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Effects of prenatal ethanol exposure and postnatal handling on cognition/behavior and hypothalamic-pituitary-adrenal… Gabriel, Kara Irene 2000

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EFFECTS OF PRENATAL ETHANOL EXPOSURE AND POSTNATAL HANDLING ON COGNITION/BEHAVIOR AND HYPOTHALAMIC-PITUITARY-ADRENAL STRESS RESPONSIVENESS IN S P R A G U E - D A W L E Y RATS.  by K a r a Irene Gabriel B . A . (Psychology, English), The University of Wisconsin-Madison, 1991 M . A . (Psychology), The University of British Columbia, 1993  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Psychology) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A June 2000 ©Kara Irene Gabriel  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  and study.  publication of this  his  or  her  Department of  r ~ s - y o ^ A <a(  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ~5~^/~N«-  "7 « "2—  that the  representatives.  may be It  thesis for financial gain shall not  permission.  requirements  I further agree  thesis for scholarly purposes by  the  is  an  advanced  Library shall make it  that permission  for extensive  granted by the  head  understood be  for  that  allowed without  of  my  copying  or  my written  ABSTRACT  The maternal consumption of alcohol during pregnancy produces a wide range of abnormalities in the offspring. The major objectives of this thesis were to investigate (1) the correspondence between prenatal ethanol-induced alterations in behavior and in hypothalamicpituitary-adrenal (HPA) activity, (2) the ability of early postnatal handling as an environmental manipulation to attenuate at least some of the adverse behavioral and physiological consequences of  prenatal  ethanol  exposure,  and  (3)  possible  mechanisms  mediating  the  HP A  hyperresponsiveness to stressors observed in animals prenatally exposed to ethanol and the possible influence of postnatal handling on those mechanisms.  Sprague-Dawley rats from  prenatal ethanol (E), pair-fed (PF) and ad libitum fed control (C) treatment groups were tested as young adults (-35-120 d of age) or mid-aged adults (13-14 months of age). The first study investigated the effects of prenatal ethanol exposure (E) and postnatal handling (H) on behavior and H P A activity during a conditioned taste aversion (CTA) task. We tested the hypothesis that E animals which underwent postnatal handling would show improved conditioned aversion learning and reduced H P A activity compared to E animals that did not experience handling (nonhandled, N H ) . We found that prenatal ethanol exposure and postnatal handling independently resulted in an increased rate of consumption of a saccharin solution over five preexposure days. In addition, we found that handling differentially affected posttoxicosis consumption of the conditioned solution as well as corticosterone (CORT) levels in E, PF and C animals. H-E animals showed increased posttoxicosis intake compared to H-PF and H-C animals during reexposure under non-deprived conditions; CORT levels were lower in PF and C than E males compared to their N H counterparts during reexposure under food- and waterdeprived conditions. Thus, E animals were less able to utilize environmental cues in the present ii  study, displaying a more rapid reduction in neophobia compared to PF and C animals and, following postnatal handling, showing a decreased acquisition of conditioned aversion and an increased CORT response during reexposure to the conditioned solution. The second study examined spatial learning and memory in young adult (2 months) and mid-aged (13-14 months of age) H and N H E and control animals utilizing a Morris water maze.  We investigated the hypothesis that postnatal handling would improve spatial  navigation in E animals compared to E animals that did not experience handling and/or attenuate differences among E and control animals, and that this effect might be age-dependent. We also examined whether performance deficits in mid-aged animals would correspond to increases in CORT levels on the last day of testing.  Young E males showed impairments in spatial  navigation compared to young PF and C animals, taking longer to find the hidden platform over the course of testing and displaying an alteration in search pattern when the platform was removed. Interestingly, differences in young E, PF and C animals in escape latency and in distance traveled prior to finding the platform were apparent in H but not in N H animals. There were no differences in performance on the Morris water maze in mid-aged E , PF and C animals, but CORT levels were elevated in mid-aged E compared to C animals, supporting previous data indicating that E animals demonstrate H P A hyperresponsiveness to stressors. Lastly, although mid-aged animals had longer escape latencies and an altered search pattern compared to young animals, handling did not appear to attenuate impairments associated with aging. The third study investigated the hypothesis that postnatal handling might attenuate stress-induced A C T H and/or C O R T differences among E , PF and C animals. Furthermore, the ability pf postnatal handling to modulate H P A feedback deficits in E animals was examined during exposure  to a restraint  stressor  following  dexamethasone ( D E X )  administration. Both E females and males showed increased A C T H and C O R T compared to PF iii  and/or C animals following saline administration. Administration of D E X to block H P A activity significantly suppressed both plasma A C T H and CORT in all animals. However, E females exhibited increased and/or prolonged elevations in A C T H and CORT compared to PF and C animals following D E X blockade. These data suggest that the insult of prenatal ethanol exposure affects both male and female offspring, but that there may be a sex-specific difference in sensitivity of the mechanism(s) underlying H P A hyperresponsiveness.  Postnatal handling  reduced A C T H levels in both females and males following saline administration. Following D E X administration, H males had lower C O R T than N H males. Postnatal handling resulted in a more rapid decrease in stress-associated C O R T elevations in C females, and attenuated differences in CORT between PF and C females. However, postnatal handling did not attenuate deficits in negative feedback inhibition in E females; E females in both the H and N H treatments showed elevated C O R T compared to their C counterparts, and H-E females also showed elevated CORT compared to H-PF females. Thus, postnatal handling did not attenuate the typical H P A hyperresponsiveness to stressors observed in E animals (saline condition), nor did it eliminate deficits in H P A feedback inhibition in E females (DEX condition). The  fourth  study  examined  whether  the  mechanisms  resulting  in H P A  hyperresponsiveness in E animals are similar to those underlying the effects of postnatal handling.  Differences in H P A responsiveness between H and N H animals appear to be  dependent upon basal CORT activity and not stress-induced elevations in CORT. Therefore, we tested the hypothesis that differences in H P A activity among E and control animals would not occur following adrenalectomy (ADX) but could be reestablished following replacement with basal levels of exogenous CORT. In the absence of a CORT feedback signal or in the presence of a constant, basal C O R T feedback signal, E , PF and C animals did not significantly differ in their abilities to regulate A C T H secretion, indicating that during the trough of the circadian iv  rhythm, E , PF and C animals are equally capable of regulating H P A activity utilizing either CORT-independent feedback or feedback mediated through basal CORT activity.  Thus, the  effects of prenatal ethanol exposure on H P A function do not appear to be dependent upon the feedback signal provided by basal CORT levels. In conclusion, handling did not attenuate the effects of prenatal ethanol exposure examined in the present experiments. This may be because the effects of postnatal handling and prenatal ethanol exposure on H P A function are mediated through different mechanisms as well as the finding that handling is, at least partly, mediated through mother-pup interactions. Therefore, postnatal handling might exert differential effects on litters in which pup behavior has already been altered by prenatal treatments, underscoring the enduring effects of prenatal ethanol exposure.  TABLE OF CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  vi  LIST OF FIGURES  ix  LIST OF T A B L E S  xi  LIST OF A B B R E V I A T I O N S  xiii  ACKNOWLEDGEMENTS  xv  C H A P T E R I: G E N E R A L INTRODUCTION  1  A . Fetal Alcohol Syndrome  1  1. Diagnosis  1  2. Clinical Features  2  3. Incidence and Epidemiology  4  4. Alcohol Teratogenesis  4  B. Animal Models of FAS  7  1. Ethanol Administration  7  2. Effects of Prenatal Ethanol Exposure in Rodent Models  9  C. The Hypothalamic-Pituitary-Adrenal (HPA) Axis 1. Negative Feedback Regulation of the H P A Axis D. The H P A Axis and Prenatal Ethanol Exposure  11 14 15  1. Mechanisms of Prenatal Ethanol Effects on H P A Hyperresponsiveness  17  2. Effects of H P A Hyperresponsiveness on Behavior  19  E. Postnatal Handling  20  1. Effects of Postnatal Handling  21  2. Mechanisms of Postnatal Handling on Alterations in H P A  22  Responsiveness F. Prenatal Ethanol Exposure and Postnatal Manipulations  23  G. Thesis Objectives  24  vi  C H A P T E R II: G E N E R A L M E T H O D S  27  A . Breeding  27  B. Diets and Feeding  27  C. Postnatal Treatment  29  D. Experimental Subjects  29  E. Blood Sampling  30  1. Decapitation  30  2. Cardiac Puncture  30  3. Jugular Cannulation  31  F. Radioimmunoassays  32  1. Plasma C O R T Levels  32  2. Plasma A C T H Levels  32  C H A P T E R III: EFFECTS OF P R E N A T A L E T H A N O L E X P O S U R E A N D P O S T N A T A L H A N D L I N G O N CONDITIONED T A S T E A V E R S I O N Introduction  34  Methods  37  Results  39  Discussion  42  C H A P T E R IV: T H E EFFECTS OF P R E N A T A L E T H A N O L E X P O S U R E A N D A G I N G O N MORRIS W A T E R M A Z E P E R F O R M A N C E A R E N O T ATTENUATED B Y POSTNATAL HANDLING Introduction  63  Methods  65  Results  68  Discussion  70  C H A P T E R V : P O S T N A T A L H A N D L I N G DOES NOT A T T E N U A T E H P A HYPERRESPONSIVENESS F O L L O W I N G P R E N A T A L E T H A N O L E X P O S U R E Introduction  87  Methods  90 vii  Results Discussion  C H A P T E R VI: VARIATIONS IN CORTICOSTERONE F E E D B A C K DO NOT R E V E A L DIFFERENCES IN H P A A C T I V I T Y F O L L O W I N G PRENATAL ETHANOL EXPOSURE Introduction Methods Results Discussion  C H A P T E R VIII: G E N E R A L DISCUSSION A . General Discussion of Studies B. Clinical Implications C. Future Directions D. Conclusions  REFERENCES  viii  LIST OF FIGURES Figure 1. Average body weight (g, Mean ± SEM) of E, PF and C females and males  56  in H and N H treatments.  Figure 2. Pretoxicosis saccharin intake (g/kg bwt) for E, PF and C females and  58  males in H and N H treatments.  Figure 3. Posttoxicosis saccharin intake (g/kg bwt/avg pretoxicosis intake,  60  Mean ± SEM) for E, PF and C females and males in H and N H treatments during non-deprived and deprived reexposure.  Figure 4. Plasma C O R T levels (pg/dl, Mean ± SEM) for E, PF and C females and  62  males in H and N H treatments during non-deprived and deprived reexposure.  Figure 5. Escape latency (avg sec/day, Mean + SEM) for E, PF and C males in H  80  and N H treatments at 2 (Young) and 13-14 (Mid-Aged) months of age.  Figure 6. Distance traveled (m/day, Mean + SEM) for E, PF and C males in H  82  and N H treatments at 2 (Young) and 13-14 (Mid-Aged) months of age.  Figure 7. Annulus crossings on day 4 for E, PF and C males in H and N H  84  treatments at 2 (Young) and 13-14 (Mid-Aged) months of age.  Figure 8 . Plasma C O R T levels (pg/dl, Mean + SEM) for E, PF and C males in  86  H and N H treatments at 13-14 (Mid-Aged) months of age.  Figure 9. Plasma A C T H levels (pg/ml, Mean ± SEM) for E, PF and C females and males in H and N H treatments three hours following S A L administration.  ix  106  Figure 10. Plasma CORT levels (ug/dl, Mean ± SEM) for E, PF and C females  108  and males in H and N H treatments three hours following S A L administration.  Figure 11. Plasma A C T H levels (pg/ml, Mean ± SEM) for E, PF and C females  110  and males in H and N H treatments three hours following D E X administration.  Figure 12. Plasma CORT levels (ug/dl, Mean ± SEM) for E, PF and C females  112  and males in H and N H treatments three hours following D E X administration.  Figure 13. Plasma A C T H levels (pg/ml, Mean ± SEM) for E, PF and C females in  139  A D X , P E L L E T and S H A M conditions.  Figure 14. Plasma A C T H levels (pg/ml, Mean ± SEM) for E, PF and C males in  141  A D X , P E L L E T and S H A M conditions.  Figure 15. Plasma C O R T levels (ug/dl, Mean ± SEM) for E, PF and C females and males in the S H A M condition.  x  143  LIST OF TABLES  Table 1. Nutritional Content of E and PF Liquid Diets from Bio-Serv, Inc.  33  Table 2. Maternal Body Weights (g, Mean ± SEM) of E, PF and C Dams during  52  Gestation (G), and of E, PF and C Dams in H and N H Treatments during Lactation (PN1, PN22) for Experiments of Chapters III-V.  Table 3. Gestational Length (d, Mean ± SEM) and Number of Live-Born Pups  53  (Mean ± SEM) of E, PF and C Dams for Experiments of Chapters III-V.  Table 4. Postnatal (PN1, PN22) Pup Body Weights (g, Mean ± SEM) of E, PF and  54  C Female and Male Pups in H and N H Treatments for Experiments of Chapters III-V.  Table 5. On Trial with Raised Platform, Escape Latency (sec, Mean ± SEM) for  78  E, PF and C Males in H and N H Treatments Tested at 2 Months (Young) or 13-14 Months (Mid-Aged) of Age for Experiment of Chapter IV.  Table 6. Adult Body Weight (g, Mean ± SEM) of E, PF and C Females and Males  104  in H and N H Treatments Tested at 90-120 Days of Age for Experiment of Chapter V .  Table 7. Maternal Body Weight (g, Mean ± SEM) of E, PF and C Dams during  132  Gestation (G) and Lactation (PN) for Experiment of Chapter VI.  Table 8. Gestational Length (d, Mean ± SEM) of E, PF and C Dams for  133  Experiment of Chapter VI.  Table 9. Postnatal (PN) Pup Body Weights (g, Mean + SEM) of E, PF and C Female and Male Pups for Experiment of Chapter VI.  xi  134  Table 10. Adult Body Weights (g, Mean ± SEM) of E, PF and C Female and  135  Male Animals at 120-150 Days of Age for Experiment of Chapter V I .  Table 11. Basal CORT Levels (ug/dl, Mean ± SEM) for E, PF and C Female and  136  Male Animals in P E L L E T and S H A M Conditions for Experiment of Chapter VI.  Table 12. Basal and Stress-Associated C O R T Levels (ug/dl, Mean ± SEM) for E, PF and C Female and Male Animals in A D X and P E L L E T Conditions for Experiment of Chapter VI.  xii  137  LIST OF ABBREVIATIONS  ACTH - adrenocorticotropin ADX - adrenalectomy ANOV A - analysis of variance B AL - blood alcohol level bw - body weight C - prenatal control diet group cm - centimeters CNS - central nervous system CORT - corticosterone CRH - corticotrophin-releasing hormone d-day DEX - dexamethasone-21 -phosphate E - prenatal ethanol diet group FAE - fetal alcohol effects FAS - fetal alcohol syndrome g - grams or gravitational force G - gestation day GABA - y-aminobutyric acid GR - glucocorticoid receptor H - postnatal handling/handled HPA - hypothalamic-pituitary-adrenal hr - hour IQ - intelligent quotient xiii  m - meters min - minutes mm- millimeters M R - mineralocorticoid receptor mRNA - messenger ribonucleic acid N H - nonhandled PF - prenatal pair-fed diet group P N - postnatal day P O M C - propiomelanocortin P V N - paraventricular nucleus RIA - radioimmunoassay SC - subcutaneously sec - second S E M - standard error of the mean V P - vasopressin  ug - micrograms  xiv  ACKNOWLEDGMENTS  I would like to thank my supervisor, Dr. Joanne Weinberg, for her patience, humor, guidance, and support.  She has created a working environment in which I was able to find  myself (although not everybody appreciated what I found), in addition to planning and conducting my research projects. Special thanks to all the individuals who were members of the Weinberg lab during my graduate career: Heather Andrews, Lawrence Chan, Glenn Edin, Linda Ellis, Maria Glavas, Pamela Giberson, Candace Hofmann, Kathy Keiver, Leslie Kerr, C. Kwon Kim, Jill Osborn, Wendy Simms, Karen Strange, Catherine Y u and Wayne Y u . The late-night brainstorming sessions (and philosophical debates), long road trips to conferences, and talking over coffee made me feel as close to normal as a graduate student can. M y years in the Weinberg lab passed more quickly because of these individuals and I managed to learn a lot about life from them, and those are the greatest compliments I can bestow. Thank you all for accepting my continual questioning and my sharing as well as my obscure references to "The Simpsons", "Star-Trek", movies and countless research papers. I also thank the members of my supervisory committee, Dr. Liisa Galea and Dr. Wolfgang Linden, as well as the professors whose have kindly donated their time to assist me in completing the requirements for my degree, Dr. James Enns and Dr. Charlotte Johnston. Special thanks to those faculty whose words of advice have helped smooth my way, Dr. Roderick Wong and Dr. Lynn Alden. Finally, I thank my husband, Cameron, and my family and friends for their support throughout my degree.  There were times my sanity may have been in question, but their  companionship kept me from permanently crossing the line. Without them, I never would have  xv  experienced the beauty of British Columbia and Washington, and my life would have been the poorer for it. Thank you one and all.  xvi  C H A P T E R I: G E N E R A L I N T R O D U C T I O N  The maternal consumption of alcohol during pregnancy has serious effects on the developing fetus.  Alcohol affects virtually every system of the organism, producing a wide  range of abnormalities that impact the individual's health and ability to function in society. The major objectives of this thesis were to investigate (1) the correspondence between prenatal ethanol-induced alterations in behavior and hypothalamic-pituitary-adrenal (HPA) activity, (2) the ability of an environmental manipulation, early postnatal handling, to attenuate at least some of the adverse behavioral and physiological consequences of prenatal ethanol exposure, and (3) the possible influence of postnatal handling on mechanisms mediating the H P A hyperresponsiveness to stressors observed in E animals.  A . Fetal Alcohol Syndrome In humans, maternal alcohol consumption during pregnancy leads to the development of a number of physical, behavioral and physiological problems in the offspring.  The  association between maternal alcohol consumption and developmental anomalies in the offspring has been formally recognized since the independent descriptions of Fetal Alcohol Syndrome (FAS) by Lemoine, Harousseau and Borteyru (1968) in France and Jones, Smith, Ulleland and Streissguth (1973) in Seattle.  The term F A S is used to describe the set of  characteristic abnormalities identified in children whose mothers chronically consumed high doses of alcohol during pregnancy (Jones & Smith, 1973). 1. Diagnosis F A S is characterized by pre- and postnatal growth retardation, craniofacial defects and central nervous system dysfunctions. Intrauterine growth retardation appears to be directly proportional to the degree of maternal alcohol intake (Streissguth et al., 1980) and children with 1  FAS are usually below the third percentile in weight, height and head circumference (Streissguth et al., 1985).  Craniofacial dysmorphology used in the diagnosis of F A S includes such  characteristics as midface hypoplasia, thin upper lip, epicanthal folds, low set ears, short palpebral fissures and a long flat philtrum. Lastly, central nervous system (CNS) anomalies such as decreased cranial size, structural brain abnormalities and neurological and behavioral problems are used in the diagnosis of F A S . FAS represents an extreme end of the spectrum of mental and physical deficits resulting from prenatal exposure to high doses of alcohol. The term fetal alcohol effects (FAE) has been used to describe cases in which children prenatally exposed to alcohol do not meet all three of the diagnostic criteria of F A S . More recently, three terms have been developed to characterize children who were affected by alcohol prenatally but do not meet all criteria for FAS (Stratton et al., 1996). The term "partial F A S " refers to children with confirmed prenatal alcohol exposure and characteristic facial anomalies but without full FAS. The term "alcohol-related birth defects" is used for children who have primarily physical malformations or physiological abnormalities. Lastly, the term "alcohol-related neurodevelopmental disorder" describes children with either physical CNS abnormalities (e.g., smaller head size or structural brain abnormalities) or with behavioral and/or cognitive abnormalities, such as deficits in memory, language skills or learning abilities. Children who do not meet all the criteria for F A S may exhibit more subtle forms of dysfunction than children with F A S , but their symptoms can be just as disabling (Sokol & Clarren, 1989). 2. Clinical Features Prenatal alcohol exposure may result in malformations in almost every system of the body; for example, cardiac, genital and renal malformations occur with high incidence in children with FAS (Qazi et al., 1981; Steeg & Woolf, 1979). Cardiac malformations occur in 2941% of infants with F A S (Sandor et al., 1981), the most common being atrial or ventricular 2  septal defects.  Genital and renal malformations occur in almost half of infants with F A S  (Clarren & Smith, 1978). Immune dysfunction has also been recorded to occur at a higher incidence in children with FAS.  Johnson et al. (1981) reported that patients with F A S had  increased rates of bacterial infections and diminished mitogen-induced lymphocyte proliferative responses. Orthopedic anomalies are found in as many as half of the children diagnosed with FAS (Goldberg, 1987). Many of the abnormalities occur at rates higher than in the general population including congenital hip dislocation, thoracic cage abnormalities, delayed skeletal maturation and clubfoot (Smith et al., 1981; Spiegel et al., 1979). In addition to physical abnormalities, numerous behavioral problems have been documented in children with FAS. Motor abnormalities, perceptual deficits, hyperactivity, poor attention span, and impaired habituation have all been described (Streissguth,  1986).  Furthermore, children with FAS display higher rates of speech and language problems (Greene et al., 1990), auditory disorders (Church & Gerkin, 1988), visual disorders (Stromland, 1990) and visual perceptual problems (Aronson et al., 1985). Many of the behavioral problems seen in children with F A S persist through adolescence and into adulthood, the most common being attention deficits, poor social adaptation and problems with comprehension and abstraction (Streissguth et al., 1991). Individuals with FAS also reportedly show marked decrements in IQ when assessed as adolescents and adults, although even when IQ is in the normal range these individuals often exhibit serious behavioral problems. Thus, FAS is not just childhood disorders; there appears to be a predictable long-term progression of these disorders into adulthood. Although the characteristic facial anomalies may become subtler with age, short stature and microcephaly are often still evident.  More importantly, the continued presence of cognitive  deficits and behavioral abnormalities into adulthood may hinder the ability of these individuals to establish independence in either housing or income (Streissguth et al., 1991).  3  3. Incidence and Epidemiology Prenatal alcohol exposure is now recognized as a leading known cause of mental retardation in the Western world with conservative estimates indicating that approximately 1,200 children are born with FAS each year in the United States (Abel & Sokol, 1991). There is no simple, objective laboratory test for diagnosing FAS and the diagnostic criteria for FAS require expertise in recognizing dysmorphic features that may change with age (Streissguth et al., 1991). In 1992, the rate of cases of FAS increased from 5.2 per 10,000 live births from the 2.2 cases per 10,000 reported in 1990, likely demonstrating the improvement over recent years in recognition and reporting of FAS at birth (Cordero et al., 1994).  While F A S / F A E have been identified in  children from all ethnic groups and socio-economic classes, F A S may be recognized and recorded in medical records at different rates in different populations. In one study examining the rate of diagnosis of F A S by pediatricians in Massachusetts, it was found that among pediatricians who reported ever making a diagnosis of FAS, nine percent also reported not recording the diagnosis in the medical record (Morse et al., 1992).  In contrast, reported  incidence of F A S / F A E in some Native Canadian villages has been as high as 25-189 per 1000 (Asante & Nelms-Matzke, 1985; Robinson et al., 1987). The resulting effects of prenatal alcohol exposure produce enormous social and financial costs, with conservative estimates of $75 million annually in the US for FAS alone (Abel & Sokol, 1991). 4. Alcohol Teratogenesis The type and extent of alcohol-induced fetal damage is partly related to the level and pattern of fetal alcohol exposure. For example, lower levels of prenatal alcohol exposure are required to induce neurodevelopmental effects than to induce physical or growth effects (Streissguth et al., 1989). Research in nonhuman primates has shown that behavioral deficits may occur without accompanying physical anomalies (Clarren et al., 1990). Moreover, those studies found that effects of prenatal alcohol exposure are clearly observable after once-per-week alcohol 4  consumption resulting in blood alcohol levels (BALs) above 140 mg/dl (i.e. the equivalent of four to six drinks by an average-sized woman).  Indeed, maternal binge drinking (i.e.  consumption of five drinks or more per occasion) during pregnancy is one of the strongest predictors of later neurodevelopmental deficits in children with alcohol-induced damage (Streissguth et al., 1989). In addition, both animal and human studies have demonstrated that binge drinking at high levels may result in more devastating effects on the developing fetus than intake of the same dose of alcohol over a longer period of time (Pierce & West, 1986). It is likely that the effects of binge drinking are particularly severe because this drinking pattern results in high B A L s in both mother and fetus, followed by repeated withdrawal episodes. Since alcohol easily crosses the placenta, fetus and mother are exposed to nearly comparable blood alcohol concentrations (Brien et al., 1983). However, the fetus is unable to metabolize the alcohol because it lacks the necessary enzymes and is dependent upon maternal elimination of alcohol following its passive diffusion across the placenta into the maternal circulation (Guerri & Sanchis, 1985). Therefore, the rate of alcohol elimination from amniotic fluid is approximately two times slower than that from maternal blood, resulting in high alcohol concentrations in the amniotic fluid even when alcohol in the blood has been eliminated (Brien et al., 1983). The type of fetal damage may also be related to the timing of alcohol exposure (i.e. whether exposure occurs during the critical period of development of a particular organ system). The critical period is the time during which an organ system undergoes crucial steps in development and/or maturation and, consequently, is most vulnerable to the disruptive effects of any agent that causes abnormal fetal development or birth defects. For example, alcohol exposure in the first trimester is more often associated with organ and musculoskeletal anomalies while exposure in the second and third trimesters is linked to growth, intellectual and behavioral deficits (Aronson & Olegard, 1987).  During the first three months of gestation in humans, the  development of the facial and skull bones occurs. Thus, alcohol exposure occurring in the first 5  trimester can result in the characteristic facial abnormalities observed in children with F A S . Conversely, alcohol exposure in the second or third trimester is more often associated with growth retardation and neurological defects because fetal growth and brain development occur more rapidly during those gestational stages. If maternal alcohol consumption occurs during all three trimesters, the fetus is exposed to alcohol during the critical periods for the development of facial characteristics, growth patterns and CNS function, and thus may develop full FAS. The exact mechanisms underlying alcohol-induced fetal damage have not been fully delineated. The tremendous impact of prenatal alcohol exposure on fetal development is not surprising, however, considering the interrelationship of the maternal and fetal endocrine systems and the numerous direct and indirect effects of maternal alcohol consumption on both the mother and the fetus. The endocrine activities of both mother and fetus change throughout gestation. For example, whereas the transfer of maternal hormones across the placenta and/or placental hormone production are essential during early pregnancy, the activity of the fetal endocrine system increases and becomes more important later in gestation.  Consequently,  alcohol's impact on fetal endocrine activity may occur through different avenues at different time points. Although numerous factors likely play a role in alcohol-induced fetal damage, the disruption of hormonal influences on the developing fetus may explain at least some of the effects of prenatal alcohol exposure. Alcohol consumption during pregnancy disrupts the normal functioning of both the maternal and fetal endocrine systems and may disturb the normal maternal-fetal endocrine balance (Anderson, 1981). These alterations may adversely affect the development and organization of multiple systems in the fetus and likely mediate some commonly observed effects of prenatal alcohol exposure.  6  B. Animal Models of FAS Animal models of prenatal ethanol exposure also provide an opportunity to isolate ethanol effects from environmental effects such as pattern and level of exposure, exposure to other substances of abuse, and nutritional status. Many animal species have been used to study the effects of prenatal ethanol exposure including the monkey, sheep, dog, chick, guinea pig, rabbit, mouse and rat (Colangelo & Jones, 1982; Driscoll et al., 1990; Streissguth et al., 1990). However, rodent models (particularly the rat) are by far the most commonly used because of the ease of handling, short gestation periods and relatively low cost to purchase, house and feed (Keane & Leonard, 1989). Although the mechanisms of ethanol metabolism are similar in humans and rats, rats have a faster metabolic rate than humans and, therefore, metabolize ethanol more quickly. The fetal development of humans and rats follow similar stages, but differ in the timing of birth. For humans, the major brain growth spurt occurs during the third trimester of gestation and growth then continues for about two years postnatally (West, 1987). In contrast, the major brain growth spurt in the rat occurs during the first 10-14 days of postnatal life (this can be called the "third trimester equivalent")(West et al., 1989). Due to this difference, both prenatal and postnatal ethanol exposure in rats have been used to address different types of research questions. In this thesis, the effects of prenatal ethanol exposure on cognition/behavior and on the H P A axis were studied since both the neuronal network for cognition and the H P A axis develop and begin to function during the prenatal life of the rat (Eguchi, 1969). 1. Ethanol Administration Many different ways of administering ethanol to the pregnant female have been utilized, including injection, inhalation, gastric intubation, and via drinking fluid or liquid diet. Although injection, inhalation and intubation methods allow for controlled doses of ethanol to be administered and high B A L s to be achieved, they require handling and/or 7  confinement, both of which are stressors, and prenatal stress has been shown to produce hyperresponsiveness of the H P A axis and behavioral abnormalities in the offspring (Suchecki & Neto, 1991). In contrast, ethanol in the drinking fluid is nonstressful, but because the taste of ethanol is aversive the animals reduce their liquid intake, resulting in low B A L s and reduced water and food intake (Weiner, 1980).  