dose of 25 ug/ml in males and 75 ug/ml in females (as determined from Experiment 1) dissolved in 0.2% ethanol and 0.9% NaCl. ADX rats were provided with 0.9% NaCl/ 0.2% ethanol for drinking water. In order to control for litter effects, no more than one animal from any one litter was used per test condition. Testing was carried out 7 d following surgery at the diurnal peak, 2 - 3 hr after lights off (2000h-2100h), under red light conditions. Experiment 2 Rats were individually removed to an adjacent room and decapitated within seconds of touching the cage. Trunk blood was collected for measurement of resting, non-stressed levels of plasma CORT and ACTH. In addition, thymus weight was measured (a peripheral target sensitive to circulating CORT levels) in order to ensure that the level of CORT replacement was adequate and that it had similar effects in E, PF and C rats. 37 Experiment 3 Rats were individually removed to an adjacent room under red light conditions and placed into polyvinyl chloride restraint tubes (7.5 cm diameter x 20 cm long for males, 5.5 cm diameter x 20 cm long for females), with 4 holes (1 cm) in the front and one at the back (1.5 cm) for the tail. After 15 min, animals were removed from the restraint tubes and immediately decapitated. Trunk blood was collected for determination of plasma CORT and ACTH levels. Statistical Analyses Developmental data were analyzed by two-way ANOVA for the factors of treatment condition (E, PF, C) and day, with days treated as repeated measures. For Expt. 1, data were analyzed by one-way ANOVA for the factor of CORT dose (25, 50, 75 ug/ml) or time of day (0700h, 1600h, 2000h). For Expts. 2 and 3, CORT and ACTH data were analyzed by two-way ANOVAs for factors of prenatal treatment group (E, PF and C) and surgical condition (SHAM, ADX, ADX+CORT). Significant main and interaction effects were further analyzed by Newman-Keuls post-hoc tests. RESULTS Experiments la & b Fluid intake (Table 2) was significantly greater overall in females compared to males (p<0.01). In addition, ADX significantly increased fluid intake in comparison to pre-surgery levels in females (p<0.01) but not males. Saline intake rose during the first hour following lights off (males: 9.3 ± 1.2 ml/kg bw; females: 15.4 ± 8.1 ml/kg bw) and continued to rise during the second hour (males: 22.2 ± 4.2 ml/kg bw; females: 25.3 ± 6.6 ml/kg bw) in comparison to the 2 hr prior to lights off (males: 3.4 ± 0.6 ml/kg bw; females: 14.6 ± 4.0 ml/kg bw). Based on these 38 findings, sampling in subsequent experiments was carried out 2 hr after lights off (unless otherwise stated) to ensure that CORT-replaced animals had received a sufficient CORT signal by the time of testing. As well, since hypothalamic drive is increased at the circadian peak, this timepoint allowed hypothalamic function to be examined. Table 2. Twenty four hour fluid intake prior to adrenalectomy (ADX) and 60 d following ADX. 24 hr fluid intake (ml/kg bw) Males Females water intake 120.0 ± 6.2 154.2 ± 11.1 prior to ADX saline intake in 145.8 ± 13.7 280.0 ± 37.2 ADX animals Main effect of sex (F(l,24)=9.58, p<0.005; females>males, p<0.01. Females only: main effect of surgery (F(l,9)=12.4; p<0.01) ADX>pre-surgery, p<0.01; n=5-9 per group In males (Fig. 3 a,c), increasing CORT doses in the drinking water (25, 50 and 75 ug/ml) resulted in a pattern of increasing plasma CORT levels, with no significant change in plasma ACTH. Although we did not measure ACTH levels in ADX animals without CORT replacement in this pilot study, the range of ACTH achieved with all three levels of CORT replacement are similar to normal ACTH levels, and are lower than ADX-induced ACTH levels seen in other studies (Akana et a l , 1985). The lowest CORT dose, 25 ug/ml, was thus chosen for males based on these findings and on other studies which have shown that this dose normalizes body weight, thymus weight and stress-induced ACTH responses following ADX (Akana et al., 1985; 39 Jacobson et al., 1988). In females (Fig. 3 b,d), plasma CORT levels were similar with all CORT doses; however, plasma ACTH levels did decrease with increasing CORT concentrations. Although even the highest concentration of CORT in the drinking water (75 ug/ml) was not fully effective in regulating ACTH levels, since greater CORT concentrations (e.g., 100 ug/ml, as mentioned in Methods) appeared to precipitate out of solution we chose 75 ug/ml CORT for females since it was the highest CORT concentration we could achieve without the concern that it would precipitate out of solution/These doses of CORT (25 [xg/ml in males, 75 ug/ml in females) resulted in mean 24 hr CORT intake of 4.8 ± 0.1 mg CORT/kg bw in males and 19.5 ± 4.1 mg CORT /kg bw in females. CORT intake via the drinking water at the chosen doses (25 and 75 ug/ml CORT for males and females, respectively) resulted in a diurnal pattern of plasma CORT levels (see Fig. 4). As expected, plasma CORT levels increased towards the evening, or active period in the rat, when food and liquid consumption (therefore CORT consumption) increase. Plasma ACTH levels were similar near the diurnal peak and trough in males, whereas females exhibited lower levels at the peak. 40 C DC O o (0 E w TO 600 25 50 75 25 50 CORT dose in drinking water ( ug/ml) Figure 3. Plasma CORT levels in adrenalectomized control a) males and b) females and plasma ACTH in c) males and d) females at the diurnal peak following 7 d of CORT replacement in the drinking water at a dose of 25, 50 or 75 ug/ml. Values represent the mean ± SEM of 5 rats per group. Note: for 25 ug/ml CORT dose in females, ACTH levels were off the standard curve (i.e., > 500 pg/ml) and were not remeasured. 41 600 0700h 1600h 2000h 0700h 1600h 2000h 600 0700h 1600h 2000h 0700h 1600h 2000h Time Figure 4. Plasma CORT and ACTH levels at three different times of day: 0700h, 1600h and 2000h in adrenalectomized control males (a,c) provided with 25 ug/ml CORT and females (b,d) provided with 75 (xg/ml CORT as drinking water. Values represent mean ± S E M of 3-5 rats per group. Note: 2000h values are taken from Fig. 3, from the 25 and 75 ug/ml doses for males and females, respectively. 42 Experiment 2 Ethanol intake of pregnant females was consistently high throughout gestation, averaging 11.6 ± 1.4, 13.0 ± 1.6 and 12.4 ± 1.1 g ethanol/ kg bw for weeks 1, 2 and 3 of gestation, respectively. We have previously shown that this level of intake results in peak blood ethanol levels of 137 - 155 mg/dl (Weinberg, 1985; Osborn et al., 1998). Developmental Data Maternal body weights during gestation and lactation are shown in Fig. 5 below (all subsequent breedings were replicates; therefore, for the current breeding all data are shown below and corresponding data for subsequent breedings is shown in the Appendix). In brief, body weight increased throughout gestation in all pregnant dams. Whereas E, PF and C dams did not differ in body weight at gestational days (G) 1 and 7, PF dams weighed significantly less than C dams on G14 (p<0.05) and both E and PF dams weighed significantly less than C dams on G21 (ps<0.05). Maternal body weights normalized during lactation, with no significant differences among E, PF and C dams. Gestation length was significantly longer in E and PF compared to C dams (ps<0.05) and there were no significant differences among treatment groups for litter size or number of stillborn pups (Table 3). Body weights of pups are shown in Fig. 6 below (for subsequent replicate breedings, corresponding figures are in the appendix). In brief, E, PF and C male and female pups did not differ in body weight on PN 1 and 8. On PN15, PF females weighed significantly less than C females (p<0.05). By PN22, E and PF male and female pups weighed significantly less than their C counterparts (ps<0.05) and E males weighed less than PF males (p<0.05). 43 500 1 7 14 21 Gestational Day 500 400 300 8 15 Postnatal Day 22 -e— P F Figure 5. Maternal body weights during a) gestation (GI to 21) and b) lactation (PN1 to 22). Values represent the mean ± SEM of 11-14 animals per group. Gestation period: significant treatment x day interaction (F(6,108)=14.11, p<0.0001); #C>PF, p<0.05. *C>E,PF, ps<0.05. Lactation period: significant treatment x day interaction: (F(6,99)=3.73, p<0.005). 44 Table 3. Gestation length, # live pups and # dead pups (mean ± SEM) of E, PF and C dams Gestation Length (d) # Live Pups # Dead Pups E 22.1 ±0 .1* 14.1 ±0.9 0.13 ± 0.09 PF 22.0 ± 0.0* 14.5 ±0.7 0 C 21.8 ±0.1 14.3 ± 1.1 0.25 ± 0.18 Gestation length: main treatment effect (F(2,38)=4.3, p<0.05); *E,PF>C, ps<0.05 (n=12-15 per group) 45 D5 •D O CO 8 15 Postnatal Day -e-Figure 6. Mean pup body weights (g) in a) males and b) females during postnatal days 1 to 22. Values represent the mean ± SEM of 11-15 rats per group. Males: significant treatment x day interaction (F(6,lll)=8.58, p<0.0001); #PF>E, p<0.05; *C>E,PF, ps<0.05. Females: significant treatment x day interaction (F(6,105)=9.13, p<0.0001); *C>E,PF, ps<0.05, APF<C, p<0.001. 46 Adult Body Weight By the time of testing (in adulthood), there were no significant differences in body weights among animals from the three prenatal treatment groups (Table 4). Briefly, body weight increased significantly in all SHAM-operated animals from the day prior to surgery to 6 d after surgery. In males, all ADX animals had significantly lower body weight 6 d after surgery whereas body weight did not differ 6 d after ADX in CORT-replaced males. Females in both the ADX and ADX+CORT condition had similar body weights prior to and 6 d following surgery. Overall, PF females weighed significantly less than E and C females. CORT Intake Mean 24 hr CORT intake for rodents consuming CORT in the drinking water (ADX+CORT condition) is summarized in Table 5. Overall, CORT intake was significantly greater in females compared to males, and there were no differences among E, PF and C prenatal treatment groups. Thymus Weight ADX significantly increased thymus weight (Fig. 7) in both males and females compared to the SHAM condition, and CORT replacement normalized thymus weight to levels in SHAM animals. There were no differences among E, PF and C animals within any surgical condition. Non-Stressed CORT Levels As expected, ADX significantly reduced CORT levels (Fig. 8) compared to the SHAM condition in both males and females (ps<0.0005) and CORT replacement significantly increased plasma CORT levels compared to the ADX condition (males: ADX+CORT>ADX, p<0.0005; females: ADX+CORT>ADX, p<0.05). However, CORT replacement at the levels utilized did not fully return plasma CORT to SHAM levels (ADX+CORT<SHAM for both males and 47 females, ps<0.0005). As well, CORT replacement produced lower plasma CORT levels in both males and females compared to the same levels in Expt. 1. This may be due to differences in the length between ADX and CORT replacement in these two studies. No differences were seen among E, PF and C animals within the three surgical conditions (SHAM, ADX, ADX + CORT). Non-stressed ACTH Levels As.expected (Fig. 9), ADX resulted in significantly greater plasma ACTH levels compared to SHAM in both males and females (ps<0.0005). In addition, in the ADX condition E males had significantly higher ACTH levels compared to ADX PF and C males (ps<0.05). There were no differences among ADX E, PF and C females. CORT replacement significantly reduced plasma ACTH below ADX levels in both males and females (ps<0.0005). However, levels remained significantly greater than in the SHAM condition for both males and females (ps<0.0005). Plasma ACTH levels did not differ among E, PF and C animals within the SHAM and ADX+CORT conditions in either males or females. 48 Table 4. Adult Body Weight (g, mean ± SEM) one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females Males (g) Females (g) Surgery Prenatal Pre-Surgery 6 d Post- Pre-Surgery 6 d Post-Treatment Surgery Surgery E 482.2 ± 11.1 495.8 ± 13.6 265.3 ± 7.5 269.8 ± 8.1 SHAM PF 47.6.5 ± 17.4 492.8 ± 19.3 256.8 ± 7.2 262.9 ± 6.1 C 499.8 ± 14.7 510.1 ± 15.7 276.6 ± 6.8 283.8 ± 6.6 E 490.3 ± 18.4 475.3 ± 18.1 267.9 ± 5.1 264.2 ± 3.1 ADX PF 472.4 ± 12.3 454.7 ± 11.8 262.0 ± 6.3 258.3 ± 4.2 C 475.4 ± 8.1 453.1 ± 9.3 274.6 ± 4.5 272.8 ± 4.7 E 476.9 ± 12.9 469.8 ± 13.3 263.8 ± 6.8 260.6 ± 6.6 ADX+ PF 462.2 ± 10.3 460.1 ± 10.1 255.7 ±7.0 251.6 ± 7.2 CORT C 486.6 ± 9.9 480.9 ± 11.3 270.8 ± 10.4 274.6 ± 9.0 Males: significant day x surgery interaction (F(2,76)=43.3, p<0.0001). SHAM post-surgery>pre-surgery, p<0.0005; ADX post-surgery<pre-surgery, p<0.0005; ADX+CORT: post-surgery=pre-surgery. Females: significant main effect of treatment (F(2,78)=5.59, p<0.01), PF<C, p<0.005; significant day x surgery interaction (F(2,78)=7.63, p<0.001; SHAM post-surgery>pre-surgery, p<0.005; ADX post-surgery=pre-surgery; ADX+CORT post-surgery=pre-surgery (n=8-10 per group) 49 Table 5. Mean CORT intake (mg CORT/kg body weight, mean ± SEM) during 24 hr period prior to testing day. CORT replacement dose: 25 ug/ml in males, 75 ug/ml in females in drinking water containing 0.2% ethanol vehicle and 0.9% NaCl. 24 hr CORT Intake (mg/kg bw) Males Females E 3.8 ± 0.3 19.8 ± 1.9 PF 3.6 ± 0.3 19.1 ± 2.4 C 3.3 ± 0.2 17.3 ± 1.2 Prenatal treatments: E: ethanol, PF: pair-fed, C: control (n=9-10/group) 50 O ) CO E h-2 h SHAM SHAM ADX ADX+CORT ADX ADX+CORT P F YZZA c Figure 7. Mean thymus weight (g/kg body weight, mean ± SEM; n =8-10 per group) in E, PF and C (a) male and (b) female rats 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Main effect of surgery in both males (F(2,75)=4.78, p<0.05) and females (F(2,74)=19.6, p<0.0001); *ADX>SHAM, ADX+CORT (ps<0.05). 51 CD D C o O cd E CO as 700 600 500 400 300 200 100 .0 700 600 500 400 300 200 100 0 a _ mm?/, SHAM ADX SHAM ADX+CORT Figure 8. Plasma CORT levels in a) male and b) female E, PF and C rats 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent the mean ± SEM of 7-10 rats per group. Males: significant main effect of surgery (F(2,72)=41.5, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.0005; ADX+CORT<SHAM, p<0.0005. Females: significant main effect of surgery (F(2,73)=66.1, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.05; ADX+CORT<SHAM, p<0.0005. 52 1200 1000 h SHAM ADX ADX+CORT M P F YZZA c Figure 9. Plasma ACTH levels in E, PF and C a) male and b) female rats 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent the mean ± SEM of 7-10 rats per group. Males: significant surgery x prenatal treatment interaction (F(4,69)=3.20, p<0.05); *ADX E>ADX PF, ADX C, ps<0.05. ADX>SHAM, p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. Females: main effect of surgery (F(2,69)=63.7, p<0.0001); ADX>SHAM, P<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. 