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Neonatal Endotoxin Exposure and Alcohol Intake of Mice in Early Adulthood


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NEONATAL ENDOTOXIN EXPOSURE AND ALCOHOL INTAKE OF MICE IN EARLY ADULTHOOD By CHERYL H. VAUGHAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Cheryl H. Vaughan

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The love and support I have received from my parents has made all the difference in my pursuit of graduate studies. Their unconditional love has made me the person I am today. This thesis is dedicated to my mother, Dahlia, and my father, Arthur.

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ACKNOWLEDGMENTS I would like to thank Dr. Neil Rowland for his support, patience and encouraging words throughout the last three years. I would also like to acknowledge my other committee members for their insights and contributions to my research at the University of Florida. I have made some close friends here at UF and their friendship has made the past few years a stronger learning experience. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................vii LIST OF FIGURES.........................................................................................................viii ABSTRACT.......................................................................................................................ix INTRODUCTION...............................................................................................................1 Alcohol Intake in Humans..............................................................................................1 Proposed Reasons behind Alcoholism............................................................................2 Genetic Basis............................................................................................................3 Trauma and Stress....................................................................................................5 Animal Models of Alcohol Intake..................................................................................6 Mouse Models..........................................................................................................7 Stress Induced Drinking...........................................................................................9 Early Life Stress and Drinking.....................................................................................11 Stress Hyporesponsive Period................................................................................11 Animal Models of Early Life Stress and Drinking................................................12 Aim of Study.................................................................................................................13 Lipopolysaccharide as a Stressor...........................................................................13 Hypothesis..............................................................................................................14 MATERIALS AND METHODS.......................................................................................15 Animals.........................................................................................................................15 Procedure......................................................................................................................15 Experiments..................................................................................................................16 Statistical Analysis........................................................................................................17 RESULTS..........................................................................................................................18 Experiment 1.................................................................................................................18 Body Weights.........................................................................................................18 Ethanol Intake........................................................................................................18 Experiment 2.................................................................................................................19 Body Weights.........................................................................................................19 Alcohol Intake........................................................................................................20 v

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Experiment 3.................................................................................................................21 Body Weights.........................................................................................................21 Alcohol Intake........................................................................................................21 DISCUSSION...............................................................................................................34 The Effects of LPS Injections on Body Weights..........................................................34 The Effects of LPS Injections on Alcohol Intake.........................................................35 Alternate Influences of Alcohol Intake.........................................................................36 Gender Differences.......................................................................................................38 The Effects of Saline Injections....................................................................................39 Conclusion....................................................................................................................40 LIST OF REFERENCES...................................................................................................42 BIOGRAPHICAL SKETCH.............................................................................................48 vi

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LIST OF TABLES Table page 1 Group weights (g) during two injection phases...........................................................23 2 Group weights (g) during ethanol phases....................................................................24 3 Number of mice showing a net preference (>50%) for ethanol over water........ 25 4 Mean water intake for treatment groups durning ethanol phases................................26 5 Ethanol intake before and after puberty,ml .................................................................27 vii vii

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LIST OF FIGURES Figure page 1. Experiment 1. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice. ................................................28 2. Experiment 1. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice.............................................29 3. Experiment 2. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice...................... 4. Experiment 2. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice.........................31 5. Experiment 3. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice......................32 6. Experiment 3. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice.............................................................33 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science NEONATAL ENDOTOXIN EXPOSURE AND ALCOHOL INTAKE OF MICE IN EARLY ADULTHOOD By Cheryl H. Vaughan May 2003 Chair: Neil E. Rowland Major Department: Psychology Hormonal manipulation during the perinatal period in rodents has been shown to result in permanent effects on brain organization and function. In the present study, we examine whether a physiological stress during early life will affect spontaneous alcohol intake in early adulthood in mice. Hsd:ICR (CD-1) mice were injected intraperitoneally (i.p.) with a dose of .05 mg/kg lipopolysaccharide (LPS) either in the first postnatal week, postnatal day (P) 3-7, or after weaning (P21-25) to induce acute immunological stress. Two other groups received either saline injections or no injection, serving as controls. Subsequent alcohol intake for the four groups was recorded at two intervals, before puberty (P30-44) and after puberty (P50-64). During these two intervals, a two-bottle choice was offered between water and beer containing either a 5% or 10% concentration of alcohol. The acute anorectic effects of LPS administration and chronic effects on growth were monitored by recording body weights throughout the experiment. The physiological stress induced by LPS administration during either the first week of life or after weaning ix

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was found to reduce body weight gain but to have an inconsistent effect on the preference for alcoholic beer during early adulthood. x

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INTRODUCTION Alcohol Intake in Humans The etiology of alcohol intake and abuse has been of importance in the past decades because of the possibility of identifying reliable predictors of the behavior. In a recent Gallup Poll (Gallup Organization, 2002) 64% of both males and females self reported consuming alcohol. In 12 year olds, the reported alcohol use is 38% (National Household Survey on Drug Abuse [NHSDA], 1999). In a survey of eighth graders, 13% report using alcohol five times or more in the past two weeks (National Institute of Drug Abuse [NIDA], 2001). The incidence of alcohol intake then doubles in the next 6 years of life. By ages 18-25, 84% of the population has reported alcohol use in his or her lifetime (NHSDA, 1999). Among high school seniors, 30% report using alcohol five times or more in the past two weeks (NIDA, 2001). Thus, there is roughly a two-fold increase from the transition from late adolescence into early adulthood. The later life increase raises questions as to why this occurs. With more than half the population trying alcohol by adulthood coupled with an increased consumption, the onset of alcohol intake appears to be linked to further alcohol use Several studies have examined the factors that contribute to adolescent drinking. Adolescence is of physiological meaning due to the documented hormonal alterations that help in developing secondary sexual characteristics (Ducharme and Collu, 1982). Frias and colleagues (2000) conducted a study on the effects of acute alcohol intoxication on the hypothalamic pituitary-gonadal (HPG) axis and hypothalamic pituitary adrenal 1