In contrast, the liquid diet method of  delivering ethanol used in the present studies provides all the required nutrients as well as a nonstressful method of administration, resulting in high B A L s while maintaining adequate nutritional status (Lieber & De Carli, 1989; Weinberg, 1985). Typically in models employing a liquid diet method of administration, there is a nutritional control group [pair-fed (PF)] in which nutritional intake is matched with that of the prenatal ethanol group. The addition of the PF group along with the prenatal ethanol and ad libitum fed control (C) groups is necessary because chronic ingestion of ethanol has marked effects on nutritional status. Primary malnutrition can result from displacement of nutrients by ethanol because of ethanol's high energy content. When consuming alcohol, humans will lose weight even i f caloric intake is sufficient to maintain body weight (Pirola & Lieber, 1972) and, in laboratory animals, ethanol consumption leads to less overall food intake compared to consumption of an ethanol-free diet (Weinberg, 1985). The inclusion of a PF group in animal models of prenatal ethanol exposure makes it possible to isolate the teratogenic effect of ethanol from the effects of primary malnutrition (i.e. the reduction of nutrient intake resulting from ethanol intake). However, ethanol can also cause secondary malnutrition by impairing the ability of an organism to digest or absorb nutrients from the gastrointestinal tract or utilize those nutrients once they have been absorbed (Lieber, 1988; Weinberg, 1984). In addition, ethanol can produce vascular changes in the placenta that may hinder the transport of nutrients and oxygen from mother to fetus (Gordon et al., 1982).  8  However, neither of these variables is controlled for by pair-feeding; there is no control for secondary malnutrition. Although malnutrition may have teratogenic effects, it is not the primary cause of the developmental abnormalities seen in F A S (Weinberg, 1985). The inclusion of the PF control group has demonstrated that although poor nutrient intake can act synergistically with ethanol, ethanol is the main teratogen causing F A S (Weinberg, 1985; Wiener et a l , 1981). However, pair-feeding itself is an experimental treatment. For example, pair-feeding can produce shifts in the circadian rhythm of a number of physiologic variables as well as alter body and organ weights and behavior of both the maternal female and offspring (Gallo & Weinberg, 1981; Weinberg & Gallo, 1982). Furthermore, PF dams may experience stress derived from their restricted meal-feeding schedule.  Although E females have ad libitum access to food, they  consume less than they would i f their diets did not contain ethanol. Thus, PF dams receive a ration that is less than they would consume ad libitum.  They typically consume this ration  within a few hours after it is presented and are food deprived until the next day's feeding. Therefore, although both groups are receiving the same number of calories, PF dams experience deprivation and possible stress, whereas E dams do not. Clearly, no method of administration is ideal and each has its advantages and drawbacks. Although animal models are not perfectly able to isolate the teratogenic effects of ethanol, they provide a large degree of control over many variables. 2. Effects of Prenatal Ethanol Exposure in Rodent Models Rodent models of prenatal ethanol exposure have replicated many of the effects found in humans prenatally exposed to alcohol (Driscoll et al., 1990; Schwetz et al., 1978).  Rodent  models have demonstrated increased fetal deaths and/or resorptions, retarded pre- and postnatal growth and development, various physical malformations, and CNS abnormalities (Abel, 1980; Abel & Dintcheff, 1978; Chernoff, 1977; Leichter & Lee, 1980; Meyer & Riley, 1986; Randall 9  et al., 1977; Streissguth et al., 1980; West & Pierce, 1986). Rodents exposed to ethanol during the third trimester equivalent (i.e. the period of most rapid brain development in humans) exhibit significant cognitive and behavioral abnormalities, whereas prenatal ethanol exposure has been shown to affect both the timing and rate of neurogenesis (Miller, 1986) and neuronal migration (Clarren & Smith, 1978). Rodents prenatally exposed to ethanol display reductions in neuronal cell numbers and decreases in the mean size of neuronal cell bodies in most cortical layers (Barnes & Walker, 1981) as well as changes in mossy fiber branching and hippocampal pyramidal cell number and arborization (West & Hamre, 1985; Wigal & Amsel, 1990). Ethanol has also been demonstrated to impair astrocyte growth and differentiation even at low doses, possibly hindering the production of trophic factors by astrocytes that are essential for normal growth and differentiation of neurons (Lokhorst & Druse, 1991). These aberrations in glial and neuronal formation, differentiation and migration may result in the delayed development of functional neuronal connections which could be responsible for behavioral abnormalities documented in animals prenatally exposed to ethanol (Pratt, 1984). In rodents, behavioral hyperactivity and hyperresponsiveness such as increased exploratory behavior (Riley et al., 1979c) and increased startle reactivity (Anandam et al., 1980) have been reported. In addition, prenatal ethanol exposure has been shown to produce deficits in passive avoidance learning (Riley et al., 1979a), taste aversion (Riley et al., 1986), and reversal learning (Lochry & Riley, 1980), reflecting possible impairments in response inhibition. Lastly, rodents prenatally exposed to ethanol have demonstrated an inability to use environmental cues (Abel, 1979; Weinberg, 1992a).  Although the neuroanatomical mechanisms underlying the  behavioral alterations observed following prenatal ethanol exposure remain to be determined, it appears that rodents exposed to ethanol prenatally or during early postnatal life may provide a valuable model for further investigations into the causes of the behavioral and cognitive deficits documented in children with F A S . 10  C . The Hypothalamic-Pituitarv-Adrenal (HPA) Axis The term stress has been defined in a variety of ways in the popular and scientific literature. The word has been used to refer interchangeably to a mental state, an external cue in the environment that is perceived as threatening or as the physiological changes that occur in response to external cues. Stress will be defined here as the state of threatened homeostasis; stressors will be defined as the threatening forces, either physical or psychological, and the adaptive or stress response will be defined as the manner in which the organism deals with the threat to homeostasis (Johnson et al., 1992). The ability to respond to stress has been acknowledged as an important basic adaptive mechanism and H P A activation is a central feature of this response. stress systems:  There are two primary  (1) the H P A system, composed of the hypothalamus which releases  corticotropin-releasing  hormone  (CRH),  the  anterior  pituitary  which  adrenocorticotrophin (ACTH) and 13-endorphin, and the adrenal cortex which corticosterone (CORT, the main glucocorticoid in rats;  Cortisol  releases releases  is the main glucocorticoid in  humans); (2) The sympathetic nervous system/locus coeruleus system, consisting of sympathetic nerves which release norepinephrine, and the adrenal medulla which releases norephinephrine and epinephrine (Chrousos & Gold, 1992; Johnson et al., 1992).  These two systems are  functionally interrelated; C R H stimulates and glucocorticoids inhibit the sympathetic nervous system/locus coeruleus system. Conversely, norephinephrine and epinephrine stimulate C R H and A C T H release from the H P A system (Johnson et al., 1992). The H P A system is the focus of the present research. occurs at several levels.  Regulation of the H P A axis  In the C N S , environmental stimuli including stressors and the  endogenous circadian rhythm (Dallman et al., 1987) act on the paraventricular nuclei (PVN) of the hypothalamus where the cell bodies of neurons that synthesize and secrete C R H are located. CRH  release/synthesis  is stimulated by epinephrine, norephinephrine, acetylcholine, 11  serotonin, dopamine, angiotension II, and enkephalins; it is inhibited by C R H , A C T H , C O R T , y-aminobutyric acid ( G A B A ) , and B-endorphin (Dunn & Berridge, 1990; Owens & Nemeroff, 1991; Reisine et al., 1986).  C R H is released into the portal hypophyseal  circulation and regulates the expression of the gene coding for propiomelanocortin (POMC), stimulating the release of POMC-derived peptides from the corticotrophs of the anterior pituitary. A C T H is a polypeptide hormone that is co-released with several other products, including B-endorphin, B-lipotropin, and y-lipotropin, by the processing of P O M C .  In certain  animal species but not in humans, A C T H may be further cleaved to form oc-melanocytestimulating hormone.  A C T H shows both a circadian rhythm and cyclic bursts regulated by  C R H . A C T H is the primary regulator of glucocorticoid secretion from the adrenal cortex and the amount of A C T H release can be altered by negative feedback control at the level of the hypothalamus ( A C T H and glucocorticoids) and the pituitary (glucocorticoids). Vasopressin (VP), better known for its role in maintaining body water balance, is another important peptide involved iri the release of A C T H .  V P is released from the  parvocellular (co-localized with neurons that release C R H ) and magnocellular divisions of the P V N (Antoni, 1993; Reisine et al., 1986) and released into the portal hypophyseal vessels. It is a weak secretagogue by itself, but acts synergistically with C R H . During some types of repeated or chronic stress, the rate of C R H release is maintained, whereas V P secretion rate and co-localization in C R H nerve terminals is markedly increased. In this way, an increase in V P synthesis and release, resulting in an increased V P : C R H ratio may be critical for maintaining pituitary responsiveness during chronic stress (Aguilera, 1994). Glucocorticoids are catabolic hormones that are released into the systemic circulation and are normally 95% bound, primarily to corticosteroid-binding globulin and to a lesser extent to albumin, in the blood (Berne & Levy, 1993).  Due to its lipophilic nature, the free fraction  readily passes through cell membranes and binds to receptors located primarily in the cytoplasm 12  of target cells. Glucocorticoids act in direct opposition to the major anabolic hormone, insulin, at target sites. At physiological concentrations, glucocorticoids promote protein mobilization and fat metabolism, facilitate vascular responsiveness and upregulate enzyme activity in metabolic pathways in the CNS and peripheral tissues. With adequate supplies of glucose, glucocorticoids promote triglyceride uptake into abdominal and facial adipose tissue while decreasing the sensitivity of target tissues to insulin. During fasting, glucocorticoids are essential because they increase protein mobilization while inhibiting its synthesis in muscle, thus sparing stores of glycogen that are necessary to supply obligate glycogen users such as the C N S . In humans, symptoms of excessive amounts of Cortisol include obesity caused by deposition of facial and abdominal fat, increased protein catabolism, loss of skeletal muscle mass, and increased susceptibility to infection (Berne & Levy, 1993). The actions of C O R T involve binding to intracellular corticosteroid receptors, consisting of mineralocorticoid (MRs) and glucocorticoid receptors (GRs), which act as gene transcription factors (de Kloet, 1991; McEwen et al., 1986). These two steroid receptor types differ in their affinities for C O R T . M R s have high affinity for C O R T , whereas GRs have a 3-10 fold lower affinity for C O R T than do M R s but have a higher affinity for the synthetic glucocorticoid,  dexamethasone-21 -phosphate (DEX).  In the  C N S , M R s are  most  concentrated in the hippocampus, septum and amygdala (de Kloet, 1991), while GRs are more widely distributed throughout the brain with high concentrations in the paraventricular nuclei of the hypothalamus, lateral septum, dentate gyrus, and central amygdala (Agnati et al., 1985). The differential binding affinities to C O R T result in differential G R and M R occupancy during the phases of the circadian rhythm and during basal versus stress conditions. M R s are extensively occupied during basal activity at the trough of the circadian rhythm. In contrast, substantial G R occupation occurs at the circadian peak and following stress, suggesting distinct roles for these receptors during negative feedback regulation of the 13  H P A axis.  C O R T inhibition of the H P A axis occurs at the level of the pituitary, the  hypothalamus [especially the P V N (Dallman et al., 1987; Rivier & Plotsky, 1986)], and at extrahypothalamic sites, most notably the hippocampus, and it is an essential element of an organism's ability to self-regulate and appropriately terminate the physiological reactions which occur in response to stress (Dallman, 1993). Increases in glucocorticoids may protect the body against its own natural defenses following exposure to stressful stimuli by preventing excess endocrine, metabolic and immune reactions during the recovery process when these systems could potentially overrespond and cause damage (Munck et al., 1984). The overall protective mechanism provided by high levels of glucocorticoids is normally self-limiting because elevations in glucocorticoids negatively feedback on the hypothalamus and pituitary, suppressing H P A activity.  Consequently, an  inability to habituate or adapt to an aversive stimulus may not only adversely affect an organism's ability to respond to new environmental challenges but prolonged increases in H P A activity may seriously impair the well-being of an organism by disrupting homeostasis of physiological systems. Dysregulation of the stress response has been implicated in a variety of psychiatric disorders such as depression, panic disorder, obsessive-compulsive disorder and anorexia nervosa (Chrousos & Gold, 1992; Johnson et al., 1992) 1. Negative Feedback Regulation of the H P A Axis There are at least three distinct time domains in which negative feedback by CORT operates during and following stress (Dallman et al., 1987; Keller-Wood & Dallman, 1984): fast feedback (seconds to minutes), early delayed or intermediate feedback (2-10 hr), and late delayed or slow feedback (hours to days). Fast feedback is sensitive to the rate of increase in CORT (Abe & Critchlow, 1977; Kaneko et al., 1981) and occurs while plasma levels of CORT are increasing. It occurs within seconds or minutes of CORT increase and is no longer effective by 20-30 min. Fast feedback inhibits release of A C T H and C R H but does not affect synthesis of 14  A C T H and C R H (Keller-Wood & Dallman, 1984). Delayed feedback (intermediate and slow) is not rate sensitive but depends on the level of CORT achieved, the dose and duration of CORT exposure and the interval since the stressor.  It requires 45-120 min to develop, with the  maximum inhibition at 2-4 hr, and an attenuation of the response at 6-12 hr after exposure to CORT (Dallman & Yates, 1969; Keller-Wood & Dallman, 1984). Intermediate feedback occurs during exposure to CORT for 2-10 hr and involves the inhibition of C R H and A C T H release as well as C R H synthesis. Slow feedback results during constant CORT exposure for 12 hr or more and involves the inhibition of the synthesis and release of both C R H and A C T H . The complex system of feedback loops controlling the H P A axis gives rise to a malleable regulatory system, allowing for varied responses in proportion to the magnitude or length of exposure to the stimuli. Fast feedback may control the rate and magnitude of CORT responses to acute stimuli, whereas intermediate feedback may limit the response of the system to repeated stimulation within a relatively short period of time (hours). Lastly, slow feedback may limit the response of the system during prolonged exposure to stressors or during pathological conditions (Keller-Wood & Dallman, 1984).  D. The H P A Axis and Prenatal Ethanol Exposure As previously discussed, ethanol exerts a potent influence on the maternal H P A axis and,  consequently, may disrupt hormonal interactions between the maternal and fetal  systems, producing long-term disturbances in fetal metabolism and physiology which may significantly alter offspring endocrine function into adulthood. During ethanol consumption by pregnant female rats, maternal adrenal weights, basal C O R T levels, and the adrenocortical response to stress have all been demonstrated to be elevated compared to PF and C females (Weinberg & Bezio, 1987; Weinberg & Gallo, 1982). These changes in maternal endocrine function persist throughout gestation and the increased CORT released by the activated maternal 15  endocrine system may cross the placenta and suppress fetal H P A activity (Weinberg & Bezio, 1987). However, the passage of ethanol across the placenta may simultaneously stimulate the fetal H P A axis. Consequently, the opposing physiological responses to both elevated maternal CORT and ethanol in utero may permanently affect the development and organization of the fetal H P A axis (Levine & Mullins, 1966). At birth, neonates prenatally exposed to ethanol have increased adrenal weights and plasma and brain concentrations of CORT, as well as elevated plasma levels and reduced pituitary content of p-endorphin compared to PF and C offspring (Taylor et al., 1983; Weinberg, 1989).  In addition, although the endocrine response to stressors such as ether and novel  environment is decreased in all offspring during approximately the first two weeks of the preweaning period, E offspring reportedly have greater reductions than PF and C offspring, showing blunted responses to stressors (Taylor et al., 1986; Weinberg, 1989). This effect is transitory,  however,  and,  from  weaning  onward,  E  animals  display  hormonal  hyperresponsiveness to stressors (Taylor et al., 1983; Weinberg, 1988; Weinberg, 1992b). H P A hyperresponsiveness in response to stressors in adult E animals is characterized by increased release of H P A hormones and/or delayed recovery to basal levels. Significantly, this difference in H P A activity is only apparent following stress; E offspring do not appear to differ from PF or C animals under basal conditions (Taylor et al., 1983; Weinberg, 1992a).  From  weaning age through adulthood, E rats display increased plasma CORT responses to various physiological and psychological stressors such as footshock, cardiac puncture, noise and shake, restraint, swim, ether and cold (Angelogianni & Gianoulakis, 1989; Nelson et al., 1986; Taylor et al., 1982; 1983; Weinberg, 1988; 1992a; Weinberg et al., 1996), and to challenges with drugs such as ethanol and morphine (Nelson et al., 1986; Taylor et al., 1981; 1983). E rats also display increased plasma A C T H responses following footshock or restraint (Lee et al., 1990; Nelson et al., 1986; Ogilvie & Rivier, 1997; Taylor et al., 1986; Weinberg et al., 1996), and increased B-EP 16  response following ether or cold stress (Angelogianni & Gianoulakis, 1989; Weinberg et al., 1996). In addition, E animals demonstrate deficits in pituitary-adrenal response inhibition or recovery from stress.  For example, E animals show prolonged CORT, A C T H , and B-EP  elevations during and following restraint stress (Weinberg, 1988; 1992a). Furthermore, deficits may exist in the ability of E animals to use or respond to environmental cues. E animals were less discriminating in their CORT responses to predictable versus unpredictable restraint stress compared to controls (Weinberg, 1992b), and displayed less attenuation in CORT responses compared to controls when allowed access to water during novel cage stress (Weinberg, 1988). Although both male and female rodents have been shown to exhibit increased H P A responsiveness following prenatal ethanol exposure, H P A hyperresponsiveness and/or the deficits in response inhibition may be manifested differentially in males and females.  For  example, following repeated exposure to restraint stress, E males showed increased (3-endorphin levels whereas E females showed elevated A C T H and CORT levels compared to their PF and C counterparts (Weinberg et al., 1996). Differential responding between males and females may depend upon the nature and intensity of the stressor, the time course measured, and the hormonal endpoint measured (Halasz et al., 1993; Weinberg, 1988; 1991; Weinberg et al., 1996). 1. Mechanisms of Prenatal Ethanol Effects on H P A Hyperresponsiveness The mechanisms underlying H P A hyperresponsiveness in E rats are unclear at present. Hyperresponsiveness may be a result of many factors including (1) hypersecretion of C R H and/or V P , (2) increase in anterior pituitary or adrenal sensitivity to their respective secretogogues, (3) altered neurotransmitter regulation of the H P A axis, and/or (4) deficits in negative feedback control of H P A activity. One or more of these mechanisms may be at work. First, there may be hypersecretion of C R H and/or V P from the P V N of E animals. Increased basal C R H m R N A levels have been demonstrated in the P V N in 21 day old pups 17  (mixed sex) of dams that were exposed to ethanol vapors during the second week of gestation (Lee et al., 1990) and in the hypothalamus of 60 day old adult male but not female offspring of dams exposed to ethanol during the last two weeks of gestation (Redei et al., 1993). In contrast, prenatal ethanol exposure has been shown to result in no changes in basal hypothalamic C R H levels (Angelogianni & Gianoulakis, 1989), and lower basal median eminence C R H levels (both E and PF) compared to C rats (Lee & Rivier, 1994). Second, there may be increased sensitivity of the anterior pituitary and adrenal cortex to their respective secretagogues in E animals. Increased basal levels of P O M C m R N A have been reported in the anterior pituitary of E rats (Redei et al., 1993). Although C R H or V P administration did not produce differential anterior pituitary A C T H release under basal conditions (Lee et al., 1990; Lee & Rivier, 1993; Taylor et al., 1988), an increased A C T H response to C R H in DEX-suppressed E animals was recently reported ( Y u et al., 1996). At the level of the adrenal cortex, exogenous A C T H administration did not produce differential C O R T release in E animals (Lee & Rivier, 1994; Osborn et al., 1994). Third, altered neurotransmitter  systems may be involved in mediating H P A  hyperresponsiveness in E animals. Norepinephrine content in the cortex and hypothalamus was found to be lower in E animals compared to controls following restraint stress (Rudeen & Weinberg, 1993). Since norepinephrine has been shown to stimulate C R H release in a dose-dependent manner (Plotsky, 1987), lower hypothalamic norepinephrine levels in E animals may be indicative of increased norepinephrine turnover which may  affect  norepinephrine regulation of C R H secretion and play a role in H P A hyperactivity. Furthermore, it has been shown that C O R T can modulate  GABAA  receptor activity and, in  turn, G A B A has a role in regulating H P A activity (Jones et al., 1984; Majewska et al., 1985). The  G A B A system appears to be altered in E animals, as demonstrated by altered  responsiveness to the anxiolytic effects of benzodiazepines (Yu et al., 1995). Thus, prenatal 18  ethanol exposure may affect the H P A axis via alterations in the G A B A system. H P A activity is also regulated by stimulatory serotonin input at the level of the hypothalamus (Feldman et al., 1987; Kageyama et al., 1998). Serotonin may be involved in circadian rhythmicity of H P A hormone secretion (Fuller, 1992) and appears to be necessary for the stress response (Feldman et al., 1987). However, decreased serotonin levels and reuptake sites have been found in E animals (Druse et al., 1991; K i m & Druse, 1996), which may result in compensatory mechanisms that alter H P A responsiveness to stressors. Fourth, there is evidence for deficits in feedback control of the H P A axis in the intermediate time domain following prenatal ethanol exposure. Following administration of D E X , E females showed increased basal C O R T levels (Nelson et al., 1985), and E males and females showed increased A C T H and/or C O R T responses to acute ether stress (Osborn et al., 1996) compared to controls.  This differential responsiveness of E animals was more  pronounced at the peak (lights off) than at the trough (lights on) of the circadian rhythm and was more apparent in E females compared to E males (Osborn et al., 1996). These findings support the hypothesis that E animals may exhibit deficits in H P A feedback inhibition and these deficits may be sex-specific. However, this alteration in H P A feedback regulation does not appear to be mediated by changes in brain corticosteroid receptors since data from our laboratory indicate that E animals do not show a long-term downregulation of hippocampal GRs or M R s either under basal conditions (Weinberg & Peterson, 1991) or following exposure to chronic intermittent stress (Kim et al., 1999b). 2. Effects of H P A Hyperresponsiveness on Behavior Not only may excessive H P A activity have profound consequences throughout the animal's life, including possible impairments in growth or immunity resulting from a redistribution of energy resources but hyperresponsiveness of the endocrine system may contribute to the behavioral deficits exhibited by E animals. Central administration of C R H 19  produces behavioral responses resembling stress-associated behaviors such as decreased feeding in familiar and novel environments, decreased sexual behavior, increased acoustic startle responses and increased defensive withdrawal (Koob et al., 1993).  These behaviors were  reportedly attenuated or reversed following the administration of C R H antagonists (Berridge & Dunn, 1987). The effects of C R H may partially explain the behavioral changes documented in E animals. During testing, elevated H P A activity in E animals may lead to increased C R H levels compared to those in PF and C animals, resulting in behavioral hyperactivity and deficits in response inhibition. Children prenatally exposed to alcohol are known to be hyperactive, uninhibited, and impulsive in behavior, particularly in challenging or stressful situations.  A recent study in  human infants whose mothers drank heavily at conception found greater increases in Cortisol levels in response to stress (e.g., having blood drawn) compared with control infants (Jacobson et al., 1999). Thus, the hormones of the H P A axis may act on the C N S to alter behavior and performance in stressful situations, indicating that altered H P A activity may underlie some of the behavioral problems seen in children prenatally exposed to alcohol.  E . Postnatal Handling. Postnatal handling is an early environmental manipulation that has been shown to produce long-term alterations in behavior and H P A activity in adult animals. Although postnatal handling procedures vary somewhat among different laboratories, the handling procedure usually involves removing rat pups from their home cage, placing the pups in small containers and, after several minutes (e.g. 3-15 min), returning the animals to their home cage and their mothers. The manipulation is generally performed daily for the first 21 days of life although the first 10-14 days appear to be the "critical period" (Denenberg & Zarrow, 1971). Recent research indicates that the effects of postnatal handling appear to be, at 20  least partly, due to altered mother-infant interactions that may occur in response to pup cues when pups are returned to the nest following handling. Early studies showed that handling increased ultrasonic vocalizations in pups which, in turn, served to increase maternal care, including licking and grooming (Bell et al., 1971). Recently, increased maternal licking and grooming of pups, in addition to increases in arched-back nursing posture as opposed to passive nursing postures during the first 10 days of life, were shown to be highly correlated with reduced plasma A C T H and CORT responses to acute stress and were the only behaviors that reliably distinguished the mothers of handled (H) and nonhandled (NH) pups (Liu et al., 1997). Furthermore, this research has found that natural variations in maternal care independent of handling may lead to differences in H P A responsiveness, suggesting that postnatal handling may artificially increase such naturally occurring behaviors. 1. Effects of Postnatal Handling H animals tested during development or as fully mature adults exhibit attenuated fearfulness in novel environments, exploring more and defecating less than N H animals in an open field (Levine et al., 1967). They learn faster and make fewer errors in some avoidance learning paradigms (Levine, 1956; Weinberg & Levine, 1977) and show less vocalization and are easier to pick up in a reaction-to-handling procedure (Ader, 1965). A generality that has emerged from this research is that H animals are less "emotionally reactive" than N H animals (Denenberg & Zarrow, 1971).  Recent work has also shown that handling may  attenuate cognitive impairments in the Morris water maze that are associated with senescence (Meaney et al., 1988; 1991). The demonstrated behavioral differences in emotionality in H animals occur alongside changes in physiology.  H animals appear to exhibit graded  responses to stressors compared to N H animals, i.e. they respond with moderate increases in C O R T in response to relatively mild stimuli such as novelty or restraint, and with larger and more rapid C O R T increases in response to more intense stimuli such as shock. In addition,  21  H animals show a faster return to basal C O R T levels during and after exposure to a variety of stressors when compared to N H animals (Denenberg & Zarrow, 1971; Levine, 1960). In contrast, N H animals appear unable to modulate their H P A activity, showing large C O R T increases in response to any environmental challenge. Significantly, adult H and N H animals do not differ in basal C O R T levels at any timepoint over the diurnal cycle (Meaney et al., 1992). 2. Mechanisms of Postnatal Handling on Alterations in HPA Responsiveness The efficiency or more appropriate modulation of H P A responsiveness in H animals following exposure to stressors may be mediated by alterations in the sensitivity of H P A negative feedback inhibition.  Administration "of exogenous C O R T and D E X is more  effective in suppressing stress-induced C O R T responses in H than in N H animals, suggesting that H animals are more sensitive to the negative feedback effects o f circulating C O R T on H P A activity (Meaney et al., 1989). Furthermore, H animals show increased corticosteroid receptor binding capacity in the hippocampus, but not the septum, amygdala, hypothalamus or pituitary compared to N H animals (Meaney et al., 1989; 1992). These differences occur in GRs but not M R s and suggest one mechanism of improved negative feedback sensitivity in H animals.  In addition, increased levels of corticosteroid receptors persist throughout the  lifespan of H animals, such that H animals do not show declines in hippocampal G R concentrations with age to the same degree as N H animals (Meaney et al., 1991; 1992). Typically, aging rats lose GRs and become insensitive to C O R T feedback regulation, resulting in C O R T hypersecretion under basal conditions and following stress. Ultimately, these increased C O R T levels may be detrimental to neuronal functioning.  In fact,  adrenalectomy ( A D X ) at 12 months of age has been shown to prevent both hippocampal neuron loss and cognitive impairments due to senescence (Landfield et al., 1981).  in standard-reared  animals  Interestingly, age-related rises in basal C O R T levels have been 22  observed in N H but not in H animals (Meaney et al., 1992).  The resulting reduction in  cumulative exposure to C O R T in H rats may lead to less pronounced hippocampal degeneration in these animals, resulting in fewer age-related cognitive impairments than in N H rats. The tonic negative feedback signal provided by basal C O R T levels may be an essential element in the differences in H P A activity between H and N H animals. Following A D X (removal of the C O R T negative feedback signal), H and N H animals do not demonstrate H P A differences in response to stressors. However, differences between H and N H animals can be reestablished in A D X animals through a steady, basal level C O R T replacement which does not permit changes in C O R T levels following stress (Viau et al., 1993). Therefore, the differences in stress-induced H P A activity between H and N H animals appear to be due to the influence of basal C O R T and not stress-induced elevations in C O R T . The importance of basal H P A activity is further underscored by the fact that basal hypothalamic C R H m R N A levels are significantly lower in H animals than in N H animals (Plotsky & Meaney, 1993).  Clearly, the effects of handling on H P A activity occur at  multiple functional levels.  