53 Experiment 3 Developmental Data The breeding for Expt. 3 was a replicate of Expt. 2 and all data, and F and p values are shown in the Appendix. Briefly, for ethanol-consuming dams ethanol intake was high throughout gestation, averaging 8.9 ± 1.1, 11.4 ± 0.8 and 11.0 ± 0.8 g/kg body weight for weeks 1, 2 and 3 of gestation, respectively. Maternal body weight (Appendix, Fig. 32) increased throughout gestation with C dams weighing significantly more than both E and PF dams on G21. Body weight normalized during lactation with no significant differences among E, PF and C dams. There were no significant differences among treatment groups for litter size or number of stillborn pups (Appendix, Table 9). However, gestation length was significantly greater in E (22.7 ± 0.2 d) compared to PF (22.1 ± 0.1 d) and C (22 ± 0.0 d) dams. Body weights of pups are summarized in the Appendix (complete data and F values in Appendix, Fig. 33). In brief, E, PF and C male and female pups did not differ in body weight on PN 1, 8 and 15. However, by PN22, C males and females weighed significantly more than their E and PF counterparts (ps<0.0005). Adult Body Weight By the time of testing (in adulthood) there were no significant differences in body weights among animals from the three prenatal treatment groups (Appendix, Table 10). Briefly, body weights did not change significantly in SHAM-operated animals from the day prior to surgery to 6 d after surgery. However, ADX as well as ADX+CORT males and females had decreased body weights 6 d post-surgery compared to pre-surgery body weights (ps<0.05). 54 CORT Intake Mean 24 hr CORT intake for rodents consuming CORT in their drinking water (ADX+CORT condition) is shown in Table 6. As in Expt. 2, CORT intake was significantly greater overall in females compared to males, with no differences among E, PF and C prenatal treatment groups. Stress CORT Levels As in Expt. 2, ADX significantly reduced CORT levels (Fig. 10) compared to the SHAM condition in both males and females (ps<0.0005) and CORT replacement significantly increased plasma CORT levels compared to the ADX condition (males: ADX+CORT>ADX, p<0.0005; females: ADX+CORT>ADX, p<0.005). However, CORT replacement at the levels utilized did not fully return plasma CORT to the levels of animals in the SHAM condition (ADX+CORT<SHAM for both males and females, ps<0.0005). Again, plasma CORT levels in ADX+CORT animals were lower than those obtained in Expt. 1. No differences were seen among E, PF and C animals in the three surgical conditions (SHAM, ADX, ADX + CORT). Stress A CTH L evels As expected (Fig. 11), ADX resulted in significantly greater plasma ACTH levels compared to SHAM in both males and females (ps<0.0005). CORT replacement significantly reduced plasma ACTH below ADX levels in both males and females (males: ADX+CORT<ADX, p<0.0005; females: ADX+CORT<ADX, p<0.05). However, levels remained significantly greater than in the SHAM condition for both males and females (ps<0.0005). Plasma ACTH levels did not differ among E, PF and C animals within any surgical condition for both males and females. 55 Table 6. Mean CORT intake (mg CORT/kg body weight, mean ± SEM) during 24 hr prior to testing after 7 days of CORT replacement at 25 ug/ml in males, 75 ug/ml in females. 24 hr CORT Intake (mg/kg bw) Males Females E 4.1 ± 0.4 15.7 ± 1.4 PF 3.2 ±0.2 17.2 ± 1.6 C 3.2 ±0.2 12.9 ±1.1 Prenatal treatments: E: ethanol, PF: pair-fed, C: control (n=9-10/group) 56 800 600 400 200 800 CD o o CD E K 6 0 0 400 200 SHAM SHAM ADX ADX+CORT WM 1^1 ADX ADX+CORT P F YZZA c Figure 10. Plasma CORT levels in E, PF and C a) male and b) female rats immediately after a 15 min restraint stress 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent the mean ± SEM of 8-10 rats per group. Males: significant main effect of surgery (F(2,71)=404.3, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.0005; ADX+CORT<SHAM, p<0.0005. Females: significant main effect of surgery (F(2,75)=1223.2, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.005; ADX+CORT<SHAM, p<0.0005. 57 CD X H O < CO E CO CO 1600 1400 a 1200 -1000 -800 -600 -400 200 0 • SHAM ADX ADX+CORT Figure 11. Plasma A C T H levels in E, PF and C a) male and b) female rats immediately after a 15 min restraint stress 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent the mean ± S E M of 8-10 rats per group. Males: significant main effect of surgery (F(2,72)=64.9, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. Females: main effect of surgery (F(2,73)=108.0, p<0.0001); A D X > S H A M , P<0.0005; ADX+CORT<ADX, p<0.05; ADX+CORT>SHAM, p<0.0005. 58 DISCUSSION The findings of the present studies demonstrate that, in contrast to intact animals in which basal plasma ACTH levels typically do not differ among E, PF and C groups (Taylor et al., 1983; Weinberg et al., 1996; Weinberg and Gallo, 1982), removal of the CORT feedback signal via ADX leads to significantly higher basal plasma ACTH levels in E males compared to their PF and C counterparts. Since HPA hyperresponsiveness is normally seen in E animals only in response to stress, hyperresponsiveness under basal conditions following ADX suggests an enhanced HPA drive in E males which is not related to any alterations in CORT feedback regulation. No differences were seen among ADX E, PF and C females. Following 15 min restraint stress, no differences were seen in ACTH responses among E, PF and C animals in the ADX condition. Although restraint is considered a mild stressor, the absence of CORT in these animals likely resulted in a maximal HPA response due to lack of feedback regulation, preventing any more subtle deficits in E animals from being observed. It is also possible that testing at the peak of the diurnal rhythm, when the set point for HPA regulation is at a higher level, may have masked any deficits. In addition, the maximal amount of ACTH available for release may be equivalent in E, PF and C animals. Therefore, a submaximal HPA response using a different stressor or different time points during and/or following recovery from stress may be required to observe stress-induced HPA hyperresponsiveness in E animals following ADX. One possible explanation for the increased ACTH following ADX is that E males may have enhanced stimulatory inputs to the PVN under basal conditions, resulting in increased CRH mRNA compared to controls. Another possibility is that ADX altered another, perhaps hormonal, system which then indirectly and differentially affected HPA function in E compared to PF and C males. For instance, ADX has been shown to alter the hypothalamic-pituitary-gonadal (HPG) axis (Viau et al., 1999), which has considerable crosstalk with the HPA axis. 59 Alternatively, E males may have increased anterior pituitary sensitivity to secretagogues such as CRH. Under such circumstances elevated POMC mRNA levels but normal CRH mRNA levels would be expected. Although E females showed no differential responses to ADX, it is possible that alterations occurred at the pituitary and/or hypothalamic level which were not reflected in ACTH levels. In males, CORT replacement following ADX resulted in plasma CORT levels approaching those of SHAM animals and well within the basal range. However, despite the higher CORT dose in females, CORT replacement did not achieve the high CORT levels observed in the SHAM condition at the diurnal peak. Yet basal ACTH levels were reduced below ADX-induced levels, indicating that the presence of a diurnal CORT signal was able to regulate HPA activity to some extent. In both males and females, replacement was only partially effective in normalizing ACTH levels under both basal and stress conditions, perhaps because testing occurred at the peak and not the trough of the diurnal rhythm, when HPA drive is higher. However, no differences were seen among E, PF and C males or females, demonstrating that E animals are as able as PF and C animals to use the exogenous CORT signal to regulate HPA activity. Al l animals exhibited thymic enlargement in response to ADX, and CORT replacement effectively normalized thymus weight. Since thymus weight is a sensitive target for circulating CORT levels, requiring a narrow window of CORT replacement for normalization (Akana et al., 1985), this suggests that CORT replacement was effective in normalizing some parameters. However, CORT replacement did not fully normalize the decreased body weight in response to ADX. As well, plasma CORT levels resulting from the same level of CORT replacement varied between experiments. With respect to the pilot study (Expt. 1), this may have been due to the longer timeframe between surgery and CORT replacement. Thus, particularly in females, CORT replacement may not have been adequate to normalize all parameters and may have prevented some more subtle alterations in E animals from being expressed. 60 As expected, body weight was lower in ethanol-consuming and pair-fed dams compared to controls during gestation but normalized during the lactation period. As well, despite a longer gestation length, both male and female E and PF offspring weighed less than their C counterparts during the preweaning period but did not differ in adulthood. Such postnatal growth deficiency is a common characteristic of prenatal ethanol exposure and normalization is typically seen in adulthood (Abel & Dintcheff, 1978; Gallo & Weinberg, 1986). Summary ADX resulted in higher basal ACTH levels in E compared to PF and C males at the diurnal peak. This suggests increased stimulatory inputs to the PVN and/or increased sensitivity of the anterior pituitary to secretagogues, such as CRH. However, no differences were seen in plasma ACTH levels among E, PF and C females as well as both males and females following 15 min restraint stress. CORT replacement via the drinking water reduced ADX-induced ACTH hypersecretion in all animals, with no differences among E, PF and C animals under either basal or stress conditions. Further investigation of key HPA components following ADX with or without CORT replacement is necessary to pinpoint the deficits occurring in E rats and the role of CORT in mediating these alterations. 61 CHAPTER IV: CHARACTERIZATION OF THE LIMBIC- HYPOTHALAMIC-PITUITARY-AD RENAL AXIS OF RATS PRENATALLY EXPOSED TO ETHANOL INTRODUCTION The present study was undertaken to determine whether the increased ACTH responses observed in E animals are mediated by alterations in central regulation of the HPA axis. Messenger RNA levels of key HPA components were measured under basal, non-stressed conditions in sham or ADX (with or without CORT replacement) animals in order to assess more fully steady-state HPA function following prenatal ethanol exposure. The experiments in Chapter III suggest that E animals (or at least E males) may exhibit enhanced stimulatory inputs under non-stressed conditions, which is unmasked by ADX. In order to pinpoint the level of enhanced activity within the HPA axis, in the present study we examined CRH and AVP mRNA as an indication of PVN activity, and CRH-R1 and POMC mRNA as a measure of anterior pituitary activity. The role of CORT in mediating any alterations in HPA activity was assessed via ADX with or without CORT replacement. We hypothesized that any CORT feedback alterations in E animals would present as elevated mRNA levels in the presence of CORT, i.e., in the sham and/or ADX+CORT condition, but would be absent in the ADX condition. As well, we hypothesized that the low level of CORT replacement may be less effective in normalizing mRNA levels in E animals, indicative of CORT feedback deficits. Conversely, enhanced mRNA levels present following ADX without CORT replacement would suggest increased stimulatory inputs independent of CORT feedback alterations. As a measure of hypothalamic activity, we measured CRH and AVP mRNA levels in the PVN, and as a measure of pituitary activity we measured CRH-R1 and POMC mRNA using in situ hybridization. As a measure of CORT feedback efficacy, MR and GR mRNA levels were determined in the hippocampus, a major neural target for CORT. Previous studies in our 62 laboratory found no differences in MR or GR receptor density among E, PF and C rats in the prefrontal cortex, hypothalamus, anterior pituitary or hippocampus (Kim et al., 1999b). As well, there were no differences in specific binding or binding affinity of either MRs or GRs in the hippocampus (Weinberg & Petersen, 1991). However, both of these previous studies utilized whole hippocampi. Since the hippocampus demonstrates wide regional differences in both MR and GR mRNA levels (Herman et al., 1989b), these techniques may not have been sensitive enough to detect alterations within regions. The present study therefore utilized in situ hybridization to measure MR and GR mRNA within hippocampal subfields as an indicator of feedback efficacy and sensitivity to CORT. We hypothesized that MR and/or GR mRNA would be decreased in E compared to control animals, indicative of CORT feedback deficits, and that receptor mRNA expression would be less sensitive to ADX with or without CORT replacement. METHODS Breeding and Animals The breeding was a replication of Chapter III Expt. 2 and 3 breedings with E, PF and C prenatal treatment groups (n=7-9 per group). Both male and female offspring were tested in all experiments and all animals were between 90 and 120 d of age at the time of testing. Testing This experiment was conducted as a replication of Chapter III Experiment 2, except that brains and anterior pituitaries were removed for in situ hybridization analysis. One day prior to surgery, rats were singly housed in clean cages and assigned to one of three surgical conditions: 1) SHAM, 2) ADX, or 3) ADX + CORT. Immediately following surgery, ADX+CORT rats were provided with CORT in the drinking water (dissolved in 0.2% ethanol and 0.9% NaCl) at a dose of 25 ug/ml in males and 75 ug/ml in females. ADX rats were provided with 0.9% NaCl/ 63 0.2% ethanol for drinking water. In order to control for litter effects, no more than one animal from any one litter was used per test condition. Animals were tested seven days following surgery at the diurnal peak (2000-2100hr, 2-3 hr after lights off). Rats were individually removed to an adjacent room under red light conditions and decapitated within seconds of touching the cage. Trunk blood was collected for measurement of resting, non-stressed levels of plasma CORT and ACTH. Brains and anterior pituitaries were rapidly removed and immediately frozen on dry ice, then stored at -70°C until sectioning on a cryostat. Brains were sectioned coronally into 15 um sections through the PVN and dorsal hippocampus. 7 slides were collected through the PVN with 4 sections per slide. Sections were rotated through the slides during collection, such that each slide had representative sections of anterior, middle and posterior portions of the PVN. Similarly for the hippocampus, 6 slides were collected with 4 sections per slide. At the time of sectioning, frozen pituitaries were embedded in Histoprep (Fisher Scientific, Edmonton, AB, Canada) which was immediately cooled to -18°C. Due to the relative homogeneity of the hybridization signals measured within the anterior pituitary, the orientation of the tissue was not accounted for and sections were 14 um in thickness. 