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2 (HPA) axis hormones. HPG axis hormones include testosterone, prolactin, luteinizing hormone and follicle stimulating hormone. One of the HPA axis hormones, adrenocorticotrophic hormone (ACTH), was included as well during assessment to determine stress levels of the adolescents. The adolescents studied (N =27; 11 males, 16 females) were admitted to the emergency department with acute intoxication. Intoxication was behaviorally defined as slurred speech and unstable walking. Twenty-one adolescents (10 males, 11 females) served as controls. Controls were admitted to the emergency room for mild traumas, like contusions or sprains. Both groups age ranged from 13-17 years. Using ACTH levels of the controls as a comparison, adolescents with acute alcohol intoxication were found to display neuroendocrine function similar to levels found in the adolescents that were not intoxicated that solely encountered the stress of the emergency room. Serum ACTH levels were higher in the intoxicated subjects in comparison to the controls in both sexes. On average, the increase was about 9-fold higher in females and 5-fold higher in males. Serum testosterone was decreased in intoxicated males and increased in intoxicated females, as compared to the controls. Serum prolactin levels were higher, as well, in both sexes. In females it increased 5-fold and in males, 2.6-fold in females. In this example, acute alcohol intoxication affected hormones that perform necessary modifications that are vital to a growing adolescent. Proposed Reasons behind Alcoholism Many reasons have been proposed to explain how moderate alcohol use can change into abuse. Exacerbating features usually include existing disorders that complicate effective coping strategies. Male alcoholics have been shown to have a concurrence of alcoholism with childhood behavioral disorders; primarily, attention deficit disorder and

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3 conduct disorder. Females usually experience alcoholism with affective disorders. A study by Glenn and Nixon (1991) studied early symptom onset (ESO; alcoholic symptoms occurring before 25), and late symptom onset (LSO; alcoholic symptoms occurring after the age of 25) of alcoholism. ESO subjects were more anxious and felt a lesser capability to interact in social situations than LSO subjects. Reportedly, female alcoholics experience more mood changes, anxiety and trouble with concentration, compared to alcoholic men. This suggests a lack of sufficient resources is a component of dealing ineffectively with psychological problems (Nixon & Glenn, 1995). Genetic Basis One argument behind the development of alcoholism is whether the problem is either learned or inherited. In trying to decipher which alternative is most accurate, findings suggest both may play a role in determining alcohol intake. There is genetic evidence in the literature of a link to alcoholism. Chromosomal linkage studies have implicated chromosomes 1, 2, 4, 7, and 11 (Enoch & Goldman, 1999). There is also evidence of heredity playing a part in alcoholism. Family histories are one way to study the influence of both the nature and nurture aspects of alcohol intake and abuse. Penick et al. (1978) performed an experiment with 155 male veterans using previous family history as a predictor of abuse and a possible treatment approach. The average age for the men was 45 years old. The men volunteered to participate in the study and agreed to return monthly for a year for evaluation at an outpatient clinic. At the beginning of the experiment a two-hour interview was conducted to determine the state of the mens family history. The men were subsequently categorized into three groups. Group I (n=61) had at least one grandparent/parent that abused alcohol. Group II (n=57) had a relative other than

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4 grandparent/parent abusing alcohol. A common other relative was reportedly brothers and uncles. Group III (n=37) had no blood relative with a drinking problem. During the monthly visits for observation, the patients participated in a variety of measures to assess mood states, 1 personality, 2 and psychiatric behavior. 3 In total, 39 variables from the different measures were studied. These variables included basic sociodemographic information and alcohol related happenings. There was a significant correlation between the onset age of drinking behavior and having an alcoholic grandparent/parent. Thirty-three percent of the men in Group I started drinking regularly at age 15 or younger. These results suggest exposure to an alcoholic family member results in a higher likelihood the subject inadvertently learns the behavior as normal and accepted. The results demonstrate that early societal and familial influences can affect drinking in adulthood. Comparable conclusions were seen in a similar study using women. Using women with driving under the influence offenses, Lex et al. (1991) found that 75% of the women had a family history of alcoholism. A majority of the women started drinking at an early age and reported failure to abstain from drinking. These studies also show that family history and early forming drinking habits have a role in the severity of alcohols effects on everyday functioning. 1 Profile of Mood States (POMS: Tension, Depression, Anger, Vigor, Fatigue and Confusion) 2 Eysenck Personality Inventory (EPI: Neuroticism, Extraversion, Lie) 3 Symptom Checklist-90 (SCL: Somatization, Obsessive-Compulsive, Interpersonal Sensitivity, Depression, Anxiety, Anger-Hostility, Phobic Anxiety, Paranoid Ideation, Psychoticism, Additional)

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5 Trauma and Stress Though trauma isnt a direct cause for alcohol abuse, there have been connections between those suffering from posttraumatic stress syndrome (PTSD) and alcoholism. In fact, 25% to 75% of survivors of abusive or violent trauma report problematic alcohol abuse. Having PTSD does increase the risk of abuse even if it doesnt have a causal relationship. In a population of Vietnam veterans diagnosed with PTSD, 60%-80% have alcohol abuse disorders as well. PTSD symptoms are often times worsened by substance abuse. About 10%-50% of adults with PTSD also have other serious disorders such as mood, anxiety, addictive and behavioral disorders (National Center for PTSD, 2000). Increases in stress have commonly been reported concurrently with increases in alcohol intake. In a population of middle-aged women, individuals whose coping abilities were classified as low problem focused consumed more alcohol per occasion than those that with better problem focused abilities in stressful circumstances (Breslin et al., 1995). This and other studies utilizing humans have uncovered some of the causes behind stress motivated drinking. Recent studies with male and female adolescents and young adults have found that one of the most common reasons for drinking is its use as a coping strategy to combat stress (Hansell et al., 1999; Laurent et al., 1997). Alcohol drinking in young adulthood has been attributed to stressful episodes and was found to decrease after college as stressors diminished and life became more stable (Perkins, 1999). Stress and the HPA axis The hypothalamic-pituitary-adrenal axis (HPA) axis is considered to be the primary system responsible for physiological responses to stress. The HPA axis consists of a negative feedback system that is triggered by a cascade of hormones. Both corticotrophic