F. Prenatal Ethanol Exposure and Postnatal Manipulations Recent research has indicated that both the behavioral and physiological impact of prenatal ethanol exposure may be, at least partly, mitigated by postnatal environmental manipulations. A n enriched environment has been shown to abolish the detrimental effects of prenatal ethanol exposure on conditioned taste aversion in E mice (Opitz et al., 1997). Rearing E rats in an enriched environment has been show to attenuate gait ataxia and improve performance in the Morris water maze when compared to E animals reared in isolation (Hannigan et al., 1993; Wainwright et al., 1993). Therapeutic motor training has 23  been shown to improve motor performance in animals postnatally exposed to ethanol and, although overall density of Purkinje neurons was decreased in E animals compared to controls, the surviving Purkinje neurons retained the capacity for synaptic plasticity (Klintsova et al., 1997). However, unlike control animals, E animals do not show increased hippocampal dendritic spine densities following environmental enrichment (Berman et al., 1996), underscoring the enduring impairments of prenatal ethanol exposure.  Previous  research in our laboratory has shown that postnatal handling eliminated deficits in preweaning weight gain often observed in E compared to PF and C pups, attenuated the hypothermic response to ethanol challenge in E and PF males, and attenuated the initial increased C O R T elevation in response to restraint stress in E females, although the more prolonged elevation following restraint was not attenuated (Weinberg et al., 1995). Further research examining the ability of postnatal handling and surrogate fostering to modulate H P A hyperresponsiveness to stress in E animals found that E pups exposed to both handling and fostering showed an A C T H response to footshock stress that was significantly larger than that of all other groups (Ogilvie & Rivier, 1997).  These results suggest that an  organism's postnatal and/or postweaning rearing environment may have a significant effect on developmental outcome and can alter the long-term effects of a prenatal insult.  G . Thesis Objectives The ability to respond appropriately to the environment is a basic mechanism for survival.  Furthermore, it is well established that maternal consumption of alcohol during  pregnancy has serious consequences for the developing fetus.  Cognitive deficits and  behavioral abnormalities are among the most detrimental consequences of prenatal alcohol exposure in clinical populations.  In addition, prenatal ethanol exposure produces H P A  hyperresponsiveness to stressors. The overall objectives of this thesis were to investigate (1) 24  the correspondence between prenatal ethanol-induced alterations in behavior and H P A activity, (2) the ability of early postnatal handling, as an environmental manipulation, to attenuate at least some of the adverse behavioral and physiological consequences of prenatal ethanol exposure, and (3) the possible influence of postnatal handling on mechanisms mediating the H P A hyperresponsiveness to stressors observed in E animals. Experiments in this thesis were designed to address four Specific Aims: Specific A i m i  (Chapter III) was designed to investigate learning deficits in E animals during a  conditioned taste aversion task as well as the correspondence between behavior and H P A activity during exposure to the conditioned solution. We tested the hypothesis that E animals which underwent postnatal handling would show improved conditioned aversion learning and reduced H P A activity compared to E animals that did not experience handling, and/or that handling might attenuate differences among E and control animals.  Specific A i m 2  (Chapter IV) was designed to assess spatial learning and memory in young adult and midaged H and N H E and control animals utilizing a Morris water maze.  We examined the  hypothesis that postnatal handling would improve spatial navigation in E animals compared to E animals that did not experience handling and/or attenuate differences among E and control animals, and that this effect might be age-dependent. We also investigated whether deficits in mid-aged animals would correspond to increases in CORT levels on the last day of testing. Specific A i m 3 (Chapter V ) was intended to test the hypothesis that postnatal handling would attenuate H P A hyperresponsiveness in E animals and to determine whether it would do so by improving H P A negative feedback inhibition during the intermediate time domain. Lastly, Specific A i m 4 (Chapter VI) examined whether the mechanisms resulting in H P A hyperresponsiveness in E animals are similar to those underlying the effects of postnatal handling.  Differences in H P A responsiveness between H and N H animals appear to be  dependent upon basal C O R T activity and not stress-induced elevations in C O R T levels. 25  Therefore, we tested the hypothesis that differences in H P A activity among E and control animals would not occur following A D X but could be reestablished following replacement with basal levels of exogenous CORT.  26  C H A P T E R II: G E N E R A L M E T H O D S  This section describes the methods common to most of the experiments.  Specific  additions or changes to this general methodology are described in the Methods section of each study.  A . Breeding Adult Sprague-Dawley male (350-375 g) and female rats (250-275 g) were obtained from the Animal Care Center, University of British Columbia, Vancouver, B.C., Canada, and were group housed for a 1-2 week adaptation period. During this period, they were maintained with ad libitum access to standard laboratory chow (Jamieson's Pet Food Distributors Ltd., Delta, B.C.) and water. The colony room had controlled temperature (21-22°C) and lighting, with lights on 0600-1800 h. A l l animal use procedures were in accordance with the Canadian Council on Animal Care and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the University of British Columbia Animal Care Committee. Following the adaptation period, males were singly housed, together with a female, in stainless-steel suspended cages (25 x 18 x 18 cm) with mesh front and floor. Waxed paper was placed under each cage containing a male and female. Cage papers were checked daily and the presence of vaginal plugs indicated day 1 of gestation (GI).  B. Diets and Feeding On G I , females were rehoused into polycarbonate cages (24 x 16 x 46 cm) and randomly assigned to one of three groups:  (1) Ethanol (E)-liquid ethanol diet (36% ethanol-derived  calories), ad libitum; (2) Pair-fed (PF)-liquid control diet with maltose-dextrin isocalorically  27  substituted for ethanol; each animal was pair-fed the amount consumed by a female in the ethanol group, g/kg bwt/d of gestation; (3) Control (C) - laboratory chow and water, ad libitum. This liquid diet method of ethanol administration is a well established standard procedure used extensively by this and other laboratories. Diets previously developed in our laboratory were prepared by Bio-Serv, Inc. (Frenchtown, NJ) and were formulated to provide adequate nutrition to pregnant females regardless of ethanol intake (Weinberg & Bezio, 1987). Although E and PF dams have somewhat reduced caloric and protein intake compared to C dams, their intake still exceeds the minimum daily nutritional requirements (Weinberg, 1985). See Table 1 for nutritional details of the E and PF diets. Fresh diet was placed on the cages daily in the late afternoon, just prior to lights off. Bottles from the previous day were removed and weighed at this time to determine amount consumed. This feeding schedule was designed to eliminate a shift in the CORT circadian rhythm, which typically occurs in animals receiving a restricted amount of food such as that received by PF females (Gallo & Weinberg, 1981). Experimental diets were replaced with ad libitum access to laboratory chow and water on G22. Birth occurred on approximately G22.524. Pregnant females were weighed once a week during gestation, i.e. G I , G7, G14, G21. At birth, designated postnatal day 1 (PN1), dam and pups were weighed and litters were culled to 10 (5 males, 5 females when possible). For litters that did receive a postnatal treatment (H, NH), dams and pups were weighed on PN1 and PN22. For litters that did not receive a postnatal treatment (standard-reared), dams and pups were weighed again on PN8, PN15 and PN22. Due to the number of subjects required for the experiments presented here, two separate breedings were undertaken. Breeding 1 supplied the animals used in experiments one, two and three, and developmental data are presented in experiment one (Chapter III).  Breeding 2  supplied the animals used in experiment four, and developmental data for that breeding is 28  presented in experiment four (Chapter VI). Animals from Breeding 1 underwent postnatal treatments (H, NH); animals from Breeding 2 did not experience postnatal treatments (standardreared).  C. Postnatal Treatment On PN1, litters within each prenatal dietary group (E, PF, C) were randomly assigned to either the handled (H) or nonhandled (NH) postnatal treatment. Handling consisted of removing the dam and then the pups from the home cage, and placing the pups into individual plastic containers for 3 min, once daily, after which the pups and then the dam, were returned to their cage. Handling occurred from PN2 to PN15 (a time that covers the critical period for the handling effect; Denenberg & Zarrow, 1971); background conditions (i.e. lighting, sound) and procedure time were kept constant across all handling days.  After PN15, dams and pups  assigned to the H condition remained undisturbed until weaning on PN22. N H animals were left completely undisturbed from PN1 to PN22. Body weights were obtained for H and N H dams and pups on PN1 and PN22. On PN22, animals from all prenatal groups x postnatal treatments were weaned and group housed by sex and by litter until testing  D . Experimental Subjects The subjects were offspring from the E , PF and C groups. They were tested as young adults (-35-150 d of age) or mid-aged adults (13-14 months of age); age at the time of testing is specified in each experiment. To control for litter effects within each experiment, no more than one male and one female per litter were randomly selected for testing for each experimental condition. Rats from the various prenatal groups and postnatal treatments were always tested in pseudorandom order. In all studies, the number of subjects per condition is reported in the figure captions or in the tables. 29  E . Blood Sampling In all studies, blood sampling occurred during the light phase. A t the time of sampling, each animal was quietly removed from the colony room to an adjacent sampling room to prevent disturbance of other animals. Three methods were used to collect blood. 1. Decapitation This method was used to collect blood for measurement of stress levels of plasma CORT. Sampling occurred within two minutes of moving the animal's cage.  This interval is rapid  enough to obtain reliable basal measures of CORT (Weinberg, 1992a; Weinberg et al., 1996), and to obtain an accurate measure of stress CORT at the time of sampling with no additional effects of the sampling procedure itself. Animals were decapitated with a guillotine and trunk blood  was  collected on ice  in plastic  tubes (12  x  75  mm)  containing  7.5  mg  ethylenediaminetetraacetic acid, an anti-coagulant, (Sigma Chemical Co., St. Louis, M O ) and 1000 K I U aprotinin, a protease inhibitor (ICN Biochemical Inc., Aurora, OH). The blood was centrifuged at 2200 x g for 10 min at 4 °C, and the plasma was stored in microcentrifuge tubes at -70 °C until assayed. 2. Cardiac Puncture This method was also used to collect blood for measurement of stress-associated plasma CORT. As for decapitation, sampling occurred within two minutes of moving the animal's cage, and the sampling procedure itself should not have affected the determination of stress-associated plasma CORT levels. Animals were lightly anesthetized with ethyl ether (Fisher Scientific Ltd., Vancouver, BC) and 0.5 cc blood was withdrawn from the heart using a 1 cc heparinized syringe with a 25G 5/8 needle (Becton Dickinson, Franklin Lakes, NJ). The blood was centrifuged at 2200 x g for 10 min at 4 °C, and the plasma was stored in microcentrifuge tubes at -70 °C until assayed.  30  3. Jugular Cannulation Animals were implanted with indwelling jugular cannulae under halothane anesthesia 4448 hr prior to testing. It has been demonstrated that catecholamines and other hormones return to basal levels by 24 hours after implantation (Wixson et al., 1987). The surgical and sampling procedures were in accordance with Rivier et al. (1982). Cannulae were cold sterilized with the Clindox-S system and implanted under semi-sterile conditions. The incision area on the rat was cleaned with 70% ethanol prior to surgery. The cannula consisted of PE50 tubing with a beveled silastic tip. Cannulae were inserted into the left internal jugular vein and secured in place with three sutures. The free end of the cannulae were then tunneled subcutaneously, and exteriorized dorsally between the scapulae. The tip of the free end of the cannula was folded over and capped with PE20 tubing until the testing day. On the testing day, a sampling cannula consisting of PE50 tubing with a blunted 22G x 1 72" needle at one end was inserted into the free exterior end of the indwelling catheter. A L U E R L O K PRN adapter attached to a needle hub for injection and sampling was attached to the other end of the sampling cannulae. Blood was collected using a cold tuberculin syringe connected to the indwelling jugular cannulae. Syringes used for collecting blood were coated with ethylenediaminetetraacetic acid and aprotinin to prevent coagulation and denaturation. Blood samples for A C T H determination were collected and stored in plastic tubes because A C T H binds to glass. Blood was centrifuged at 3500 g for 10 min at 4 °C, and plasma was transferred to plastic Eppendorf tubes using plastic pipettes and stored at -70 °C until assayed.  31  F. Radioimmunoassays 1. Plasma C O R T Levels. Total CORT (bound plus free) was measured by radioimmunoassay (RIA) in plasma extracted in absolute ethanol (1:10 v/v), using our adaptation (Weinberg & Bezio, 1987) of the method of Kaneko et al. (1981). Antiserum was obtained from Immunocorp (Montreal, PQ, Canada); tracer, [l,2,6,7- H]CORT was obtained from Mandel Scientific (Guelph, ON, Canada); 3  and unlabelled CORT for standards was obtained from Sigma Chemical Co. (St Louis, M O ) . Dextran-coated charcoal was used to absorb and precipitate free steroids after incubation. Samples were counted in Formula 989 (Dupont). Intra- and interassay coefficients of variation were 1.45% and 3.9%, respectively. 2. Plasma A C T H Levels. Plasma A C T H was assayed using a modification of the Incstar A C T H Equilibrium R I A kit (Incstar Inc, Stillwater, M N , USA) with all reagent volumes halved and 50ul plasma per tube. The antiserum cross-reacts 100% with Porcine  ACTH1.39  and Human  ACTH1.24  but shows less  than 0.01% cross-reactivity with melanocyte-stimulating hormone, endorphin, lipotropin, leucine enkaphalin, methionine enkephalin, bombesin, calcitonin, parathyroid hormone,  follicle-  stimulating hormone vasopressin, oxytocin, and substance-P (Orth, 1979). The mid-range intraand interassay coefficients of variation were 3.9% and 6.5%, respectively.  32  Table 1. Nutritional Content of E and PF Liquid Diets from Bio-Serv, Inc.  E Diet (kcal/L)  PF Diet (kcal/L)  Protein  258  258  Fat  255  255  Carbohydrate  118  486  Ethanol  368  0  Total  999  999  The actual values have a variability of less than or equal to 10% of the listed theoretical values due to analytical and sampling variability, and moisture levels.  33  C H A P T E R III; E F F E C T S OF P R E N A T A L E T H A N O L EXPOSURE AND POSTNATAL HANDLING ON CONDITIONED T A S T E AVERSION.  Introduction Among the most detrimental consequences of prenatal alcohol exposure in clinical populations are cognitive deficits and behavioral abnormalities (Coles, 1992; Conry, 1990; Streissguth, 1986; Streissguth et al., 1980; 1991). Intellectual functioning measured by IQ scores is typically below normal; a recent comprehensive study showed individuals with F A S and F A E to have mean IQ scores of 66 and 73, respectively (Streissguth et al., 1991). CNS dysfunction may also be manifested in a variety of behavioral abnormalities, including hyperactivity, poor attention span and inhibition skills, cognitive and perceptual problems, impaired habituation, and poor sensitivity to social cues (Streissguth, 1986).  Consistent  with these findings, experiments in laboratory animals have demonstrated that E animals show behavioral hyperactivity and hyperresponsiveness, including increased exploratory behavior (Riley et al., 1979c) and increased startle reactivity (Anandam et al., 1980) as well as deficits in passive avoidance learning (Gallo & Weinberg, 1982; Riley et al., 1979a; 1979b) and in taste aversion learning (Clausing et al., 1995; Driscoll et al., 1990; Riley et al., 1984), the latter suggesting that E animals are unable to inhibit consummatory behavior (withhold responses) in such tasks. These deficits in response inhibition following prenatal ethanol exposure may have analogies to the hyperactivity, attention deficits, and deficits in response inhibition observed in children with F A S . Conditioned taste aversion tasks are based on the finding that i f an animal consumes a novel fluid or food, and subsequently experiences illness, it will avoid that particular substance in the future (Garcia & Koelling, 1966). Conditioned taste aversion is a unique 34  phenomenon in that it can be learned following just one exposure to an association between the novel substance and the illness, and the delay between consumption and illness can be considerable.  In addition, the conditioned aversion is very difficult to extinguish,  suggesting that organisms may be predisposed to learn the association between taste and illness (Seligman, 1970). Upon first exposure to a novel substance, animals show neophobia and will consume only a small amount of the substance. decrease neophobia and increase intake.  Repeated exposures serve to  Therefore, pretoxicosis familiarity with the  conditioned substance may alter posttoxicosis consumption such that when animals are more familiar with the taste of the solution used for conditioned taste aversion (i.e. more pretoxicosis exposures and reduced neophobia) their posttoxicosis consumption is increased compared to that of animals for whom the taste is totally novel (Archer & Sjoden, 1979; Braveman & Crane, 1977).  Although this effect is most apparent when pretoxicosis consumption exceeds  approximately five grams, increased posttoxicosis consumption could be misinterpreted as weakened aversion in these conditions, complicating the assessment of conditioned taste aversion learning. Pituitary-adrenal responses upon reexposure to the conditioned substance may provide another means of measuring conditioned taste aversion learning.  If animals are  reexposed to the conditioned substance under non-deprived conditions, which is the typical procedure, they avoid ingesting it and typically do not show increased C O R T levels on the reexposure day. In contrast, i f animals are reexposed under conditions of food and water deprivation, they are deprived to ingest a limited amount of the conditioned substance upon reexposure and show significant C O R T elevations.  The deprivation paradigm is in fact a  model of conflict and the plasma CORT response appears to represent not an index of aversion but rather an index of conflict (Weinberg et al., 1978). 35  When animals are food and water  deprived, they are in a strong conflict situation; they are motivated to drink but presented only with a substance that has been paired with illness. Animals that show the greatest behavioral aversion (measured by consummatory behavior) also tend to show the largest elevations in C O R T in such conflict situations (Smotherman et al., 1976). As with measures of intake, pituitary-adrenal responses may also be altered by manipulating the number of exposures to the novel substance prior to conditioning. As familiarity with the substance increases, the magnitude of posttoxicosis intake tends to increase and the rise in plasma C O R T is attenuated (Weinberg et al., 1978). Postnatal handling appears to alter both performance on behavioral tasks and H P A responses to stressors (Denenberg & Zarrow, 1971; Levine et al., 1967; Meaney et al., 1989; Weinberg & Levine, 1977). H animals exhibit attenuated fearfulness or emotionality in novel environments such as the open field (Levine et al., 1967), and learn faster and make fewer errors in some avoidance learning paradigms (Weinberg & Levine, 1977). Postnatal handling has also been shown to reduce neophobia in adult animals (Ferre et al., 1995; Weinberg et al., 1978) as well as reduce the magnitude of an initial taste aversion and increase the rate of recovery of drinking to pretoxicosis levels (Weinberg et al., 1978). However, when taste aversion learning was tested following food deprivation, H animals showed lower C O R T levels than N H animals (Weinberg et al., 1978), indicating that the conflict of the situation may have been greater for N H animals. Although previous studies have found that E animals are less able to inhibit consummatory behavior in conditioned taste aversion tasks than controls (Clausing et al., 1995; Driscoll et al., 1990; Riley et al., 1984), no investigations of pituitary-adrenal function in E animals during conditioned taste aversion have been undertaken. Furthermore, previous studies have not assessed posttoxicosis intake in relation to pretoxicosis intake. 36  Both of  these variables may be important since E animals display H P A hyperresponsiveness to a variety of stressors (Taylor et al., 1983; 1984; Weinberg, 1992a), and E males show increased water consumption (Dow-Edwards et al., 1989; McGivern et al., 1998) compared to control animals. The current experiment investigated the hypothesis that E animals which underwent postnatal handling would show improved conditioned aversion learning and reduced H P A activity compared to E animals which did not experience handling and/or that postnatal handling might attenuate differences among E and control animals.  Methods Subjects Subjects were female and male rats from E, PF and C groups that were either handled or nonhandled from 1-15 days of age. Subjects were tested from 35 to 43 days of age. Pretoxicosis Exposure At 35 days of age, animals were weighed, singly-housed and water-deprived overnight. Animals were exposed to a sodium saccharin solution (30% by weight) for 30 min daily for five consecutive days, beginning at approximately 0900 hr each day (lights on 0830-2030 h). Animals were not water deprived after the first day of saccharin exposure. This number of preexposures has been shown to insure stabilized pretoxicosis drinking with the fewest number of preexposures possible (Weinberg et al., 1978).  This is important  because increased pretoxicosis intake may affect the strength of conditioning (Archer & Sjoden, 1979; Braveman & Crane, 1977).  A t approximately 1600 hr on day 4 of  pretoxicosis exposure, animals were weighed for determination of lithium chloride dose. On day 5 of pretoxicosis exposure (i.e. the conditioning day), animals received an injection of lithium chloride (0.40 M ; 7.5ml/kg i.p.) within 5-10 min after removal of the saccharin 37  solution.  Animals were then randomly assigned to either the non-deprived or deprived  reexposure condition. Posttoxicosis Exposure Non-Deprived. Animals were maintained with ad libitum access to food and water for 72 hr after lithium chloride injection. A l l animals were then reexposed to the saccharin solution for 30 min and blood samples were collected by decapitation immediately after reexposure. Decapitation was performed as discussed in the General Methods section. Deprived.  After the lithium chloride injection, animals were allowed 48 hr with ad  libitum access to food and water in order to recover from the illness.  Animals were then  subjected to 24 hr food plus water deprivation, and then reexposed to the saccharin solution for 30 min. Blood samples were collected by decapitation immediately after exposure. Statistical Analysis Pretoxicosis intake (ml/100 g bwt) was analyzed by four-way, repeated-measures A N O V A for sex (M, F), prenatal group (E, PF, C), postnatal treatment (H, N H ) and day (1-5). Posttoxicosis intake (as a percentage of pretoxicosis intake) and CORT levels for each reexposure condition were analyzed using three-way A N O V A s for sex, prenatal group, and postnatal treatment. When appropriate, data for each sex, postnatal treatment and/or prenatal group were analyzed by A N O V A s and were followed by Tukey post-hoc comparison tests. Dependent, one-tailed t-tests comparing pre- and posttoxicosis intake were used to assess the strength of the conditioned aversion.  38  Results Developmental Data. Maternal Data. Daily ethanol intake during gestation averaged 10.3 ± 0.98, 13.4 ± 1.13, and 12.2 ± 0.97 g/kg body weight for gestation weeks 1, 2, and 3, respectively.  Repeated-  measures A N O V A on maternal weight gain during gestation revealed significant main effects of prenatal group, F(2, 72) = 16.33, p < 0.01, and day, F(3, 216) = 1747.99, p < 0.01, and a prenatal group x day interaction, F(6, 216) = 18.35, p < 0.01 (Table 2). E and PF females had lower body weights than C females from G7 through G21 (p's < 0.01). Analysis of maternal body weight during lactation revealed significant main effects of prenatal group, F(2, 73) = 6.50, p < 0.01, and day, F ( l , 73) = 360.92, p < 0.01, and a postnatal treatment x day interaction, F ( l , 73) = 4.44, p < 0.04 (Table 2). Dams with pups in both the H and N H treatments gained weight across lactation (p's < 0.01), and E and PF females had lower body weights than C females during lactation (p's < 0.03). There was also a significant effect of prenatal group on gestation length, F(2, 73) = 5.71, p < 0.01 (Table 3), with E females having longer pregnancies than C females (p < 0.01). Pup Data. There was a significant effect of prenatal group, F(2, 76) = 4.52, p < 0.01, on the number of live born pups (Table 3); E dams had smaller litters than PF and C dams (p's < 0.04). Analysis of pup body weights revealed significant effects of prenatal group, F(2, 146) = 31.11, p < 0.01, sex, F ( l , 146) = 4.20, p < 0.05, and day, F ( l , 146) = 10305.56, p < 0.01, and a prenatal group x day interaction, F(2, 146) = 27.10, p < 0.01 (Table 4). At birth, E, PF and C pups did not differ in weight. However, at PN22, E pups weighed less than PF and C pups (p's < 0.01), and PF pups weighed less than C pups (p < 0.01). Overall, male pups weighed more than female pups. There were no significant effects of postnatal treatment on offspring body weight from PN1 to PN22.  39  Offspring Body Weight During Pretoxicosis Testing Analysis of body weight during testing (mean of weights on days 35 and 39 of age) revealed main effects of sex, F ( l , 219) = 266.12, p < 0.01, prenatal group, F(2, 219) = 21.51, p < 0.01, and postnatal treatment, F ( l , 219) = 9.05, p < 0.01, as well as a prenatal group x postnatal treatment interaction, F(2, 219) = 4.19, p < 0.02 (Figure 1). Males had higher body weights than females. Post-hoc analyses of the interaction revealed that H-E animals weighed more than N H E animals (p < 0.01). In addition, N H - E animals weighed less than NH-PF and N H - C animals (p's < 0.01), and NH-PF animals weighed less than N H - C animals (p < 0.02). Pretoxicosis Intake Due to differences in body weight among animals in different prenatal groups and postnatal treatments, measures of saccharin intake were adjusted for body weight (g/kg bwt). Overall analysis of pretoxicosis intake revealed significant main effects of sex, F ( l , 206) = 35.10, p < 0.01, postnatal treatment, F ( l , 206) = 18.53, p < 0.01, and day, F(4, 824) = 21.91, p < 0.01, as well as sex x day, F(4, 824) = 3.39, p < 0.01, prenatal group x day, F(8, 824) = 5.70, p < 0.01, and postnatal treatment x day, F(4, 824) = 6.75, p < 0.01, interactions. Posthoc analyses revealed that both females and males increased their intake over days (females: day 2 < days 4-5, p's < 0.03; males: days 1-3 < days 4-5, p's < 0.01), and males had higher intake than females on all days (p's < 0.03). Furthermore, E animals had higher intake on days 4 and 5 than PF and C animals (p's < 0.03), and higher intake on day 3 than PF animals (p < 0.04). Similarly, H animals had higher intake on days 3-5 than N H animals (p's < 0.01). Posttoxicosis Non-Deprived Reexposure Intake.  A dependent t-test showed a significant reduction in posttoxicosis intake  compared to the last day of pretoxicosis intake for all animals in the non-deprived reexposure condition, t(,108) = 13.00, p < 0.01, one-tailed. A n overall A N O V A on posttoxicosis intake 40  revealed significant main effects of sex, F ( l , 99) = 15.53, p < 0.01, prenatal group, F(2, 99) = 3.41, p < 0.04, and postnatal treatment, F ( l , 99) = 6.18, p < 0.01, as well as sex x postnatal treatment, F ( l , 99) = 5.18, p < 0.02, and prenatal group x postnatal treatment, F(2, 99) = 4.13, p < 0.02, interactions. Due to these interactions, reexposure intake (as a percentage of mean pretoxicosis intake) for females and males within each postnatal treatment were analyzed by separate one-way A N O V A s for prenatal group. Separate A N O V A s revealed significant main effects of prenatal group [F(2, 25) = 6.14, p's < 0.01, and F(2, 24) = 4.63, p's < 0.02, for females and males, respectively] (Figure 3) with E females and males in the H treatment having higher posttoxicosis intake than their PF and C counterparts (females: p's < 0.01; males: p's < 0.03). There were no significant effects of prenatal group on posttoxicosis intake in N H females or males. CORT Levels. As expected, females had significantly higher CORT than males, F ( l , 101) = 8.79, p < 0.01 (Figure 4). For females, there were no significant effects of prenatal group or postnatal treatment. For males, there was a marginal effect of postnatal treatment, F ( l , 52) = 3.46, p = 0.065 (Figure 4), with H males showing a trend toward lower C O R T than N H males. Posttoxicosis D e p r i v e d Reexposure  Intake.  A dependent t-test showed a significant reduction in posttoxicosis intake  compared to the last day of pretoxicosis intake, t(109) = 9.74, p < 0.01, one-tailed, for all animals in the deprived reexposure condition. In order to facilitate comparisons with data on intake during non-deprived reexposure, intake during deprived reexposure for females and males was analyzed by one-way A N O V A s for prenatal group within each postnatal treatment. For females in the H treatment, there was a main effect of prenatal group, F(2, 23) = 9.25, p < 0.01, with H PF females having higher posttoxicosis intake than H-E and H-C females (p's < 0.01), indicating an effect of pair-feeding (Figure 3). For females in the N H treatment and for males in the H and 41  NH treatments, there were no effects of prenatal group or postnatal treatment on posttoxicosis intake during deprived reexposure (Figure 3). CORT. As expected, females had significantly higher CORT than males, F(l, 110) = 29.51, p < 0.01. For females, there was a main effect of postnatal treatment on CORT during reexposure, F(l, 55) = 5.99, p < 0.02 (Figure 4), with H females having lower CORT than NH females across all prenatal groups. For males, there was also an effect of postnatal treatment, F(l, 55) = 5.84, p < 0.02. However, when data were analyzed for males in each prenatal group, one-way ANOVAs for postnatal treatment revealed that H marginally lowered CORT in PF and C but not in E males compared to their NH counterparts [F(l, 17) = 3.75, p = 0.07; F(l, 20) = 3.30, p = 0.08, for PF and C, respectively] (Figure 4).  