5 slides were collected with 8 sequential sections per slide. All sections were mounted on poly-L-lysine/gelatin-coated slides at -18°C, briefly warmed, then stored at -70°C until analysis by in situ hybridization. For each probe, all slides were hybridized at the same time and one slide per animal was included. Probes and Labeling Antisense oligonucleotide probes for AVP (5'-GTA GAC CCG GGG CTT GGC AGA ATC CAC GGA CTC TTG TGT CCC AGC CAG-3'; complementary to the last 16 amino acids of the AVP glycopeptide region) (Young et al., 1986b; Ivell & Richter, 1984), CRH (5'- CAG TTT CCT GTT GCT GTG AGC TTG CTG AGC TAA CTG CTC TGC CCT GGC-3'; 64 complementary to amino acids 22-37 of rat CRH proper) (Young et al., 1986a; Jingami et al., 1985) and POMC (5'- CTT GCC CCA GCG GAA GTG CTC CAT -3' ; complementary to region coding for ACTH 4" 1 1) (Farquharson et al., 1990) were tail-labeled with 3 5S d-ATP (Amersham Biosciences, Piscataway, NJ, USA) using terminal deoxytransferase (New England Biolabs Inc., Beverly, MA, USA) for in situ hybridizations. For northern hybridization, the POMC oligonucleotide was tail-labeled with digoxigenin (DIG) using the DIG Oligonucleotide Tailing Kit (Roche Diagnostics, Laval, PQ, Canada). Oligonucleotides were synthesized at the Oligonucleotide Synthesis Laboratory, University of British Columbia, Vancouver, Canada. MR and GR cRNA probes (kindly provided by Dr. James P. Herman, University of Kentucky, Lexington, KY) were 550-bp and 456-bp EcoRI fragmented inserts, respectively. The CRH-1 receptor cRNA probe (kindly provided by Dr. Victor Viau, University of British Columbia, Vancouver, BC, Canada) was a 1.3Kb BamHI fragmented insert. Following transformation, plasmid DNA was isolated using the Promega Wizard Plus SV Miniprep System (Promega Corp., Madison, WI, USA), cut by restriction digest, and transcribed using the Promega Riboprobe System (Promega Corp., Madison, WI, USA) and 3 5S-UTP. All probes were purified using Quick Spin Columns (Roche Diagnostics, Laval, PQ, Canada). Northern Hybridization Specificity of the POMC oligonucleotide probe was confirmed by northern hybridization. Blots consisted of total RNA extracted from whole anterior pituitary glands in control male rats which had undergone sham surgery or ADX (with or without CORT replacement) 7 d prior to termination. Briefly, total RNA was isolated from anterior pituitaries using TRIzol reagent (Invitrogen Life Technologies, Burlington, ON, Canada). Total RNA was separated in a 1% agarose/10% formalin gel then transferred by northern blotting to a nylon membrane overnight in 20X SSC, air-dried, then UV cross-linked. The membrane was prehybridized at 43°C in DIG 65 Easy Hyb Solution (Roche Diagnostics, Laval, PQ, Canada) then hybridized in DIG Easy Hyb Solution containing 2 pmol/ml DIG-labeled POMC oligonucleotide at 43°C overnight. The membrane was then washed in 2X SSC/0.1% SDS at RT for 15 min (2 washes), followed by two washes in 0.1X SSC/0.1% SDS at 43°C for 15 min. DIG label was detected using the DIG Luminescent Detection Kit (Roche Diagnostics, Laval, PQ, Canada) and the membrane was then exposed to X-ray film for 30 min. In Situ Hybridization Oligonucleotides: CRH, AVP and POMC Slides were removed from the -70°C freezer and sections were thawed for 15 min, followed by fixation in formalin for 60 min. Sections were then washed twice for 10 min in 1XPBS, 10 min in 0.1M triethanolamine containing 0.25% acetic anhydride, 5 min in 2XSSC, then dehydrated through 50% (1 min), 75% (1 min), 95% (2 min) and 100% (1 min) ethanol, followed by 5 min in chloroform, 1 min 100% ethanol, then dried for 30 min. 25 ul hybridization buffer (10% dextran sulfate, 50% formamide, 3XSSC, IX Denhardt's solution, 100 ug/ml yeast tRNA, 25 mM sodium phosphate buffer (pH 7.4), 55 mM dithiothreitol, 30% deionized water), was applied to each section (probe activity for AVP: 5xl0 4 cpm/section; CRH: 105 cpm/section; POMC: 3.07xl05/slide) and covered with glass coverslips. Slides were incubated overnight at 37°C in humidified chambers. Sections were washed in 2XSSC for 60 min then coverslips were removed. Sections were then washed twice in 2XSSC for 20 min; 2XSSC/0.01M DTT, 40°C, 15 min; 1XSSC, 40°C, 15 min; lXSSC/50% formamide, 40°C, 30 min; 1XSSC, 10 min; 0.5XSSC, 10 min, rinsed in deionized water, then 70% ethanol for 5 min. Sections were dried overnight then exposed to X-ray film for 3 hr for the AVP probe, 8 days for the CRH probe, and 11.5 hr for POMC. In order to allow accurate identification and analysis of the parvocellular region of the 66 PVN, CRH and AVP slides were dipped in Kodak NTB2 autoradiography emulsion (Eastman Kodak Co., Rochester, NY), exposed for 1 day (AVP) or 18 days (CRH), then developed. Due to the homogeneity of the POMC mRNA signal in the anterior pituitary, coronal brain sections at the level of the hypothalamic arcuate nucleus, a brain region expressing significant levels of POMC, were included in in situ hybridizations as a positive control in order to validate specificity of the probe. cRNA Probes: MR, GR and CRH-R1 Slides were removed from the -70°C freezer and sections allowed to thaw for 15 min. Sections were fixed in formalin for 60 min followed by 2 washes in IX PBS for 10 min, then digested by proteinase K (100 ug/L, at 37°C for 9 min). Sections were rinsed in DEPC water for 5 min, 0.1M triethanolamine / 0.25% acetic anhydride for 10 min, 2 washes in 2XSSC for 5 min, dehydrated through 50% (1 min), 75% (1 min), 95% (2 min) and 100% (1 min) ethanol, chloroform for 5 min, and 1 min in 100% ethanol. Sections were then air-dried for 30 min. 30 ul of hybridization buffer (75% formamide, 3X SSC, IX Denhardt's solution, 200 ug/ml yeast tRNA, 50 mM sodium phosphate (pH 7.4), 10% dextran sulfate, 10 mM dithiothreitol) was applied to each section (probe activity: 6xl0 5 cpm/section for MR, 3.8xl05 cpm/section for GR, 1.8xl06 cpm/slide for CRH-R1), and slides were covered with a glass coverslip. Sections were incubated overnight at 55°C in a chamber saturated with 75% formamide. Slides were washed in 2X SSC for 1 hr then coverslips were removed, followed by a second wash in 2X SSC for 10 min. Slides were then transferred to a 25 ug/ml RNase A solution (37°C, 45 min) followed by washes in 2XSSC, 10 min; IX SSC, 10 min; 0.5XSSC, 55°C, 60 min; 0.5X SSC, 5 min; 0.2X SSC, 10 min. Sections were dehydrated through 50%, 70%, 95% and 100% ethanol, then air-dried for 30 min. For the CRH-R1 in situ hybridization, 0.1M DTT was added to all SSC washes 67 after the RNase wash in order to minimize background. Slides were exposed to X-ray film for 6 d for the GR probe, 3 d for the MR probe, and 7 d for the CRH-R1 probe. Due to the homogeneity of CRH-R1 mRNA in the anterior pituitary, coronal brain sections at the level of the central nucleus of the amygdala, a brain region expressing significant levels of CRH-R1, were included in in situ hybridizations as a positive control, to ensure specificity of the probe. Densitometric Analysis In situ hybridization data was analyzed using Scion Image 4.0.2 software (National Institutes of Health, USA). Images of X-ray autoradiographs (MR, GR, CRH-R1 and POMC) were digitized and grey level measurements were taken over regions of interest. Background measurements were taken from adjacent areas lacking signal. Al l grey level measurements were compared to C-l4 standards exposed with the slides, to ensure that grey levels were below saturation point. Background levels were subtracted from measurements to obtain corrected grey level measurements. In the dorsal hippocampus, a representative section, in which the two arms of the dentate gyrus were equal in length, was chosen from among the four sections on each slide. In some cases, two different sections were required in order to obtain measurements on both sides of the brain. Al l values represent the mean corrected grey values of the two sides. For the MR hybridization, measurements were taken from the subfields CAla, CAlb, CA2, CA3 and the dentate gyrus. This division was established based on the visible pattern of signal intensity. For the GR hybridization, measurements were taken from the subfields CA1, CA2, CA3 and dentate gyrus. Background signal for both MR and GR analysis was sampled from the molecular layer of the hippocampus. For the anterior pituitary, all 8 sections per slide were analyzed. Since the anterior pituitary contains a relatively homogenous signal for both POMC and CRH-R1 throughout the tissue, no 68 background measurements could be taken within the tissue. Therefore, background readings were taken over the slide where no tissue was present, in order to control for any differences within the film. This background measurement was subtracted from the grey level for each section and the mean corrected grey measurement was obtained from the average of all 8 sections on each slide. In some cases, some intermediate pituitary tissue remained adhered to the anterior pituitary. In the case of POMC, the signal within the intermediate pituitary is significantly greater than in the anterior pituitary and could easily be excluded from analysis. Since the intermediate pituitary also expresses CRH-R1, it is possible that in some sections this tissue was included in analysis. However, due to the minimal difference in mRNA signal between the intermediate and anterior lobes, this likely did not have a significant impact on measurement. For nuclear-emulsion dipped slides (CRH, AVP), images were digitized under dark field illumination (50X magnification) using a Leica optical system and Improvision OpenLab 3.0.8 software. For AVP, sections were matched for rostrocaudal level based on the signal pattern, and CRH measurement was carried out on the section adjacent to the chosen AVP section. Densitometric analysis was conducted over the dorsomedial parvocellular region of the PVN. For AVP, any ectopic magnocellular clusters which fell within this region were included in analysis. CRH densitometry was measured concurrently with AVP, such that the area of measurement used for AVP was then superimposed on the corresponding CRH image. Therefore, CRH and AVP measurements were done in equivalent areas on adjacent sections. Background signal was sampled over a region immediately lateral to each side of the PVN and this measurement was subtracted from the measurement taken of the ipsilateral PVN. The corrected grey levels from the two sides of the PVN were averaged to obtain a mean corrected grey level measurement. 69 Statistical Analyses Developmental data were analyzed by two-way ANOVAs for the factors of treatment condition (E, PF, C) and day, with days treated as repeated measures. Plasma hormone and in situ hybridization data were analyzed by two-way ANOVAs for factors of prenatal treatment group (E, PF and C) and surgical condition (SHAM, ADX, ADX+CORT). Further analysis was conducted by one-way ANOVA for prenatal treatment within each surgical condition. Due to the inherent sexual dimorphism of the HPA axis, data from males and females were analyzed separately. Since our a priori hypothesis was that hippocampal MR or GR mRNA levels would differ within specific regions among E, PF and C rats, statistical analysis was conducted within hippocampal subfield. All significant main and interaction effects were further analyzed by Newman-Keuls post-hoc tests. RESULTS Developmental Data This breeding was a replicate of the Expt. 2 and 3 breedings and therefore all data are shown in the Appendix. Briefly, for ethanol-consuming dams ethanol intake was high throughout gestation, averaging 12.8 ± 0.2, 13.4 ± 0.4 and 12.6 ± 0.3 g/kg body weight for weeks 1, 2 and 3 of gestation, respectively. Maternal body weight (Appendix, Fig. 34) increased throughout gestation with C dams weighing significantly more than both E and PF dams on G7, G14 and G21. In addition, E dams weighed significantly more than PF dams on G14. During lactation, C dams continued to weigh more than E and PF dams on PN1. However, by PN8 body weight no longer differed among E, PF and C dams. There were no significant differences among treatment groups for gestation length, litter size or number of stillborn pups (Appendix, Table 11). 70 Pup body weights increased significantly throughout the pre-weaning period (complete data and F values in Appendix, Fig. 35). In brief, there was a significant main effect of treatment in male pups, with C males weighing significantly more than E and PF males overall. Female pups did not differ in body weight on PN 1. However, on PN8, 15 and 22, C females weighed significantly more than both E and PF (ps<0.0005). As well, PF females weighed significantly more than E females on PN 22 (p<0.0005). Adult Body Weight By the time of testing (in adulthood, 90-120 d), there were no significant differences in body weights among males from the three prenatal treatment groups (see Table 12 in Appendix for complete data). However, E females weighed significantly less than PF females (p<0.05). Body weight did not change significantly in SHAM-operated animals from the day prior to until 6 d after surgery. However, ADX as well as ADX+CORT males and females had decreased body weight 6 d post-surgery compared to their pre-surgery body weight (ps<0.0005). CORT Intake Mean 24 hr CORT intake for rodents consuming CORT in their drinking water (ADX+CORT condition) is summarized in Table 7. As in Chapter III, Expts. 2 and 3, overall CORT intake was significantly greater in females compared to males, and there were no differences in intake among animals in E, PF and C prenatal treatment groups. Plasma CORT Levels As expected, and consistent with data in Chapter III, Expts. 2 and 3, ADX significantly reduced CORT levels (Fig. 12) compared to those in the SHAM condition in both males and females (ps<0.0005). CORT replacement significantly increased plasma CORT levels compared 71 to the ADX condition (males: ADX+CORT>ADX, p<0.0005; females: ADX+CORT>ADX, p<0.001). CORT replacement returned plasma CORT to levels in the SHAM condition in males but not in females (females: ADX+CORT<SHAM, p<0.001). No differences were seen among E, PF and C animals in the three surgical conditions (SHAM, ADX, ADX + CORT). Plasma A CTH L evels As expected, ADX resulted in significantly greater plasma ACTH levels compared to SHAM in both males and females (ps<0.0005) (Fig. 13). As in Chapter III Expt. 2, within the ADX condition, E males had significantly higher ACTH levels compared to ADX C males (p<0.05). In addition, in this replication, ADX PF males also showed higher ACTH levels compared to ADX C males (p<0.05). There were no differences among ADX E, PF and C females. CORT replacement significantly reduced plasma ACTH below ADX levels in both males and females (ps<0.0005). However, levels remained significantly greater than in the SHAM condition for both males and females (ps<0.0005). As before, plasma ACTH levels did not differ among E, PF and C animals within the SHAM and ADX+CORT conditions for both males and females. 72 Table 7. Mean CORT intake (mg CORT/kg body weight, mean ± SEM) during 24 hr prior to testing, after 7 days of CORT replacement at 25 (xg/ml in males, 75 ug/ml in females. 24 hr CORT Intake (mg/kg bw) Males Females E 3.6 ± 0.4 10.5 ± 2.1 PF 2.8 ± 0.5 11.7 ± 1.4 C 3.1 ±0.4 12.6 ± 1.3 Prenatal treatments: E: ethanol, PF: pair-fed, C: control (n=5-6/group) 73 800 600 400 200 CD I-cc o o 03 E CO 800 7T 600 400 200 a SHAM SHAM X ADX ADX+CORT ADX ADX+CORT P F YZZA c Figure 12. Plasma CORT levels in a) male and b) female E, PF and C rats 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Data represent the mean ± SEM of 5-6 rats per group. Males: significant main effect of surgery (F(2,41)=19.5, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.0005; ADX+CORT=SHAM. Females: significant main effect of surgery (F(2,43)=27.7, p<0.0001); ADX<SHAM, p<0.0005; ADX+CORT>ADX, p<0.001; ADX+CORT<SHAM, p<0.001. 