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6 releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN). Normally AVP alone is a weak secretagogue of ACTH, however when secreted in conjunction with CRH it works synergistically to boost the release of ACTH from corticotrope cells in the anterior pituitary. AVP works at the molecular level on cells of the anterior pituitary. Once ACTH is released it regulates the release of glucocorticoids from the adrenal cortex. Corticosterone (CORT) in rodents and cortisol in humans acts via the circulatory system, providing negative feedback at the level of the hypothalamus and the pituitary to modify further secretion of hormones (Whitnall, 1993). Animal Models of Alcohol Intake Alcohol intake in rats is generally tested using a two bottle choice procedure. When given the choice between alcohol and water, the net choice for alcohol is regarded as preference for the fluid. Exposure to alcohol is presented in incrementing concentrations so a range of preference can be established. For instance, the range can be used to characterize a particular line of subjects. Further ways to explore voluntary alcohol intake are conducting self-administration and fluid discrimination studies. Rhesus monkeys can be trained to self-administer alcohol through an intravenous catheter via a lever press. Deneau and colleagues (1969) demonstrated that rhesus monkeys would self administer to the point of intoxication. During intoxication, monkeys exhibited poor motor coordination and stupor. Monkeys reduced food intake and would self-administer alcohol throughout the day for consecutive days. Animal models utilizing selective breeding and genetic manipulations are beneficial to the study of alcohol intake. Alcohol preferring (P) and alcohol non-preferring (NP) rats are one selectively bred strain used to study the physiology and behavior of alcohol

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7 seeking behavior. P rats voluntarily consume alcohol in concentrations of 10%-30%. They acquire neural tolerance to and physical dependence of alcohol. NP rats avoid alcohol and show no physical tolerance or dependence (Li et al., 1987). McMillan and Li (2001) reported that another selectively bred line, high-alcohol drinking (HAD) rats can be trained to discriminate between three different fluids in a three lever choice procedure. Rats were trained to lever press for food then began drug discrimination training. Before discrimination training sessions, rats received saline, a low dose (0.75 g/kg) of ethanol or a high dose (1.5 g/kg) of ethanol. HAD rats were able to discriminate between ethanol and saline and responded at high rates for the highest ethanol dose administered. The high response rate reflects the control of the high dose of ethanol on behavior. Mouse Models One of the results of the completion of the mouse genome, is the ability to use mice in alcohol studies to test the genetic basis for alcoholism. Quantitative trait locus (QTL) mapping has been helpful in locating specific genes that may contribute to physiological disorders. QTLs for ethanol consumption have been identified. To locate a QTL, firstly, strains of mice with largely different phenotypes are inbred. Secondly, the offspring or F 2 generation, are phenotyped and then genotyped using genetic markers. Once the identity of the F 2 generation is known, behavioral tests can be performed and a correlation between the behavior and genes can be established (Crabbe et al., 1999). Mice created by transgenic procedures have been used to study the role of specific hormones or neuropeptides that may be involved in mediating alcohol intake. Recently, the involvement of neuropeptide Y (NPY) in alcohol consumption has been reported.

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8 NPY is usually associated with the control of food intake because of its excitatory effect on the PVN resulting in an increase in food intake (Levine & Morley, 1984). The NPY subtype 1 receptor (Y1R), in particular is involved in the response of NPY to ethanol (Thiele et al., 2002). In alcohol tests Y1R knockouts (Y1 -/) drank more of 3%, 6% and 10% ethanol than wild type mice (Y1 +/+ ). Additional experiments were done to rule out alcohol drinking due to caloric and taste influences. The drinking was not a result of high ethanol metabolism because Y1R knockouts had no significant differences in plasma ethanol concentrations than wild type mice. Therefore, Y1 receptors are suggested to be involved in voluntary ethanol consumption (Thiele et al., 2002). C57BL/6 (B6) and DBA/2J (D2) are two strains of mice used commonly in alcohol studies due to their opposing profiles for alcohol preference and intake. In a test of taste preferences, B6 mice have high preferences for ethanol at 5%, 10%, 15%, and 20% concentrations, while D2s show low preferences in this range. In B6 mice, the peak of preference occurs at 10% (Bachmanov et al., 1996). Extracellular recordings from brain slices of the ventral tegmental area were taken from both D2 and B6 mice. Results indicate that D2 mouse neurons were more responsive to excitation by ethanol. One of the reasons for the difference in intake in these two strains is proposed to be difference in the effects of ethanol in the brain. This heightened sensitivity could be why D2 mice will drink less during alcohol intake studies (Brodie & Appel, 2000). In a separate set of studies, B6 and D2 mice were crossed to produce B6D2 F 2 mice. The new recombinant strain was tested for free choice ethanol consumption. Genomic DNA was collected from mouse spleens at the termination of ethanol testing. Genetic

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9 markers corresponding to high ethanol drinkers were located on loci on both chromosomes 2 and 9 (Phillips et al., 1998). Identification of these functional genes helps to answer some of the questions of the influence of the environment on behavior. Marking the location additionally will aid in finding genes and gene products and their homologs in humans. Stress Induced Drinking One factor that has been confirmed in both human and animal data to have an effect on alcohol drinking is stress. Young adult college graduates reported consuming more alcohol during college to relieve stress in both social and academic situations. After college, subjects reported a decrease in stress encounters and this highly correlated with the decrease reported in alcohol intake (Perkins, 1999). Rats stressed with either immobilization or isolation in adulthood show significantly higher ethanol intake than non-stressed controls. Alcohol intake was increased three weeks after cessation of the stressors, showing that stress can have a lasting effect on inducing coping behavior (Nash & Maickel, 1985). Genetic models have also been used to test stress induced alcohol intake. CRH is one of the hormones involved in the cascade of activation during stress induced HPA axis activation. CRH binds to two types of receptors in the anterior pituitary, CRH1 and CRH2. The CRH1 receptor (CRH1R) is highly expressed in the anterior pituitary, cortex, hippocampus, amygdala and cerebellum (Potter et al., 1994). Chronic psychosocial stress significantly reduces the binding sites of CRH in anterior pituitary, dentate gyrus, and hippocampus (Fuchs & Flugge, 1995). Due to the importance of CRH, its receptor also has a large role in regulating the bodys response to stress. Mice CRH1R knockouts have an impaired stress response and an increased ACTH/CORT response after stress