Discussion The present study extends previous work in our lab (Weinberg et al., 1995) and others (Hannigan et al., 1993; Ogilvie & Rivier, 1997) examining the long-term adverse effects of prenatal ethanol exposure and the role of early postnatal experience in modulating the adverse behavioral and physiological effects of prenatal ethanol exposure. These experiments also extend prior investigations of conditioned taste aversion by assessing intake over repeated pretoxicosis exposures as well as hormonal responsiveness to conditioned taste aversion under different reexposure conditions. In the present study, prenatal ethanol exposure had direct effects in lowering weights and increasing pretoxicosis intake in E compared to PF and C animals. Postnatal handling differentially affected E, PF and C animals on several measures, attenuating weight deficits in E animals at the time of testing, reducing posttoxicosis intake in H-PF and H-C compared to H-E animals during non-deprived reexposure, and reducing posttoxicosis intake of H-E and H-C compared to H-PF animals during deprived reexposure. Furthermore, in the 42  deprived reexposure condition, handling reduced CORT levels in PF and C but not E males compared to their N H counterparts. Postnatal handling also had effects across prenatal groups, increasing pretoxicosis intake, and reducing C O R T levels in males during non-deprived reexposure and in females during deprived reexposure. The data demonstrating adverse effects of maternal ethanol consumption on maternal weights, pregnancy outcome (i.e. length of gestation and litter size), and offspring weight gain are consistent with previous data both from our laboratory (Weinberg et al., 1995; Weinberg, 1985) and from others (Abel & Dintcheff, 1979; Chernoff, 1977). Maternal ethanol intake was consistently high throughout gestation, ranging from 10.3 to 13.4 over the 3 weeks of gestation. We have shown previously that this range of ethanol intake results in blood ethanol levels of approximately 145-155 mg/dl (Weinberg, 1985; Osborn et al., 1996). The finding that E and PF dams were similar in body weights through most of gestation and lactation, and that pup weights at weaning were reduced in both E and PF compared to C animals indicates that the suppressed maternal weight gain in E dams during gestation and lactation, and the reduced body weights of E pups were in part mediated by ethanol-induced nutritional effects. However, direct effects of ethanol were also observed. E dams had longer gestation lengths than C dams and had smaller litters than both PF and C dams. In addition, although PF pups had lower weights at weaning compared to C pups, E pups weighed significantly less than both PF and C pups. Previous work from our laboratory has shown that handling may, under some conditions, attenuate the adverse effects of prenatal ethanol exposure on pup weight gain during the preweaning period (Weinberg et al., 1995). In the present study, there were no significant effects of handling on offspring preweaning body weights, but handling did attenuate weight differences in postweaning E animals. Manipulations such as postnatal handling (Ogilvie & Rivier, 1997) and environmental enrichment (Hannigan et al., 1993) have not consistently produced differences 43  in body weight gain, perhaps due to differences in techniques or in the age of the animal when body weight was assessed. Altered suckling behaviors in rat pups prenatally exposed to ethanol have been shown, with E pups displaying longer latencies to attach to the nipple, as well as failure to nipple shift (Chen et al., 1982; Rockwood & Riley, 1990). Postnatal manipulations may not always be able to ameliorate weight differences at all ages given such feeding deficiencies. In the present study, both prenatal ethanol exposure and postnatal handling resulted in increased consumption of the saccharin solution over the five preexposure days, suggesting that neophobia decreased at a faster rate in animals in these conditions. Typically, rats exhibit innate neophobic response to novel foods, consuming only a small amount initially, and thus testing its palatability (Franchina & Dyer, 1985; Franchina & Slank, 1989).  This phenomenon has an  obvious survival value for an omnivorous species; individuals with a strong neophobic response may survive, even i f the substance is poisonous.  Alternatively, it has been proposed that  neophobia may be one means by which an animal learns the metabolic consequences of eating a particular food and, therefore, how much it can comfortably ingest at one time. A neophobic reaction might enable an animal to minimize homeostatic disturbances during its initial encounter with an unfamiliar diet (Kronenberger & Medioni, 1985; Woods, 1991). In the present study, differences in neophobia may have been due, in part, to differences in emotional reactivity or in response inhibition. Animals that have been preexposed to novel tastes are similar to those that have been preexposed to novel environmental stimuli in that they are generally less emotionally reactive than animals that have not been preexposed (Braveman, 1978). Thus, repeated exposure to the saccharin solution over the five pretoxicosis exposure days may have led to more rapid reductions in emotional reactivity in H compared to N H animals and, thus, reductions in 44  neophobia. Previous studies have shown that H animals tend to be less emotionally reactive (Ader, 1965) and exhibit reduced neophobia as adults (Weinberg et al., 1978). In fact, levels of emotional reactivity in H and N H animals have been directly linked to neophobia. Ferre et al. (1995) reported that H animals showed reduced emotional reactivity and neophobia compared to N H animals, and animals with the highest emotionality scores (preferentially N H animals) were also those that showed the longest latencies to begin eating and spent less time eating in a neophobic situation. In contrast, prenatal ethanol exposure typically does not result in decreased emotionality. E animals show behavioral hyperactivity in novel situations such as the open field (Osborne et al., 1980) and hyperactivity to acoustic startle stimuli (Anadam et al., 1980) as well as deficits in response inhibition in passive avoidance tasks (Riley et al., 1979a; 1979b) and in a punished step-down task (Gallo & Weinberg, 1982), although preweaning handling eliminated this latter deficit. Thus, the increased pretoxicosis intake observed in E animals in the present study may have been due to deficits in response inhibition. Weakened taste aversion learning in E animals compared to controls in standard conditioned taste aversion tasks (Driscoll et al., 1990; Riley et al., 1984) and when lithium chloride was presented directly in the drinking water (Riley et al., 1979a) have been interpreted as further evidence of deficits in response inhibition. Even under conditions in which E animals acquired conditioned aversions, retention of the aversion was impaired, suggestive of an inability to inhibit consummatory behavior even when the association between the solution and illness had been learned.  Thus, it is possible that the increase in  pretoxicosis intake observed in E and H animals compared to their counterparts may have been mediated through two distinct mechanisms.  That is, elevations in pretoxicosis intake in H  animals may have been due to general reductions in emotional reactivity while the elevations in pretoxicosis intake in E animals may have been the result of deficits in response inhibition. 45  Handling differentially affected E, PF and C animals, reducing posttoxicosis intake in PF and C but not E females and males during non-deprived reexposure and reducing posttoxicosis intake in E and C but not PF females during deprived reexposure. It is possible that handling differentially affected maturation in E, PF and C animals. Increased weight (Weinberg et al., 1995) and earlier maturation of sensorimotor skills (Chapillon et al., 1998) have been reported in handled animals, suggesting that handling may increase the rate of maturation of some systems. Previous research has found that adrenergic excitatory areas in the brainstem mature more rapidly than cholinergic inhibitory areas in the forebrain (Campbell et al., 1969). In the present experiment, handling may have accelerated the maturation of inhibitory systems in C animals while not producing a similar effect in PF and E animals, resulting in greater inhibition of consummately behavior (i.e. lower posttoxicosis intake) in C animals.  Alternatively, both  handling and prenatal ethanol exposure increased pretoxicosis consumption, thereby decreasing the novelty of the conditioned substance and increasing the difficulty of associating the saccharin solution with subsequent illness. Increased intake following safe preexposures may reduce the general salience of the solution, suggesting a form of learned meaningless or learned irrelevance (Best, 1975). However, pretoxicosis intake was also elevated in N H - E animals compared to N H PF and N H - C animals and in H-C animals compared to N H - C animals, and these groups did not show differences in posttoxicosis consumption, indicating that increased pretoxicosis intake need not necessarily result in increased posttoxicosis consumption. Therefore, while the task may have been more difficult for H - E animals and H-PF females due to their elevated pretoxicosis intake, these animals still displayed difficulty in inhibiting further intake of the solution compared to H C animals, possibly indicating a failure to discriminate the relevance of the saccharin solution. As previously mentioned, postnatal handling also resulted in increased posttoxicosis intake in PF females during deprived reexposure, indicating an effect of pair-feeding. Pair46  feeding provides a necessary nutritional control group; however, PF dams may experience stress derived from their restricted meal-feeding schedule. Although E females have ad libitum access to food, they consume less than they would i f their diets did not contain ethanol. Therefore, PF dams receive a ration that is less than they would normally consume, and typically consume this ration within a few hours after it is presented. possible stress, whereas E dams do not.  Thus, PF dams experience deprivation and  Although previous investigators have found that  neonatal handling reversed behavioral abnormalities on the open field and plus maze induced by prenatal random noise and light stress (Wakshlak & Weinstock, 1989), the current finding indicates that PF animals, with their possible exposure to prenatal stress, may respond differentially to postnatal handling. Handling differentially affected CORT levels in E , PF and C males; handling lowered CORT in PF and C but not in E males compared to their N H counterparts during deprived reexposure.  This difference in CORT activity was not associated with alterations in  posttoxicosis intake; E , PF and C males in the H treatment all showed similar posttoxicosis consumption.  It has been previously reported that animals which drink the least during  reexposure also show the greatest elevations of CORT, indicating that these animals may experience increased conflict in the situation. Conversely, as consumption increases, elevations in CORT decrease, indicating a lower level of conflict (Weinberg et al., 1978). Thus, it appears that H - E males experienced more conflict during deprived reexposure than did H-PF and H-C males even though their level of consumption did not differ. Dissociations between intake and CORT levels were also demonstrated in H - E animals during non-deprived reexposure and H-PF females during deprived reexposure; as just discussed, these animals showed increased posttoxicosis intake but they did not display alterations in C O R T activity. Previously it has been shown that elevations in CORT levels occur only under deprivation conditions and not under 47  non-deprived conditions (Weinberg et al., 1978). Therefore, the fact that H-E animals displayed increased posttoxicosis consumption but no decrease in CORT activity during non-deprived reexposure is not surprising, especially considering the low, basal CORT levels that were present in all animals during non-deprived reexposure. However, increased posttoxicosis consumption in H-PF females during deprived reexposure was not associated with alterations in CORT levels compared to H-E and H-C females, emphasizing the importance of assessing both behavior and H P A function when examining an animal's response to a task. Prenatal ethanol exposure has been shown to produce abnormalities in the hippocampus (Sutherland et al., 1997; West & Hamre, 1985; Wigal & Amsel, 1990) including altered hippocampal theta activity (Cortese et al., 1997). Theta activity has been associated with information processing of sensory stimuli (Bland,  1986) and may be important in  associational learning such as conditioned taste aversion. The hippocampus has also been postulated to play a role in response inhibition and in delayed associative learning (Gray & McNaughton, 1983), further indicating that it may play a role in conditioned taste aversion (Gaston, 1978; Riley et al., 1986). Since hippocampectomized animals have been shown to display attenuated taste aversion learning (Krane et al., 1976) and prenatal ethanol exposure produces abnormalities in the hippocampus, it has been proposed that impaired conditioned taste aversion in E animals may be related to hippocampal damage (Driscoll et al., 1985). Although postnatal treatments have been shown to improve performance in E animals on behavioral tasks mediated by the hippocampus such as the Morris water maze (Hannigan et. al., 1993), environmental enrichment has not been found to attenuate alterations in hippocampal structure in E animals (Berman et al., 1996), indicating that postnatal manipulations may be limited in their effectiveness.  The present finding that handling did not attenuate impaired  conditioned taste aversion performance in E animals further underscores the lasting effect of 48  prenatal ethanol exposure, especially in regard to behaviors associated with hippocampal function. It is possible that the differential effects of handling observed among E , PF and C animals may be mediated by differences in mother-pup interactions. The effects of postnatal handling appear to be, at least partly, due to specific maternal behaviors that occur in response to pup cues when pups are returned to the nest following handling (Smotherman & Bell, 1980). It has been reported that briefly separating pups from their mothers results in more frequent feeding bouts (of reduced duration) coupled with increased grooming and licking of pups (Liu et al., 1997), resulting in greater stimulation for H pups. H pups also show increased ultrasonic vocalizations (Bell et al., 1971) and such calls may elicit increased maternal care (e.g. retrieval). In contrast, reductions in the rate of ultrasonic calling by pups have been reported following exposure to behavioral teratogens such as ethanol (Kehoe & Shoemaker, 1991). Prenatal ethanol exposure has been shown to decrease vocalizations during a five minute isolation test and attenuate the normal increase in calling observed following injection with the opiate antagonist, naltrexone (Kehoe & Shoemaker, 1991). In contrast, prenatal stress has been shown to increase ultrasonic vocalizations during isolation although this may vary depending upon the gestational period in which the stressor was applied (Williams et al., 1997). Prenatal ethanol exposure has also been shown to produce deficits in homing development (Gallo & Weinberg, 1982) that may alter dam retrieval behavior (Fernandez et al., 1983). Thus, it appears that maternal behaviors arise from complex interactions with the pup, and, therefore, it is possible that postnatal handling might exert differential effects on litters in which pup behavior has already been altered by exposure to prenatal stress or to prenatal ethanol. Across prenatal groups, postnatal handling reduced "CORT responses in females during deprived reexposure, and in males during non-deprived reexposure. 49  These findings extend  previous research showing reductions in H P A activity in H compared to N H animals during exposure to stressors (Durand et al., 1998; Meaney et al., 1989; Vallee et a l , 1997). This study also extends previous investigations of conditioned taste aversion (Weinberg et al., 1978) to include findings of reduced C O R T in H females as well as reductions in C O R T levels in males during non-deprived reexposure. The data demonstrating effects of postnatal handling in females is particularly interesting considering that many studies examine handling only in males. This may be important since both the H and N H treatments differ from normal rearing conditions and data regarding their effects on sex-typical behaviors are lacking. The sexually dimorphic nucleus of the preoptic area is sensitive to hormone action from G18 through PN5 (Rhees et al., 1990), creating a timeframe when postnatal handling may impact on the development of sex-typical behaviors. Furthermore, maternal behavior, particularly anogenital licking (Melniczek & Ward, 1994; Moore, 1992), has been shown to differ between male and female offspring, playing a role in future sexual development (Moore, 1992), and the effects of postnatal handling also appear to be mediated by maternal behavior.  In the present experiment, males had higher pretoxicosis  intake of saccharin than females, a finding that contrasts with previous reports that females typically consume more saccharin solution than males (Beatty, 1979), and indicates the importance of research examining the effects of postnatal treatments on sex-typical behaviors. In summary, prenatal ethanol exposure and postnatal handling independently resulted in an increased rate of consumption of a saccharin solution over five days of exposure, suggesting that neophobia decreased at a faster rate in these animals. However, findings from previous research suggest that the increases in pretoxicosis intake observed in the present study may be mediated through two distinct mechanisms; elevations in pretoxicosis intake in H animals may have been due to general reductions in emotional reactivity "and elevations in pretoxicosis intake in E animals may have been the result of deficits in response inhibition. Handling differentially 50  affected conditioned taste aversion acquisition in E, PF and C animals.  It is possible that  handling accelerated the maturation of inhibitory systems in C compared to E and PF animals, resulting in greater inhibition of consummatory behavior (i.e. lower posttoxicosis intake). Handling also differentially affected CORT levels in E, PF and C animals; lowering CORT in PF and C but not in E males compared to their N H counterparts. Because handling may be mediated through mother-pup interactions, postnatal handling might exert differential effects on litters in which pup behavior has already been altered by prenatal treatments such as exposure to ethanol or pair-feeding, underscoring the importance of further research into the effects and mechanisms of such postnatal manipulations.  51  Table 2 Maternal Body Weights (g, Mean ± SEM) of E, PF and C Dams during Gestation (G), and of E, PF and C Dams in H and N H Treatments During Lactation (PN1, PN22).  Gestation  GI  G7  G14  G21  E  232.3 ± 2 . 3  241.2 ± 2 . 8 *  275.0 ± 3 . 6 *  335.8 ± 5 . 0 *  PF  236.0±3.0  243.1 ± 3 . 7 *  273.5 ± 4.2*  350.0 ± 5 . 7 *  C  242.3 ± 5.3  267.1 ± 3 . 4 *  302.7 ± 3.7*  391.2 ± 5 . 9 *  NH  H PN22  PN1  PN22  +  Lactation  PN1  E  293.4 ± 9.3  327.0 ± 5 . 8  274.4 ± 5.8  332.3 ± 5.6  A  288.0 ± 8 . 4  332.9 ± 4 . 1  285.8 ± 6 . 5  339.6 ± 5 . 2  A  299.1 ± 5 . 1  347.6 ± 3.8  304.7 ± 6.7  351.7 ± 5 . 8  A  PF  C  Gestation:  +  +  +  E (n = 28); PF (n = 26); C (n = 25). • A t G7, G14 & G21, E - PF < C, p's < 0.05  Lactation:  H-E (n = 14); N H - E (n = 14); H-PF (n = 13); NH-PF (n = 13); H-C (n = 13); N H - C (n= 12). +  A  A t PN1 & PN22, H > N H , p's < 0.05 A t PN1 & PN22, E = PF < C, p's < 0.05  52  Table 3 Gestation Length (d, Mean ± SEM) and Number of Live-Born Pups (Mean ± SEM) of E, PF and C Dams. Gestational Length E  23.2 ± 0.09®  PF  23.1 ±0.08  C  22.9 ± 0.06®  Live-Born Pups E  13.0 ± 0 . 5 "  PF  14.6±0.3  C  14.6 ± 0.5  E (n = 28); PF (n = 26); C (n = 25). Gestation Length: Live-Born Pups:  ® E > C, p < 0.05 b  E < C, p < 0.05  53  b  Table 4 Postnatal (PN1, PN22) Pup Body Weights (g, Mean ± SEM) of E, PF and C Female and Male Pups in H and N H Treatments.  II  N  Female*  PN1  PN22  E  5.5 + 0.1  40.9 ± 1.5  PF  5.5 ± 0 . 1  46.4 ± 2.3  C  5.9 ± 0 . 1  47.8 ± 1.3  A  A  A  PN1  PN22  5.5 ± 0 . 1  41.1 ± 1.2  5.5 ± 0 . 1  46.6 ± 0.6  6.0 ± 0 . 1  49.5 ± 1.1  II  A  N  Male*  PN1  PN22  E  5.7 ± 0 . 1  43.4 ± 1.4  PF  5.8 ± 0 . 1  47.4 ± 2.2  C  6.2 ± 0 . 1  49.1 ± 1.1  A  A  A  H  A  A  H  PN1  PN22  5.7 ± 0 . 1  42.5 ± 1.2  5.8 ± 0 . 1  47.3 ± 0.9  A  6.4 ± 0 . 1  51.1 ± 1.1  A  A  H-E (n = 14 litters); N H - E (n = 14 litters); H-PF (n = 13 litters); NH-PF (n = 13 litters); H - C (n = 13 litters); N H - C (n = 12 litters).  Pup Weights: *Female < Male A  AtPN22, E<PF<C,p's<0.05  54  Figure 1. Average body weight (g, Mean ± SEM) of E , PF and C females (left panels) and males (right panels) in H and N H treatments (n's = 9-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). Males > Females (p < 0.01); N H - E < NH-PF < N H - C (p's < 0.02); H-E > N H - E (p < 0.01).  55  56  Figure 2. Pretoxicosis saccharin intake (g/kg bwt) for E, PF and C females (left panels) and males (right panels) in H and N H treatments (n's = 9-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). Males > Females on all days (p's < 0.03). E d 4-5 > PF and C d 4-5 (p's < 0.03), and E d 3 > PF d 3 (p < 0.04). H d 3-5 > N H d 3-5 (p's < 0.01).  57  Day  1  NH - +-  Ethanol  - ° ~ Pair-Fed  58  Control  Figure 3. Posttoxicosis saccharin intake (g/kg bwt/avg pretoxicosis intake, Mean ± SEM) for E, PF and C females (left panels) and males (right panels) in H and N H treatments during nondeprived (top panels) and deprived (bottom panels) reexposure (n's = 8-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). During non-deprived reexposure, H-E females and males > H-PF, H-C females and males (p's < 0.05). During deprived reexposure, H PF females > H-E, H-C females (p's < 0.05).  59  Males  H  NH  H  NH  Non-Deprived  Non-Deprived  H  H  NH  NH  Deprived  Deprived Pair-Fed  Ethanol  60  Control  Figure 4. Plasma C O R T levels (p-g/dl, Mean ± SEM) for E, PF and C females (left panels) and males (right panels) in H and N H treatments during non-deprived (top panels) and deprived (bottom panels) reexposure (n's = 8-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). During non-deprived reexposure, H males < N H males (p < 0.05). During deprived reexposure, H females < N H females (p < 0.05); H-PF males < NH-PF males; H-C males < N H - C males (p's < 0.05).  61  Nil  H  H  Non-Deprived  NH  Non-Deprived Males  H  H  NH  Deprived  Deprived Ethanol  NH  H Pair-Fed  62  Control  CHAPTER IV: EFFECTS OF PRENATAL ETHANOL EXPOSURE AND AGING ON MORRIS WATER M A Z E PERFORMANCE ARE NOT ATTENUATED BY POSTNATAL HANDLING  Introduction Prenatal ethanol exposure has been associated with performance deficits on a number of spatial learning and memory tasks such as the radial arm maze (Omoto et al., 1993; Reyes et al., 1989), T-maze (Lochry et al., 1985; Nagahara & Handa, 1997; Zimmerberg et al., 1991), spatial lever response (Zimmerberg et al., 1989) and Morris water maze (Blanchard et al., 1987; 1990; Gianoliakos, 1990; K i m et al., 1997). In the Morris water maze, rats are placed in a circular pool filled with opaque water from which they can escape by locating and climbing onto a submerged platform. Distal spatial cues in the testing room are used by the rat to locate the submerged, hidden platform, and normal animals readily learn to perform this task (Morris, 1984). Importantly, an enriched postweaning environment has been show to attenuate deficits in motor performance in E animals, such as gait ataxia, and to improve performance in the Morris water maze in E animals when compared to E animals reared in isolation (Hannigan et al., 1993; Wainwright et al., 1993). Aging has also been shown to impair performance in tasks requiring the retention of spatial information (Gallagher & Burwell, 1989; Pelleymounter et al., 1987). Age-related deficits in memory, particularly in tasks that require the retention and/or integration of spatial information such as in the Morris water maze, have been reported as early as 12 months of age (Aitken & Meaney, 1989; Frick et al., 1995; Schukitt-Hale et al., 1998) and decrements in performance are commonly reported by 24 months of age (Fischer et al., 1990; Frick et al., 1995).  However, the rate at which spatial deficits appear may be 63  differentially affected by early experience. In particular, postnatal handling has been shown to attenuate performance deficits on the Morris water maze that are associated with aging. Whereas H animals show little or no spatial memory impairments at 12- or 24 months compared to 6 months of age, N H animals show Morris water maze deficits at 12- and 24 months of age compared with either age-matched H animals or 6 month old N H animals (Meaney et al., 1988; 1991). A possible mechanism mediating deficits in both E and aged animals on spatial tasks involves alterations in the hippocampus. Performance on the Morris water maze is disrupted by bilateral lesions to the hippocampus. Hippocampal-lesioned rats take longer to learn to reach the platform and travel more circuitous routes (Morris et al., 1982), thereby supporting a role for the hippocampus in spatial learning and memory. Rats that have been prenatally exposed to ethanol display hippocampal abnormalities, including changes in mossy fiber branching and hippocampal pyramidal cell number and arborization, and reductions in hippocampal synaptic plasticity (Sutherland et al., 1997; Ward & West, 1992; West & Pierce, 1986). There is also evidence that the aging hippocampus undergoes progressive changes in calcium homeostasis (Landfield & Eldridge, 1994), in the plasticity of responses to glucocorticoids (Landfield et al., 1978; Sapolsky et al., 1985) and in the expression of markers related to neuroprotection and damage (Sugaya et al., 1996).  Furthermore, the  attenuation of spatial deficits in mid-aged and aged H animals occurs alongside reductions in hippocampal cell loss compared to N H animals. It has been hypothesized that reductions in the rate of cognitive aging observed in H animals may be due to a lifetime of lower H P A reactivity (Meaney et al., 1991). In normal animals, hippocampal cell loss can be prevented by adrenalectomy at mid-age (Landfield et al., 1981) or accelerated by chronic exposure to C O R T (Sapolsky et al., 1985), indicating 64  that the decreased hippocampal degeneration observed in aged H animals may be mediated by their lower cumulative glucocorticoid exposure compared to N H animals. In contrast to the effects of handling, prenatal ethanol exposure results in prolonged and/or enhanced A C T H and C O R T responses to a variety of stressors (Angelogianni & Gianoulakis, 1989; Taylor et al., 1982; 1983; Weinberg, 1992a; 1993).  Therefore, not only may impaired  performance on spatial learning and memory tasks in E animals be related to stress-induced performance deficits associated with increased H P A activity, but aging E animals may also experience an accelerated rate of impairment in hippocampal-mediated functions. Although this remains to be investigated, research demonstrating accelerated aging in E animals, including a shortened lifespan in E animals (Abel et al., 1987) and an earlier loss of reproductive function in E female rats (McGivern et al., 1995) alludes to the possibility that E animals may experience an increased rate of aging for physiological functions. The current experiment was designed to assess spatial learning and memory in young adult and mid-aged H and N H E and control animals using a Morris water maze. We examined the hypothesis that postnatal handling would improve spatial navigation in E animals compared to E animals that did not experience handling and/or attenuate differences among E and control animals and that this effect might be age-dependent. We also investigated whether deficits in mid-aged animals would correspond to increases in CORT levels on the last day of testing.  Methods Subjects Subjects were adult male rats from E, PF and C groups that were either handled or nonhandled from 1-15 days of age. Separate subjects were tested at 2- or 13-14 months of age.  65  M o r r i s Water Maze A circular pool (180 cm diameter x 60 cm high), filled to the 22-cm level with water made opaque with nontoxic powdered white paint, was installed in a 3 x 3 m room with no windows, diffuse light sources, and numerous salient and distinct extramaze visual cues. The pool was divided into quadrants, apparent only to the experimenters, and marked by the four compass points (N, S, E , W). A circular platform (12 cm diameter x 18 cm high) was placed within the pool, submerged 3-4 cm below the level of the water, into the center of the "target" quadrant.  During testing, the experimenters remained concealed behind a screen  and observed the pool by means of a video monitor mounted above the pool. Experimental Procedure A l l testing occurred 1-4 hrs after lights on (lights on 0700-1900). One day prior to testing, each animal was placed in the pool and allowed to swim for 90 sec in order to familiarize the animals with the apparatus.  Each animal received 20 trials with the  submerged platform over 4 days of testing (5 trials per day). Each trial consisted of placing the rat in the water at one of four starting points (designated by the compass points), facing away from the center of the pool.  The rats were required to swim until they found, and  climbed onto the submerged platform or until a limit of 90 sec was reached, at which point the animals were directed to the location of the platform. Once the rat had climbed onto the platform, the rat was left there for a period of 10 sec before being removed to a holding cage for a minimum of 60 sec before the next trial. After testing on the third day, two additional trials were conducted. First, the platform was removed and animals were allowed to swim freely for 90 sec while their search pattern was recorded. Then, the platform was returned to the "target" quadrant but elevated above the surface of the water. Animals could use visual cues (the platform) instead of distal spatial cues, and the swimming ability (latency to reach 66  elevated platform) of each animal was assessed. On the last (4 ) day of testing, the platform th  was shifted to a new "target" quadrant on the opposite side of the pool. After 5 trials, the platform was removed in order to record the search pattern for the new "target" quadrant. Immediately after this probe trial (approximately 20-25 minutes after the start of testing), blood was collected via cardiac puncture for determination of C O R T levels from mid-aged animals. Video recordings were used to determine escape latency (sec) and distance travelled to reach the submerged platform (m) on each trial. For the trials without a platform, search pattern was assessed for the first 30 seconds of the trial, including the number of annulus crossings and time spent in that day's "target" quadrant.  For the visible platform trial,  latency to reach the platform was determined. Blood Sampling A l l blood sampling was by cardiac puncture, as discussed in the General Methods section. Statistical Analysis. Mean escape latency and distance to locating the platform were calculated for each animal on each of the four testing days. Means were then analyzed by four-way repeatedmeasures A N O V A s for age (2 month, 13-14 months), prenatal group (E, PF, C), postnatal treatment (H, N H ) and day of testing (1-4). Data on search pattern and latency to reach the visible platform were analyzed using 3-way A N O V A s for age, prenatal group and postnatal treatment. When appropriate, data for each age, postnatal treatment and/or prenatal group were analyzed by A N O V A s and were followed by Tukey post-hoc comparison tests.  67  Results Developmental Data Maternal and pup data were discussed in Chapter III. Separate analysis of data for male pups is presented in this section. Male Pup Data.  Analysis of male pup body weights revealed significant effects of  prenatal group, F(2, 73) = 14.74, p < 0.01, and day, F ( l , 73) = 5486.65, p < 0.01, and a prenatal group x day interaction, F(2, 73) = 12.57, p < 0.01 (Table 4). At birth, E, PF and C male pups did not differ in weight. However, at PN22, E males weighed less than PF and C males (p's < 0.01), and PF males showed a trend toward lower body weights than C males (p - 0.07). There were no significant effects of postnatal treatment on body weight for male pups from PN1 to PN22. Escape Latency Importantly, a repeated-measures A N O V A on mean latency revealed significant main effects of age, F ( l , 103) = 5.63, p < 0.02, prenatal group, F(2, 103) = 3.76, p < 0.03, and day, F(3, 309) = 173.55, p < 0.01, as well as age x day, F(3, 309) = 2.69, p < 0.04, and postnatal treatment x day, F(3, 309) = 3.74, p < 0.01, interactions. Post-hoc analyses revealed that midaged animals had longer escape latencies than young animals on days 1 (p < 0.01) and 2 (p = 0.07), and that escape latencies for both H and N H animals decreased after day 1 (p's < 0.01). Due to these interactions, data for young and mid-aged animals within each postnatal treatment were analyzed by separate one-way, repeated-measures A N O V A s for prenatal group. Young Animals.  Analysis of data from H animals revealed main effects of prenatal  group, F ( l , 27) = 4.21, p < 0.03, and day, F(3, 81) = 47.08, p < 0.01 (Figure 5). Young H - E animals had longer escape latencies than H-C (p < 0.04) animals and marginally longer than H PF (p = 0.08) animals. Generally, escape latencies decreased over the first three days (day 1 > days 2-4; day 2 > day 3) but increased on the last day (day 4 > day 3)(p's < 0.05). In contrast, 68  analysis of data from N H animals revealed a main effect of day, F(3, 81) = 43.87, p < 0.05 (Figure 5), with escape latencies decreasing after the first day (day 1 > days 2-4; p's < 0.01). Mid-Aged Animals. Separate analysis of mid-aged animals in H and N H treatments revealed significant main effects of day for both [F(3, 72) = 48.30, p < 0.01, and F(3, 75) = 37.84, p < 0.01, for H and NH animals, respectively] (Figure 5). Escape latencies decreased after day 1 (day 1 > days 2-4; p's < 0.01) for H and NH animals. Raised Platform. On latency to reach the raised platform, there were no significant differences due to age, prenatal group or postnatal treatment (Table 5). Distance  There were no effects of age on distance to reach the platform. However, data for young and mid-aged animals within each postnatal treatment were analyzed separately in order to facilitate comparisons with data on escape latency. Young Animals. Analysis of data from H animals revealed main effects of prenatal group, F(l, 27) = 5.31, p < 0.01, and day, F(3, 81) - 41.11, p < 0.01 (Figure 6). Young H-E animals traveled longer distances than H-PF (p < 0.02) and marginally longer distances than H-C (p = 0.07) animals. Across all prenatal groups, distance traveled decreased after day 1 (day 1 > days 2-4; p's < 0.01). In the NH treatment, there was only a main effect of day, F(3, 81) = 53.78, p < 0.01, with distance decreasing after day 1 (day 1 > days 2-4; p's < 0.01). Mid-Aged Animals. Separate analysis of mid-aged animals in H and N H treatments revealed significant main effects of day for both [F(3, 72) = 68.74, p < 0.01, and F(3, 75) = 40.15, p < 0.01, for H and NH animals, respectively] (Figure 6). Distance travelled decreased over days in the H treatment (day 1 > days 2-4; day 2 > days 3-4; p's < 0.04), and after day 1 in the NH treatment (day 1 > days 2-4; p's < 0.01).  