74 CD CL I h-o *5 1600 CO § 1400 CO £ 1200 1000 800 600 400 200 0 SHAM ADX ADX+CORT SHAM ADX ADX+CORT PF ?m c Figure 13. Plasma ACTH levels in E, PF and C a) male and b) female rats 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent the mean of 5-6 rats per group. Males: significant main effect of treatment (F(2,41)=4.30, p<0.05), E,PF>C, ps<0.05; main effect of surgery (F(2,41)=58.0, p<0.0001), ADX>SHAM, p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005; ADX only (one-way ANOVA): main effect of treatment (F(2,14)=4.59, p<0.05. *ADX E,PF>ADX C, ps<0.05. Females: main effect of surgery (F(2,44)=82.3, p<0.0001); ADX>SHAM, P<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. 75 Confirmation of Probe Specificity The specificity of the POMC oligonucleotide was confirmed by northern analysis (Fig. 14). The oligonucleotide was specific for a single band consistent with the size of POMC mRNA (1.1 kb) as estimated from the location of 18S (1874 nucleotides) and 28S (4718 nucleotides) rRNA bands. In addition, signal intensity was greater in the ADX condition compared to SHAM, and the ADX+CORT condition had an intermediate signal intensity. This pattern is consistent with that expected for POMC mRNA. Signal intensity of the 18S and 28S rRNA bands were of similar intensity as visualized under UV light (image not shown), indicating equal loading in all lanes. Brain sections at the level of the arcuate nucleus included in the POMC in situ hybridization revealed a stronger signal in the arcuate nucleus compared to background, confirming specificity of the probe. Similarly for in situ hybridization with the CRH-R1 riboprobe, brain sections at the level of the central nucleus of the amygdala revealed a stronger signal within the amygdala compared to surrounding regions. Since remaining signals (CRH, AVP, MR, GR) localize to specific regions and produce a distinctive pattern, positive controls for these peptides were not included. CRH mRNA in the PVN Fig. 15 demonstrates the pattern of CRH mRNA signal in the PVN in SHAM, ADX and ADX+CORT surgical conditions, indicating the area of densitometric analysis. CRH mRNA levels (Fig. 16) did not differ among E, PF and C males in the SHAM condition. However, SHAM E females had significantly higher levels of CRH mRNA compared to SHAM C females. In both males and females, ADX significantly increased CRH mRNA compared to SHAM (ps<0.0005). In addition, ADX E males had significantly higher CRH levels compared to ADX C males (p<0.05). However, there were no differences among ADX E, PF and C females. CORT replacement resulted in a significant reduction in CRH mRNA compared to the ADX condition 76 in both males and females (ps<0.0005) but levels remained elevated compared to the SHAM condition (ps<0.0005). In addition, PF females in the ADX+CORT condition had significantly greater CRH levels compared to their C counterparts (p<0.05). AVP mRNA in the PVN Fig. 17 shows the pattern of AVP mRNA signal in the PVN in SHAM, ADX and ADX+CORT surgical conditions, indicating the area of densitometric analysis. Semi-quantitative analysis of AVP mRNA in the dorsomedial (dm) pPVN is shown in Fig. 18. Overall, ADX significantly increased AVP mRNA compared to that in the SHAM condition in both males and females (ps<0.0005). CORT replacement significantly reduced AVP mRNA compared to that in the ADX condition (males: ADX+CORT<ADX, p<0.0005; females: ADX+CORT<ADX, p<0.005); however, mRNA levels remained greater than that in the SHAM condition (ps<0.0005). In addition, E males had significantly lower AVP mRNA overall compared to both PF and C males (ps<0.05) whereas PF females had greater mRNA compared to E and C females. Within the SHAM condition, PF males had significantly greater AVP mRNA compared to E males (p<0.05), whereas PF females had higher levels than C females (p<0.05). There were no significant differences among ADX E, PF and C animals. Within the ADX+CORT condition, both PF males and females had significantly greater AVP mRNA levels compared to their E and C counterparts (ps<0.05). CRH-R1 mRNA in the Anterior Pituitary Fig. 19 shows the pattern of CRH-R1 mRNA signal in the anterior pituitary gland in SHAM, ADX and ADX+CORT surgical conditions. CRH-R1 mRNA in the anterior pituitary (Fig. 20) did not differ among E, PF and C males or females in the SHAM condition and ADX significantly decreased CRH-R1 mRNA levels compared to the SHAM condition in both males 77 and females (ps<0.005). In males, CORT replacement increased CRH-R1 mRNA above ADX levels (p<0.0005) but did not restore receptor mRNA to SHAM levels (ADX+CORT<SHAM, p<0.00005). In females, CORT replacement did not increase mRNA levels compared to the ADX condition and CRH-R1 mRNA levels remained significantly below SHAM levels (ADX+CORT<SHAM, p<0.05). There were no differences among E, PF and C females in any surgical condition. However, in males there was an overall treatment effect such that E males had significantly lower mRNA than C males (p<0.05). POMC mRNA in the Anterior Pituitary Fig. 21 shows the pattern of POMC mRNA signal in the anterior pituitary gland in SHAM, ADX and ADX+CORT surgical conditions. Semi-quantitative densitometric analysis of POMC mRNA (Fig. 22) revealed that overall, ADX significantly elevated POMC mRNA in both males and females (ps<0.0005) and CORT replacement decreased mRNA below ADX levels (ps<0.005) but not quite to sham levels (ADX+CORT>SHAM, ps<0.0005). In addition, there were no significant differences among E, PF and C animals within any surgical condition. 78 Figure 14. Northern analysis of anterior pituitary gland total R N A from (lane 1) S H A M , (lane 2) A D X and (lane 3) ADX+CORT (25 ug/ml CORT in drinking water) control males hybridized with digoxigenin-labeled POMC oligonucleotide. Location of 18S and 28S rRNA size markers, visualized under U V light on membrane, are indicated. 79 a b e d Figure 15. Location of pPVN within hypothalamus (a) and representative nuclear emulsion-dipped sections hybridized with 3 5S-dATP-labeled C R H oligonucleotide demonstrating C R H mRNA pattern in the right P V N of S H A M (b), A D X (c), and ADX+CORT (d) (25 ug/ml CORT in drinking water) control (C) male rats 7 d following surgery. Area outlined in (b) indicates the region of densitometric analysis. Note the increased signal in A D X compared to S H A M and the effect of CORT replacement in decreasing the signal toward S H A M levels. 80 C/5 c CD Q "co o 4—» o SHAM SHAM ADX ADX+CORT ADX ADX+CORT PF VZZA c Figure 16. C R H mRNA in the dorsomedial pPVN in E, PF and C a) male and b) female rats 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent mean ± S E M of densitometric analysis in emulsion-dipped slides hybridized with a 3 5S-dATP-labeled oligonucleotide for C R H (n=5-6 rats per group). Males: significant main effect of surgery (F(2,43)=44.6, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. Within A D X condition, significant main effect of treatment (F(2,15)=3.78, p<0.05) *E>C, p<0.05. Females: significant main effect of surgery (F(2,45)=50.7, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, P<0.0005; ADX+CORT>SHAM, p<0.0005; significant main effect of treatment (F(2,45)=6.22, p<0.005); E, PF>C, ps<0.01. Within S H A M condition, significant main effect of treatment (F(2,15)=4.10, p<0.05); #E>C, p<0.05. Within ADX+CORT condition (F(2,15)=4.80, p<0.05); +PF>C, p<0.05. 81 b e d Figure 17. Location of parvocellular PVN (pPVN) and magnocellular PVN (mPVN) within 35 hypothalamus (a) and representative nuclear emulsion-dipped sections hybridized with S-dATP-labeled AVP oligonucleotide demonstrating AVP mRNA pattern in the right PVN of SHAM (b), ADX (c), and ADX+CORT (d) (25 ug/ml CORT in drinking water) control (C) male rats 7 d following surgery. Area outlined in (b) indicates the region of densitometric analysis within the pPVN. Note the increased signal in ADX compared to SHAM and the effect of CORT replacement in decreasing the signal toward SHAM levels. 82 SHAM ADX ADX+CORT PF YZZZ C Figure 18. A V P mRNA in the dorsomedial pPVN in E, PF and C a) male and b) female rats 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 ug/ml in females). Values represent mean ± S E M of semi-quantitative densitometric analysis in emulsion-dipped slides hybridized with a 3 5S-dATP-labeled oligonucleotide for A V P (n=4-6 rats per group). Males: significant main effect of surgery (F(2,38)=41.2, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. Significant main effect of treatment (F(2,38)=4.36, p<0.05) E<C, p<0.05; E<PF, p<0.005. Within S H A M condition, significant main effect of treatment (F(2,12)=3.91, p<0.05) *PF>E, p<0.05. Within ADX+CORT condition, significant main effect of treatment (F(2,13)=9.50, p<0.005) tPF>E,C, ps<0.05. Females: significant main effect of surgery (F(2,41)=68.0, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, P<0.005; ADX+CORT>SHAM, p<0.0005; significant main effect of treatment (F(2,41)=5.82, p<0.01); PF>E,C, ps<0.05. Within S H A M condition, significant main effect of treatment (F(2,14)=4.13, p<0.05); APF>C, p<0.05. Within ADX+CORT condition (F(2,15)=4.58, p<0.05); #PF>E,C, ps<0.05 83 a b c Figure 19. Representative autoradiography of anterior pituitary sections hybridized with S-dUTP-labeled cRNA probe for CRH-R1 in a) SHAM, b) ADX and c) ADX+CORT (25 ug/ml CORT in drinking water) control (C) male rats 7 d following surgery. Note the decreased signal in ADX compared to SHAM and the effect of CORT replacement in increasing the signal toward SHAM levels. 84 Q SHAM ADX ADX+CORT O 60 Figure 20. CRH-R1 mRNA in the anterior pituitary in E, PF and C a) male and b) female rats 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 u.g/ml in females). Values represent mean ± S E M of semi-quantitative densitometry (7 d exposure) of slides hybridized with a 3 5S-dUTP-labeled riboprobe for CRH-R1 (n-4-6 rats per group). Males: significant main effect of surgery (F(2,42)=42.2, p<0.0001); A D X < S H A M , p<0.0005; ADX+CORT>ADX, p<0.0005; SHAM>ADX+CORT, p<0.0005. Significant main effect of treatment (F(2,42)=3.75, p<0.05) C>E, p<0.05. Females: significant main effect of surgery (F(2,41)=8.13, p<0.005); A D X < S H A M , p<0.005; ADX=ADX+CORT; ADX+CORT<SHAM, p<0.05. 85 a b c Figure 21. Representative autoradiography of anterior pituitary sections hybridized with S-dATP-labeled oligonucleotide probe for POMC in a) SHAM, b) ADX and c) ADX+CORT (25 ug/ml CORT in drinking water) control (C) male rats 7 d following surgery. Note the increased signal in ADX compared to SHAM and the effect of CORT replacement in decreasing the signal toward SHAM levels. 86 CO c CD Q "cc o '•4—» Q. o SHAM ADX ADX+CORT SHAM ADX ADX+CORT PF WZA c Figure 22. POMC mRNA in the anterior pituitary in E, PF and C a) male and b) female rats 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 |xg/ml in females). Values represent mean ± S E M of semi-quantitative densitometry of slides hybridized with a 3 5S-dATP-labeled oligonucleotide for POMC (n=5-6 rats per group). Males: main effect of surgery: (F(2,42)=117.5, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.0005. Females: main effect of surgery: (F(2,44)=82.7, p<0.0001) ADX>SHAM,p<0.0005; ADX+CORT<ADX, p<0.005; ADX+CORT>SHAM, p<0.0005. 87 Hippocampal MR mRNA Fig. 23 shows the MR mRNA pattern in the dorsal hippocampus of SHAM, ADX and ADX+CORT C males and indicates the subdivisions used for densitometric analysis, i.e., CAla, CAlb, CA2, CA3 and dentate gyrus. Semiquantitative analysis of MR mRNA in these different subfields revealed that mRNA levels vary across subfields, with CA2>CAla>dentate gyrus>CAlb>CA3 in both males (Fig. 24) and females (Fig. 25), and this pattern was similar to that observed by Herman et al. (1989b). ADX significantly increased MR mRNA in all subfields compared to the SHAM condition (ps<0.0005). Overall, CORT replacement reduced MR mRNA below ADX levels in all subfields in males (ps<0.005). Similarly in females, CORT replacement reduced MR mRNA compared to the ADX condition in the CAla, CAlb, CA2 and CA3 subfields (ps<0.005), but not in the dentate gyrus. In nearly all subfields, CORT replacement did not normalize MR mRNA to SHAM levels (ADX+CORT>SHAM, ps<0.005) with the exception of the CA3 subfield in males. Within CAla and the dentate gyrus, E males had significantly greater MR mRNA in the ADX+CORT condition compared to their PF and C counterparts (ps<0.05). In addition, in the CA3 subfield E females had overall greater MR mRNA compared to PF and C females (significant main effect of treatment, ps<0.01). Hippocampal GR mRNA Fig. 26 shows the GR mRNA pattern in the dorsal hippocampus of SHAM, ADX and ADX+CORT C males and indicates the subdivisions used for densitometric analysis, i.e., CA1, CA2, CA3 and dentate gyrus. Semiquantitative analysis of GR mRNA in these different subfields revealed an intensity pattern of CAl>dentate gyrus>CA3>CA2 in both males (Fig. 27) and females (Fig. 28), similar to the pattern of mRNA reported by Herman et al. (1989b). ADX significantly increased GR mRNA in all subfields compared to the SHAM condition 88 (ps<0.0005). CORT replacement reduced GR mRNA levels below A D X levels in all subfields (ps<0.0005). In males, CORT replacement effectively normalized GR mRNA to S H A M levels in the CA2 subfield and dentate gyrus, whereas mRNA levels remained greater than S H A M levels in the CA1 and CA3 subfields (ps<0.05). In addition, within the ADX+CORT condition MR mRNA was significantly greater in E males compared to C in the CA2 subfield (p<0.05) and greater than PF and C in the CA3 subfield (ps<0.05). In females, CORT replacement normalized GR mRNA to S H A M levels in CA2, CA3 and the dentate gyrus, whereas levels remained greater than S H A M in the CA1 subfield (p<0.05). There were no significant differences among E, PF and C females; however, within the CA3 subfield in males, there was a significant main effect of treatment, with E overall greater than PF and C (ps<0.05). a b c Figure 23. Representative autoradiography of dorsal hippocampus sections hybridized with S-dUTP-labeled cRNA probe for M R in a) S H A M , b) A D X and c) ADX+CORT (25 ug/ml CORT in drinking water) C male rats 7 d following surgery. Divisions utilized for image analysis are indicated in (a). Note the increased signal in A D X compared to S H A M and the effect of CORT replacement in decreasing the signal toward S H A M levels. 