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10 (Timpl et al. 1998). Therefore the binding abilities of CRH 1Rs are important in stress and can be indicators of the stress response in the presence of alcohol. Mice lacking functional CRH1Rs were used in an experiment by Sillaber et al. (2002) to test their importance to stress induced drinking. CRH1R knockouts and wild type mice were offered a choice between increasing concentrations of ethanol and water. Baseline intake was recorded for eight weeks. Baseline intake of alcohol did not differ significantly for the two groups of mice. After the eight weeks, mice were exposed to social defeat stress for three consecutive days. During and immediately following the stressor, there was no difference in alcohol intake. Three weeks after the repeated social defeat exposure, CRH1R knockout mice showed an increase in alcohol intake compared to baseline. This increase was not seen in the wild type mice. Following the alcohol testing, CRH1R knockout mice were then exposed to a forced swim test for three consecutive days. There were no significant differences in alcohol intake during the stress days. Three weeks after the forced swim alcohol intake the intake of the knockout mice rose significantly higher than the wild type mice. The post stress response of CRH1R knockout mice lasted six months after the second stressor, the forced swim. Alcohol metabolism did not play a role in the difference in alcohol intake between wild type and CRH1R knockout mice. Blood alcohol concentrations did not differ significantly between the two groups. The prolonged drinking of alcohol in CRH1R knockout mice may be due to an increase in NR2B subunit of the NMDA receptor in the nucleus accumbens and hippocampus. Neurons with mostly NMDA receptors have been

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11 implicated in the reinforcing effects of alcohol and the increased expression of the NR2B subunits have been linked to a stressful experience (Bartanusz et al., 1995). Early Life Stress and Drinking Stress experienced at specific times during development has been shown to have long lasting effects on the responsivity of the HPA axis in adult rodents. Thus, prenatal (Reul et al., 1994) and postnatal studies (Shanks et al., 1995) have shown that immunological stress during critical periods of development produces increased HPA responsiveness. Early maternal separation and handling are examples of processive stressors that have been also been reported to play a part in a heightened stress response. Rats that have experienced maternal separation once a day during days 2 to 14 of life demonstrate an altered HPA axis in adulthood. The result of this manipulation is an adult rat that is characterized by increased CRF mRNA and plasma corticosterone levels (Plotsky & Meaney, 1993). Levine (1967) was one of the first to report that rat pups that were reared by non-handled dams exhibited a high adrenocortical response. Early experience in the dam can affect the offsprings response to a novel stimulus. This evidence illustrates there is an important influence of early maternal interaction between pups and dams. The results of early life stress affecting adult behavior have been replicated in other studies using other stressors indicating that the effect can be generalized (i.e., Weinberg, 1987). Stress Hyporesponsive Period The behavioral response to early life stress is pertinent because during the first two weeks of life the stress axis activation is thought to be hyporesponsive. Beginning on postnatal day 2, rat pups would fail to respond or do so weakly if challenged with a stressor. Circulating basal levels of plasma CORT are low for the first two weeks of life.

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12 The levels start to appear around postnatal day (P) 7 and reach adult levels by P15. The CORT response to stress does not develop fully until after P16. The hyporesponsive period, between P 2-14, has an adaptive function because if excessive glucocorticoids were released during this time, they would promote decreased cell division, protein synthesis and uptake of amino acids. All of these actions are vital for growth and development (Sapolsky & Meaney, 1986). The responsivity of the HPA axis during this period indicates that there are key points in development. Butte and colleagues (1973) tested for the specificity and implemented a stressor (subcutaneous injection of histamine) at various times throughout the stress hyporesponsive period. There were significant increases in plasma CORT secretion at days 1, 2, 3, 16 and 21. Subsequent studies have also verified the idea of critical periods. Using this information, administration of a stressor in early life can result in adult behavioral and neuroendocrine reactivity to stress. Animal Models of Early Life Stress and Drinking In animal models, physical (i.e. early handling & immobilization) and psychological stressors have been employed to examine the subsequent effects on alcohol intake (Weinberg, 1987). Early handling in BALB/cJ mice performed during the first three weeks of life increased a preference for alcohol as compared to non-handled mice of the same strain (Jones et al., 1985). Lancaster (1998) found that handling as well increased a preference for alcohol in rats, specifically during 32-45 days of age. This evidence suggests this period is influential in preparing the individuals level of stress responsiveness. This work and the large literature on the neuroendocrine response of stress itself suggest that there is a possible interaction between stress and increased

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13 alcohol preference. Weaning age has also been shown to affect HPA axis activity in response to stress during adulthood (Cook, 1999). Aim of Study This study asks whether perinatal stress of another sort, an immunological challenge, would affect alcohol intake in young adult mice. Mice have been widely used and studied as a model for alcohol preference (Belknap et al., 1993) and have previously been founded to exhibit an increased preference following of an early life stressor (Jones et al., 1985). Hsd:ICR (CD-1) mice were used in this experiment because this outbred strain does not consume large amounts of ethanol when presented with a free choice situation (McMillen & Williams, 1998). Lipopolysaccharide as a Stressor To induce the immunological stress, lipopolysaccharide (LPS) was chosen because it has been used previously and reliably as an agent to modify the HPA axis (Reul et al., 1994) and it can be administered in controlled doses. LPS is a bacterial endotoxin that produces an elevation in temperature signaling the release of cytokines. Abdominal temperature starts to rise 1 hour after injection and peaks at 2hours post injection (Konsman et al., 1999). LPS exposure proves to be a potent stressor because it increases CRH, ACTH, AVP and plasma CORT (Shanks et al., 1995, 2000; Laugero & Moberg, 2000a). Dent et al. (1999) found that LPS begins to exert these effects 2 hours after injection. Plasma ACTH and CORT were increased significantly in rats aged 6, 12 and 18 days old. LPS affects metabolism and it is known to depress energy deposition. Both factors are important in maintaining the energy requirement of young adult mice. LPS significantly reduced energetic efficiency, the ability of the animal to allocate energy into tissues from

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14 an equal unit of energy intake. These changes are indicative of alterations in growth and deposition of lean and fat tissues. The decreased energy intake helps to explain the depression in weight that occurs after LPS injection. After an immune challenge LPS increases IL-1 (Laugero & Moberg, 2000a). IL-1 increases energy expenditure and lean and fat tissue catabolism resulting in anorexia (Benson et al., 1993) LPS activation is found in the PVN as shown by significant c-fos, an early gene marker, expression in neurons linked to the induction of the neuroendocrine cascade of the HPA axis. In addition to altering HPA activity, LPS administered to mice in the first week of life was found to increase social reactivity in adulthood (Granger et al., 1996). Hypothesis We hypothesize that receiving this immunological stress in either the first week of life or after the weaning period will increase alcohol preference in adulthood. Recording the intake of alcohol in adolescence will be a reflection of the physiological changes taking place during puberty and may also suggest a coping response, which has been the common explanation for alcohol intake in human adolescents.