69  Search Pattern There were no effects of age, prenatal group or postnatal treatment on annulus crossings when the platform was removed on day 3 or on time spent in the "target" quadrant on either day 3 or 4 of testing. Analysis of data on annulus crossings when the platform was removed on day 4 revealed significant main effects of age, F ( l , 103) = 5.53, p < 0.02 (Figure 7); mid-aged animals had fewer annulus crossings than did young animals. Data for young and mid-aged animals were analyzed separately in order to facilitate comparisons with data on escape latency. For young animals, there was an effect of prenatal group, F(2, 54) = 3.71, p < 0.04 (Figure 7); E animals had fewer annulus crossings than did PF and C animals (p's < 0.04).  For mid-aged animals, there were no effects of prenatal group or  postnatal treatment on annulus crossings. CORT  Mid-Aged Animals. There was a significant effect of prenatal group, F(2, 45) = 3.77, p < 0.03 (Figure 8); overall, E animals had higher plasma C O R T levels than C animals (p < 0.03). There were no significant effects of postnatal treatment on C O R T levels.  Discussion The present study extends previous work in our lab (Weinberg et al., 1995) and others (Hannigan et al., 1993; Ogilvie & Rivier, 1997; Wainwright et al., 1993) examining the long-term adverse effects of prenatal ethanol exposure and the role of postnatal experience in modulating these effects.  The present findings also continue previous work in our laboratory  assessing the relationship between H P A activity and performance on behavioral tasks in E animals (Osborn et al., 1998; Weinberg, 1988). Investigation of spatial abilities in the Morris water maze revealed that young H-E males had longer latencies and traveled greater distances 70  prior to locating the submerged platform than did their PF and C counterparts.  Furthermore, E  animals displayed an altered search pattern compared to PF and C animals, supporting previous findings that prenatal ethanol exposure produces deficits in spatial navigation (Blanchard et al., 1987; 1990; Gianoulakis, 1990; K i m et al., 1997). Although mid-aged E, PF and C animals did not differ in performance on the Morris water maze, CORT levels were elevated in mid-aged E compared to C animals, indicating H P A hyperresponsiveness in E males. Finally, there were overall effects of age; mid-aged animals showed longer escape latencies and an altered search pattern compared to young animals. Handling did not appear to attenuate impairments associated with either prenatal ethanol exposure or aging In the present experiment, handling revealed deficits in spatial navigation in young E animals compared to PF and C animals.  These performance deficits appear to result from  impairments in spatial learning and memory and not from extraneous factors such as swimming ability or visual acuity, since there were no significant differences among groups in latency to reach a visible platform.  These findings correspond with previous research demonstrating  impaired spatial learning and memory in E animals on such tasks as the radial arm maze (Omoto et al., 1993; Reyes et al., 1989), T-maze (Lochry et al., 1985; Nagahara & Handa, 1997; Zimmerberg et al., 1991), spatial lever response (Zimmerberg et al., 1989) and Morris water maze (Blanchard et al., 1987; 1990; Gianoliakos, 1990; K i m et al., 1997). Deficits in spatial ability have also been shown following neonatal ethanol exposure (Goodlett & Johnson, 1997; Goodlett & Peterson, 1995; Pauli et al., 1995; Tomlinson et al., 1998).  Although deficits  following prenatal ethanol exposure may not always appear in relatively simple spatial tasks such as the T-maze (Abel, 1979), the effects of prenatal ethanol exposure on spatial learning and memory tasks appear to be robust.  71  The appearance of deficits in spatial learning and memory in E animals may be mediated by such factors as pretest exposure and the age of the animal. Prior swimming experience has been shown to reveal impairments in E compared to PF and C animals that were not observed in animals lacking prior experience. However, effects of prior experience were only apparent at 24 days of age; 21 day old E animals displayed deficits regardless of prior experience (Blanchard et al., 1990). Because normal animals begin to exhibit spatial navigation abilities around 20-22 days of age (Rudy et al., 1987; Schenck, 1985), the differential effects of prior experience in E, PF and C animals shown in previous research may be due to an alcohol-related delay in the development of spatial learning in E animals. Thus, the effects of prenatal ethanol exposure on spatial abilities in young E animals may be due to developmental delays, whereas the appearance of deficits in older E animals may be more dependent on the influence of prior experience or on the difficulty of the testing procedure. Findings of impaired Morris water maze performance in adult E animals have been varied. Although deficits have been reported in standard-reared E animals at 20-24 days (Blanchard et al., 1987; 1990), at 40, 60 and 90 days (Gianoulakis, 1990; Minetti et a l , 1996) and at 16-17 months of age (Kim et al., 1997), others assessing environmental enrichment and isolation have not found deficits in E compared to control animals at 2 months (Hannigan et al., 1993) and 5 months (Wainwright et al., 1993) of age. Although the neural changes underlying the deficits induced by prenatal ethanol exposure remain ambiguous, the deficits in spatial navigation demonstrated in E animals may be attributable, at least partly, to hippocampal damage. Prenatal ethanol exposure has been shown to produce alterations in hippocampal structure and function, changing mossy fiber branching, hippocampal pyramidal cell number and arborization (Hammer, 1986; Ward & West, 1992; West & Pierce, 1986), and to reduce the affinity of hippocampal N M D A receptors for binding glutamate (Fair et al., 1988; Savage et al., 1991). In addition, in response to high-frequency 72  stimulation, E animals show a smaller increase in field EPSPs and population spikes compared to control animals, demonstrating a deficit in a neural pathway believed to be critical in certain forms of learning and memory (Sutherland et al., 1997). It has also been suggested that animals prenatally exposed to ethanol display behavioral similarities to animals with hippocampal lesions.  Compared to controls, both E and hippocampal-lesioned animals show increased  activity, exploration and reactivity in the open field as well as deficits in spontaneous alternation and poorer passive avoidance (Riley et al., 1986). Furthermore, deficits in Morris water maze performance occur in both E and hippocampal-lesioned animals although deficits are more pronounced and prolonged in hippocampal-lesioned animals.  Hippocampal-lesioned animals  also show reductions in novelty-induced grooming (Reinstein et al., 1982) while E animals display increased stress-induced grooming (Hannigan et al., 1987). However, previous research has shown that the effects of neonatal hippocampal damage on spatial memory tend to be graded, rather than all-or-none (Altemus & Almli, 1997). Therefore, although E animals may display alterations in hippocampal-mediated functions, prenatal ethanol exposure will not always result in behavioral or cognitive deficits precisely paralleling those observed in hippocampal-lesioned animals. Exposure to environmental  enrichment  has  been  shown to produce  general  improvements in Morris water maze performance in both control and ethanol-exposed rats (Hannigan et al., 1993; Wainwright et al., 1993). Although deficits were not observed in enriched or isolated E animals compared to their control counterparts in either of these previous studies, these findings indicate that E and control animals may respond similarly to such postnatal manipulations. However, the ability of postnatal manipulations to attenuate the effects of prenatal ethanol exposure may vary depending on the species tested or the task measured. For example, group-housing of dams and their offspring did not attenuate the effects 73  of prenatal ethanol exposure on radial maze performance compared to E mice raised by individually-housed dams (Opitz et al., 1997), nor did postweaning group-housing in cages with varied stimuli (e.g. "toys" consisting of wood blocks, glass jars and cans) alter open-field and Y maze behavior in Sprague-Dawley E rats compared to E rats that were individually-housed in standard conditions (Osborne et al., 1980). In addition, exposure of adult E animals to ten weeks of environmental enrichment did not attenuate alterations in hippocampal dendritic spine densities compared to E that spent ten weeks in isolation (Berman et al., 1996). In the present study, deficits in spatial navigation were not observed in N H animals; rather, postnatal handling revealed differences in spatial navigation following prenatal ethanol exposure.  These data  underscore the lasting impact of such a prenatal insult especially in regard to behaviors associated with hippocampal function although, in the current experiment, such findings were dependent upon the age of the animal at testing. Postnatal manipulations such as handling may be dependent for their effects on numerous variables (i.e. pup ultrasonic vocalizations, dam-pup interaction), and it is possible that postnatal handling might exert differential effects on litters in which pup behavior has already been altered by prenatal treatments such as exposure to ethanol. Previous research has shown that handling may attenuate both stress-induced and senescence-associated alterations in Morris water maze performance in mice and rats. BALB/cByJ  mice,  postnatal  handling  attenuated  both  the  stress-induced  In  CORT  hyperresponsiveness and the learning impairments in the Morris water maze typically seen in standard-reared animals (Zaharia et al., 1996). In addition, postnatal handling has been shown to attenuate certain neuroendocrine, anatomical and cognitive dysfunctions associated with aging in rats. Compared to 6, 12 and 24 month old N H and 6 month old H animals, 12 and 24 month old H animals show little or no senescence-associated hippocampal neuron loss or spatial memory impairments (Meaney et al., 1988; 1991). 74  In the present study, we had  expected that postnatal handling would be most effective in attenuating the effects of prenatal ethanol exposure in mid-aged animals, because both stress and aging may lead to impairments in spatial ability. It has been argued that cognitive and behavioral abnormalities produced by prenatal ethanol exposure may be masked in young adult animals because of compensatory mechanisms or strategies, but that these compensatory mechanisms may break down due to stressful situations, complex testing procedures, or aging (Riley, 1990).  Of  particular relevance to the performance of mid-aged E animals in the present study, previous research has shown that E animals exhibit both behavioral and hormonal hyperactivity to stress (Angelogianni & Gianoulakis, 1989; Lee et al., 1990; Nelson et al., 1986; Osborne et al., 1980; Taylor et al., 1982; Weinberg, 1988; 1992a; 1993), and accelerated aging, including a shortened lifespan (Abel et al., 1987) and an earlier loss of reproductive function in E female rats (McGivern et al., 1995). In the present study, there were no effects of prenatal ethanol exposure on escape latency or distance traveled prior to locating the submerged platform.  However, mid-aged E males  displayed elevated CORT levels compared to mid-aged C males.  Elevations in C O R T were  apparent on the last day of testing, extending previous findings of H P A hyperresponsiveness to repeated stress in E animals (Weinberg et al., 1996). It is possible that alterations in H P A activity in mid-aged E animals may have masked deficits in spatial learning and memory. Previous research examining the influence of H P A activity on spatial learning has reported that while prolonged elevations in CORT impair spatial abilities, moderate increases may enhance performance on spatial tasks. Researchers have found that restraint stress in young rats produces varying spatial learning performance on a radial arm maze depending on the number of days of restraint; for example, 7 days has no effect, 13 days produces an enhancement, and 21 days results in impaired performance (Luine et al., 1994; 1996). Interactions between C O R T levels 75  and cognitive performance may be further complicated when assessing aged animals. Very old Fischer-344 x Brown Norway hybrid rats that received CORT injections for 15 days performed as well as young animals and demonstrated early spatial learning when compared to their noninjected counterparts (Hebda-Bauer et al., 1999). High CORT levels may make aged animals more aware of their surroundings, possibly helping them to integrate elements more quickly into a cognitive map (Hebda-Bauer et al. 1999). Therefore, it is possible that short-term increases in CORT activity in E animals in the present study enhanced their Morris water maze performance compared to PF and C animals, masking deficits in spatial navigation in mid-aged E animals. Contrary to our expectations, we found that postnatal handling did not improve performance on the Morris water maze in mid-aged animals. general reductions in emotional reactivity in H animals.  However, this may be due to  When tested as adults, H animals  explore or investigate novel stimuli or novel environments more than N H animals.  This  phenomenon has been found in simple tasks such as the open field (Levine et al., 1967; Reboucas & Schmidek, 1997) and in complex tasks involving novelty-seeking (Denenberg & Grota, 1964), conflict (Nunez et al., 1996), and stimulus-seeking (Weinberg et al., 1978) behaviors. H animals may differ in their reactivity to stimuli encountered in a testing situation, therefore it is difficult on many learning tasks, including the Morris water maze, to separate the effects of learning from motivational or emotional variables (Wong & Wong, 1978). Thus, the finding that postnatal handling did not alter escape latency and distance in the present experiment may have been due to emotionality differences, making it difficult to interpret the effects of handling on learning. Differences in search pattern indicate that young animals more effectively searched the target area compared to mid-aged animals, exhibiting a significant spatial bias for the region of the testing apparatus where the platform was positioned during the last day of testing. Mid-aged animals also had longer escape latencies than young animals even though similarities in distance 76  indicate that swimming speed may have differed between the two ages.  Although we had  examined mid-aged animals in an attempt to isolate possible effects of accelerated aging in E animals, the presence of any senescence-associated deficits may not have been extensive enough to reveal effects of postnatal handling. The ability of postnatal handling to attenuate age-related neuroendocrine, anatomical and cognitive dysfunctions in male animals has been primarily observed in old (i.e. 24 months of age) rather than in mid-aged (i.e. 12 month of age) animals. Furthermore, the effects of aging on spatial learning and memory in mid-aged rats are not well established. Impaired place navigation has been shown to develop progressively with age. In one study, 8% of the 12 month old, 45% of the 18 month old, 53% of the 24 month old and over 90% of the 30 month old rats showed impaired place navigation (Fischer et al., 1991), with the appearance of morphological alterations such as alterations in the forebrain cholinergic system tending to develop later than behavioral deficits (Fischer et al., 1991). Due to the individual nature of aging, many studies using aged rats divide them into two groups, "impaired" and "nonimpaired" as only "impaired" rats (i.e. rats with a learning decrement) are likely to improve from treatment (Rapp & Amaral, 1992). Impairments in spatial learning have been demonstrated in adolescents and children with fetal alcohol syndrome (Streissguth et al., 1994; Uecker & Nadel, 1996; 1998), consistent with the finding of impaired spatial learning and memory in rodents. Although previous research has found  that postnatal  manipulations  such  as  environmental  enrichment  may  produce  improvements in spatial abilities in E animals compared to isolation-reared E animals, the current findings suggest that the nature of the postnatal manipulation is critical in determining whether such deficits can be attenuated.  Furthermore, H P A activity and early postnatal  experience may complicate the assessment of cognitive/behavioral testing in mid-aged animals, making the identification of deficits in E animals more difficult. 77  Table 5 On Trial with Raised Platform, Escape Latency (sec, Mean ± SEM) for E, PF and C Males in and N H Treatments Tested at 2 Months (Young) or 13-14 Months (Mid-Aged) of Age.  MID-AGED  YOUNG H  NH  H  NH  E  12.35 ± 1.1  12.23 ± 1.7  11.70+1.7  14.04 + 1.4  PF  1.1.92 ± 1.8  11.35 ± 1.4  8.86+1.7  12.06+1.7  C  9.67 ± 1.7  11.74 ± 1.9  9.40+1.7  11.81 ± 2 . 1  There were no differences in latency on the raised platform trial due to age, prenatal group or postnatal handling (n's = 8-10 for each of E, PF and C prenatal groups, in each of H and N H treatments).  78  Figure 5. Escape latency (avg sec/day, Mean ± SEM) for E, PF and C males in H and N H treatments at 2 months (Young) (top panels) and 13-14 (Mid-Aged) (bottom panels) months of age (n's = 8-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). Young < Mid-aged on d 1 (p < 0.01) and 2 (p = 0.07). For young H animals, E > PF (p = 0.08) and C (p < 0.04); and d 1 > d 2-4; d 2 > 3; d 4 > d 3 (p's < 0.05). For young N H animals, d 1 > d 2-4 (p's < 0.01). For both H and N H mid-aged animals, d 1 > d 2-4, (p's < 0.01).  79  DAY 1  +-  Ethanol  - °-  Pair-Fed  80  Control  Figure 6. Distance traveled (avg m/day, Mean ± SEM) for E , PF and C males in H and N H treatments at 2 months (Young) (top panels) and 13-14 (Mid-Aged) (bottom panels) months of age (n's = 8-10 for each of E, PF and C prenatal groups, in each of H and N H treatments). For young H animals, E > PF (p < 0.02) and C (p = 0.07). For both H and N H young animals, d 1 > d 2-4 (p's < 0.01). For mid-aged H animals, d 1 > d 2-4; d 2 > d 3-4 (p's < 0.01). For N H animals, d 1> d 2-4 (p's < 0.01).  81  1  D A Y  I  D A Y  1  O  oo  NH  H  20  Mid-Aged Animals  • T—I  Q 15 10 5  D A Y  1  0  D A Y  1  NH  H +-  Ethanol  o-  Pair-Fed  82  Control  Figure 7. Annulus crossings on day 4 (Mean ± SEM) for E, PF and C males in H and N H treatments at 2 months (Young) (top panels) and 13-14 (Mid-Aged) (bottom panels) months of age (n's = 8-10 for each of E , PF and C prenatal groups, in each of H and N H treatments). Young > Mid-aged (p < 0.02). Young E < Young PF and C (p's < 0.04).  83  84  Figure 8. Plasma CORT levels (pg/dl, Mean ± SEM) for E , PF and C males in H (left panel) and N H (right panel) treatments at 13-14 (Mid-Aged) months of age (n's = 7-8 for each of E, PF and C prenatal groups, in each of H and N H treatments). E > C (p < 0.04).  85  50  86  C H A P T E R V: POSTNATAL HANDLING DOES NOT A T T E N U A T E H P A HYPERRESPONSIVENESS FOLLOWING PRENATAL ETHANOL EXPOSURE  Introduction The ability to respond to stress is an important basic adaptive mechanism, and H P A activation is known to be a central feature of this response.  Following the termination of  stress, there is a recovery process by which the endocrine, metabolic, immune and neural defensive reactions that were mobilized in response to stress are terminated (Chrousos & Gold, 1992; Johnson et al., 1992).  Increased and/or prolonged H P A activation may have adverse  physiological and behavioral consequences which could compromise health and possibly even survival of the organism.  These adverse consequences include gastrointestinal ulceration,  immunosuppression, weight loss, fatigue, steroid diabetes, hypertension, psychogenic dwarfism, and reproductive dysfunction (Stratakis & Chrousos, 1995).  Maintenance of high levels of  CORT either pharmacologically or by subjecting rats to chronic stress paradigms is also associated with alterations in neuronal function including fewer neurons in the hippocampus, reduced dendritic branching in the neurons that remain and impairment of hippocampal plasticity (de Kloet et al., 1998; Magarinos et al., 1996).  Because the hippocampus is an important  component of H P A feedback regulation, such changes in neuronal substrate might result in dysregulation of the H P A response to stress. Although clinical studies have established that alcohol consumption markedly alters H P A function in chronic alcoholics (Merry & Marks, 1973), few clinical studies have investigated the effects of drinking during pregnancy on the H P A axis of the developing child. A recent study found that maternal drinking at conception and during pregnancy was associated with higher poststress Cortisol levels in infants (Jacobson et al., 1999). Animal 87  studies in our laboratory and others have shown that prenatal ethanol exposure produces H P A hyperresponsiveness to stressors.  In adulthood, E animals display increased H P A  responsiveness (i.e. elevated and/or prolonged plasma A C T H and/or C O R T responses) to a variety of stressors including shock (Nelson et al., 1986), restraint (Weinberg, 1988; Weinberg et al., 1995; 1996), cardiac puncture, noise (Taylor et al., 1982), and challenges with drugs such as morphine (Nelson et al., 1986) and ethanol (Taylor et al., 1981) when compared with controls. In addition, while H P A hyperresponsiveness in E animals is a robust phenomenon, E males and females may exhibit differential H P A responsiveness to aversive stimuli, depending on the nature and intensity of the stressor, as well as the time course and the hormonal endpoint measured (Weinberg, 1988; 1992a; Weinberg et al., 1996). Thus, prenatal ethanol exposure can produce long-term disturbances in physiology, significantly altering offspring endocrine function into adulthood. H P A hyperresponsiveness in E animals has been shown to be mediated in part by deficits in the negative feedback regulation of the H P A axis (Nelson et al., 1985; Osborn et al., 1996). Previous work from our laboratory investigating inhibitory components of the H P A axis showed that E animals demonstrate deficits in feedback inhibition in the intermediate (Osborn et al., 1996) but not in the fast (Hofmann et al., 1999) feedback time domain.  Thus, E animals had elevated stress C O R T levels three hours after D E X (a  glucocorticoid analogue) administration compared to control animals.  This deficit was  shown in E females but not E males during the trough, and in both E males and females during the peak of the H P A circadian rhythm (Osborn et al., 1996). Although the extent of these deficits in E animals requires further investigation, impaired negative feedback regulation of the H P A axis in E animals does not appear fo be mediated by decreased levels of corticosteroid receptors at H P A feedback sites. Work in our laboratory has demonstrated 88  that under both basal conditions and following stress, there were no significant effects of prenatal ethanol exposure on hippocampal G R and M R densities or binding affinities ( K i m et al., 1999b; Weinberg & Peterson, 1991). Previous research has shown that early postnatal events may permanently alter hormonal responses to stressors. In rodents, postnatal handling (comprising brief periods of maternal separation over the first 14-21 postnatal days) results in greater modulation of H P A functioning following exposure to stressors, and these effects may still be apparent in rats at 24-26 months of age (Meaney et al., 1988; 1991).  This contrasts with the adverse  consequences of protracted maternal deprivation (three hours or more each day).  While  adult animals that were exposed to maternal deprivation show increased plasma A C T H in response to stressors such as restraint, footshock and exposure to novel environments (Ladd et al., 1996; Plotsky & Meaney, 1993), postnatal handling results in modulated H P A responses to stressors with blunted A C T H and C O R T responses to various types of stressors and a faster return to basal C O R T levels following stressor termination compared to N H animals (Hennessy, 1997; Meaney et al., 1988; 1991). Data suggest that these differences are related to more efficient negative feedback in handled animals, possibly due to elevations in the number of glucocorticoid receptors (Meaney et al., 1989; 1991; 1996; Tees et al., 1990). In support of the contention that H animals show improved negative feedback inhibition of the H P A axis, it has been demonstrated that H animals which received either C O R T or D E X three hours prior to restraint stress exhibited greater A C T H suppression relative to N H animals (Meaney et al., 1989; 1991). Previous research in our laboratory has shown that postnatal handling can, under some conditions, eliminate the deficits in preweaning weight gain often observed in E compared to control pups, attenuate the hypothermic response to ethanol challenge in E 89  males, and attenuate the initial C O R T elevation in response to restraint stress observed in E females (Weinberg et al., 1995). In contrast, handling and cross-fostering after birth in E animals has been shown to increase A C T H response to footshock compared to handled controls, and nonhandled, cross-fostered E and control animals (Ogilvie & Rivier, 1997). These findings suggest that an organism's postnatal and/or postweaning rearing environment may have a significant effect on developmental outcome and may alter some aspects of the long-term effects of a prenatal insult, although the nature and extent of these effects remain poorly understood.  The current study investigated the hypothesis that postnatal handling  might attenuate stress-induced A C T H and/or C O R T differences among E , PF and C animals. Furthermore, the ability of postnatal handling to modulate H P A feedback deficits in E animals was examined during exposure to a restraint stressor following D E X administration.  Method Blood Sampling At 90-120 days of age, male and female offspring from each prenatal group (E, PF, C) x postnatal handling treatment (H, NH) were randomly assigned to either the saline (SAL) or the D E X experimental condition. Two days prior to testing, animals were weighed and underwent brief surgery to implant indwelling jugular cannulae under Halothane anesthesia, as discussed in the General Methods section. Animals were then singly-housed for the remainder of the study. On the test day, three hours prior to testing, animals were injected intraperitoneally with either S A L or D E X at doses that have previously been shown to be effective in suppressing the H P A axis in control animals [30.0ug/100g body weight for females and 15.0ug/100g body weight for males (Osborn et al., 1996)]. A l l testing occurred between 1230 and 1530 (lights on 0330-1530), within the peak of the circadian rhythm. Blood samples (-150 pi) were taken from each animal 90  via the jugular cannulae prior to restraint (0 min), during (10, 30, 60 min) a 60 min restraint and 30 min post-restraint (90 min). Blood was collected and stored as discussed in the General Methods section. Statistical Analyses Data on body weight were analyzed by a three-way A N O V A for sex, prenatal group and postnatal treatment. Due to expected sex and experimental condition differences, A C T H and CORT concentrations were analyzed by three-way, repeated-measures A N O V A s for prenatal group (E, PF, C), postnatal treatment (H, NH) and sampling time (0, 10, 30, 60, 90 min). Missing hormone levels for a single sampling timepoint were replaced with the mean for that timepoint from that animal's sex x prenatal group x postnatal treatment x adult experimental condition. If more than two samples were missing for an animal, that animal was dropped from overall analysis but its A C T H and C O R T values were used to calculate means for each timepoint. When appropriate, A N O V A s were followed by Tukey's paired comparison tests.  Results Adult Body Weights Analysis of adult body weights at the time of testing revealed significant main effects of sex, F(2, 251) = 16.38, p < 0.01, prenatal group, F ( l , 251) = 3892.35, p < 0.01, and a prenatal group x postnatal handling interaction, F(2, 251) = 3.16, p < 0.04 (Table 6). Females weighed less than males. Furthermore, N H - E animals weighed less than NH-PF (p < 0.03) and N H - C (p < 0.01) animals. Hormone Levels Separate analysis of A C T H and C O R T levels revealed significant main effects for all between (sex, prenatal group, postnatal treatment, and experimental condition) and within91  subject (sampling time) factors as well as multiple two- and three-way interactions. Post-hoc analyses of significant sex x experimental condition x sampling time interactions for both A C T H , F(4, 720) = 13.16, p < 0.01, and CORT, F(4, 720) = 7.26, p < 0.01, showed that, in general, D E X administration resulted in lower A C T H and CORT than did S A L administration and females had higher A C T H and CORT levels than did males.  Due to these expected  differences, separate analyses were conducted on data for each sex within each experimental condition. S A L Condition Females. Following S A L administration, there were significant main effects of prenatal group, F(2, 54) = 4.84, p < 0.01, postnatal treatment, F ( l , 54) = 4.08, p < 0.048, and sampling time, F(4, 216) = 85.85, p < 0.01, as well as a prenatal group x sampling time interaction, F(8, 216) = 2.30, p < 0.02, on A C T H levels (Figure 9). A l l females showed an increase in A C T H levels following the initiation of restraint (0 < 10-60 min; p's < 0.01) and a decrease toward basal levels following restraint termination. During exposure to restraint (30 and 60 min), A C T H levels were higher in E than in C females (p's < 0.04). In addition, H females had lower A C T H than N H females. For CORT, there was a significant main effect of sampling time, F(4, 216) = 258.45, p < 0.01, and a prenatal group x time interaction, F(8, 216) = 3.21, p < 0.01 (Figure 10). A l l females showed an increase in CORT over basal levels during and after restraint (0 < 10, 30, 60, 90 min; p's < 0.01). At the end of restraint (60 min), E females had elevated CORT levels compared to C females (p < 0.01). Males. Following S A L administration, there were significant effects of prenatal group, F(2, 36) = 3.69, p < 0.03, postnatal treatment, F ( l , 36) = 8.09, p < 0.01, and sampling time, F(4, 144) = 46.33, p < 0.01, as well as a postnatal treatment x time interaction, F(4, 144) = 3.00, p < 92  0.02, on A C T H levels (Figure 9). Overall, E males had higher A C T H than PF (p < 0.04) males and marginally higher A C T H than C (p = 0.09) males. In addition, H males had lower A C T H than N H males during restraint (10 min; p < 0.02) and after restraint (90 min; p < 0.04). For CORT, there were main effects of prenatal group, F(2, 36) = 5.86, p < 0.01, and sampling time, F(4, 144) = 89.59, p < 0.01 (Figure 10). Overall, E males had elevated CORT compared to PF and C males (p's < 0.01). In addition, all males showed increases in CORT during restraint [0 < 10-60 min (p's < 0.01); 10 < 30-60 min (p's < 0.01); 30 < 60 min (p < 0.01)] that did not return toward basal levels after the termination of restraint (0 < 90 min, p < 0.01). D E X Condition Females. Following D E X administration, there were significant main effects of prenatal group, F (2, 54) = 8.11, p < 0.01, and sampling time, F(4, 216) = 22.59, p < 0.01, as well as a prenatal group x time interaction, F(8, 216) = 2.39, p < 0.02, on A C T H levels (Figure 11). During exposure to restraint (10 and 30 min), A C T H levels were higher in E than in C females (p's < 0.01). In addition, A C T H levels remained elevated longer in E than in both PF and C females and longer in PF than in C females [E: 0 < 10-60 min (p's < 0.04); PF: 0 < 10-30 min (p's < 0.01); C: 0 = 10-60 min]. After the termination of restraint, A C T H levels returned toward basal levels in all females. For CORT, there was a significant main effect of sampling time, F(4, 216) = 148.57, p < 0.01, and a prenatal group x sampling time interaction, F(8, 216) = 6.53, p < 0.01, as well as a marginal prenatal group x postnatal treatment x sampling time interaction, F(8, 216) = 1.73, p = 0.09.  