89 Figure 24. M R mRNA in the dorsal hippocampus in E, PF and C males measured in hippocampal subfields C A l a , C A l b , CA2, CA3 and dentate gyrus (DG) 7 d following sham surgery or A D X with or without CORT replacement (CORT in drinking water: 25 ug/ml in males, 75 u,g/ml in females). Values represent mean ± S E M of semi-quantitative densitometry of slides hybridized with a 3 5S-dUTP-labeled riboprobe for M R (n=4-6 rats per group). CAla: significant surgery x treatment interaction (F(4,40)=4.14, p<0.01); *CORT E>CORT PF, CORT C, ps<0.05; overall A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.005; ADX+CORT>SHAM, p<0.0005. CAlb: significant main effect of surgery (F(2,40)=30.3, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.005. CA2: significant main effect of surgery (F(2,40)=35.0, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.001; ADX+CORT>SHAM, p<0.0005. CA3: significant main effect of surgery (F(2,40)=14.4, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.001; ADX+CORT=SHAM. D G : significant surgery x treatment interaction (F(4,40)=4.54, p<0.005); # CORT E>CORT PF, CORT C, ps<0.005; overall A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.005. 90 Figure 25. M R mRNA in the dorsal hippocampus in E, PF and C females measured in hippocampal subfields C A l a , C A l b , CA2, CA3 and dentate gyrus (DG) 7 d following sham surgery or A D X with or without CORT replacement (CORT in drinking water: 25 fig/ml in males, 75 ug/ml in females). Values represent mean ± S E M of semi-quantitative densitometry of slides hybridized with a 3 5S-dUTP-labeled riboprobe for M R (n=5-6 rats per group). C A l a : significant main effect of surgery (F(2,44)=38.9, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.001; ADX+CORT>SHAM, p<0.0005. C A l b : significant main effect of surgery (F(2,44)=23.4, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.005; ADX+CORT>SHAM, p<0.001. CA2 : significant main effect of surgery (F(2,44)=19.7, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.01; ADX+CORT>SHAM, p<0.005. C A 3 : significant main effect of surgery (F(2,44)=27.7, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.005; significant main effect of treatment (F(2,44)=5.79, p<0.01) E>PF=C, ps<0.01. D G : significant main effect of surgery (F(2,44)=11.7, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT=ADX; ADX+CORT>SHAM, p<0.005. 91 a b c Figure 26. Representative autoradiography of dorsal hippocampus sections hybridized with J : )S-dUTP-labeled cRNA probe for GR in a) SHAM, b) ADX and c) ADX+CORT (25 ug/ml CORT in drinking water) C male rats 7 d following surgery. Divisions utilized for image analysis are indicated in (a). Note the increased signal in ADX compared to SHAM and the effect of CORT replacement in decreasing the signal toward SHAM levels. 92 CA1 CA2 SHAM CO c CD Q "cO o "•4—» Q. o ADX ADX+CORT CA3 100 80 60 40 20 0 I fcf/l SHAM ADX ADX+CORT SHAM ADX ADX+CORT PF SHAM ADX ADX+CORT YZZA c Figure 27. GR mRNA in the dorsal hippocampus in E, PF and C males measured in hippocampal subfields CA1, CA2, CA3 and dentate gyrus (DG) 7 d following sham surgery or ADX with or without CORT replacement (CORT concentration in drinking water: 25 u,g/ml in males, 75 ug/ml in females). Values represent mean ± SEM of semi-quantitative densitometry of slides hybridized with a 35S-dUTP-labeled riboprobe for GR (n=4-6 rats per group). CA1: significant main effect of surgery (F(2,38)=42.8, p<0.0001); ADX>SHAM, p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.05. CA2: significant main effect of surgery (F(2,38)=23.9, p<0.0001)ADX>SHAM,p<0.0005; ADX+CORT<ADX,p<0.0005; ADX+CORT=SHAM. Within ADX+CORT: significant main effect of treatment (F(2,12)=4.03, p<0.05) *E>C, p<0.05. CA3: significant main effect of surgery (F(2,38)=31.4, p<0.0001) ADX>SHAM, p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.01; significant main effect of treatment (F(2,38)=4.42, p<0.05) E>PF=C, ps<0.05. Within ADX+CORT, significant main effect of treatment (F(2,12)=6.56, p<0.05) #E>PF=C, ps<0.05. DG: significant main effect of surgery (F(2,38)=34.1, p<0.0001); ADX>SHAM, p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT=SHAM. 93 CA1 CA2 0) c CD Q "cc o Q. o SHAM ADX ADX+CORT CA3 100 80 60 40 20 0 100 80 60 40 20 0 100 SHAM ADX ADX+CORT SHAM ADX ADX+CORT SHAM 4 P F ADX ADX+CORT YZZA c Figure 28. GR mRNA in the dorsal hippocampus in E, PF and C females measured in hippocampal subfields CA1, CA2, CA3 and dentate gyrus (DG) 7 d following sham surgery or A D X with or without CORT replacement (CORT concentration in drinking water: 25 ug/ml in males, 75 u.g/ml in females). Values represent mean ± S E M of semi-quantitative densitometry of slides hybridized with a 3 5 S-dUTP-labeled riboprobe for GR (n=4-6 rats per group). CA1: significant main effect of surgery (F(2,44)=48.1, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT>SHAM, p<0.05. CA2: significant main effect of surgery (F(2,44)=33.3, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT=SHAM . CA3: significant main effect of surgery (F(2,44)=26.9, p<0.0001) A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT=SHAM. D G : significant main effect of surgery (F(2,44)=29.4, p<0.0001); A D X > S H A M , p<0.0005; ADX+CORT<ADX, p<0.0005; ADX+CORT=SHAM. 94 DISCUSSION Examination of mRNA levels by in situ hybridization revealed differential HPA activity in intact (sham-operated) E compared to PF and/or C animals as well as alterations in E animals which are revealed only with ADX or ADX with low-level CORT replacement. Alterations following ADX are indicative of CORT-independent mechanisms. As in Chapter III, the level of CORT intake in ADX+CORT animals was equivalent in E, PF and C animals and resulted in similar plasma CORT levels at the time of testing. Therefore, any differences in gene regulation in CORT-replaced animals are likely not a result of differences in available CORT but rather differential sensitivity to CORT. Paraventricular Nucleus: CRH and AVP mRNA E animals appear to have enhanced drive to the PVN, mediated mainly through CRH activation. That is, E males exhibit elevated CRH compared to C following ADX suggesting that a deficit in CORT feedback regulation probably does not play a role, and that the increased CRH may be due to increased stimulatory or decreased inhibitory neural inputs to the PVN. E females, on the other hand, exhibit elevated CRH mRNA in the SHAM condition, which leaves the possibility that CORT feedback deficits to the PVN or higher brain regions may play a role. Alternatively, enhanced CRH mRNA in E animals may be caused by a higher level of steady-state mRNA levels without differential neural inputs. Enhanced activity in the PVN appears specific to CRH for E animals, since AVP mRNA in E females did not differ from controls and was lower overall in E males. However, PF males and females show notable increases in AVP mRNA, in both the SHAM and CORT-replaced but not the ADX condition. This suggests a possible CORT-mediated deficit in the regulation of AVP in PF animals. Interestingly, since PF animals do occasionally demonstrate HPA hyperresponsiveness in response to certain stressors, these findings suggest that the mechanism 95 of hyperresponsiveness may differ in E and PF animals. Since AVP appears to be the major target of CORT negative feedback, glucocorticoid-mediated inhibitory inputs to the PVN may be deficient in PF animals (Kovacs et al., 2000). Thus, AVP-mediated alterations may play a larger role in PF animals whereas CRH may be more important in E animals with respect to HPA dysregulation. Evidence of enhanced stimulatory inputs to the HPA axis has been reported previously in E animals. In response to footshock and endotoxemia, c-fos, NGFI-B and CRH hnRNA responses are greater in the pPVN of E compared to control animals (Lee et al., 2000). As well, Rudeen and Weinberg (1993) found that E females had significantly lower hypothalamic norepinephrine (NE) content following restraint stress compared to PF and C females. If decreased NE levels are indicative of greater depletion and therefore higher turnover of NE, this would suggest that enhanced catecholaminergic activity may be involved in HPA hyperresponsiveness. Since elevated basal PVN activity has not been a consistent finding in the literature, HPA hyperresponsiveness may occur in response to PVN activation. Although some previous studies have shown no differences in basal CRH mRNA levels (Kim et al., 1999b; Lee et al., 2000), interestingly animals in these studies were examined at the circadian trough, whereas the present studies were conducted at the circadian peak. It is possible, therefore, that the elevated CRH mRNA seen in sham E females in the present study relate to the enhanced circadian inputs to the PVN at this time of day. Thus, HPA hyperactivity at the PVN may be due to enhanced drive in response to stimulation, be it stress, circadian drive or loss of CORT feedback inhibition with ADX. Anterior Pituitary: CRH-R1 and POMC At the level of the anterior pituitary, there appear to be some compensatory responses in E animals such that the enhanced activation seen at the PVN does not translate directly into 96 enhanced pituitary activity. E males, overall, had lower CRH-R1 mRNA compared to C males. As well, CORT replacement was less effective in increasing CRH-R1 mRNA towards sham levels in E males, suggesting decreased sensitivity to CORT regulation at the pituitary level. Alternately, this may reflect enhanced sensitivity to CRH-mediated receptor down-regulation. Either way, the decreased responsiveness of CRH receptors to changes in CRH levels may be protective in E animals, minimizing enhanced pituitary activity in the face of enhanced hypothalamic drive. Levels of POMC mRNA did not differ among E, PF and C animals, which strengthens the argument that decreased CRH-R1 levels in E males may compensate for enhanced PVN activity. However, as mentioned above, E males had higher ACTH levels following ADX compared to C, but this was not reflected in altered POMC mRNA levels. Thus it appears that release of ACTH is enhanced without increased POMC synthesis, suggesting alterations in translational and posttranslational processes and/or rate of constitutive ACTH release. Since the AVP receptor Vlb was not assessed, it is possible that the AVP pathway is enhanced with ADX in E males. In fact, there are suggestions that AVP, while having no significant effect on POMC mRNA synthesis, can mobilize a rapid turnover pool of ACTH (Antoni, 1993). Measurement of not only Vlb receptors but also ACTH content in the pituitary are necessary to answer these questions. In both E and PF males, CORT replacement was not effective in reducing ADX-induced increases in POMC mRNA as it was in C males. This may be due either to decreased sensitivity of POMC synthesis to CORT and/or to the lower mRNA levels of CRH-R1, at least in E males. MR and GR mRNA Hippocampal MR and GR mRNA levels in E males and females did not differ from their respective PF and C counterparts in either the SHAM or ADX surgical conditions. This would suggest that, at least at the receptor level, CORT feedback via the hippocampus is normal in E 97 animals. CORT replacement, however, was less effective in normalizing ADX-induced increases in MR and GR mRNA in E males in several hippocampal subfields. It is not known at present whether different hippocampal subfields have differing effects on HPA activity, therefore the significance of this regional variation is unclear. What it does suggest is that MR and GR mRNA may be less sensitive to CORT regulation in E compared to PF and C males. Although this effect may not be apparent in a non-stressed, intact animal, it is possible that such a decreased sensitivity may impair E animals when faced with excessive challenge such as chronic stress, when receptor down-regulation is vital to protect against the damaging effects of elevated CORT levels. Although previous studies found no differential down-regulation of MRs or GRs among E, PF and C animals in response to chronic intermittent stress, these studies utilized cytosolic binding assays of whole hippocampi (Kim et al., 1999b). Thus, it is possible that this method was not sensitive enough to detect regional differences within the hippocampus. Also, it is possible that the differences at the mRNA level do not translate into differences in binding. Unlike the present study, other studies found no significant effect of ADX on MR or GR mRNA levels, whereas binding was significantly increased (Chao et al., 1989). Although these studies utilized whole hippocampi and therefore may not have detected regional effects on mRNA, this demonstrates the importance of measuring both mRNA and protein levels or function. Therefore, determination of MR and GR binding following ADX with or without CORT replacement is necessary to confirm the changes seen at the mRNA level in E animals. CORT Replacement: Effects on mRNA Expression As in Chapter III, plasma CORT levels did not differ among E, PF and C animals in the SHAM condition. This is consistent with previous studies suggesting that basal, non-stressed CORT levels are typically normal in E animals (Taylor et al., 1983; Weinberg and Gallo, 1982; 98 Weinberg et al., 1996). The low CORT levels following ADX indicate that the surgery effectively removed circulating CORT to near zero, allowing the hypothalamic-pituitary system to be investigated without possible confounding effects of differential CORT responsiveness among E, PF and C animals. In CORT-replaced animals, both 24 hr CORT intake and plasma CORT levels were similar among E, PF and C animals. Since plasma CORT levels in these animals are a direct reflection of exogenous CORT intake and since CORT has a half-life of approximately 20 min in the circulation, these levels likely reflect CORT intake within the hour prior to termination. Expression of CORT-regulated genes as a result of this exogenous CORT intake therefore does not correlate directly with the CORT level at the time of termination, but rather the CORT environment present over a longer time frame. Akana et al. (1985) showed that CORT intake during the prior evening is important in regulating HPA responses the following morning, and that a phasic exogenous CORT signal, such as that produced with CORT in drinking water, is more effective than a tonic signal in regulating HPA activity. Therefore, baseline mRNA levels and thus HPA responsiveness may be related more to the daily pattern and overall level of CORT exposure rather than the CORT level at time of testing. Since daily CORT intake did not differ among E, PF and C animals, it is likely that any differences seen in mRNA expression in response to CORT replacement are not due to differences in CORT levels, but rather to different sensitivity to CORT. However, plasma CORT levels attained as a result of similar CORT concentrations in the drinking water were not consistent between studies, making comparisons between studies difficult and questioning the reproducibility of this method of CORT replacement. In addition, plasma CORT levels were much lower in CORT-replaced females compared to their sham counterparts. Thus, this may have limited our ability to interpret CORT-regulated mechanisms in E females. In the ADX+CORT condition, the plasma CORT levels attained should have resulted in >90% occupation of mineralocorticoid receptors (MRs) and likely minimal occupation of 99 glucocorticoid receptors (GRs) (Reul and de Kloet, 1985). This level of CORT replacement resulted in similar plasma CORT and