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MATERIALS AND METHODS Animals Untimed pregnant female Hsd:ICR mice were purchased from Harlan Labs (Indianapolis, IN). After the dams gave birth, litters were culled to eight (approximately 4 of each sex) and left with the mother. Each litter was randomly assigned to experimental groups by order of birth date. In these experimental groups mice received no injection, saline injections, LPS injections in the first postnatal week or LPS injections after weaning. Litters were weaned at 21 days of age and were then group housed until the beginning the alcohol intake testing at postnatal day (P) 30. Animals were housed in shoebox cages with SaniChips bedding. Food (PMI Rodent Chow) was available ad libitum throughout the experiment and tap water was available ad libitum prior to and after ethanol testing. Animal rooms were kept at 22 2 C and had a 12 hr light/dark cycle with lights off at 1900 h. Procedure The LPS and saline groups were injected intraperitoneally (i.p.) with 5l of LPS or saline during the light cycle. LPS injected groups received 0.05 mg/kg Salmonella entiritidis, (Sigma Chemical Co.) either on P 3 & 5 or P 21 & 23. Mice in the saline injected group received equivalent saline injections on P 3, 5, 21 & 23. Weights were recorded for all groups on P 3, 5, & 7. Injections were given immediately after weighing to monitor each groups body weight. Non-injected control mice were only handled for weighing. Animals were returned to their mothers immediately after injections. 15

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16 Weights were recorded again on P 21, 23 & 25 during the second round of injections. Once animals reached P 30, all were housed singly and given a choice between 5% or 10% ethanol (ethanol + non-alcoholic beer) and water. These fluids were presented in 20 ml pipettes, fitted with metal drinking spouts and rubber stoppers. Pipettes were mounted to home cages by wedging them firmly into the bars of the cage lid. In the first week of testing (P 30-37) mice had a choice between water and 5% ethanol. In week two (P 38-44) of testing mice had a choice between water and 10% ethanol. The same sequence was repeated at P 50-57 and P 58-64, respectively. Daily intake was recorded and data are presented as mean ml SE per week for each ethanol concentration. Experiments All three experiments followed the same procedure. Experiment one consisted of 2 control litters (9 female, 7 male), 3 saline injected control litters (11 female, 13 male), 3 litters injected with LPS in the first postnatal week (11 female, 12 male) and 3 litters injected with LPS after weaning (12 female, 10 male). All animals in experiment one were weaned at P 21, with the exception of one litter in the non-injected group that was weaned 2-3 days early. Experiment two and three were conducted in an attempt to replicate experiment one. Experiment two consisted of 4 non-injected control litters (11 female, 21 male), 4 saline injected control litters (14 female, 17 male), 4 litters injected with LPS in the first postnatal week (15 female, 16 male) and 4 litters injected with LPS after weaning (12 female, 17 male). Experiment three consisted of 3 non-injected control litters (14 female, 10 male), 4 saline injected control litters (14 female, 16 male), 3 litters injected with LPS

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17 in the first postnatal week (6 female, 18 male) and 3 litters injected with LPS after weaning (11 female, 13 male). Statistical Analysis One-way ANOVA (Sigma Stat 1.0, SPSS) was used to test the relationship between treatment and mean intake and Bonferronis method was used for multiple comparisons between groups. One-way repeated measures ANOVA was used to compare before puberty drinking to after puberty drinking. Preference ratios were derived from dividing the total alcohol consumed by total fluid consumed (ml alcohol/ml water + ml alcohol). A chi-squared test was used to compare preference ratios. A t-test was used to compare male and female alcohol intake. A p-value of < 0.05 was considered significan

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RESULTS Experiment 1 Body Weights The body weights are shown in the top panels of Table 1. The non-injected (NON) mice weighed consistently more than the other groups throughout the study. Early and late treatment with LPS (L1 and L2) decreased significantly the weights during P3-P7 and P21-25. By the weaning period (P21-25), groups L1 and L2 weighed less than the saline injected mice. Non injected animals weighed more than group L2 during 5% and 10% alcohol phases (see Table 2). The other LPS injected group (L1) only showed significant differences at P 30 and P38. Ethanol Intake In intake tests before puberty for 5% and 10% EtOH, groups L1 and L2 drank a significantly greater mean volume of ethanol than non-injected animals (see Figure 1). During the 5% alcohol phase, L2 drank more ethanol than saline injected animals. All animals decreased total volume intake during the 10% alcohol phase, however both LPS injected groups drank more than the SAL and NON groups (p< .05). Figure 2a shows 5% ethanol intake after puberty. Groups L1 and L2 drank more 5% alcohol than NON animals. Group L2 was the only LPS injected group that drank significantly more than the SAL group (p < .05). Table 3 shows the ethanol preference by group, expressed as the number of animals showing a net preference (>50%) for alcohol. There were significant differences between 18

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19 groups during both the prepubertal (P30-37) and postpubertal (P50-57) periods. The water intakes during the two-bottle choice are shown in Table 4 and were significantly higher for the NON group than other treatment groups during intervals P30-37 and P 50-57. Ethanol intakes before (P30-44) and after (P50-64) puberty are compared in Table 5. The non-injected control group showed no difference in either 5% or 10% ethanol intake between the two phases of testing, and the saline-injected controls showed a modest increase in only 5% intake after puberty compared with before puberty. In contrast, both LPS groups had significantly lower 10% ethanol intakes after puberty than before, although no such change was evident for the 5% ethanol. The results suggest that the LPS injections did have an effect not only on the animals weights but also on the preference and intake of alcohol in adulthood. Experiment 2 attempts to build on these findings by replicating the study with a larger set of animals. Experiment 2 Body Weights The middle panel of Table 1 shows group weights during the two injection phases. In the first week of life, group L1 weighed significantly less than NON and SAL groups. By P7, contrary to experiment one, the SAL group weighed significantly more than the other three groups. This result shows there was no suppressant effect of the saline injection on body weight gain. After injections in the weaning period, group L1 weighed significantly less than the NON and SAL groups. An effect of the second LPS injection was seen only on P25, when L1 weighed significantly less than the NON group (p < .05).