Data within each postnatal treatment were analyzed separately by one-way, repeated  measures A N O V A s for prenatal group. Within the H treatment, there were significant main effects of prenatal group, F(2, 27) = 25.84, p < 0.01, and sampling time, F(8, 108) = 86.29, p < 0.01, as well as a prenatal group x sampling time interaction, F(8, 108) = 7.38, p < 0.01, on 93  CORT levels. During exposure to restraint, CORT levels were higher in H-E than in H-PF and H-C females (30 and 60 min; p's < 0.01), and higher in H-PF than in H-C females (60 min; p < 0.01). In addition, C O R T levels remained elevated longer in H-E and H-PF than in H-C females [H-E and H-PF: 0 < 10-60 min (p's < 0.01); H-C: 0 < 10-30 min (p's < 0.01)]. Within the N H treatment, there were significant main effects of prenatal group, F(2, 27) = 5.01, p < 0.01, and sampling time, F(4, 108) = 66.35, p < 0.01, on CORT levels. N H - E and NH-PF females had elevated C O R T compared to N H - C females (p's < 0.03). Furthermore, all N H females showed elevations in C O R T following the initiation of restraint (0 < 10-60 min; p's < 0.01) which did not return to basal levels after the termination of restraint (0 < 90 min; p < 0.03). Males. Following D E X , there was a significant main effect of sampling time, F(4, 144) = 24.79, p < 0.01, and a postnatal treatment x sampling time interaction, F(4, 144) = 2.96, p < 0.02, for A C T H levels (Figure 11). During restraint (60 min), H males had lower A C T H than did N H males (p < 0.01), but A C T H levels returned toward basal levels after the termination of restraint in both H and N H males. For CORT, there were significant main effects of postnatal treatment, F ( l , 36) = 5.71, p < 0.02, and sampling time, F(4, 144) = 56.65, p < 0.01 (Figure 12). During restraint, CORT levels increased in all males and were elevated over basal levels (0 < 10 < 30, 60 min, p's < 0.01); CORT levels decreased after restraint (30, 60 > 90 min, p's < 0.01), but did not return to basal levels (0 < 90 min, p < 0.01). In addition, H males had lower C O R T levels overall than N H animals.  Discussion  The results from the present study support and extend data from previous studies demonstrating adverse effects of prenatal ethanol exposure on H P A responsiveness to stressors in 94  both females and males (Weinberg, 1988; 1992a; 1992b; Weinberg et a l , 1996), and further support the suggestion that H P A hyperresponsiveness and/or delays in recovery from stressors in E animals may result, at least in part, from deficits in feedback inhibition of the H P A axis. In the present study, E females and males showed increased A C T H and C O R T compared to PF and/or C animals in the S A L condition, extending previous findings of H P A hyperresponsiveness in E animals (Taylor et al., 1982; Weinberg et al., 1995; 1996). Administration of D E X to block HPA activity significantly suppressed both plasma A C T H and C O R T in all animals. However, E females exhibited increased and/or prolonged elevations in A C T H and C O R T compared to C animals following D E X blockade. These data suggest that the insult of prenatal ethanol exposure affects both female and male offspring, but that there may be a sex-specific difference in sensitivity of the mechanism(s) underlying H P A hyperresponsiveness. As previously discussed in Chapter III, adverse effects of maternal ethanol consumption on maternal weights, pregnancy outcome (i.e. length of gestation and litter size), and offspring weight gain were observed in animals in this experiment, and these effects are consistent with previous data both from our laboratory (Weinberg, 1985; Weinberg et al., 1995) and from others (Abel & Dintcheff, 1979; Chernoff, 1977).  In the present study, prenatal ethanol exposure  resulted in lower body weights at the time of testing (i.e. 90-120 day of age) in N H but not in H animals.  Previous work from our laboratory has shown that handling may, under some  conditions, attenuate the adverse effects of fetal ethanol exposure on pup weight gain during the preweaning period (Weinberg et al., 1995) and the early postweaning period (Chapter III). Therefore, the findings of the present study extend these results to include adult animals, indicating that postnatal handling may be able to attenuate weight deficits in E animals at varying ages.  95  Early experience in the form of postnatal handling lowered A C T H levels in both females and males in the S A L condition compared to their N H counterparts, extending previous research on the effects of postnatal handling on H P A functioning in male animals (Meaney et al., 1989; 1991). Furthermore, our data demonstrating that following D E X administration H males had lower CORT levels than N H males, supports previous findings of enhanced DEX-mediated negative feedback inhibition in H males (Meaney et al., 1989).  Interestingly, handling  differentially affected feedback inhibition in E, PF and C females. Postnatal handling truncated stress-associated C O R T elevations in C females, and attenuated differences in CORT between PF and C females. However, postnatal handling did not attenuate deficits in negative feedback inhibition in E females; E females in both the H and N H treatments showed elevated CORT compared to their C counterparts, and H-E females also showed elevated C O R T compared to H PF  females.  Therefore,  postnatal  handling  did  not  attenuate  the  typical H P A  hyperresponsiveness to stressors observed in E animals (SAL condition), nor did it eliminate deficits in H P A feedback inhibition in E females (DEX condition). Neither prenatal ethanol exposure (Kim et al., 1999a) nor postnatal handling (Meaney et al., 1989; 1992) has been shown to alter levels of corticosteroid-binding globulin. Thus, differences  in plasma C O R T levels observed in the present study likely represent  functionally significant changes in free C O R T . Furthermore, differences in total C O R T are likely predictive of differences in brain uptake of the steroid as well as its overall physiological effect on the body. No differences in basal A C T H or C O R T activity were found in the present study or in previous studies following prenatal ethanol exposure (Kim et al., 1999a; Weinberg et al., 1995; 1996) or postnatal handling (Meaney et al., 1989; Weinberg et al., 1995), indicating that differences in A C T H and/or C O R T levels observed during stress cannot be accounted for by altered basal glucocorticoid levels. 96  Feedback inhibition of the H P A axis through the actions of glucocorticoids occurs within three time domains and through multiple sites in the brain (Keller-Wood & Dallman, 1984). Fast feedback inhibition occurs within seconds to one hour of the initiation of a stress-induced rise in CORT and is sensitive to the rate of CORT increase. Fast feedback prevents further release of CORT by inhibiting release of A C T H from the pituitary and C R H from the hypothalamus. Intermediate feedback occurs at 2-10 hours after a stress-induced rise in C O R T and inhibits the release of A C T H as well as the release and synthesis of C R H . Slow feedback occurs after 12 or more hours of prolonged CORT exposure and inhibits both A C T H and C R H synthesis and release (Keller-Wood & Dallman, 1984). However, unlike fast and intermediate feedback, slow feedback occurs most often in pathological conditions.  Investigations of H P A feedback  sensitivity in E animals during the fast feedback time domain have yielded conflicting results. Taylor et al. (1986) showed elevated A C T H levels in E females ten minutes after footshock stress, which is consistent with possible deficits during the fast feedback time domain. However, a recent study in our lab (Hofmann et al., 1999) systematically examined male and female E, PF and C offspring using a validated fast feedback paradigm in which exogenous CORT administered five minutes prior to stress simulates a specific fast feedback signal. Under these conditions, differences among groups were not observed, suggesting that feedback deficits in E animals within the fast feedback domain may not be robust or may occur only under specific conditions and/or timepoints. Consistent with these findings in E animals, it appears that H and N H animals also do not differ in glucocorticoid fast feedback sensitivity, since inhibitory signals associated with a rapid increase in plasma CORT (administered immediately prior to exposure to a stressor) do not distinguish between H and N H animals (Viau et al., 1993). The finding that E females show greater stress-induced H P A activation than controls at three hours post-DEX injection supports previous findings in our laboratory (Osborn et al., 1996) 97  and others (Nelson et al., 1985), suggesting that alterations in H P A responsiveness in E animals may be mediated through a feedback deficit in the intermediate time domain.  However, it  appears that there may be sex-specific differences in feedback sensitivity of the H P A axis in E females and males. Nelson et al. (1985) found elevated basal C O R T levels in E females (males not tested) four hours after D E X administration when testing occurred one hour prior to lights off (early in the circadian rise). Osborn et al. (1996) found that at three hours following D E X injection, E females had elevated stress-induced CORT at the trough (three hours after lights on) and elevated stress-associated A C T H and CORT levels at the peak (two hours after lights off) of the CORT circadian rhythm, whereas E males showed elevated stress-induced CORT compared to PF and C males only at the circadian peak.  In the present study, E females showed a  breakthrough of the D E X blockade during restraint stress whereas E males did not show increased A C T H or CORT compared to control animals. It is possible that because testing in the present study occurred early in the CORT rise, it was not the optimal time to detect deficits in E males. The H P A axis exhibits a circadian rhythm, which in rodents, is fully established by postnatal day 28 (Jones & Gillhan, 1988). The peak activity of the H P A rhythm occurs within approximately two hours after lights off for nocturnal animals, and it has been shown that greater levels of CORT are required in the P M (lights off) than in the A M (lights on) to suppress A C T H secretion (Bradbury et al., 1994). This may be due to decreased sensitivity of the H P A axis to CORT feedback in the P M compared to the A M , and deficits in feedback inhibition in E animals, particularly in E males, may be more readily revealed during this period of reduced H P A sensitivity. It is also possible that the sexual dimorphism of the H P A stress response underlies the differences in sensitivity to D E X suppression seen in males and females. Females have greater diurnal variations in plasma C O R T (Ottenweller et al., 1979), higher basal C O R T 98  and transcortin levels, and show greater C O R T responses to stress than males (Critchlow et al., 1963; Weinberg, 1988). Such differences are likely due to effects of gonadal hormones on the H P A axis (Handa et al., 1994; Patchev et al., 1995). Burgess and Handa (1992) demonstrated that estrogen elevates and prolongs activation of the H P A axis after ether and footshock stress.  In addition, females require higher doses of D E X to produce H P A  suppression than males.  Since D E X is a selective G R agonist, these differences may be  related to differences in hippocampal glucocorticoid receptors concentration and/or binding affinity between females and males although reports of the direction of the difference vary (Kim et al., 1999b; Turner & Weaver, 1985; Weinberg & Peterson, 1991). Furthermore, prenatal ethanol exposure may differentially alter glucocorticoid receptor in females and males. Redei et al. (1993) reported that male E rats showed elevations in G R m R N A in the hypothalamus but not the anterior pituitary, while E females displayed no changes in G R m R N A levels at either site. The finding that postnatal handling resulted in decreased or less prolonged elevations in CORT levels in all males and in PF and C females following D E X administration, suggests that handling may alter H P A responsiveness or regulation. Levine and Mullins (1966) were the first to propose that differences in pituitary-adrenal responsiveness between H and N H animals are the result of steroid hormone effects during sensitive periods of development that permanently modify the organization of the central nervous system, resulting in greater modulation of the H P A response to stressors. Recently, Meaney et al. (1989) have proposed that the differences between H and N H animals in H P A responsiveness may be related to differential H P A negative feedback sensitivity that occurs within the intermediate feedback time domain. When H and N H males were injected with one of five doses of either C O R T or D E X three hours prior to a 20 minute period of restraint, both glucocorticoids were more effective in suppressing 99  stress-induced H P A responses in H than in N H males (Meaney et al., 1989). The present study has extended these findings to include females and to examine a more prolonged period of restraint than was previously used. Importantly, handling differentially altered DEX-mediated feedback inhibition in E , PF and C females. Postnatal handling reduced or truncated the stress-induced rise in C O R T levels in PF and C but not in E females; postnatal handling reduced stress-associated C O R T elevations in C females, and attenuated differences in C O R T between PF and C females. However, postnatal handling did not attenuate adverse effects of prenatal ethanol exposure on negative feedback inhibition; H - E females continued to show elevated CORT compared to H-PF and H-C females. In the N H condition, both N H - E and NH-PF females had higher levels of C O R T than N H - C females. The finding that postnatal handling could not attenuate deficits in E animals supports previous research (Ogilvie & Rivier, 1997) that found that H - E animals that were surrogate fostered or non-fostered at birth had elevated peak A C T H levels after 10 min of footshock compared to controls. In fact, postnatal handling and fostering in E animals resulted in an A C T H response that was elevated compared to all other groups (Ogilvie & Rivier, 1997). Previous work in our laboratory has also shown that fostering may be a treatment in itself, altering the percentage of splenocytes and antigen expression in both E and C offspring (Giberson & Weinberg, 1997) although the effects of fostering may depend on the endpoint being measured. As previously discussed, the differential effects of postnatal handling on DEX-mediated feedback inhibition in E, PF and C females may have been due to alterations in mother-pup interactions in E pups which resulted in differential effects of postnatal handling in E , PF and C animals. It is also possible that the differential effects of handling among E, PF and C females may have been mediated by differences in the mechanisms underlying the effects of prenatal ethanol exposure and postnatal handling on H P A responsiveness. 100  Although the mechanisms  underlying the H P A feedback deficits in E animals remain to be elucidated, hippocampal glucocorticoid receptor concentrations appear to be unchanged following prenatal ethanol exposure when assessed either under basal conditions (Weinberg & Peterson, 1991) or following exposure to chronic intermittent stress (Kim et al., 1999b). In contrast, postnatal handling results in increased hippocampal glucocorticoid receptor binding capacity in the hippocampus (Meaney et al., 1989; 1992), indicating a possible mechanism of increased negative feedback sensitivity in H animals.  These differential effects of prenatal ethanol exposure and  postnatal handling on hippocampal glucocorticoid receptors and, possibly other mechanisms of H P A regulation, may explain the differential effects of handling on E females compared to controls. Previous studies have shown that pair-feeding, in addition to providing an essential nutritional control condition, may serve as an experimental treatment in itself. Although the inclusion of a PF group in animal models of prenatal ethanol exposure makes it possible to isolate the teratogenic effect of ethanol from the effects of primary malnutrition, pair-feeding can produce shifts in the circadian rhythm of a number of physiologic variables as well as alter body and organ weights and behavior of both the maternal female and offspring (Gallo & Weinberg, 1981; Weinberg & Gallo, 1982). Furthermore, PF dams may experience stress derived from their restricted meal-feeding schedule. Although E females have ad libitum access to food, they consume less than they would i f their diets did not contain ethanol. Thus, PF dams receive a ration that is less than they would consume ad libitum.  Therefore, although both groups are  receiving the same number of calories, PF dams experience deprivation and possible stress, whereas E dams do not. PF dams demonstrate prolonged corticoid levels following stress and decreased C B G binding capacity on day 21 of gestation compared with E and C dams (Weinberg & Bezio, 1987; Weinberg & Gallo, 1982). It has been proposed that the effects of prenatal stress 101  on H P A activity are mediated by feedback deficits (Weinstock, 1996) and, importantly, PF offspring display deficits in fast feedback regulation (Hofmann et al., 1999). In contrast to prenatal ethanol exposure, prenatal stress has been shown to increase ultrasonic vocalizations during isolation although this may depend on the gestational period in which the stressor was applied (Williams et al., 1997). Previous research has shown that postnatal handling reverses behavioral abnormalities in the open-field and plus-maze as well as decreasing emotionality in adulthood in prenatally stressed animals (Denenberg et al., 1978; Wakshlak & Weinstock, 1989). It appears that the effects of postnatal handling arise from complex mother-pup interactions, and, therefore, postnatal handling may exert differential effects on litters in which pup behavior has already been altered by prenatal manipulations. Activation of the adrenocortical/sympathomedullary responses to stress is both essential for survival and metabolically costly. Stress increases susceptibility to infection by the common cold (Kiecolt-Glaser & Glaser, 1991) as well as mediating increases in asthmatic attacks (Mrazek & Klinnert, 1991) and myocardial infarction (Muller & Tofler, 1990) in vulnerable individuals. Stress elevates both glucocorticoid and insulin levels, and in animal studies, repeated stress in adulthood increases expression of the diabetic state in diabetes-prone rats (Lehman et al., 1991). Furthermore, increased glucocorticoid responses to stress are associated with an increased risk for neuropathology in later life (McEwen, 1994; Sapolsky et al., 1985), and dysregulation of the H P A axis has been implicated in a variety of psychiatric disorders such as depression, panic disorder, obsessive-compulsive disorder and anorexia nervosa (Chrousos & Gold, 1992; Johnson et al., 1992). Thus, while it is clearly in the animal's best interest to activate neuroendocrine systems in response to threat, exaggerated or unnecessary activity such as that observed in E animals and in N H animals may be damaging in the long-term. 102  Indeed, studies in our laboratory have  demonstrated stress-associated immune alterations in E animals, such as increased mitogeninduced proliferation in E females after one day of cold stress compared to controls (Giberson et al., 1997), and reduced numbers of pan T-cells in the thymus and peripheral blood in E males after 21 days of a chronic intermittent stress regimen compared with nonstressed E males (Giberson & Weinberg, 1995). In conclusion, we have shown that prenatal alcohol exposure and postnatal handling permanently alter H P A function into adulthood, although they do not interact as expected.  It  appears that although postnatal handling may, under some conditions, attenuate some of the adverse effects of prenatal ethanol exposure (Weinberg et al., 1996), postnatal handling is not sufficient in itself to reduce H P A hyperresponsiveness to stress or to attenuate feedback deficits in E animals. The differential effect of postnatal handling on E, PF and C females may be mediated by alterations in pup behavior or changes in H P A activity either during the handling procedure itself or due to altered mother-pup interactions following the return to the nest. Both the H and N H treatments used in the present experiment involved postnatal manipulations that differ from the normal rearing experience. N H animals remained totally undisturbed from PN2 to weaning, without even the typical disturbances involved in weighing or cage cleaning, whereas H animals were separated daily from their dams during the first two weeks of life. Postnatal handling is often inadvertently introduced into developmental studies during routine procedures such as weighing, cage cleaning or early experimental manipulations. In addition to underscoring the enduring impairments that may result from prenatal ethanol exposure, the present findings clearly indicate that postnatal handling is not inconsequential for the developing organism and its possible impact on the animal should be considered.  103  Table 6 Adult Body Weight (g, Mean ± SEM) of E , PF and C Females and Males in H and N H Treatments Tested at 90-120 Days of Age.  Male*  Female* H  NH  E  275.6 ± 4 . 0  269.0 ± 3 . 7  A  PF  294.6 ± 4.7  285.0 ± 6 . 6  A  C  292.6 ± 4.2  292.4 ± 4.3  A  H  NH  521.6 ± 11.6  508.2 ± 12.1  534.7 ± 7 . 7  534.4 ± 9.9  A  535.1 ± 8 . 2  561.6 ± 6 . 5  A  Females: n = 10 per each group; Males: n = 7 per each group. * Females < Males, p < 0.01 A  N H - E < NH-PF = N H - C , p's < 0.03  104  A  Figure 9. Plasma A C T H levels (pg/ml, Mean ± SEM) for E, PF and C females (left panels) and males (right panels) in H and N H treatments three hours following S A L administration (females: n = 10 per each point; males: n = 7 per point). Females: Overall, 0 < 10-60 min (p's < 0.01); E > C at 30 and 60 min (p's < 0.04); H < N H (p < 0.05). Males: H < N H at 10 and 90 min (p's < 0.04); E > PF (p < 0.04) and C (p = 0.09).  105  106  Figure  10. Plasma CORT levels (ug/dl, Mean ± SEM) for E, PF and C females (left panels) and  males (right panels) in H and N H treatments three hours following S A L administration (females: n = 10 per each point; males: n = 7 per point). Females: Overall, 0 < 10-90 min (p's < 0.01); E > C at 60 min (p < 0.01). Males: Overall, 0 < 10-90 min (p's < 0.01); 10 < 30-60 min (p's < 0.01); 30-60 min (p < 0.01); E > PF and C males (p's < 0.01).  107  NH  - +-  Ethanol  NH  ~ ° ~ Pair-Fed  108  Control  Figure 1 1 . Plasma A C T H levels (pg/mL, Mean ± SEM) for E , PF and C females (left panels) and males (right panels) in H and N H treatments three hours following D E X administration (females: n = 10 per each point; males: n = 7 per point). Females: E > C at 10 and 30 min (p's < 0.01). For E females, 0 < 10-60 min (p's < 0.04); for PF, 0 < 10-30 min (p's < 0.01). A l l females, 0 = 90 min. Males: H < N H at 60 min (p < 0.01). A l l males, 0 = 90 min.  109  10  0  30  60  90 min  Ethanol  30  60  90 min  NH  NH  +-  10  - o - Pair-Fed  no  Control  Figure 12. Plasma C O R T levels (ug/dl, Mean ± SEM) for E , PF and C females (left panels) and males (right panels) in H and N H treatments three hours following D E X administration (females: n = 10 per each point; males: n = 7 per point). Females: Overall, H-C: 0 = 90 < 10-30 min (p's < 0.01); all other females, 0 = 90 < 10-60 min (p's < 0.01). For H females, E > PF and C at 30 and 60 min (p's < 0.01), and PF > C at 60 min (p < 0.01). For N H females, N H - E and NH-PF > N H - C (p's < 0.01).  Males: H < N H (p < 0.02); 0 < 10 < 30-60 min (p's < 0.01); 30-60 > 90  min (p's < 0.01).  Ill  NH  - +-  Ethanol  N  - °-  Pair-Fed  112  H  Control  C H A P T E R VI: VARIATIONS IN CQRTICOSTERONE F E E D B A C K DO NOT R E V E A L DIFFERENCES IN HPA ACTIVITY F O L L O W I N G P R E N A T A L E T H A N O L EXPOSURE  Introduction In humans, maternal alcohol consumption during pregnancy may disrupt hormonal interactions between the maternal and fetal systems, producing long-term disturbances in fetal physiology that may significantly alter offspring endocrine function into adulthood (Anderson, 1981). In pregnant female rats, ethanol consumption has been shown to increase maternal adrenal weights, basal C O R T levels, and the adrenocortical response to stress compared to PF and ad libitum-fed C females (Weinberg, 1989; Weinberg & Bezio; Weinberg & Gallo, 1982). The increased C O R T released by the activated maternal H P A axis may cross the placenta and suppress fetal H P A activity (Weinberg, 1989). However, ethanol also readily crosses the placenta and may simultaneously stimulate the fetal H P A axis. The opposing physiological responses to both indirect (elevated maternal CORT) and direct effects of ethanol may permanently affect the development and organization of the fetal H P A axis (Weinberg et al., 1986). In adulthood, E animals display H P A hyperresponsiveness to a variety of stressors (Angelogianni & Gianoulakis, 1989; Taylor et al., 1986; Weinberg, 1989). Significantly, this difference is only apparent following stress. E offspring do not appear to differ from PF and C offspring when tested in adulthood under basal conditions (Nelson et al., 1986; Weinberg et al., 1996). Furthermore, the hormonal hyperresponsiveness demonstrated by E animals following exposure to stressors may be limited to specific types of stimuli. Neurogenicallymediated stressors such as restraint, cardiac puncture, noise and shake, and challenges with drugs such as morphine or ethanol elicit greater plasma C O R T response in E offspring than 113  in PF and C animals (Nelson et al., 1986; Taylor et al., 1988; 1981; Weinberg, 1988; 1992; Weinberg et al., 1996).  However, findings of differential responding in E animals in  response to metabolic stressors are more varied, with reports of increased H P A responses in E animals to cold (Angelogianni & Gianoulakis, 1989; K i m et al., 1999a) but not to prolonged fasting (Taylor et al., 1982).  In addition, E males and females may exhibit  differential H P A responsiveness to aversive stimuli. While H P A hyperresponsiveness in E animals is a robust phenomenon, prolonged and/or enhanced A C T H and C O R T responses to stressors have been more consistently observed in E females (Weinberg, 1988; 1992a; 1992b).  Sex differences in H P A responsiveness may be dependent on the nature and  intensity of the stressor, the time course and the hormonal endpoint measured (Weinberg et al., 1996). The H P A axis plays a key role in mediating an organism's response to stress (Herman et al., 1996).  After a stressful stimulus, C R H , produced in the hypothalamic P V N , is  delivered to the portal vascular system of the median eminence. C R H induces A C T H release from the anterior pituitary, which, in turn, is responsible for the regulation of the C O R T production in the adrenal cortex.  Elevated plasma C O R T is central to an organism's  response to stress; physiological effects of C O R T include mobilization of glucose from the liver, increased cardiovascular tone, and inhibition of nonessential physiological and endocrine functions (Munck et al., 1984).  The H P A axis is also regulated by circadian  influences such that A C T H and C O R T levels exhibit a diurnal pattern, with maximal secretion at the onset of the organism's active period [in the AM/lights on in diurnal species such as humans and in the PM/lights off in nocturnal species such as rats (Dallman et al., 1987)]. During the circadian peak, the actions of C R H on pituitary corticotropes may be potentiated through the co-release of V P from the hypothalamus (Bradbury et al., 1994). In contrast, during the trough of the circadian rhythm, there appears to be no hypothalamic 114  stimulation of the resting, basal system because hypothalamic-lesioned animals and normal animals do not differ in basal A C T H and C O R T concentrations during the circadian trough (Dallman et al., 1985; Kaneko et al., 1980; Levin et al., 1988). Corticosteroids also influence their own production through negative inhibition of C R H and A C T H activity. CORT-mediated regulation of H P A activity occurs mainly via binding to corticosteroid receptors at the anterior pituitary, P V N of the hypothalamus, and hippocampus. Type I, or mineralocorticoid receptors (MRs), are localized primarily in the hippocampus and lateral septum, whereas Type II, or glucocorticoid receptors (GRs), are more widespread in the brain with large densities in the hippocampus, lateral septum, amygdala and P V N (de Kloet, 1991; Reul & de Kloet, 1985).  As high affinity C O R T  receptors, M R s are thought to provide tonic inhibition of the H P A axis although they may also play a role in feedback regulation following stress (Bradbury et al., 1994). In contrast, GRs have lower C O R T affinity and G R occupancy increases during stress and at the circadian peak. GRs are thought to mediate stress-induced feedback inhibition of the H P A axis as well as tonic H P A inhibition during the circadian peak (Reul et al., 1987; Reul & de Kloet, 1985). In addition, they may mediate the peripheral effects of circulating C O R T on such tissues as the thymus, spleen and liver (Ballard et al., 1974; Lowy, 1989; Spencer, 1991). The mechanisms underlying H P A hyperresponsiveness in E rats are unclear at present.  Data suggest that one possible mechanism involves deficits in CORT-mediated  negative feedback regulation of the H P A axis. Although previous work in our laboratory (Hofmann et al., 1999) indicates that E animals do not show robust deficits in fast feedback, deficits have been demonstrated in E males and females during the intermediate feedback domain (Osborn et al., 1996).  Three hours after  administration of the  synthetic  glucocorticoid, D E X , E males and females showed significantly higher stress-induced levels 115  of C O R T and/or A C T H than PF and C animals.  This differential responsiveness of E  animals to DEX-mediated H P A inhibition was more pronounced at the peak than at the trough of the circadian rhythm. Furthermore, feedback deficits were seen in E males only at the circadian peak, whereas deficits were seen in E females at both the peak and trough of the circadian rhythm (Osborn et al., 1996). We have also shown that this deficit in intermediate feedback inhibition is not attenuated in E females following postnatal handling, a procedure shown to enhance intermediate feedback in control animals (Chapter V ; Gabriel et al., 1998). However, differential responsiveness of E animals to D E X , a selective G R agonist, cannot be excluded.  Although previous studies from our laboratory using cytosolic binding assays  found no deficits in M R and G R densities or binding affinities in E animals in the hippocampus and other feedback sites (Kim et al., 1999b; Weinberg & Petersen, 1991), it is possible that there are differences in downstream effects of selective corticosteroid receptor occupancy. Previous results from our laboratory have shown that adrenalectomy (ADX) results in significantly greater basal A C T H levels in E males compared to PF and C males during the peak of the circadian rhythm (Glavas et al., 1998a). Thus, complete removal of the CORT feedback signal unmasked deficits in E males, indicating that alterations in H P A activity in E animals may occur via CORT-independent mechanisms.  We have also shown that A D X and C O R T  replacement via the drinking water (which results in diurnal variations in plasma CORT) normalize basal A C T H levels and reduce A C T H responses to stress to the same extent in E , PF and C animals (Glavas et al., 1998a; 1998b). Therefore, it appears that E animals are similar to controls in their ability to utilize an exogenous phasic CORT feedback signal.  The current  experiment was designed to evaluate further whether alterations in H P A responsiveness in E animals are due to CORT-independent mechanisms, stress-induced changes in C O R T activity or alterations in H P A feedback under basal conditions. The current study utilized C O R T 116  replacement via CORT-cholesterol pellets (which provide a constant, basal CORT signal) and tested animals during the trough of the circadian rhythm.  Consequently, we were able to  examine whether the decreased hypothalamic drive during the trough of the circadian rhythm or the lack of a phasic CORT feedback signal would unmask further deficits in E animals. In addition, the present study assessed multiple timepoints prior to, during and after exposure to a restraint stressor in order to investigate more thoroughly differences amdng E, PF and C animals. Lastly, this experiment was designed to examine whether the mechanisms resulting in H P A hyperresponsiveness in E animals are similar to those underlying the effects of postnatal handling.  Differences in H P A responsiveness between H and N H animals appear to be  dependent upon basal C O R T activity and not stress-induced elevations in CORT. Therefore, we tested the hypothesis that differences in H P A activity among E and control animals would not occur following A D X but could be reestablished following replacement with basal levels of exogenous C O R T .  