ACTH levels. This suggests that MR function is as effective in E as in PF and C animals in regulating HPA activity, at least at the pituitary level. Since MRs are thought to maintain basal HPA activity by mediating tonic inhibition, this is consistent with the finding that E animals have normal basal HPA activity, showing hyperresponsiveness only in response to stressors when CORT levels are increased and therefore a larger percent of GRs are occupied. Any feedback deficits in E animals may therefore involve mainly the GRs which, with the level of CORT replacement used in the present study, were likely minimally occupied. This hypothesis is supported by our previous studies which found that E animals exhibit higher stress CORT and/or ACTH levels following suppression by DEX, a GR agonist (Osborn et al., 1996). Feedback deficits may also be specific to the intermediate time domain. Interpretations based on mRNA measurements come with the caveat that protein levels may not show the same pattern. Alternatively, measurement of protein levels may reveal additional or different results than those seen with mRNA as a result of differential translational and posttranslational processing. In control animals, CRH, GR and MR mRNA species have been shown to correlate well with protein levels (Herman & Morrison, 1996; Herman & Spencer, 1998; Herman et al., 1999). Whether this applies to E animals as well remains to be determined and will require measurement of both mRNA and proteins in the same tissue. Such studies are currently underway in our laboratory. Sexual Dimorphism In addition to the inherent sexual dimorphism of the HPA axis, prenatal ethanol exposure differentially affects the HPA axis in males and females. Previous studies indicate that both E males and females show alterations in HPA regulation, but the pattern of HPA 100 hyperresponsiveness may differ between the sexes depending on the time of day, type of stressor and hormonal endpoint measured (Weinberg et al., 1996), likely due to interactions between the HPA and HPG axes. In the present study, we found that both E males and females show enhanced drive to the PVN as well as decreased sensitivity to CORT replacement. However, the balance between these two may differ between the sexes resulting in different patterns of mRNA expression and HPA responsivity. Summary -This study confirms the presence of enhanced HPA drive in E animals at the level of the PVN and specifically, enhanced CRH mRNA levels. Although this appears to be a CORT-independent effect in E males, a deficit in CORT feedback regulation cannot be ruled out in females. There appears to be some compensatory mechanisms in place at the level of the anterior pituitary via CRH-R1 down-regulation. In addition, measures of CRH-R1, POMC, MR and GR mRNA suggest that E animals may have a decreased sensitivity to CORT feedback regulation of these genes in response to perturbation such as ADX. 101 CHAPTER V: EFFECTS OF MINERALOCORTICOID AND GLUCOCORTICOID RECEPTOR BLOCKADE ON HYPOTHALAMIC-PITUITARY-ADRENAL FUNCTION IN RATS PRENATALLY EXPOSED TO ETHANOL INTRODUCTION The data described in Chapter IV provide support for the suggestion that CORT feedback, or responsivity of various components of the HPA axis to CORT, may be deficient in E animals. However, mRNA alterations do not necessarily translate into parallel functional alterations. The present study investigated CORT action mediated by the two CORT receptor subtypes, MRs and GRs, with the use of selective receptor antagonists, in order to assess functional CORT feedback. Since plasma CORT and ACTH responses are elevated in E rats only under stress and not under basal conditions, this might suggest GR-mediated deficits and normal MR function. However, the previous studies (Chapter IV) demonstrate that HPA hyperactivity does occur in E rats, at least in E females, under basal conditions and that this hyperactivity is at the hypothalamic level. Whether this hyperactivity is mediated in part by feedback deficits or is mainly due to increased HPA drive is not clear at present. Evidence of Feedback Deficits in E Rats Tonic regulation of basal HPA activity appears to be adequate in E rats, at least at the pituitary level, since basal plasma CORT and ACTH levels are typically normal throughout the circadian cycle. As well, CORT replacement in ADX rats (to a level occupying mainly MRs) resulted in similar basal plasma ACTH levels as well as CRH and AVP mRNA in E compared to control rats (Chapters III & IV), indicating a similar ability to utilize an exogenous CORT signal. Previous studies in our laboratory suggest that fast feedback does not appear to be deficient in E rats. In these studies we utilized CORT injection to produce a rapid rise in plasma CORT that 102 could serve as a negative feedback signal for subsequent stressors. In response to both swim and ether stress, the exogenous CORT signal resulted in an equally blunted ACTH response in E compared to control rats at 5 or 30 min after the CORT injection, i.e., during the fast feedback time domain (Hofmann et al., 1999). This suggests that E rats do not exhibit impairments in fast feedback and therefore do not have alterations in non-genomic CORT-mediated mechanisms. Thus, any feedback alterations in E rats are likely mediated by MRs and/or GRs. In contrast, intermediate feedback, which acts through genomic mechanisms, may be altered in E rats. Our laboratory has previously shown that compared to control animals, both E males and females demonstrate significantly greater CORT and/or ACTH responses to an ether stress 3-6 hr (i.e. within the intermediate feedback time domain) following HPA suppression by the synthetic glucocorticoid dexamethasone (DEX) (Osborn et al., 1996). Since DEX does not readily cross the blood-brain barrier and therefore mainly activates GRs at the anterior pituitary, this suggests possible GR-mediated intermediate feedback deficits at the anterior pituitary. Alternatively, it is possible that E rats have greater pituitary HPA activity in response to a stressor which can overcome the suppressive effects of DEX. Effects of MR and GR Antagonists The respective roles of MRs and GRs in HPA regulation have been elucidated partly based on studies using selective antagonists. Subcutaneous injection of either the MR antagonist RU28318 or the GR antagonist RU38486 at the circadian trough has been shown to increase and prolong the CORT response to a novel environment stressor compared to that in vehicle-injected rats, without affecting basal CORT levels (Ratka et al., 1989). However, a similar study using a higher dose of RU28318 found elevated basal CORT levels at the circadian trough (Spencer et al., 1998). In contrast, at the circadian peak, basal ACTH levels were increased in response to icv injection of either RU38486 or RU28318; however, sc RU28318 had no effect (van Haarst et al., 103 1997; Spencer et al., 1998). Together, these studies suggest that MRs are important regulators of basal HPA tone at the circadian trough but that both MRs and GRs may be needed at the circadian peak. In addition, both MRs and GRs appear necessary to inhibit the stress-induced HPA response. Purpose Although binding studies suggest that E rats do not appear to have decreased MR or GR levels, it is possible that CORT feedback alterations occur in sites downstream of receptor expression and binding. The present study was conducted in order to investigate the function of CORT-mediated feedback through both MRs and GRs by examining HPA function in response to MR or GR blockade. We hypothesized that MR and/or GR blockade would differentially affect ACTH and/or CORT levels under basal, stress and/or recovery conditions in E compared to control animals, indicative of an altered balance in HPA drive and feedback. METHODS Surgery and Testing Testing was conducted on male and female E, PF and C rats at 90-120 d of age. In order to control for litter effects, no more than one animal from any one litter was used per test condition. All animals were implanted with indwelling jugular cannulae (see General Methods) which were externalized dorsally between the scapulae. Body weight was determined immediately after surgery while the animal was anaesthetized. Two days after surgery, rats were removed from the animal room to an adjacent testing room at the circadian trough (0600h). At this time a 75 cm piece of PE50 tubing filled with saline/1% heparin was attached to the externalized cannula by way of a blunted 22GIV2" needle attached to the end. The opposite end of this tubing was connected to an intermittent infusion plug which allowed for the attachment of a syringe for 104 blood collection. The patency of the cannula was checked and the rat was placed into an opaque cylindrical bucket (height: 43 cm; diameter: 29 cm) with bedding on the bottom. The lid contained a hole in the middle (5 cm diameter) through which the sampling cannula was threaded, and the cannula was hung over the side of the bucket, allowing for blood samples to be taken without disturbing the animal. Drug Injections Two hr after being placed into the bucket, rats were individually removed, injected sc with either a GR antagonist, an MR antagonist, or vehicle, then placed back into the bucket. The GR antagonist used in this study was mifepristone (RU38486) (Sigma Chemical Co., St. Louis, MO, USA), which is the most selective, potent GR antagonist currently available. This compound does, however, also act as an antagonist at the progesterone receptor (PR); therefore, it is possible that some of its effects may have been via actions at the PR. Although RU28318 has been the most commonly used MR antagonist in rodent studies, this compound was no longer available at the time of this study. Thus, we used the MR antagonist spironolactone (SPIRO; Sigma Chemical Co., St. Louis, MO, USA). RU38486 and SPIRO were suspended in absolute ethanol (5% final volume) and propylene glycol. SPIRO was prepared at a concentration of 10 mg/ml for males and 7 mg/ml for females. Mifepristone was prepared at a concentration of 10 mg/ml for males and 28 mg/ml for females. Animals were injected subcutaneously with either SPIRO (30 mg/kg bw for both males and females), mifepristone (RU38486; 30 or 120 mg/kg bw for males and females, respectively), or propylene glycol/ethanol vehicle at a volume of 5 and 3 ml/kg bw for males and females, respectively. These concentrations were chosen based on a pilot study which showed that these levels significantly increased ACTH levels either prior to, during or following a 1 hr restraint stress (data not shown). 105 Blood Sampling and Novel Confinement Blood samples (0.4 ml maximum) were taken beginning 3 hr after the rat was first placed into the bucket (1 hr post antagonist/vehicle injection). This procedure involved using a heparin/saline-filled syringe to draw blood up until it just reached the syringe tip, filling the cannula and the infusion plug with blood. An ice-cold EDTA/aprotinin-coated syringe was then used to withdraw 0.4 ml of blood. The blood volume was then replaced with an equal volume of saline (i.e. 0.4 ml), followed by infusion of 0.3 ml of heparin/saline to fill the cannula and maintain patency, with care taken to ensure that no heparin reached the animal. At the start of sampling, a 0.4 ml pre-stress blood sample was taken via the sampling cannula. The animal was then immediately removed to an adjacent room and restrained in a rectangular container with a wire mesh bottom (8x11x13 cm for males, 8x11x11 cm for females). The entire bucket with the animal inside was carried to the adjacent room in order to minimize disturbance to the other animals. Sampling cannulae were threaded through a hole in the middle of the lid (1 cm) which contained 4 additional breathing holes (0.5 cm each). This container was then replaced into the bucket, the sampling cannula threaded through the lid, and the bucket was placed back in the testing room for sampling during stress. Although the containers provided a tight fit for the animals, the square shape allowed the animals some ability to turn; therefore, this stressor was a mild restraint. Animals remained restrained for 60 min, during which time additional 0.4 ml blood samples were taken at 15, 30 and 60 min. Immediately after the 60 min blood sample, animals were again moved to an adjacent room in the bucket, removed from the smaller container, replaced in the bucket and returned to the testing room. Sixty min after the end of the stressor, a final "recovery" blood sample (0.4 ml) was taken. As noted, all blood samples were collected in ice-cold EDTA/aprotinin-coated syringes and immediately upon collection the sample was transferred into a polypropylene tube on ice. 106 Samples were then centrifuged and plasma was transferred to another polypropylene tube and stored at -70°C until assayed for CORT and ACTH levels, as described under General Methods. Statistical Analyses Since the present study examined whether E, PF and C animals exhibit differential MR or GR function, statistical analyses were conducted within prenatal treatment conditions. Thus, to assess the effects of MR or GR blockade on ACTH and CORT levels following stress, two-way ANOVAs were conducted for each of E, PF and C for the factors of drug condition (vehicle and SPIRO or vehicle and RU38486) and time (0, 15, 30, 60 and 120 min), with time treated as a repeated measure. Due to differing patterns of ACTH responses prior to, during and following the stressor, one-way ANOVAs were also conducted at each timepoint for the factor of drug condition (vehicle & SPIRO or vehicle & RU38486). Al l significant main and interaction effects were further analyzed by Newman-Keuls post-hoc analysis. RESULTS Developmental Data Ethanol intake of pregnant females was consistently high throughout gestation, averaging 11.5 ± 0.5, 12.3 ± 0.5 and 13.0 ± 0.3 g/kg body weight for weeks 1, 2 and 3 of gestation, respectively. Maternal body weights during gestation and lactation are shown in the Appendix, Fig. 37. In brief, body weight increased throughout gestation in all pregnant dams. E, PF and C dams did not differ in body weight at G l , whereas E and PF dams weighed significantly less than C dams on G7, 14 and 21 (ps<0.0005). As well, PF dams weighed significantly less than E dams on G14 and 21 (ps<0.005). During lactation (PN1 to 22), both E and PF dams continued to weigh less than C on PN1 (ps<0.0005) and PF weighed less than E dams (p<0.01). By PN8, E dams did not differ in body weight from C, although PF dams weighed significantly less than 107 both E and C dams (ps<0.05). By PN15, There were no differences in body weight among E, PF and C dams. There were no significant differences among treatment groups for gestation length, litter size or number of stillborn pups (Appendix, Table 13). Pup body weights increased significantly throughout the pre-weaning period (complete data and F values in Appendix, Fig. 38). In brief, there were no differences in body weight among E, PF and C male pups on PN1. However, C males weighed significantly more than E and PF males on PN8, 15 and 22 (ps<0.01). Similarly, E, PF and C female pups did not differ in body weights on PN1, whereas by PN8, PF weighed significantly less than C dams (p<0.005). On PN15 and 22, both E and PF females weighed significantly less than C females (ps<0.05). By adulthood (Table 8), there were no significant differences in body weight among E, PF and C males. However, PF females weighed significantly less than C females (p<0.05). Table 8. Body weight of adult E, PF and C males and females following jugular cannulation, 2 d prior to testing. Body Weight (g, mean ± SEM) Males Females E 518.6 ±8.1 297.7 ± 3.9 PF 506.0 ± 5.8 292.8 ± 3.6* C 519.9 ± 6.0 308.3 ± 4.0 For females only, significant main effect of treatment (F(2,95)=4.18, p<0.05) *PF<C, p<0.05 (n=29-35 per group). 