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20 Table 2 shows weights of mice during alcohol testing done before and after puberty. SAL and NON groups weighed significantly more than L1 before the 5% alcohol phase. The NON group weighed more than L1 after 10% alcohol phase. The NON group weighed significantly more than L1 throughout the second phase of 5% and 10% alcohol testing (p < .05). Alcohol Intake Figure 3 shows 5% and 10% intake for all treatment groups before puberty. Both non-injected and saline injected groups of mice drank significantly more 5% alcohol than group L2 during P30-37. For 10% alcohol consumption, only the CON group drank significantly more than L2 (p < .05). Table 3 shows the number of animals that preferred alcohol to water. L1, SAL and NON mice showed a net preference (>50%) for 5% alcohol over water both before and after puberty. The NON group had the largest percentage of animals that showed a net preference for 5% alcohol at 23 out of 32 (72%) mice. Comparison of preference ratios during the four intervals of ethanol exposure, revealed a significant difference in P 30-37 (p < .005) and P 38-44 (p < .005). Figure 4 shows alcohol intake after puberty. During 10% alcohol testing, group L2 drank less than the non-injected group (p < .05) (see Figure 4b). About 1/3 of mice in groups L1, SAL and CON prefer 10% alcohol to water after puberty (see Table 3). The water intake values during the two-bottle choice are shown in Table 4. During P50-57, L2 mice drank more water than NON and L1 mice (p<.05). Table 5 contains the comparison of alcohol testing before (P30-44) and after (P50-64) puberty. There were significant differences for both LPS groups. L1 and L2 mice consumed the 5% alcohol during P50-57 as compared to P30-37 (p<.02).

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21 The weight loss of the LPS injected groups was a trend that was evident in experiments one and two. However, the increased alcohol intake of groups L1 and L2 in experiment one was not replicated in experiment two. A third replication was done in hopes of a definitive answer to whether an immune challenge would increase alcohol intake in adulthood. Experiment 3 Body Weights The bottom panel of Table 1 shows the weights for the third set of mice during the two injection phases. In the first week of life the NON group weighed more than all three other groups. This trend was significant on P 3 and 5. After the second round of injections group L2 weighed significantly less than the other three groups (p<.05). In the period before puberty, the weights of L2 remained lower than NON and L1 mice (see Table 2). This trend occurred until the end of the 10% alcohol phase; the L2 group weighed significantly less than L1 mice. Alcohol Intake Figure 5 shows alcohol intake for all treatment groups before puberty. Although there were no significant differences between the groups, Table 3 shows that during the 5% alcohol phase more than half the animals preferred alcohol to water. The number of alcohol preferring animals decreased considerably during the 10% phase. Alcohol intake after puberty shows was significantly different during the 10% alcohol phase (see Figure 6). The L2 group drank significantly more 10% alcohol than L1 and SAL group, and 75% of these mice preferred the 10% alcohol to water (see Table 3). A comparison of preference ratios during the four ethanol phases revealed a significant difference from chance ratios for consumption of 5% and 10% alcohol after

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22 puberty. The water intakes during the two-bottle choice are shown in Table 4; there were no significant differences. Table 5 contains the comparison of alcohol testing before puberty (P30-44) and after puberty (P50-64). All four groups of mice drank significantly more 10% the second time it was offered; this was not evident in the other two experiments.

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23 Table 1. Group weights (g) during the two injection periods. First week of life Weaning period Experiment one P3 P5 P7 P21 P23 P25 L1 3.1 .1 a 4.3 .1 a 5.7 .1 a 14.1 .4 a b 16.7 .5 a b 19.1 .5 a b L2 3.0 .1 a 4.2 .1 a 5.6 .1 a 13.4 .3 a b 15.4 .4 a b 17.7 .4 a b SAL 3.3 .1 4.6 .1 a 5.9 .1 a 15.9 .3 a 18.9 .4 21.6 .4 NON 3.6 .1 5.1 .1 6.5 .1 17.6 .6 19.2 .4 22.2 .6 Experiment two P3 P5 P7 P21 P23 P25 L1 2.2 .1 a b 3.2 .1 a b 4.2 .1 a b 8.9 .3 a b 11.0 .3 a b 13.2 .4 a b L2 2.5 .1 3.6 .1 4.4 .1 b 10.5 .5 12.5 .6 14.7 .6 a SAL 2.7 .1 3.8 .1 4.8 .1 10.7 .1 13.0 .2 15.3 .2 NON 2.5 .1 3.6 .1 4.5 .1 b 11.1 .3 13.8 .4 16.6 .4 Experiment three P3 P5 P7 P21 P23 P25 L1 2.4 .1 a 3.4 .1 a 4.6 .1 a 11.0 .2 13.1 .3 15.6 .3 L2 2.5 .1 a 3.4 .1 4.5 .1 a 9.5 .3 a b c 11.4 .3 a b c 13.5 .3 a b c SAL 2.5 .1 a 3.4 .1 4.4 .1 a 11.5 .2 13.1 .3 15.1 .3 NON 2.7 .1 3.7 .1 5.0 .1 11.7 .4 13.7 .5 16.5 .6 L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group a Significantly less than non-injected group b Significantly less than saline group c Significantly less than LPS group injected on P3 & 5 Significance level at p<.05; numbers expressed are mean SE grams