Methods Breeding and Feeding Breeding and diet administration were conducted as discussed in the General Methods section. Surgeries and Sampling One day prior to surgery, 120-150 day old male and female offspring from prenatal E , PF and C groups were singly housed and assigned to one of three adult experimental conditions: (1) A D X , removal of the C O R T negative feedback signal; (2) P E L L E T , A D X and replacement with a tonic, basal C O R T signal. Animals in this condition lacked the increased negative feedback signal associated with stress-induced increases in C O R T as well as the phasic signal associated with a C O R T circadian rhythm; and (3) S H A M , intact, sham-operated, 117  possessing basal and stress-induced C O R T levels as well as a phasic C O R T signal. No more than one male and one female per litter were assigned to any one experimental condition. Bilateral adrenalectomies were carried out via the dorsal approach under Halothane anesthesia. For animals in the P E L L E T group, fused CORT+cholesterol pellets (males: one pellet of 75% C O R T and 25% cholesterol; females: two pellets, one pellet of 100% C O R T and one pellet of 25% C O R T and 75% cholesterol) were implanted subcutaneously at the base of the neck.  Replacement levels were chosen based on previous literature on males  (Meaney et al., 1989) and on pilot studies indicating that these levels were sufficient to reduce basal A C T H toward S H A M levels.  Corticosterone was obtained from Sigma  Chemical Co., St. Louis, M O , U S A . Immediately following surgery, all A D X and P E L L E T animals were provided with 0.9% NaCl drinking water. S H A M animals were anesthetized with Halothane and bilateral incisions were made over the adrenal glands but the adrenals were not removed. Three days after A D X , P E L L E T , or S H A M surgery (e.g. two days prior to testing), indwelling jugular cannulae were implanted in all animals under Halothane anesthesia, as discussed in the General Methods section. Testing occurred between 1200 and 1430 (lights on 0900-2100), within the trough of the circadian rhythm.  Blood samples (-150 pi) were taken from each animal via the jugular  cannulae prior to restraint (0 min), during (10, 30, 60 min) a 60 min restraint and 30 min postrestraint (90 min). Blood was collected and stored as discussed in the General Methods section. Statistical Analysis Plasma A C T H concentrations were analyzed by a four-way, repeated-measures A N O V A for sex, prenatal group (E, PF, C), adult experimental condition ( A D X , P E L L E T , S H A M ) and sampling time (0, 10, 30, 60, 90 min).  Due to expected sex and experimental condition  differences, A C T H levels for males and females from each experimental condition were then analyzed by two-way, repeated measures A N O V A s for prenatal group and sampling time. 118  Basal CORT concentrations were analyzed by a three-way A N O V A for sex, prenatal group, and adult experimental condition. For the S H A M condition, CORT levels for males and females were then analyzed by two-way, repeated-measures A N O V A s for prenatal group and sampling time.  For animals in the A D X and P E L L E T conditions, basal and stress CORT  concentrations were compared by dependent sample, one-tailed t-tests to ensure the absence of endogenous steroid. Missing data on hormone levels for a single sample were replaced with the mean for that timepoint from that animal's sex x prenatal group x adult experimental condition. If more than two timepoints were missing for an animal, that animal was dropped from overall analysis but its CORT and/or A C T H levels were used to calculate means for each timepoint. When appropriate, A N O V A s were followed by Tukey paired comparison tests.  Results Developmental Data For the experiments presented in this thesis, two separate breedings were undertaken. Breeding 1 supplied the animals used in experiments one, two and three, and developmental data were presented in experiment one (Chapter III). Breeding 2 supplied the animals used in this experiment, and developmental data for that breeding are presented here.  Animals from  Breeding 1 underwent postnatal treatments (H, NH); animals from Breeding 2 did not experience postnatal treatments (standard-reared). Maternal Data. Ethanol intake of pregnant females was consistently high throughout gestation, averaging 9.38 + 1.06, 10.40 ± 1.06, and 9.61 ± 0.67 g/kg body weight for weeks 1, 2 and 3 of gestation, respectively. Blood ethanol levels of approximately 125 mg/dl were found in the one pregnant dam that was sampled. Repeated-measures A N O V A on maternal weight gain during gestation revealed significant main effects of prenatal group, F(2, 42) = 9.47, p < 0.01, 119  and day, F(3, 126) = 1224.62, p < 0.01, and a prenatal group x day interaction, F(6, 126) = 34.24, p < 0.01 (Table 7). E, PF and C dams did not differ in body weight at GI and maternal body weight increased throughout gestation in all groups. However, E and PF dams had lower body weights than C dams from G7 through G21 (p's < 0.01). Analysis of maternal body weights during lactation indicated a significant main effect of day, F(3, 129) = 110.15, p < 0.01, and a prenatal group x day interaction, F(6, 129) = 6.50, p < 0.01 (Table 7). E and PF dams weighed significantly less than C dams on PN1 (p's < 0.04) but did not differ thereafter. There was a significant effect of prenatal group on gestation length, F(2, 43) = 9.64, p < 0.01 (Table 8), with E dams having longer pregnancies than PF and C dams (p's < 0.01). There were no significant differences among prenatal groups for litter size or number of stillborn pups. Pup Data. Analysis of pup body weights revealed a significant main effect of sex, F ( l , 84) = 7.41, p < 0.01, prenatal group, F(2, 84) = 22.68, p < 0.01, and day, F(3, 252) = 6556.79, p < 0.01, as well as sex x day, F(3, 252) = 3.50, p < 0.02, and prenatal group x day, F(6, 252) = 7.45, p < 0.01, interactions. Both females and males gained weight over days (p's < 0.01), however, females weighed significantly less than males on PN15 and PN22 (p's < 0.01). There were no differences in body weights among E , PF and C groups at birth. However, E and PF pups weighed significantly less than C pups during the preweaning period (p's < 0.01). At weaning (PN22), E and PF animals weighed less than C animals (p's < 0.01), and PF animals weighed marginally more than E animals (p = 0.08). By the time of testing (120-150 days of age; Table 10), there was a main effect of sex, F ( l , 227) = 2115.03, p < 0.01, on body weight. Males weighed more than females. There were no effects of prenatal group on body weight in adulthood. A C T H Levels A four-way, repeated-measures A N O V A revealed significant main effects of sex, F ( l , 171) = 46.02, p < 0.01, experimental condition, F(2, 171) = 218.23, p < 0.01, and sampling time, 120  F(4, 684) = 92.09, p < 0.01, as well as sex x sampling time, F(4, 684) = 4.98, p < 0.01, and experimental condition x sampling time, F(8, 684) = 10.66, p < 0.01, interactions. As expected, females had higher A C T H levels than males during the restraint stress (10-60 min) and recovery (90 min)(p's < 0.01).  Data for females and males were analyzed by separate three-way,  repeated-measures A N O V A s for prenatal group (E, PF, C), experimental condition ( S H A M , A D X , PELLET), and sampling time (0, 10, 30, 60, 90 min). For females (Figure 13) and males (Figure 14) there were significant main effects of experimental condition [F(2, 90) = 135.37, and F(2, 81) = 87.87, p's < 0.01, respectively] and sampling time [F(4, 360) = 61.82, and F(4, 324) = 33.46, p's < 0.01, respectively] as well as experimental condition x time interactions [F(8, 360) = 7.95, and F(8, 324) = 3.77, p's < 0.01, respectively]. Prior to the initiation of restraint (0 min), A C T H levels were significantly greater in animals in the A D X than in both the P E L L E T and S H A M conditions (p's < 0.01) and greater in the P E L L E T than in the S H A M condition (p's < 0.01). After the initiation of restraint (10 min), females and males in the A D X condition showed significantly greater A C T H elevations than animals in both the P E L L E T and S H A M conditions (p's < 0.01). During restraint (30, 60 min), however, females and males in the P E L L E T condition had a markedly increased A C T H response, thus eliminating differences in A C T H levels between animals in the A D X and P E L L E T conditions. For females, further analysis by A N O V A s for prenatal group and sampling time within each experimental condition revealed main effects of prenatal group, F(2, 30) = 4.86, p < 0.01, on A C T H levels for females in the S H A M but not in the A D X and P E L L E T conditions. In the S H A M condition, E females had higher A C T H than C females (p < 0.01). In addition, there were effects of sampling time for females in all three conditions [F(4, 120) = 31.17, p < 0.01, F(4, 120) = 19.48, p < 0.01, and F(4, 120) = 51.26, p < 0.01, in A D X , P E L L E T and S H A M conditions, respectively]. As expected, females in all conditions had elevations in A C T H after 121  the initiation of restraint (0 < 10, 30, 60 min; p's < 0.01) although females in the A D X condition showed more prolonged elevations in A C T H during restraint (10 > 30 and 60 min; p's < 0.01). Females in the A D X and P E L L E T conditions showed reduced A C T H levels following the termination of restraint compared to levels during restraint ( A D X : 10 > 90 min; P E L L E T : 10, 60 > 90 min; p's < 0.01), but A C T H did not recover to basal levels (0 < 90 mi; p's < 0.01). In contrast, females in the S H A M condition showed recovery to basal A C T H levels after the termination of restraint (0 = 90 < 10, 30 and 60 min; p's < 0.01). For males, further analyses for prenatal group and sampling time within each experimental condition revealed effects of sampling time [F(4, 108) = 8.52, p < 0.01, F(4, 108) = 14.73, p < 0.01, and F(4, 108) = 41.56, p < 0.01, in the A D X , P E L L E T and S H A M conditions, respectively]. As expected, males in all conditions had elevations in A C T H after the initiation of restraint [ A D X : 0 < 10, 30 min; and 10 > 60 min (p's < 0.04); P E L L E T and S H A M : 0 < 10, 30, 60 min (p's < 0.01)]. Males in the A D X condition showed recovery to basal A C T H levels after the termination of restraint.  Males in the P E L L E T and S H A M conditions showed reduced  A C T H levels following the termination of restraint [PELLET: 60 > 90 min (p < 0.02); S H A M : 10, 30, 60 > 90 min (p's < 0.05)], but did not recover to basal levels (0 < 90 min, p's < 0.01). C O R T Levels Analysis of basal CORT levels (0 min) revealed significant main effects of sex, F ( l , 165) = 51.57, p < 0.01, and experimental condition, F(2, 165) = 87.37, p < 0.01 as well as a sex x experimental condition interaction, F(2, 165) = 18.31, p < 0.01. Females had higher basal CORT levels than males in the P E L L E T and S H A M conditions (p's < 0.01), but not in the A D X condition (Table 11). In addition, basal C O R T levels were significantly reduced in females and males in the A D X compared to the P E L L E T and S H A M conditions (p's < 0.01), with CORT levels near zero for animals in the A D X condition. Basal CORT levels were elevated in females in the P E L L E T compared to the S H A M condition (p < 0.02), whereas basal CORT levels in 122  males in the P E L L E T condition did not differ from those of males in the S H A M condition. Furthermore, dependent, one-tailed t-tests showed no differences between basal and stressassociated C O R T levels for animals in the A D X or P E L L E T conditions. Analysis of basal and stress-associated C O R T levels for females within the S H A M condition by a repeated-measures A N O V A for prenatal group and sampling time revealed effects of prenatal group, F(2, 30) = 4.86, p < 0.01, and sampling time, F(4, 120) = 119.56, p < 0.01 (Figure 15). As expected, CORT levels increased in all females after the initiation of restraint (0 < 10, 30, 60 min; p's < 0.01) and decreased after the termination of restraint (10, 30, 60 > 90 min; p's < 0.01), although they did not return to basal levels (0 < 90 min; p < 0.01). Furthermore, E and PF females had higher CORT than C females (p's < 0.04). For males, in contrast, there were no effects of prenatal group but there was an effect of sampling time, F(4, 108) = 105.04, p < 0.01, on CORT levels (Figure 15). CORT levels increased after the initiation of restraint (0 < 10, 30, 90 min; p's < 0.01) and over the course of restraint (10 < 30, 60 min; p's < 0.01) and did not recover to basal levels after the termination of restraint (0 < 90 min; p < 0.01).  Discussion There are a number of major findings in this study.  As expected, basal and stress-  induced A C T H levels were significantly increased in animals in the A D X compared to the S H A M condition, across all prenatal groups.  C O R T replacement with pellets producing a  constant basal level (PELLET) reduced basal A C T H levels in all prenatal groups compared to levels in their A D X counterparts (although not to the level of S H A M animals), but was minimally effective at reducing A C T H levels during stress, supporting previous findings that constant basal C O R T replacement permits sustained stress-induced A C T H hypersecretion in A D X rats (Akana et al., 1988; Jacobsen et al., 1988; Kaneko et al., 1980). These findings 123  indicate that in the absence of a CORT feedback signal or in the presence of a constant, basal CORT feedback signal, E, PF and C animals did not significantly differ in their abilities to regulate ACTH secretion. These data further indicate that during the trough of the circadian rhythm, E, PF and C animals are equally capable of regulating HPA activity utilizing either CORT-independent feedback or feedback mediated through basal CORT activity. In addition, we found that in the SHAM condition, E females but not males showed elevated ACTH and CORT levels compared to C females, extending previous work demonstrating the differential appearance of HPA hyperresponsiveness in E female and male animals (Weinberg, 1988; 1992a; Weinberg et al., 1996). The data demonstrating adverse effects of maternal ethanol consumption on maternal weights, pregnancy outcome and offspring weight gain are consistent with previous data both from our laboratory and from others (Abel & Dintcheff, 1979; Ogilvie & Rivier, 1997; Weinberg, 1992a; 1992b; Weinberg et al., 1995). Maternal ethanol intake was consistently high throughout gestation, ranging from 9.38 to 10.4 g/kg bw over the 3 weeks of gestation. We have previously shown that this level of intake results in peak blood ethanol levels of 145-155 mg/dl (Osborn et al., 1996; Weinberg, 1985).  In the present study, blood ethanol levels of  approximately 125 mg/dl were found in the one pregnant dam that was sampled. The suppressed maternal weight gain in E dams during gestation and reduced body weights of E pups were, in part, mediated by ethanol-induced nutritional effects since both E and PF groups showed similar effects.  However, direct effects of ethanol were also observed, with E dams having longer  gestation lengths than PF and C dams and E pups weighing less than both PF and C pups at weaning. These weight differences did not persist into adulthood; by the time of testing, there were no differences in body weights among E, PF and C animals. We have previously shown that A D X is associated with increased basal ACTH levels in E males during the peak of the circadian rhythm, indicating that alterations in HPA activity in E 124  animals may be, at least partly, mediated through CORT-independent mechanisms (Glavas et al., 1998a; 1998b). The results of the present experiment indicate that this altered regulation of the H P A axis in E animals may occur differentially depending upon the phase of the circadian rhythm. During the peak of the circadian rhythm, increases in CORT appear to be driven by the hypothalamus; C O R T increases do not occur in rats with lesions of the medial basal hypothalamus or the P V N (Dallman et al., 1985; Kaneko et al., 1980). In contrast, there is normally no hypothalamic stimulation of the resting, non-stressed animal during the trough of the diurnal rhythm as evidenced by the lack of effect of hypothalamic lesions (Dallman et al., 1985; Kaneko et al., 1980; Levin et al., 1988). In the present experiment, no differences were seen in A C T H levels among A D X E, PF and C females and males. This lack of differences during the trough of the circadian rhythm compared to previous findings indicating significant differences during the peak may be due to differences in hypothalamic stimulation of the pituitary between the trough and peak.  Since H P A activity during the trough is driven by  constitutive pituitary activity, the similarity of E, PF and C animals may be indicative of equal releasable pools of pituitary A C T H . These findings, therefore, suggest that differences in basal A C T H activity among E, PF and C animals in the P M but not the A M may be, at least partly, mediated by differences in hypothalamic activity. Extreme disequilibrium in central components of the H P A axis occurs during the first days after A D X with removal of the CORT feedback signal and results in both increased basal A C T H levels and augmented A C T H responses to stress (Akana et al., 1985; Keller-Wood & Dallman, 1984). Feedback mechanisms may be reestablished with steroid replacement, thereby reducing the hyperresponsiveness of A D X animals (Akana et al., 1985; Keller-Wood & Dallman, 1984; Levin et al., 1988). Previous research has shown that plasma CORT levels between 4.5 and 7.4 pg/dl best restore body weight, thymus weight, and basal A C T H levels during the trough of the circadian rhythm to normal in juvenile male rats (Akana et al., 1985). 125  Furthermore, while corticosteroid-binding globulin and thymus weight require 4.4-4.6 pg/dl of CORT, A C T H is more sensitive to inhibition by CORT, requiring levels of only 1.5-3.0 u.g/dl (Levin et al., 1987) in the A M . The very low concentration of C O R T required to normalize A C T H secretion in the A M indicates that its effect may be mediated through high affinity, type I glucocorticoid receptors (i.e. MRs). In contrast, the effects of C O R T on the liver and thymus may be mediated by lower affinity, type II glucocorticoid receptors (i.e. GRs; Dallman et al., 1987; Reul & de Kloet, 1985).  Thus, the liver and thymus may be protected from daily  perturbations in CORT activity such as occurs during the peak of the circadian rhythm when negative feedback sensitivity is lower and higher concentrations of C O R T are required to normalize A C T H . In the present experiment, levels of CORT replacement were chosen that would control H P A activity during the trough of the circadian rhythm when there is increased sensitivity to CORT feedback (Akana et al., 1986; Dallman et al., 1987). Levels of C O R T in the animals in the P E L L E T condition were 13.7 u,g/dl for females and 5.0 pg/dl for males. These levels of CORT replacement resulted in plasma CORT levels in females that were elevated compared to S H A M , whereas for males plasma CORT levels in P E L L E T and S H A M animals did not differ. Thus, CORT feedback signals differed in P E L L E T and S H A M females but were similar in P E L L E T and S H A M males. In response to these CORT signals, E , PF and C animals did not differ in their basal A C T H levels.  Thus, the lack of a circadian C O R T rhythm did not  differentially affect E, PF and C animals. This finding extends previous work in our laboratory that examined the ability of a phasic CORT signal (CORT replacement via the drinking water) to regulate A C T H activity. These previous data demonstrated that E, PF and C animals had similar basal and stress-associated A C T H levels during the peak of the circadian rhythm (Glavas et al., 1998a; 1998b). Together, these results indicate that E, PF and C animals are equally able to utilize an exogenous CORT feedback signal to regulate basal A C T H activity, regardless of the 126  phase of the circadian rhythm ( A M or P M ) or the type of CORT feedback signal (tonic or phasic). As under basal conditions, A C T H levels during and after restraint did not differ among CORT-replaced (i.e. P E L L E T ) E, PF and C animals. However, A C T H levels were very high in these P E L L E T groups, approaching the levels observed in A D X animals over the course of restraint. Although this level of CORT replacement was sufficient to maintain low basal A C T H levels, levels were too low to control the stress-induced A C T H response, and may have resulted in a maximal response in all animals. In contrast, a phasic CORT signal provided by CORT replacement in the drinking water has been shown to be sufficient for normal termination of A C T H responses to stress (Jacobson et al., 1988).  Although animals who receive CORT  replacement in the drinking water are still unable to mount a C O R T response to a stressor, CORT in the water does produce circadian variations in steroid levels (Jacobson et al., 1988) since rats consume approximately 70-75% of their daily fluid intake during the dark period (Kakolewski et al., 1971). This differential effect of CORT replacement via drinking water versus pellets in inhibiting stress-induced A C T H hypersecretion may be mediated by differences in receptor occupancy.  Due to the presence of a CORT circadian rhythm, animals in both  S H A M and A D X conditions that receive CORT in the water have higher glucocorticoid receptor occupancy during lights off (Reul et al., 1987). In contrast, constant levels of CORT in animals replaced with CORT pellets may result in full occupation of M R s and continual partial occupation of GRs throughout the 24-hour cycle that could, perhaps, lead to corticosteroid receptor downregulation (Sapolsky et al., 1984). However, the similarity in results from studies in our laboratory examining A C T H responses following CORT replacement with either a phasic or a tonic CORT signal suggests that receptor downregulation in the P E L L E T treatment did not mask differences among E, PF and C animals in the present study.  127  The level of CORT replacement achieved in the present study should have resulted primarily in M R occupation (Reul & de Kloet, 1985). Since MRs are thought to maintain basal H P A activity by mediating tonic inhibition, this suggests that a low C O R T signal, acting mainly via MRs, is equally effective in regulating H P A activity in E , PF and C animals. This is not unexpected since E animals have normal basal H P A activity, showing hyperresponsiveness only to stressors when C O R T is increased to a level which would occupy a large percent of GRs. Feedback deficits in E animals may therefore involve mainly the GRs that were likely minimally occupied with the level of CORT replacement used in the present study. While studies from our laboratory using cytosolic binding assays found no deficits in M R and G R densities or binding affinities in E animals in the hippocampus and other feedback sites (Kim et al., 1999b; Weinberg & Peterson, 1991), Redei et al. (1993) found that E males had significantly greater levels of GR mRNA in the P V N of the hypothalamus under basal conditions compared to control males, suggesting that glucocorticoid receptor expression may be altered by prenatal ethanol exposure.  Increased glucocorticoid receptor expression may be an indication of  enhanced feedback in E males occurring as a compensatory response to deficits downstream of glucocorticoid binding and/or feedback deficits elsewhere in the H P A axis. It is also possible that the balance of MRs and GRs at various feedback sites may be altered as a result of prenatal ethanol exposure. Such a possibility is supported by the finding that E animals exhibit higher stress A C T H and/or CORT levels following suppression by D E X , a selective G R agonist (Nelson et al., 1985; Osborn et al., 1996). In the present study, prenatal ethanol exposure resulted in increased A C T H and C O R T levels in response to restraint although this H P A hyperresponsiveness was only observed in E females. It is becoming increasingly clear that the type and duration of the stressor, the sex of the animal, and the animal's previous experiences all influence the response to stress, and that such influences may be mediated through multiple pathways 128  (Lightman, 1994; Sapolsky, 1994).  Research has shown that hypothalamic responses to  stressful stimuli may be mediated through input by the hippocampal-hypothalamic pathway (Herman et al., 1989), ascending catecholamine system from the brainstem (Plotsky et al., 1989), other cell groups including the amygdala (Beaulieu et al., 1986), the bed nucleus of the stria terminalis, and frontal cortical regions (Feldman & Conforti, 1985), as well as through activated cells of the immune system which have been shown to release C R H and A C T H (Harbuz & Lightman, 1992; Rivier, 1993). A n example of the complexity of the H P A axis may be found in the differential response of the hypothalamus to acute and chronic stress. In response to acute stress, both V P and C R H are released from the hypothalamus; however, in response to chronic stress, V P release is selectively increased while C R H release remains unchanged or is reduced, resulting in an increased V P : C R H ratio, and demonstrating the flexibility and adaptability of the H P A response to stressful stimuli (Dallman, 1993). It seems likely that the widely distributed corticosteroid feedback sites in the brain may mediate H P A activity in a stressor-specific manner (Lightman, 1994). Lesions of the ventral subiculum, a principal source of hippocampal efferents, appear to alter H P A activity in a stressor-specific manner, increasing H P A activity in response to restraint or open-field exposure but not affecting responses to ether inhalation or basal H P A activity (Herman et al., 1998).  Transsection of the lateral fimbria-fornix, which carries a large proportion of  efferents from the ventral subiculum, does not result in A C T H or C O R T hypersecretion in response to hypoxia (Bradbury et al., 1993). In contrast, a site in the prefrontal cortex that contains M R s and GRs may respond to feedback inhibition during exposure to restraint stress but not during ether stress or under basal conditions, suggesting that this area of brain exerts C O R T feedback with great stressor specificity (Diorio et al., 1993). Thus, the regulation of the H P A axis may be a distributed network covering many areas of the brain.  Therefore,  perturbations resulting from prenatal ethanol exposure at any number of sites may influence 129  HPA activity and the effects may be specific to a particular type or duration of stressor or may be revealed in a sex-specific manner. Our laboratory has been assessing the ability of the early manipulation, postnatal handling, to attenuate some of the adverse effects of prenatal ethanol exposure on endocrine function.  We have previously demonstrated that E animals show deficits in intermediate  feedback inhibition of H P A activity (Osborn et al., 1996), and Meaney et al. (1989) have shown that postnatal handling results in enhanced intermediate feedback inhibition compared to N H animals.  However, postnatal handling does not appear to eliminate deficits in  feedback inhibition in E females, nor does it attenuate H P A hyperresponsiveness to restraint stress in E animals (Chapter V ; Gabriel et al., 1998). Interestingly, previous research has found that H P A differences between H and N H animals are dependent on circulating basal C O R T , but not on the elevated C O R T levels associated with exposure to stressors (Viau et al., 1993). Specifically, H P A differences between H and N H animals can be reestablished in A D X animals through a steady, basal level C O R T replacement (Viau et al., 1993) in contrast to the differences among E , PF and C animals. The present finding that H P A differences following prenatal ethanol exposure do not appear to be mediated by basal C O R T feedback signals indicates that the effects of handling and prenatal ethanol exposure on H P A function may occur through different mechanisms. Therefore, the ability of handling to attenuate the adverse effects of prenatal ethanol exposure may be limited. In conclusion, CORT replacement reduced ADX-induced A C T H hypersecretion equally among E , PF and C animals, indicating that in the presence of a constant, basal C O R T feedback signal E, PF and C animals did not significantly differ in their abilities to regulate A C T H secretion during the trough of the circadian rhythm. The finding that tonic inhibition of the H P A axis does not differentiate among E, PF and C animals during the circadian trough supports previous findings that M R function may be normal in E rats. 130  However, further studies to  determine changes in C R H , V P and M R / G R m R N A levels following A D X with or without CORT replacement may be necessary to pinpoint the sites or mechanisms of H P A alterations in E animals.  131  Table 7 Maternal Body Weights (g, Mean ± SEM) of E, PF and C Dams during Gestation (G) and Lactation (PN).  Gestation  GI  G7  G14  G21  E  232.4 + 5.2  237.3 +4.8*  264.5 + 5.1*  318.2 + 5.0*  PF  236.8 + 4.5  237.0 + 4.9*  261.7 + 5.2*  325.1 +5.6*  C  233.1 +4.7  262.8 ± 5.0*  295.6 + 5.4*  378.6 + 8.1*  Lactation  PN1  PN8  PN15  PN22  E  267.0 ± 5.3  A  304.5 ± 5.9  323.4 + 5.9  310.3 + 5.1  PF  269.4 ± 6.0  A  307.3 +4.2  332.7 + 5.1  327.7 + 4.9  C  302.6+ 10.4  310.9 + 4.7  333.1 +4.8  325.4 + 4.4  Gestation:  A  E (n = 16); PF (n = 16); C (n = 14). *At G7, G14 & G21, E = PF < C, p's < 0.05  Lactation:  E (n = 15); PF (n = 16); C (n = 16). A  A t PN1, E = PF < C, p's < 0.05  132  Table 8 Gestation Length (d, Mean ± SEM) of E, PF and C Dams.  Gestational Length E  23.6 ± 0.20®  PF  22.9 ± 0.06®  C  23.1'±0.07®  E (n = 16); PF (n = 16); C (n = 14). Gestation Length:  ® E < PF = C, p's < 0.01  133  Table 9 Postnatal (PN) Pup Body Weights (g, Mean ± SEM) of E, PF and C Female and Male Pups.  Female  PN1  PN8  PN15*  E  5.2 ± 0 . 1  12.1 ± 0 . 6 "  26.5 ± 0.9"  PF  5.4 ± 0 . 1  C  6.5 ± 0 . 1  12.9 ±6.5® 15.9 ±6.3®  27.5 ± 29.3 ±  6.8® 6.7®  PN22* .. 39.9 ± 1.3  A  42.8 ± 0.8  A  47.2 ± 0.9  A  Male  PN1  PN8  PN15*  PN22*  E  5.5 ± 0 . 1  13.0 ± 0 . 5 "  28.4 ± 0.9"  43.6 ± 1.5  A  PF  5.7 ± 0 . 1  13.6 ± 0 . 5 "  43.8 ± 0.9  A  C  6.8 ± 0 . 1  28.7 ± 6.5®  48.1 ± 1.0  A  16.3 ±  6.4®  31.3 ±6.7®  For both females and males: E (n = 15 litters); PF (n = 16 litters); C (n = 16 litters). Pup Weights: *Female < Male on PN15 & PN22, p's < 0.01 ®At PN8 & PN15, E = PF < C animals, p's < 0.05. A  A t PN22, E < PF < C animals, p's < 0.05  134  Table 10 Adult Body Weights (g, Mean ± SEM) of E , PF and C Female and Male Animals at 120-150 Days of Age.  Female*  Male*  E  270.9 ± 4.4  489.7 ± 7.7  PF  277.8 ± 3.9  491.6 ± 7 . 5  C  280.5 ± 4 . 0  492.6 ± 4.8  Adult Weights:  Female (E: n = 41; PF: n = 38; C: n = 38); Male (E: n = 35; PF: n = 45; C: n = 45); *Female < Male, p < 0.01  135  Table 11 Basal CORT Levels (ug/dl, Mean ± SEM) for E, PF and C Female and Male Animals in A D X , P E L L E T and S H A M Conditions.  SHAM  Female  ADX  PELLET  E  0.60 + 0.17^  15.6 ± 1.5  11.5 ± 2 . 3 "  PF  0.35 ± 0.03*  12.5 ± 1.3  12.0 ± 2 . 3  C  0.36 ± 0.03*  13.3 + 1.2*  Male  ADX  E  0.94 ± 0.45  PF C  CORT Levels:  9  9  A  69  129  9  A  Q9  8.5 + 1.4^  SHAM  PELLET  A  +  5.1 ± 0 . 6  +  0.32 ± 0.03  +  5.3 ± 0.6  +  4.5 + 1.2  0.47 +0.18  T  4.6 ± 0.7  +  6.5 + 1.6  A  8.4+ 1.5  +  +  +  Female: A D X (E = 11, PF = 10, C = 11), P E L L E T (E = 11, PF = 11, C = 11), S H A M (E = 11, PF = 11, C = 11). Male: A D X (E = 8, PF = 10, C = 7), P E L L E T (E = 10, PF = 10, C = 10), S H A M (E = 10, PF = 10, C = 10) A  In P E L L E T & S H A M conditions, female > male, p's < 0.01.  ®For females, A D X < S H A M < P E L L E T , p's < 0.02. +  For males, A D X < S H A M = P E L L E T , p's < 0.01  136  Table 12 Basal and Stress-Associated CORT Levels (u,g/dl, Mean ± SEM) for E, PF and C Female and Male Animals in A D X and P E L L E T Conditions.  PEL L E T  A I )X Female  Basal  Stress  Basal  Stress  E  0.60 ± 0 . 1 7  0.63 ± 0 . 1 9  15.6 ± 1.5  14.2 ± 1.1  PF  0.35 ± 0 . 0 3  0.48 ± 0 . 1 0  12.5 ± 1.3  14.0 ± 1.9  C  0.36 ± 0 . 0 3  0.39 ± 0 . 0 6  13.3 ± 1.2  16.4 ± 1.9  PEL L E T  A I )X Male  Basal  Stress  Basal  Stress  E  0.94 ± 0.45  1.06 ± 0 . 5 1  5.1 ± 0 . 6  5.8 ± 1.0  PF  0.32 ± 0 . 0 3  0.35 ± 0 . 0 8  5.3 ± 0 . 6  4.6 ± 0 . 8  C  0.47 ± 0 . 1 8  0.59 ± 0.24  4.6 ± 0 . 7  4.5 ± 0.6  CORT Levels: Female: A D X (E = 11, PF = 10, C = 11), P E L L E T (E = 11, PF = 11, C = 11). Male: A D X (E = 8, PF = 10, C = 7), P E L L E T (E = 10, PF = 10, C = 10). Within A D X and P E L L E T conditions, there were no significant differences between basal and stress CORT levels.  