108 Plasma ACTH Following MR or GR Blockade Animals in all conditions demonstrated increased ACTH levels in response to stress and decreased levels during recovery. In addition, C males exhibited a significantly greater ACTH response overall compared to both PF and C males. However, in males (Fig. 29) neither SPIRO nor RU38486 had any effect on the ACTH response of E, PF or C animals at any time during the stress response. In contrast, SPIRO significantly increased ACTH levels prior to stress (0 min) in E females (p<0.05) and during restraint (15 and 30 min) in C females (ps<0.05) compared to their vehicle-injected counterparts (Fig. 30). RU38486 significantly increased the ACTH response in E females prior to (0 min) and during restraint (15 min timepoint only) compared to vehicle-injected E females (ps<0.05), whereas C females had an increased ACTH response to RU38486 during restraint (30 min) and following recovery (120 min) (ps<0.05). Neither SPIRO nor RU38486 affected ACTH levels in PF females at any timepoint. Plasma CORT Following MR or GR Blockade Animals in all conditions demonstrated increased CORT levels in response to stress and decreased levels during recovery. SPIRO significantly increased the CORT response to restraint at 60 min in E and PF (ps<0.05) but not C males in comparison to their vehicle counterparts (Fig. 31). As well, compared to vehicle, RU38486 significantly increased CORT levels at 60 min in E males (p<0.05), but had no effect in PF and C males. In females, SPIRO significantly increased the CORT response to restraint in E at 15 min (p<0.05) but had no effect in PF or C in comparison to their vehicle counterparts (Fig. 32). As well, compared to vehicle, RU38486 significantly increased CORT levels in E females at 120 min (p<0.05) and in PF females at 15 and 120 min (ps<0.05) but had no effect in C females. 109 Ethanol 400 400 Pair-Fed 300 200 100 0 30 60 90 120 vehicle 400 Control 300 200 100 restraint 0 30 60 90 120 Time (min) -©— SPIRO 0 30 60 90 120 RU38486 Figure 29. Plasma ACTH in a) ethanol b) pair-fed and c) control males following sc injection of either propylene glycol vehicle, spironolactone (MR antagonist; 30 mg/kg bw) or RU38486 (GR antagonist; 30 mg/kg bw). Vehicle/antagonist was injected 60 min prior to the 0 min blood sample, and animals were then immediately restrained. Rats were removed from restraint immediately following the 60 min blood sample but remained in their holding bucket until the final "recovery" sample (n=8-12 per group). There were no significant effects of drug condition within E, PF or C prenatal groups. 110 ethanol 400 300 en a. O 200 < CO E _co 0. 100 o w A restraint _i i_ 0 30 60 90 120 400 pair-fed control 300 200 100 400 300 h 200 100 vehicle 0 30 60 90 120 Time (min) -e— SPIRO — A — RU38486 0 30 60 90 120 Figure 30. Plasma ACTH in a) ethanol b) pair-fed and c) control females following sc injection of either propylene glycol vehicle, spironolactone (MR antagonist; 30 mg/kg bw) or RU38486 (GR antagonist; 120 mg/kg bw). Vehicle/antagonist was injected 60 min prior to the 0 min blood sample, and animals were then immediately restrained. Rats were removed from restraint immediately following the 60 min blood sample but remained in their holding bucket until the final "recovery" sample (n=8-13 per group). Ethanol Vehicle & SPIRO: at 0 min, main effect of drug (F(l,18)=5.00, p<0.05) #SPIRO>vehicle. Vehicle & RU38486: at 0 min, main effect of drug (F(l,17)=6.37, p<0.05) *RU38486>vehicle; at 15 min, main effect of drug (F(l,20)=7.56, p<0.05) *RU38486>vehicle, p<0.05. Pair-fed: no significant effects of drug. Control: Vehicle & SPIRO: at 15 min, main effect of drug (F(l,16)=8.41, p<0.05) ASPIRO>vehicle, p<0.05; at 30 min, main effect of drug (F(l,18)=4.97, p<0.05) ASPIRO>vehicle, p<0.05. Vehicle & RU38486: at 30 min, main effect of drug (F(l,17)=4.70, p<0.05) +RU38486>vehicle, p<0.05; at 120 min, main effect of drug (F(l,17)=6.07, p<0.05) +RU38486>vehicle, p<0.05. Il l ethanol 1 0 0 0 8 0 0 o o co E a. 6 0 0 4 0 0 2 0 0 restraint" 0 3 0 6 0 9 0 120 1 0 0 0 8 0 0 6 0 0 4 0 0 2 0 0 pair-fed control restraint J I I I L_ vehicle 0 3 0 6 0 9 0 120 Time (min) - © — S P I R O 1 0 0 0 8 0 0 6 0 0 4 0 0 2 0 0 0 3 0 6 0 9 0 120 R U 3 8 4 8 6 Figure 31. Plasma CORT in a) ethanol b) pair-fed and c) control males following sc injection of either propylene glycol vehicle, spironolactone (MR antagonist; 30 mg/kg bw) or RU38486 (GR antagonist; 30 mg/kg bw). Vehicle/antagonist was injected 60 min prior to the 0 min blood sample, and animals were then immediately restrained. Rats were removed from restraint immediately following the 60 min blood sample but remained in their holding bucket until the final "recovery" sample (n=8-12 per group). Ethanol Vehicle & SPIRO: at 60 min, main effect of drug (F(l,22)=4.80, p<0.05) #SPIRO>vehicle. Vehicle & RU38486: at 60 min, main effect of drug (F(l,21)=11.19, p<0.005) *RU38486>vehicle. Pair-fed: at 60 min, main- effect of drug (F(l,17)=8.88, p<0.01) +SPIRO>vehicle. Control: no significant effects of drug condition. 112 ethanol 1000 800 DC o o CO E w _co Q. 600 400 200 0 30 60 90 120 1000 800 600 400 200 pair-fed control vehicle 0 30 60 90 120 Time (min) - e — SPIRO 1000 800 600 400 200 0 30 60 90 120 RU38486 Figure 32. Plasma CORT in a) ethanol b) pair-fed and c) control females following sc injection of either propylene glycol vehicle, spironolactone (MR antagonist; 30 mg/kg bw) or RU38486 (GR antagonist; 120 mg/kg bw). Vehicle/antagonist was injected 60 min prior to the 0 min blood sample, and animals were then immediately restrained. Rats were removed from restraint immediately following the 60 min blood sample but remained in their holding bucket.until the final "recovery" sample (n=8-13 per group). Ethanol Vehicle & SPIRO: at 15 min, main effect of drug (F(l,19)=4.60, p<0.05) *SPIRO>vehicle. Vehicle & RU38486: at 120 min, main effect of drug (F(l,18)=4.47, p<0.05) #RU38486>vehicle. Pair-fed: vehicle & RU38486: at 15 min, main effect of drug (F(l,20)=4.67, p<0.05) ARU38486>vehicle; at 120 min, main effect of drug (F(l,20)=7.41, p<0.05) +RU38486>vehicle. Control: no significant effect of drug condition. 113 DISCUSSION E females exhibited altered HPA responses to blockade of both MRs and GRs, as revealed using the selective antagonists SPIRO and RU38486. Blockade of either MRs or GRs significantly increased ACTH levels in E and C but not PF females compared to their vehicle-injected counterparts. However, the pattern of this responsivity differed between E and C females, suggesting a differential balance in HPA drive and feedback regulation. In E females, blockade of either MRs or GRs significantly increased ACTH levels prior to restraint. Although MR blockade has been shown to increase non-stressed HPA responses at the circadian trough (Spencer et al., 1998), indicative of this receptor's role in mediating tonic HPA inhibition, under the conditions and doses of the current study, SPIRO had no effect on pre-stress ACTH levels in PF and C females. This suggests that CORT feedback via MRs may play a greater role in E than in control females in maintaining HPA tone at the circadian trough. During stress, MR blockade significantly increased the ACTH response in C females and the CORT response in E and PF females compared to that in vehicle-injected animals. This is consistent with suggestions that CORT acts through MRs to facilitate GR-mediated effects (Ratka et al., 1989; Spencer et al., 1998; Pace et al., 2001), particularly with low intensity stressors such as the one used here. Since the pattern of ACTH and CORT responses to MR blockade differs between E and C females, this suggests a differential balance between HPA drive and feedback in response to the stressor. Previous studies have shown no effect of GR blockade on tonic HPA regulation (Ratka et al., 1989; Spencer et al., 1998), consistent with the hypothesized role of GR in mediating mainly stress-induced HPA feedback inhibition. However, E females demonstrated significantly elevated pre-stress ACTH levels in response to GR blockade. This may be indicative of enhanced HPA drive in E females which is unmasked following loss of CORT feedback (via GRs), much in the same way that ADX revealed elevated basal ACTH levels in E males (Chapters III and IV). Both E and C females exhibited elevated ACTH levels during restraint 114 following GR blockade; however, E females showed a peak response earlier than C. Again, this suggests a differential pattern of drive and feedback. Although GR blockade did not affect the ACTH stress response in PF females, the CORT response was elevated. Furthermore, GR blockade prolonged ACTH recovery following stress in C but not E and PF females. This is indicative of the role GR-mediated CORT feedback typically plays in recovery from stress. The fact that E and PF females showed no response to GR blockade during recovery may be indicative of a GR-mediated CORT feedback deficit. However, since E, PF and c females showed no differential ACTH response to the stressor and since E and PF females in all drug conditions showed recovery from the stressor, it may be that E and PF animals recovered more rapidly from the stressor than C. However, E and PF but not C females exhibited prolonged CORT elevations following stress, indicating a possible dissociation between ACTH and CORT responsiveness. Alternately, effects seen in CORT and not ACTH may be due to adrenal effects of the GR antagonist. Plasma ACTH and CORT levels measured immediately prior to restraint stress were not true basal levels, since these animals were in a novel environment and had received an injection one hour earlier. Although it is possible that E animals responded differentially to these conditions, this was controlled for by examining drug compared to vehicle effects within prenatal treatment condition. Nevertheless, the fact that GR blockade significantly increased pre-stress ACTH levels in E females could be a reflection of a greater stress response to these conditions compared to that in controls, thereby requiring GR-mediated feedback regulation. Another possibility that cannot be ruled out is that the MR or GR antagonists were differentially absorbed and/or metabolized in E, PF and C females. Thus, differences between prenatal treatment groups may have been due to different levels of the circulating antagonists. In males, blockade of neither MRs or GRs had any effect on plasma ACTH levels at any timepoint compared to vehicle-injected animals. Although the doses of antagonists utilized 115 effectively elevated ACTH responses in a pilot study under the same parameters, using control animals of similar age and weight, these doses were clearly inadequate in the present study for unknown reasons. In addition, E males did not exhibit hyperresponsiveness with the stressor and in fact exhibited a significantly lower HPA response. Although we have previously observed HPA hyperresponsiveness with restraint stress (Weinberg, 1988, 1992), we have not previously utilized the form of restraint used in this study. In addition, the numerous manipulations these animals had received, including cannula implantation, drug injection and being placed in a novel opaque bucket prior to the restraint, this may have differentially affected HPA activity prior to blood sampling in E animals. CORT responses during restraint stress were significantly elevated in E males with both MR and GR blockade and in PF males with MR blockade. However, the fact that C males showed no differences in ACTH or CORT responses with either antagonist confirms that the doses were ineffective. Due to the high sensitivity of the adrenal glands to ACTH, small differences in ACTH can translate into large CORT responses. Thus, although the antagonist effects on CORT observed in E and PF males may suggest that the antagonists were somewhat effective in these animals, this may again have been a local effect at the adrenal glands. Summary A deficit in CORT feedback does not appear to be the principal mediator of HPA hyperresponsiveness in E females. Although E females do show altered responsiveness to both MR and GR blockade, the results suggest that CORT feedback may sufficient to regulate HPA activity, at least under non-stressed and/or acute stress conditions, in order to counterbalance enhanced HPA drive. Since our previous studies have shown evidence of feedback deficits in the intermediate time domain (Osborn et al., 1996), feedback deficits may occur only under certain stress conditions and time frames. 116 The altered balance between HPA drive and CORT feedback in E females suggests a dysregulated HPA axis. Since a proper balance between MRs and GRs is vital in order to maintain neuronal excitability and appropriate stress responses (De Kloet, 1995), a disruption between CORT feedback and drive may have detrimental effects on the animal, particularly in response to excessive challenges. 