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24 Table 2 Group weights (g) during ethanol phases Before puberty After puberty Experiment one 5% ethanol 10% ethanol 5% ethanol 10% ethanol P30 P38 P44 P50 P58 P64 L1 24.5 .7 a b 27.5 .7 a 29.9 .8 31.2 .8 33.5 .9 34.4 .9 L2 23.2 .6 a b 26.9 .6 a 28.8 .7 a 30.0 .8 a 32.1 .9 a 33.7 .9 a SAL 27.2 .5 29.2 .6 31.4 .8 32.6 .8 34.8 .7 36.5 .8 NON 27.6 .9 30.2 .9 32.5 1.1 34.0 1.1 36.0 1.2 37.5 1.3 Experiment two 5% ethanol 10% ethanol 5% ethanol 10% ethanol P30 P38 P44 P50 P58 P64 L1 19.5 .5 a b 25.0 .6 26.7 .8 a 28.1 .8 a 29.2 .9 a 30.8 .9 a L2 21.0 .9 26.7 .7 29.0 .8 30.4 .8 31.7 .9 33.4 .9 SAL 21.7 .4 26.1 .9 29.3 .8 30.7 .8 31.6 .9 33.0 .9 NON 23.0 .5 27.4 .5 30.2 .6 31.6 .6 33.3 .6 34.7 .7 Experiment three 5% ethanol 10% ethanol 5% ethanol 10% ethanol P30 P38 P44 P50 P58 P64 L1 22.4 .5 27.0 .6 30.0 .7 31.5 .7 32.6 .8 34.0 .8 L2 19.4 .4 a c 24.2 .5 a c 26.5 .7 c 27.5 .8 c 28.7 .8 c 29.6 .9 c SAL 21.1 .4 25.2 .4 27.3 .6 28.3 .6 c 29.9 .6 30.5 .7 c NON 22.3 .7 26.7 .8 29.0 1.0 30.2 1.0 31.6 1.1 33.0 1.1 L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group a Significantly less than non-injected group b Significantly less than saline injected group c Significantly less than LPS group injected on P3 & 5 Significance level at p<.05; numbers expressed are mean SE in grams

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25 Table 3 Number of mice showing a net preference (>50%) for ethanol over water Experiment one Ethanol concentration P L1 (n=23) L2 (n = 22) SAL (n= 24) NON (n=16) 5% 30-37 a 20 19 19 8 10% 38-44 12 16 12 5 5% 50-57 b 18 18 20 6 10% 58-64 10 11 11 4 Experiment two Ethanol concentration P L1 (n=31) L2 (n = 29) SAL (n= 31) NON (n=32) 5% 30-37 c 20 8 16 23 10% 38-44 d 5 1 6 7 5% 50-57 24 18 21 21 10% 58-64 10 4 11 10 Experiment three Ethanol concentration P L1 (n=24) L2 (n = 24) SAL (n= 30) NON (n=24) 5% 30-37 19 15 20 15 10% 38-44 1 6 5 5 5% 50-57 e 21 18 21 11 10% 58-64 f 11 18 9 6 L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group a Significant difference from chance during this period ( 2 = 8.73, p< .05) b Significant difference from chance during this period ( 2 = 12.83, p<.01) c Significant difference from chance during this period ( 2 = 13.2, p< .005) d Significant difference from chance during this period ( 2 = 19.4, p<.005) e Significant difference from chance during this period ( 2 = 10.4, p<.025) f Significant difference from chance during this period ( 2 = 15.3, p<.005)

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26 Table 4 Mean water intake for treatment groups during ethanol phases, ml Experiment one P L1 L2 SAL NON 30-37 1.9 .3 1.9 .3 2.3 .3 3.5 .6 a b 38-44 3.5 .4 2.6 .4 3.4 .3 4.3 .6 50-57 2.2 .2 2.1 .5 2.0 .3 3.9 .5 a b c 58-64 3.3 .3 3.3 .3 2.9 .3 4.3 .5 Experiment two P L1 L2 SAL NON 30-37 3.9 .4 4.8 .4 4.3 .4 3.5 .3 38-44 4.6 .3 5.8 .3 5.5 .3 6.3 .7 50-57 2.4 .3 3.6 .4 a d 2.6 .3 2.4 .3 58-64 3.7 .3 4.7 .3 3.9 .3 3.7 .3 Experiment three P L1 L2 SAL NON 30-37 2.7 .3 3.2 .4 3.2 3.1 .4 38-44 5.3 .3 5.2 .4 4.8 .3 5.6 .5 50-57 3.2 .3 3.0 .4 3.2 .2 3.7 .4 58-64 4.1 .3 3.3 .4 4.1 .3 4.5 .4 L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group a Significantly more than LPS group injected on P3 & 5 b Significantly more than LPS group injected on P21 & 23 c Significantly more than saline injected group d Significantly more than non injected group

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27 Table 5 Ethanol intake before and after puberty, ml Experiment one Concentration Stage L1 L2 SAL NON P30-37 5.8 .4 6.1 .4 4.4 .3 a 3.7 .5 5% P50-57 5.5 .4 6.5 .4 4.9 .3 a 3.7 .4 P38-44 4.0 .2 a 4.3 .4 a 2.9 .2 2.3 .3 10% P58-64 3.4 .2 a 3.5 .3 a 3.1 .2 2.6 .3 Experiment two Concentration Stage L1 L2 SAL NON P30-37 4.4 .2 a 3.5 .3 a 4.7 .3 5.2 .3 5% P50-57 5.1 .3 a 4.4 .3 a 5.2 .4 5.3 .4 P38-44 2.7 .2 2.1 .2 2.5 .2 3.1 .2 10% P58-64 2.9 .2 2.3 .2 2.8 .3 3.4 .3 Experiment three Concentration Stage L1 L2 SAL NON P30-37 5.2 .3 a 5.3 .4 4.2 .3 5.4 .4 5% P50-57 4.5 .3 a 5.5 .5 4.0 .3 4.7 .6 P38-44 2.1 .2 a 2.9 .3 a 2.2 .2 a 2.4 .4 a 10% P58-64 3.3 .2 a 4.6 .3 a 2.8 .2 a 3.6 .4 a L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group a Significant difference between before puberty and after puberty intake Significance level at p<.05; Expressed as mean SE ml/day