137  Figure 13. Plasma A C T H levels (pg/ml, Mean ± SEM) for E , PF and C females in A D X , P E L L E T and S H A M conditions (n = 11 per each point). A D X > P E L L E T > S H A M at 0 and 10 min (p's < 0.01), but A D X = P E L L E T > S H A M at 30 and 60 min (p's < 0.01). For all females, 0 < 10, 30, 60 min (p's < 0.01). In the A D X condition, 10 > 30, 60 min; 10 > 90 min (p's < 0.01). In the P E L L E T condition, 10, 60 > 90 min (p's < 0.01). In both the A D X and P E L L E T conditions, 0 < 90 min (p's < 0.01). In the S H A M condition, 0 = 90 < 10, 30, 60 min (p's < 0.01);andE>C(p<0.01).  138  Ethanol - ° ~ Pair-Fed —*- Control  139  Figure 14.  Plasma A C T H levels (pg/ml, Mean ± S E M ) for E, PF and C males in A D X ,  P E L L E T and S H A M conditions (n = 10 per each point). A D X > P E L L E T > S H A M at 0 and 10 min (p's < 0.01), but A D X = P E L L E T > S H A M at 30 and 60 min (p's < 0.01). In the A D X condition, 0 < 10, 30 min (p's < 0.01); and 0 = 90 min. In the P E L L E T condition, 0 < 10, 30, 60, 90 min; 60 > 90 min (p's < 0.02). In the S H A M condition, 0 < 10, 30, 60 min; 10, 30, 60 > 90 min; 0 < 90 min (p's < 0.01).  140  1500  SHAM  PELLET  1200 h  900  600  300 h  Restraint 0  10 30 60 90  Ethanol  0 —G--  10 30 60 90  Pair-Fed  141  4  Restraint  0  10 30  60 90  Control  Figure 15. Plasma CORT levels (ug/dl, Mean ± SEM) for E, PF and C females and males in the S H A M condition (n = 10-11 per each point). For females, 0 < 10, 30, 60, 90 min; 10, 30, 60 > 90 min; E > PF and C (p's < 0.04). For males, 0 < 10 < 30, 60 min; 0 < 90 min (p's < 0.01).  142  100  Females ^  80  H P4  60  o O §  40  20  Restraint 10  30  + - Ethanol  60  90  - ° ~ Pair-Fed  143  10  30  60  •  Control  90  C H A P T E R VII: G E N E R A L DISCUSSION  The major objectives of this thesis were to investigate (1) the correspondence between prenatal ethanol-induced alterations in behavior/cognition and in H P A activity, (2) the ability of early postnatal handling as an environmental manipulation to attenuate at least some of the adverse behavioral/cognitive and physiological consequences of prenatal ethanol exposure, and (3) possible mechanisms mediating the H P A hyperresponsiveness to stressors observed in E animals and the possible influence of postnatal handling on those mechanisms.  A . General Discussion of Studies The first study (Chapter III) investigated learning deficits in E animals during a conditioned taste aversion task as well as the correspondence between behavior and C O R T levels during reexposure to the conditioned solution.  We tested the hypothesis that E  animals which underwent postnatal handling would show improved conditioned aversion learning and reduced H P A activity compared to E animals that did not experience handling, and/or that handling might attenuate differences among E and control animals. We found that prenatal ethanol exposure and postnatal handling independently resulted in an increased rate of consumption of a saccharin solution over five preexposure days, suggesting that neophobia decreased at a faster rate in E and in H animals. Based on previous research, we suggest that increases in pretoxicosis intake may have been mediated through two distinct mechanisms; elevations in pretoxicosis intake in H animals may have been due to general reductions in emotional reactivity (Ferre et al., 1995; Weinberg et al., 1978) whereas elevations in pretoxicosis intake in E animals may have been the result of deficits in response inhibition (Riley et al., 1979a; 1979b).  In addition, we found that handling differentially affected acquisition of  conditioned taste aversion in E, PF and C animals; H - E animals showed increased posttoxicosis 144  intake compared to H-PF and H-C animals during non-deprived reexposure, indicating that H - E animals did not acquire the conditioned aversion to the same extent as H-PF and H-C animals. However, H-PF females also displayed deficits in conditioned aversion during deprived reexposure compared to H-E and H-C females, suggesting that deficits may be revealed in E and PF animals under different testing procedures.  Although the acquisition of the association  between the solution and subsequent illness may have been affected by the increased pretoxicosis intake in E animals, handling increased pretoxicosis intake across all prenatal groups and acquisition was not impaired in H - C compared to N H - C animals. Thus, although increased pretoxicosis intake may have affected posttoxicosis intake, H-E animals and H-PF females still exhibited deficits in their ability to discriminate the significance of the saccharin solution compared to H-C animals. Handling also differentially affected CORT levels in E , PF and C animals; CORT levels in response to deprived reexposure were lower in PF and C than E males compared to their N H counterparts. However, increases in CORT were not associated with increases in consumption, indicating dissociation between hormonal and behavioral measures.  Importantly, this study  demonstrates that behavior and H P A activity may not always correspond, emphasizing the importance of assessing them both when examining E animals' responses during testing.  E  animals were less able to utilize environmental cues in the present study, displaying a more rapid reduction in neophobia compared to PF and C animals regardless of postnatal handling treatments and, following postnatal handling, showing a decreased acquisition of the conditioned aversion. Importantly, handling appeared to unmask differences among E, PF and C animals rather than improving the performance of E animals. The second study (Chapter IV) examined spatial learning and memory in young adult (2 months of age) and mid-aged (13-14 months of age) H and N H E and control animals utilizing a Morris water maze. We investigated the hypothesis that postnatal handling would 145  improve spatial navigation in E animals compared to E animals that did not experience handling and/or attenuate differences among E and control animals, and that this effect might be agedependent.  We also examined whether deficits in mid-aged animals would correspond to  increases in CORT levels on the last day of testing.  Data demonstrate that young E males  showed impairments in spatial navigation compared to young PF and C animals, taking longer to find the hidden platform over the course of testing and displaying an alteration in search pattern when the platform was removed. Interestingly, differences in E, PF and C animals in escape latency and in distance traveled prior to finding the platform were apparent in H but not in N H animals. Thus, as in conditioned taste aversion, it appears that postnatal handling was associated with deficits in H - E animals compared to H-PF and H-C animals. There are at least two possible explanations for the differences in performance in young E males in the Morris water maze. First, the brain area underlying spatial cognitive abilities may be vulnerable to the teratogenic effects of prenatal ethanol exposure, and postnatal handling may be ineffective in attenuating the effects of such a prenatal insult. Second, E animals, which display H P A hyperresponsiveness to stressors, may be differentially responsive to the swim stress associated with performance in the Morris water maze, and postnatal handling may not attenuate this hyperresponsiveness. When mid-aged E , PF and C animals were tested, there were no differences in performance on the Morris water maze but CORT levels were elevated in mid-aged E compared to C animals, supporting previous data that E animals demonstrate H P A hyperresponsiveness to stressors or to the aversive aspects of a task.  While increased H P A activity in young animals may reflect  increased stress and possibly impair performance, recent research suggests that elevations in CORT level may produce more complex effects on behavior in aged animals (Hebda-Bauer et al., 1999) and may not necessarily conform to theories that elevations in CORT must necessarily be associated with learning deficits in aged animals. It is becoming increasingly apparent that HPA  activity and  early  postnatal  experience 146  may  complicate  the  assessment  of  cognitive/behavioral testing in mid-aged animals, making the identification of deficits in E animals and the interpretation of alterations in hormone levels and behavior more difficult. Lastly, although mid-aged animals had longer escape latencies and an altered search pattern compared to young animals, handling did not appear to attenuate impairments associated with aging, indicating that the ability of postnatal handling to ameliorate  senescence-associated  impairments may be difficult to demonstrate in mid-aged animals. The third and fourth studies (Chapters V and VI) of this thesis examined H P A hyperresponsiveness following prenatal ethanol exposure.  The third study (Chapter V )  investigated the hypothesis that postnatal handling might attenuate stress-induced A C T H and/or C O R T differences among E , PF and C animals. Furthermore, the ability of postnatal handling to modulate H P A feedback deficits in E animals was examined during exposure to a restraint stressor following D E X administration. The data demonstrate that both E females and males showed increased A C T H and CORT compared to PF and/or C animals following saline administration, extending previous findings of H P A hyperresponsiveness in E animals (Taylor et al., 1982; Weinberg et al., 1995; 1996). Administration of D E X to block H P A activity significantly suppressed both plasma A C T H and CORT in all animals. However, E females exhibited increased and/or prolonged elevations in A C T H and C O R T compared to PF and C animals following D E X blockade.  These data suggest that the insult of prenatal ethanol  exposure affects both female and male offspring, but that there may be a sex-specific difference in sensitivity of the mechanism(s) underlying H P A hyperresponsiveness. Early experience in the form of postnatal handling reduced A C T H levels in both females and males compared to their N H counterparts, and attenuated the elevation in CORT levels in H compared to N H males following saline administration, extending previous research on the effects of postnatal handling on H P A functioning in male animals (Meaney et al., 1989; 1991).  Furthermore, our data  demonstrate that following D E X administration H males had lower C O R T than N H males, 147  supporting previous findings of enhanced DEX-mediated negative feedback inhibition in H males (Meaney et al., 1989). Interestingly, handling differentially affected feedback inhibition in E, PF and C females. Postnatal handling resulted in a more rapid decrease in CORT levels in C compared to E and PF females, and attenuated differences in C O R T between PF and C females. However, postnatal handling did not attenuate deficits in negative feedback inhibition in E females; E females in both the H and N H treatments showed elevated C O R T compared to their C counterparts, and H - E females also showed elevated CORT compared to H-PF females. Therefore, postnatal handling did not attenuate the typical H P A hyperresponsiveness to stressors observed in E animals (saline condition), nor did it eliminate deficits in H P A feedback inhibition in E females (DEX condition). The fourth study (Chapter VI) examined whether the mechanisms resulting in H P A hyperresponsiveness in E animals are similar to those underlying the effects of postnatal handling.  Differences in H P A responsiveness between H and N H animals appear to be  dependent upon basal CORT feedback signals and not elevations in C O R T associated with stress. Therefore, we tested the hypothesis that differences in H P A activity among E and control animals would not occur following A D X but could be reestablished following replacement with basal levels of exogenous CORT. However, the hypothesis that basal CORT feedback signals underlie the H P A hyperresponsiveness following prenatal ethanol exposure was not supported. The findings indicate that in the absence of a C O R T feedback signal or in the presence of a constant, basal C O R T feedback signal, E , PF and C animals did not significantly differ in their abilities to regulate A C T H secretion. These data further indicate that during the trough of the circadian rhythm, E , PF and C animals are equally capable of regulating H P A activity utilizing either CORT-independent feedback or feedback mediated through basal CORT activity. In addition, we found that in the S H A M condition, E females but not males showed elevated A C T H and CORT levels compared to C females, extending previous work demonstrating the 148  differential appearance of H P A hyperresponsiveness in E female and male animals (Weinberg, 1988; 1992a; Weinberg et al., 1996). Because the current work indicates that the effects of prenatal ethanol exposure on H P A function are not mediated by basal C O R T activity, it appears that the effects of postnatal handling and prenatal ethanol exposure on H P A function occur through different mechanisms. Therefore, the ability of handling to attenuate the adverse effects of prenatal ethanol exposure may be limited. These findings extend previous research demonstrating that prenatal ethanol exposure results in impaired performances on behavioral/cognitive tasks including conditioned aversion learning and spatial learning and memory as well as resulting in H P A hyperresponsiveness to stressors. In addition, these data provide valuable insight into the mechanisms mediating H P A hyperresponsiveness in E animals; differences among E, PF and C animals do not appear to be mediated by alterations in basal CORT feedback signals, but are, at least partly, due to deficits in intermediate feedback inhibition in E animals. Although a differential appearance of age-related performance impairments in E , PF and C animals was not observed on the Morris water maze, mid-aged E animals did demonstrate H P A hyperresponsiveness to the aversive aspects of the testing situation, indicating a possible mechanism through which differential aging in E, PF and C animals might occur. Furthermore, these findings extend previous investigations of the ability of environmental manipulations such as postnatal handling and environmental enrichment to mitigate alterations in behavior/cognition and H P A activity in E animals. Data indicate that postnatal handling may, in fact, reveal differences among E, PF and C animals, and suggest that animals already altered through prenatal manipulations may have different requirements during environmental interventions in order to show improvement and, even then, their improvements may be limited.  Furthermore, E animals may be less likely to respond appropriately to  manipulations in which a specific response or behavior is necessary (i.e. increased pup  149  vocalizations following postnatal handling), resulting in further deficits or alterations in behavior and/or H P A functioning. B . Clinical Implications Cognitive deficits and behavioral abnormalities have been demonstrated in both clinical populations and animal models of prenatal alcohol exposure. Behavioral abnormalities such as hyperactivity, distractibility, response perseveration, impaired habituation and poor attention span (Coles, 1992; Streissguth, 1986; Streissguth et al., 1991) are among the most detrimental consequences in individuals diagnosed with F A S , resulting in a compromised ability to function in society. Data from this thesis indicate that E animals displayed performance deficits on spatial navigation in the Morris water maze, mirroring clinical studies which have shown that cognitive deficits in individuals with F A S / F A E are most apparent on spatial and complex tasks (Streissguth et a l , 1994). Furthermore, the H P A hyperresponsiveness in E animals shown in the present studies may be important when assessing clinical populations. Recent research has found that maternal drinking at conception and during pregnancy is associated with higher poststress Cortisol levels in infants (Jacobson et al., 1999). Data from studies of laboratory animals have demonstrated that central administration of C R H produces behavioral responses resembling stress-induced behaviors such as decreased  feeding in familiar and novel  environments, decreased sexual behavior, increased acoustic startle responses and increased defensive withdrawal (Koob et al., 1993). It is possible that C R H may increase the sensitivity of the organism to stressful aspects of their environment; thus, individuals with F A S may have difficulty in complex testing situations due to the behavioral results of enhanced H P A responsiveness. Although clinical studies have established that alcohol consumption markedly alters H P A function in chronic alcoholics (Merry & Marks, 1973), there is limited clinical information on how drinking during pregnancy effects the H P A axis of the developing child, indicating the importance of this issue for further study. Given that H P A hyperresponsiveness 150  has been shown to have adverse physiological and behavioral consequences which could compromise health and possibly even survival of an organism, it is not surprisingly that H P A hyperresponsiveness following prenatal alcohol exposure has been proposed to underlie, at least in part, some of the behavioral abnormalities (Kim et al., 1997) and immune deficits (Giberson et al., 1997; Giberson & Weinberg, 1995) observed in humans and animals. Several recent critiques of fetal alcohol research have emphasized the absent or limited scientific research in humans on early intervention, or treatment (Morse & Weiner, 1996; Norman et al, 1995; Stratton et al., 1996). In part, this reflects the difficulties in identifying individuals affected by prenatal alcohol exposure (Stratton et a l , 1996). In research utilizing animal models of prenatal ethanol exposure, there has been a growing emphasis on identifying treatments that can be administered to the alcohol-affected individual as well as determining which of the many physiological and/or behavioral consequences of prenatal ethanol exposure may be attenuated through the use of such treatments. The identification of conditions in animal models under which postnatal treatments can produce significant functional or neuroanatomical improvements may speed the development of therapeutic treatments in children affected by prenatal alcohol exposure. For example, rearing E rats in an enriched environment has been shown to attenuate gait ataxia and improve spatial learning and memory (Hannigan et al., 1993; Wainwright et al., 1993). However, unlike control animals, E animals do not show increased  hippocampal dendritic spine densities  following  environmental  enrichment  (Berman et al., 1996). Previous research in our laboratory has shown that postnatal handling eliminated deficits in preweaning weight gain often observed in E compared to PF and C pups and attenuated the initial increased C O R T elevation in response to restraint stress in E females although the more prolonged elevation following restraint was not attenuated (Weinberg et al., 1995). These findings suggest that postnatal interventions may be capable of altering some of the long-term effects of a prenatal insult. However, the results of the 151  studies reported in this thesis and previous work by Berman et al. (1996) indicate that the nature of the postnatal manipulation and the endpoints measured are critical in determining whether deficits in E animals can be attenuated.  Although caution must be used in  extrapolating data obtained from rodents to that of humans, given the large phylogenetic gap between the two species, animal models are useful in providing insight into those behaviors or physiological responses which may benefit for intervention. It has been proposed that the identification of possible treatments for children exposed prenatally to alcohol may be complicated by the variability in timing and amount of alcoholexposure, resulting in the lack of a common behavioral phenotype (Goodlett & Johnson, 1999). Commonly, after pregnancy is recognized, alcohol consumption declines as the pregnancy progresses with occasional episodes of binge drinking interspersed over the second and third trimesters (Rosett et al., 1983; Smith et al., 1986). Thus, the first trimester may be marked by relatively heavy drinking, but more variability in exposure may occur as the pregnancy progresses. Therefore, there may be a subset of effects resulting from differences in the timing and extent of exposure, affecting different brain structures or cell populations and their associated behaviors. For example, binge drinking at high levels has been shown to produce more devastating effects on the developing fetus than intake of the same dose of alcohol over a longer period of time (Pierce & West, 1986).  Thus, animal models have been critical in  demonstrating that the pattern and timing of drinking have significant effects on outcome. In many ways, the studies in this thesis examined behaviors and functions associated with the hippocampus. The hippocampus is a brain structure that is capable of considerable structural and functional plasticity, and the key to its plasticity is also a clue to its vulnerability to a variety of insults and to the wear and tear associated with the aging process (McEwen, 1994). Children with F A S appear to display problems with hippocampal-related functions such as spatial learning, response inhibition, and memory/attention deficits (Streissguth et al., 1994). As this 152  population ages, they may be particularly vulnerable to senescence-associated impairments in hippocampal function. The inability of postnatal handling to ameliorate deficits in hippocampalrelated behaviors in the present studies does not necessarily reflect permanent impairments in hippocampal plasticity following prenatal ethanol exposure. For humans, the major brain growth spurt including hippocampal development occurs during the third trimester of gestation and growth then continues postnatally (West, 1987). In contrast, the major brain growth spurt in the rat occurs during the first 10-14 days of postnatal life (this can be called the "third trimester equivalent")(West et al., 1989). Although we used a postnatal manipulation that overlapped with the brain growth spurt in the rat, we may not have accurately assessed the ability of postnatal manipulations to attenuate deficits in humans prenatally exposed to alcohol who may display hippocampal plasticity during a longer time in development.  C . Future Directions Despite two and a half decades of research on prenatal alcohol exposure, many questions remain unanswered.  Foremost among these questions concerns the manner in which animals  prenatally exposed to ethanol perceive and respond to stimuli in their environments, especially during stressful situations. In the current work, deficits were observed in E animals during both conditioned taste aversion and the Morris water maze. While these findings correspond with previous reports that E animals exhibit deficits in response inhibition (Riley et al., 1979a; 1979b) and spatial navigation (Blanchard et al., 1987; 1990; K i m et al., 1997), such deficits also may indicate that E animals have difficulty utilizing or responding to environmental cues.  In other words, it is  possible that E animals were less able than controls to associate the saccharin solution with their subsequent illness during conditioned taste aversion or to construct a cognitive spatial map from visual cues in the room during Morris water maze testing. Unlike PF and C animals, E animals 153  do not respond differentially to predictable versus unpredictable restraint stress (Weinberg, 1992a), although such deficits may occur selectively in either males or females depending on the nature of the task. Furthermore, exposure to cues associated with eating and drinking has been shown to be as effective as drinking in reducing adrenocortical activity (Coover et al., 1977); however, E animals do not use cues associated with consummatory behavior to reduce arousal (CORT responses) in a novel situation, whereas PF and C animals demonstrate significant attenuation of the C O R T response when allowed to drink (Weinberg, 1988). Impairments in utilizing environmental cues in E animals are especially troubling when the ecological relevance of conditioned taste aversion is considered. Previous research has suggested that animals may be predisposed to learn an association between food and subsequent illness because it is such a critical association for survival (Seligman, 1970). The finding that E animals display learning deficits in such situations suggests that the effects of prenatal ethanol exposure may produce impairments that can threaten the survival of the organism. Future research should examine cognitive/behavioral tasks which animals may be predisposed to learn due to their adaptive function (e.g. defensive burying). Such research may reveal the extent to which prenatal ethanol exposure impairs an organism's ability to utilize and respond to environmental cues, and whether such deficits are limited to particular stimuli or components of such stimuli. A n inability to accurately assess the environment when stressful stimuli are present may have severe consequences on H P A functioning in E animals.  There is a great deal of  interindividual variability in the magnitude and quality of responses to psychological stressors, with the perception of stress affected by variables such as the degree of control an organism has over the experience, the predictability of the stressor, or the availability of arousal-reducing responses.  For example, in a yoked shock condition, where one animal has control of the  termination of shocks by pressing a lever and another raf is passively "yoked" to the shock pattern of the first rat, the "yoked" rat has higher CORT levels and is more vulnerable to gastric 154  erosions (Swenson & Vogel, 1983; Weiss, 1971a; 1971b). Furthermore, the removal of the lever controlling shock can trigger CORT secretion, even in the absence of shocks (Coover et al., 1973). Predictability may also affect the experience of stress. The presence of a warning signal prior to the onset of shock has been shown to lower the CORT response compared to animals that receive no warning (Dess-Beech et al., 1983). The ability of variables such as control and predictability to attenuate the H P A response to stress is, however, dependent on an organism's ability to attend to and utilize such environmental cues. As noted, it appears that E animals have deficits in the ability to interpret cues providing information about such factors. Unlike PF and C animals, E animals do not respond differentially to predictable versus unpredictable restraint stress (Weinberg, 1992a). The ability to make an arousal-reducing response may also reduce the impact of psychological stress. Studies have shown that the magnitude of the CORT response to shock decreases in rats allowed any of a broad array of arousal-reducing behaviors, including a consummatory event such as eating or drinking (Levine et al., 1979), access to a running wheel (Levitsky & Collier, 1968), or fighting (Weinberg et al., 1980). However, as noted, E animals do not show reductions in CORT responses to a novel environment when allowed access to water, whereas PF and C animals demonstrate significant attenuation of the CORT response when allowed to drink (Weinberg, 1988). Therefore, further research exploring the manner in which E animals interpret stimuli in their environment, and whether the presence of specific variables may alter their experience of stress may provide important information on how E animals respond to a typical testing situation. The correspondence between cognitive/behavioral impairments and alterations in H P A functioning should also be further examined. High stress responses have been shown to impair cognitive function, with high CORT levels impairing performance on memory tasks and induction of long-term potentiation (McEwen, 1994).  Previous research has shown that E  animals exhibit both behavioral and hormonal hyperactivity to stress (Angelogianni & 155  Gianoulakis, 1989; Lee et al., 1990; Nelson et al., 1986; Osborne et al., 1980; Taylor et al., 1982; Weinberg, 1992a; 1993), suggesting that cognitive/behavioral impairments in E animals may be most apparent during stressful situations or in complex testing procedures. Therefore, cognitive/behavioral measures should be compared with stress hormone levels measured during or after testing. In the present studies, impairments in behavioral/cognitive performance in E animals were not associated with elevations in C O R T during either conditioned taste aversion or Morris water maze testing. During testing on the Morris water maze, mid-aged E animals showed elevated C O R T compared to C animals, indicating that E animals may have been more responsive to the aversive aspects of the task although increased CORT activity was not associated with deficits in spatial learning or memory. Previous research has shown that older animals may show improved function with moderate increases in H P A activity (Hebda-Bauer et al., 1999), complicating the interpretation of data in the present study and emphasizing the need for further investigations on the effects of aging in E animals. In the present study, the hypothesis of accelerated aging in E animals was not supported. However, animals were examined at only 13-14 months of age, an age range that may be considered relatively young in research on aging, and it is possible that studies examining more advanced ages may reveal differences among E, PF and C animals. Research evaluating various types of environmental manipulations for their effectiveness in attenuating the effects of prenatal ethanol exposure has yielded mixed results. A n enriched environment has been shown to abolish the detrimental effects of prenatal ethanol exposure on conditioned taste aversion in E mice (Opitz et al., 1993). In rats, an enriched environment has been show to attenuate gait ataxia and improve performance in the Morris water maze in E animals when compared to E animals reared in isolation (Hannigan et al., 1993; Wainwright et al., 1993). However, unlike control animals, E animals do not show increased hippocampal dendritic spine  densities  following  environmental 156  enrichment  (Berman et  al., 1996),  underscoring the enduring impairments of prenatal ethanol exposure. In contrast, Klintsova et al. (1997) demonstrated that adult behavioral rehabilitation in the form of acrobatic motor training resulted in synaptic neuroplasticity as well as behavioral improvement in rats with alcoholinduced cerebellar damage. Future research should focus on identifying conditions under which postnatal treatments can produce significant functional or neuroanatomical improvements in E animals. To date, the examination of postnatal handling as a treatment to attenuate the effects of prenatal ethanol exposure is the only manipulation that has investigated effects on H P A hyperresponsiveness in E animals (Gabriel et al., 1998; Ogilvie & Rivier, 1997); further research examining the correspondence between behavior and H P A activity as well as the influence of postnatal interventions on both are necessary. E animals differ in their behavioral and hormonal responses to stressful stimuli and complex environmental cues, therefore it is imperative that investigations of possible interventions consider differences in the manner in which E animals respond to their environment as well as their capacity for dealing with change in that environment. The results of such research may aid in the development of cues that better provide E animals with information concerning control, predictability, and/or the possibility of arousal-reducing activity in their environment. The ability to utilize such cues may reduce the stressful nature of testing for E animals, and we may be able to more accurately assess their behavioral/cognitive abilities. Furthermore, the behavioral, neuroanatomical and hormonal effects of prenatal ethanol exposure may vary across the lifespan, and research that examines animals at older ages is essential. Given such information, we may be better able to assess the effectiveness of postnatal interventions across the lifespan as well as be able to predict the care requirements of aging individuals with F A S / F A E .  157  D. Conclusions In conclusion, the experiments of this thesis demonstrate that prenatal ethanol exposure has long-term effects on cognitive/behavioral function and on H P A responsiveness to stressors. Prenatal ethanol exposure produced deficits on conditioned taste aversion and spatial navigation, as well as resulting in H P A hyperresponsiveness to restraint stress, an effect likely mediated through deficits in feedback inhibition during the intermediate time domain and not through basal CORT feedback signals. Postnatal handling altered pretoxicosis consummatory behavior and differentially  affected conditioned taste averison acquisition, C O R T levels during  conditioned taste aversion, and spatial learning and memory in young E , PF and C animals. However, postnatal handling did not attenuate performance impairments in the Morris water maze in mid-aged animals. Nor did postnatal handling attenuate H P A hyperresponsiveness to restraint stress or enhance H P A feedback inhibition during the intermediate time domain in E animals. The effects of prenatal alcohol exposure are entirely preventable. 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