117 CHAPTER VI: GENERAL DISCUSSION Summary and Discussion The major objective of this thesis was to elucidate the mechanisms underlying HPA hyperresponsiveness in E rats. We hypothesized that 1) HPA hyperresponsiveness in E rats is mediated, in part, by enhanced HPA drive and 2) HPA hyperresponsiveness in E rats is mediated, in part, by alterations in CORT feedback regulation. These hypotheses were based on previous studies which assessed HPA function in E rats mainly by measuring plasma CORT and ACTH responses to various stressor paradigms. The current studies have expanded these investigations using manipulations of the HPA axis, particularly via ADX and CORT receptor blockade, to examine the role of CORT feedback in mediating HPA hyperresponsiveness. In addition, the limbic-HPA axis was characterized through measurements of mRNA at the levels of the pituitary, hypothalamus and hippocampus. The results provide us with more detailed information on HPA regulation and the interplay between drive and feedback in E animals, providing a solid foundation for further mechanistic studies. In the first set of studies (Chapter III), ADX revealed that E males exhibit enhanced HPA activity, independent of CORT alterations. In addition, low level CORT replacement was equally effective in normalizing ACTH levels in E, PF and C animals under both basal conditions and following a 15 min restraint stress. These findings provide a clear indication that HPA hyperresponsiveness in E animals is not due solely to altered CORT feedback. As a result of our findings in ADX E males, we further examined HPA function following ADX with or without CORT replacement using in situ hybridization (Chapter IV). This study revealed the presence of enhanced HPA drive in E animals at the level of the PVN and specifically, enhanced CRH mRNA levels. However, in E males this only appeared following ADX, suggesting that CORT feedback may be sufficient to regulate CRH mRNA. This study 118 also suggests that there may be some compensatory mechanisms in place at the level of the anterior pituitary in E males via CRH-R1 down-regulation. As well, measures of MR and GR mRNA determined that E males may have a decreased sensitivity to CORT feedback regulation of these genes in response to perturbation such as ADX. The study detailed in Chapter V was undertaken to investigate CORT action mediated by the two CORT receptor subtypes, MRs and GRs, with the use of selective receptor antagonists, in order to assess functional CORT feedback. Both E males and females exhibited enhanced HPA responsiveness to both antagonists compared to controls, and the pattern of this responsiveness differed. That is, enhanced responsiveness to the antagonists was seen in E females both prior to and during restraint but only during restraint in E males. These studies suggest that CORT feedback, via both MRs and GRs, may be enhanced in E animals, particularly females, to counteract the enhanced drive in these animals, since loss of CORT feedback reveals enhanced HPA drive. As well, since the pattern of the HPA response to the antagonists differed between E animals and controls, the balance between HPA drive and feedback appears to be altered in E animals. Taken together, these studies indicate that prenatal ethanol exposure permanently reprograms the HPA axis of the rat such that HPA drive is enhanced into adulthood. Although some compensatory mechanisms appear to be in place to maintain normal basal ACTH and CORT levels, in response to challenge, such as stress, ADX (with or without low level CORT replacement), and CORT receptor blockade, these mechanisms break down and alterations are revealed. HPA dysregulation in E rats appears to be mainly suprapituitary, mediated by enhanced drive to the PVN as reflected by enhanced CRH mRNA levels. This may be due to a shift towards enhanced stimulatory inputs to the PVN, although enhanced CORT feedback regulation may partially counteract this effect. 119 Although previous studies suggest that HPA hyperresponsiveness in E animals may be mediated in part by CORT feedback deficits, the present studies suggest that CORT feedback is adequate and even enhanced in some instances. Following ADX, MR or GR blockade, E animals demonstrate enhanced HPA activity, suggesting that CORT feedback is restraining HPA hyperactivity in these animals. As there appears to be reduced sensitivity to CORT replacement (following ADX) in normalizing various measures, including MR and GR mRNA in E males, and since we have previously shown evidence of feedback deficits in the intermediate time domain (Osborn et al., 1996), a deficit in CORT feedback may be revealed under certain conditions but does not appear to be a major mediator of the HPA hyperresponsiveness to an acute stressor. Finally, our data indicate that there is a sexual dimorphism in the effects of prenatal ethanol exposure on HPA development. Numerous earlier studies have shown differential patterns of ACTH and CORT responses to a variety of stressors in E males and females. This not only reflects the inherent sexual dimorphism of the HPA axis, but differential sensitivity to the type of stressor, time of day and hormonal endpoint measured between E males and females. The current studies extend these findings to reveal a differential pattern of mRNA expression throughout the limbic-HPA axis, reflected ultimately in differential responses to various manipulations including ADX, CORT receptor blockade and stress. Previous studies have shown that HPA hyperresponsiveness appears to be more robust in E females compared to males, and similarly in the current studies, intact E females showed greater basal CRH mRNA as well as exaggerated ACTH responses following MR or GR blockade. Clinical Relevance HPA hyperresponsiveness is a robust phenomenon in E rats, persisting into adulthood. In humans with FAS, the occurrence of HPA hyperresponsiveness has not been adequately studied. 120 Only one human study has been conducted in which 13 month old infants with a history of prenatal alcohol exposure were found to exhibit higher Cortisol responses to a stressor compared to non-exposed infants (Jacobson et al., 1999). Since most of the effects of ethanol seen in rats also appear in humans with FAS, and since HPA hyperresponsiveness also occurs in an adult primate model of FAS (Schneider et al., 2002), it is likely that this phenomenon also persists into adulthood in humans with FAS. Behavioural abnormalities and learning deficits are among the most detrimental consequences of FAS, and it has been suggested that HPA hyperresponsiveness may underlie some of these problems (Kim et al., 1996). E rats exhibit increased stress-induced ethanol consumption and an inability to adapt to a stressful swimming paradigm (Nelson et al., 1983; Taylor et al., 1983), suggesting that stress may exacerbate problems. Although the relationship between HPA activity and other abnormalities seen in individuals with FAS has not been examined, behavioural and/or pharmacological interventions which may minimize HPA dysregulation may decrease further harm. HPA dysregulation is not merely a symptom of prenatal ethanol exposure, but may increase susceptibility to further disease. Inappropriate HPA responses may lead to allostatic load, resulting in maladaptation and impaired responses to further challenges (Pacak & Palkovits, 2001). Since children with FAS have alcoholic mothers who often live in poverty, they are typically born into a difficult environment and many end up in foster homes. Learning deficits and behavioural problems, in addition to problems at home, can make school a difficult and stressful situation for children with FAS. These problems typically continue into adult life and may lead to secondary disabilities such as difficulties finding a job, trouble with the law and alcohol and drug problems (Streissguth et al., 1996). Overall, a life with FAS is likely a stressful life and, compounded with impaired allostatic responses, may result in increased susceptibility to allostatic load in these individuals. Impaired recovery from a stressor, resulting in prolonged 121 glucocorticoid, elevations, can have numerous detrimental consequences, including immunosuppression, alteration of other neuroendocrine systems, affective disorders and neuronal degeneration (Habib et al., 2001; Magarinos et al., 1996; De Kloet et al., 1998). HPA dysregulation in individuals with FAS may therefore make them even more prone to disease. Future Directions Further investigation of HPA hyperresponsiveness in E rats is necessary to clearly elucidate the underlying mechanisms. Measurement of proteins is required to confirm that the alterations in mRNA expression we observed translate into similar alterations in protein levels. Examining the HPA axis in fetal and pre-weaning rats at the level of mRNA expression will allow us to determine how prenatal ethanol exposure affects development. As well, the temporal pattern of any changes will indicate whether some alterations seen in adulthood are in fact compensatory mechanisms which develop after an initial ethanol-induced effect. Although HPA dysregulation typically occurs with old age in normal animals, it is possible that E animals exhibit further dysregulation and that age-related changes may occur at an earlier age. Thus, assessment of HPA function should be extended to aged E rats. Since HPA hyperresponsiveness is evident under challenge, characterization of HPA activity in response to acute compared to chronic stressors is needed. Measurement of mRNA, heteronuclear RNA and protein levels will provide a temporal pattern of how E animals respond to a stressor at all levels of the HPA axis. Investigation into the function of both MRs and GRs at the cellular level is also warranted. This can be assessed through mobility shift assays, to determine if CORT effectively binds to MRs and GRs, and immunohistochemistry, to determine if MR and GR effectively translocate to the cell nucleus upon CORT binding. Since sex differences are a consistent finding in our studies, the interaction between the HPA and HPG axes in E animals needs further investigation. Gonadectomy with or without sex 122 hormone replacement will allow us to determine whether the sexual dimorphism of prenatal ethanol effects on HPA function is mediated by HPG hormones. Conclusions In conclusion, the experiments outlined in this thesis demonstrate that prenatal ethanol exposure has long-term effects at all levels of the HPA axis. HPA hyperresponsiveness in E rats appears to be mediated primarily by enhanced hypothalamic drive; however, this enhanced drive is not solely in response to a stressor but is present under non-stressed conditions. 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Brain Res 387: 231-241. 139 APPENDIX CHAPTER III Experiment 3 500 400 D3 300 CD CO 200 100 a * J / J. / Zk Zr i i 1 7 14 21 Gestational Day + 500 400 300 200 100 8 15 Postnatal Day —e— P F Figure 33. Maternal body weights during a) gestation (Gl to 21) and b) lactation (PN1 to 22). Values represent the mean ± SEM of 12-14 rats per group. Gestation: *C>E,PF, ps<0.05. Significant treatment x day interaction (F(6,108)=10.9, p<0.0001). Lactation: Significant treatment x day interaction (F(6,108)=5.48,p<0.0001) 140 Table 9. Gestation length, # live pups and # dead pups (mean ± SEM) of E, PF and C dams Gestation Length (d) # Live Pups # Dead Pups E 22.7 ± 0.1* 14.1 ± 1.0 0.29 ± 0.29 PF 22.1 ± 0.1 13.7 ± 0.8 0.15 ±0.10 C 22.0 ± 0.0 15.0 ± 0.6 0.07 ± 0.07 *E>PF,C, ps<0.0005. Gestation length: significant main effect of treatment (F(2,38)=21.5, p<0.0001) (n=ll-14/group) 141 T3 O CO 8 15 Postnatal Day Figure 34. Mean pup body weights (g) in a) males and b) females during postnatal days 1 to 22. Values represent the mean ± SEM of 11-14 rats per group. Males: significant treatment x day interaction (F(6,105)=6.88, p<0.0001); *C>E,PF, ps<0.0005. Females: significant treatment x day interaction (F(6,108)=5.84, p<0.0001); #C>E,PF, ps<0.0005. 142 Table 10. Adult Body Weight (g, mean ± SEM) one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females Males (g) Females (g) Surgery Prenatal Treatment Pre-Surgery 6 d Post-Surgery Pre-Surgery 6 d Post-Surgery E 520.4 ± 16.6 519.0 ± 19.6 266.0 ± 7.0 265.8 ± 5.9 SHAM PF 519.2 ± 12.9 521.1 ± 16.2 276.0 ± 9.4 277.9 ± 7.5 C 546.3 ± 20.9 550.7 ± 23.7 292.4 ± 7.9 294.2 ± 5.8 E 519.3 ± 14.3 496.9±11.2 276.9 ± 9.5 264.4 ± 8.4 ADX PF 516.8 ± 14.1 477.7 ± 12.9 281.8 ± 12.8 264.1 ± 9.6 C 542.3 ± 19.4 507.0 ± 20.7 277.2 ± 8.4 267.4 ± 7.3 E 508.9 ± 10.7 489.4 ± 10.2 272.1 ± 6.0 265.7 ± 5.3 ADX+ PF 546.1 ± 14.9 528.4 ± 13.1 275.6 ± 6.0 272.6 ± 6.6 CORT C 537.1 ± 15.5 514.1 ± 11.7 283.7 ± 14.5 273.8 ± 13.0 Males: significant day x surgery interaction (F(2,76)=28.31, p<0.0001). SHAM: post-surgery=pre-surgery; ADX: post-surgery<pre-surgery, p<0.0005; ADX+CORT: post-surgery<pre-surgery, p<0.0005. Females: significant day x surgery interaction (F(2,76)=7.04, p<0.005; SHAM: post-surgery=pre-surgery; ADX: post-surgery<pre-surgery, p<0.0005; ADX+CORT post-surgery<pre-surgery, p<0.05 (n=8-10 per group). 143 Chapter IV 500 1 7 14 21 Gestational Day 500 400 300 200 100 h 8 15 Postnatal Day 22 -e— P F Figure 35. Maternal body weights during a) gestation (GI to 21) and b) lactation (PN1 to 22). Values represent the mean ± SEM of .7-9 rats per group. Gestation: Significant treatment x day interaction (F(6,57)=22.6, p<0.0001); *C>E,PF, ps<0.0005; AE>PF, p<0.05.. Lactation: Significant treatment x day interaction (F(6,54)=2.82, p<0.05); #C>E,PF,ps<0.005. 144 Table 11. Gestation length, # live pups and # dead pups (mean ± SEM) of E, PF and C dams Gestation Length (d) # Live Pups # Dead Pups E 22.4 ± 0.2 14.9 ±0.5 0.1 ±0.1 PF 22.0 ± 0.0 14.6 ± 0.4 0.1 ±0.1 C 22.1 ± 0.1 12.5 ± 1.1 0.3 ± 0.3 No significant differences among E , PF and C (n=7 :9 per group). 145 Postnatal Day Postnatal Day — i — E — e— P F — A — c Figure 36. Mean pup body weights (g) in a) males and b) females during postnatal days 1 to 22. Males: main effect of treatment (F(2,20)=102, p<0001); C>E,PF, ps<0.005; main effect of day (F(3,60)=2979, p<0.0001. Females: significant treatment x day interaction (F(6,60)=6.00, p<0.0001); *C>E,PF, ps<0.05; #PF>E, p<0.05 (n=6-9 per group). 146 Table 12. Adult Body Weight (g, mean ± SEM) one day prior to and 6 d following sham surgery or ADX with or without CORT replacement in E, PF and C males and females Males (g) Females (g) Surgery Prenatal Treatment Pre-Surgery 6 d Post-Surgery Pre-Surgery 6 d Post-Surgery E 537.6 ± 21.2 546.2 ± 23.9 282.5 ± 8.3 279.2 ± 9.1 SHAM PF 489.8 ± 9.7 486.3 ± 7.9 313.7 ± 15.4 312.0 ± 14.7 C 535.8 ± 11.5 536.8 ± 12.2 280.5 ± 7.1 279.2 ±6.7 E 559.3 ± 20.1 ,519.3 ±19.8 277.7 ± 3.3 269.3 ± 4.2 ADX PF 540.3 ± 25.7 504.7 ± 24.3 292.5 ± 6.0 282.5 ± 5.9 C 541.7 ± 8.9 514.3 ± 9.9 287.3 ± 8.7 275.5 ± 11.2 E 518.8 ± 22.6 496.4 ± 22.6 279.2 ± 9.9 273.3 ± 11.0 ADX+ PF 498.5 ± 16.8 473.2 ± 13.7 294.3 ± 12.2 287.5 ± 13.4 CORT C 503.5 ± 16.0 488.3 ± 16.5 305.2 ± 6.3 297.0 ± 5.5 Males: significant day x surgery interaction (F(2,43)=14.4, p<0.0001). SHAM post-surgery=pre-surgery; ADX post-surgery<pre-surgery, p<0.0005; ADX+CORT: post-surgery<pre-surgery. Females: significant main effect of treatment (F(2,45)=3.48, p<0.05), PF>E, p<0.05; significant day x surgery interaction (F(2,45)=7.24, p<0.005; SHAM post-surgery=pre-surgery; ADX post-surgery<pre-surgery, p<0.0005; ADX+CORT post-surgery <pre-surgery, p<0.0005 (n=5-6 per group). 147 Chapter V 500 400 g> 300 CD T3 O CO 200 100 1 7 14 21 Gestational Day 500 400 300 200 100 h 8 15 Postnatal Day PF Figure 37., Maternal body weights during a) gestation (Gl to 21) and b) lactation (PN1 to 22). Values represent the mean ± SEM of 10-12 rats per group. Gestation: Significant treatment x day interaction (F(6,87)=17.8, p<0.0001); *C>E,PF, ps<0.0005, AE>PF, ps<0.005. Lactation: Significant treatment x day interaction (F(6,84)=6.48,p<0.0001). #C>E, p<0.0005; +E>PF, p<0.05; @C>PF, p<0.005. 148 Table 13. Gestation length, # live pups and # dead pups (mean ± SEM) of E, PF and C dams Gestation Length (d) # Live Pups # Dead Pups E 22.2 ± 0.1 15.1 ±0.6 0.3 ±0.1 PF 22.0 ± 0.1 16.3 ± 0.4 0.2 ±0.1 C 22.0 ± 0.0 15.8 ±0.3 0.1 ±0.1 No statistical differences among E, PF and C (n = 11-12 per group) 149 'CD O CO 8 15 Postnatal Day —e— Figure 38. Mean pup body weights (g) in a) males and b) females during postnatal days 1 to 22. Values represent the mean ± SEM of 11-12 rats per group. Males: significant treatment x day interaction (F(6,81)=5.27, p<0.0005); *C>E,PF, ps<0.01. Females: significant treatment x day interaction (F(6,84)=4.39, p<0.001); #C>E, ps<0.05; AC>PF, p<0.005. 150 

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