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28 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON **# 012345678 L1 L2 SAL NON ##**(a) 5% EtOH(b) 10% EtOH Figure 1. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. L1 and L2 consumed more alcohol than NON mice at both concentrations. Fluid intakes are expressed as mean SE ml/day Significantly greater than NON. # Significantly greater than SAL. L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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29 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON **# 012345678 L1 L2 SAL NON (b) 10% EtOH(a) 5% EtOH Figure 2. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. L1 and L2 consumed more ethanol than NON mice during 5% exposure. No significant differences for 10% ethanol intake. Fluid intakes are expressed as mean SE ml/day Significantly greater than NON. # Significantly greater than SAL. L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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30 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON (a) 5% EtOH (b) 10% ETOH 012345678 L1 L2 SAL NON #** Figure 3. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. L2 mice drank less than NON mice at both concentrations and less than SAL mice at the 5% concentration. Fluid intakes are expressed as mean SE ml/day Significantly less than NON. # Significantly less than SAL. L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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31 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON (a) 5% ETOH (b) 10% ETOH 012345678 L1 L2 SAL NON Figure 4. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. No significant differences occurred for 5% ethanol intake. L2 intake for 10% alcohol was less than NON mice. Fluid intakes are expressed as mean SE ml/day Significantly less than NON. L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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32 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON (a) 5% EtOH (b) 10% ETOH 012345678 L1 L2 SAL NON Figure 5. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, before puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. No significant differences occurred for 5% and 10% ethanol intake. Fluid intakes are expressed as mean SE ml/day L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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33 mean alcohol consumed/day (ml) 012345678 L1 L2 SAL NON (a) 5% ETOH (b) 10% ETOH 012345678 L1 L2 SAL NON @@ Figure 6. Mean ( + SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice with water, after puberty in CD-1 mice. The four treatment groups received each concentration for 1 week each. Intake is shown as the average over the 1 week period. No significant differences occurred for 5% ethanol intake. L2 drank significantly more 10% alcohol than L1 and SAL. Fluid intakes are expressed as mean SE ml/day @ Significantly less than L2. L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

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DISCUSSION LPS is an endotoxin that produces an immune and HPA axis response. It is believed to function as a stressor because of its activation of the HPA axis (Reul et al., 1994; Granger et al., 1996). The first week of life and the week during weaning have been reported to be critical periods in development (Dent et al., 1999). There is evidence of manipulations that were done during these critical periods that have affected behavior in adulthood. We chose LPS injections as a stressful manipulation and alcohol intake as our behavioral measure in adulthood. Alcohol intake is a problem in adult youth and one hypothesized reason as to why that is occurring could be an early life immune challenge. In our experiments we saw differing results after testing this hypothesis. The Effects of LPS Injections on Body Weights As previously reported, LPS did produce characteristic lowering of body weight in all three experiments. In experiments one and three, the L2 group remained at a significantly lower weight than controls until the end of the study (P64). In experiment two, the L1 group was the LPS injected group that showed suppressed body weight gain until the end of the study. This suppression of body weight gain is due to the short and long-term metabolic effect of LPS. After acute injection of LPS, body weight gain and energy deposition decrease in the first 24 hours (Laugero & Moberg, 2000). Short term effects last up to 48 hours, during which the endotoxin functionally activates the HPA axis (Shanks et al., 34

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35 1995; Konsman et al., 1999). Other short-term effects of the endotoxin include the increase of central production of stress hormones, abdominal temperature and brain IL-1 (Shanks et al., 1995; Konsman et al., 1999). LPS increases circulating CORT, which also contributes to decreased weight gain via its catabolic effects and inhibition of caloric efficiency (Laugero & Moberg, 2000a). The slowed weight gain, that can last indefinitely, seems to occur as a result of the release of interleukin-1 (IL-1) that happens when LPS triggers an inflammatory response to sickness (Inui, 2001). There is an endogenous IL-1 receptor antagonist (IL-1ra) that inhibits the effects of IL-1 on cells, therefore decreasing a full immune response (Arend, 1993). In mice lacking a naturally occurring IL-1ra, Inui (2001) showed that there was decreased weight of the mice with a deficient IL-1 receptor antagonist in comparison to control littermates. The weight difference was apparent at six weeks of age and continued until 13 weeks of age. This lasting weight difference suggests that IL-1 has a large part in mediating the long term effects of LPS. Although there was a reliable weight loss observed following LPS exposure in Experiments 1,2, and 3, the degree of weight loss did not serve as a reliable predictor of alcohol intake in adulthood in our studies. The Effects of LPS Injections on Alcohol Intake In Experiment 1, LPS administered in the first week of life and around weaning influenced an increased preference for ethanol before puberty (P 30-44) and through a portion of early adulthood (P 50-57). LPS injections, as seen in prior studies, contributed to a modified stress response later in life (Granger et al., 1996; Laugero & Moberg, 2000b; Plotsky and Meaney, 1993; Shanks et al., 1995, 2000). The results of experiments

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36 2 and 3 did not however confirm this influence of LPS. Though methodologically all three experiments were the same, the results were inconsistent. Physiological changes, environmental changes and taste properties of the alcohol itself could be possible contributors to why the results did not consistently support our hypothesis. Alternate Influences of Alcohol Intake In Experiment 1, both LPS injected groups demonstrated significantly high ethanol intake until P 57. A lack of an increase in alcohol intake was observed in Experiments 2 and 3. This lack of an effect could be due to hormonal changes that occur around puberty. In Experiment 3, during P 58-64, the L2 group 10% intake was significantly higher than the L1 and SAL groups. Lancaster et al. (1996) found similar results in rats. Under no-stress conditions, increased voluntary ethanol intake was observed around P 52. In our experiment, it is possible that changes in circulating pubertal hormones affected high ethanol intake in adulthood. Ethanol intake, whether high or low, could be due to these hormonal changes independent of LPS administration at critical periods in development. The preference for 5% alcohol over 10% alcohol is in agreement with a study done by McMillen & Williams (1998). McMillen and Williams found CD-1 mice to prefer ethanol at a 6.8% concentration in a free choice situation with no physiological manipulation in comparison to the commonly high alcohol preferring B6 strain. The inconsistent alcohol intake results of alcohol in Experiments 1, 2, and 3 cannot be readily attributed to low taste reactivity. CD-1 mice can learn a LiCl induced taste aversion to alcohol as well as prefer water to a bitter quinine solution in a two bottle choice test (McMillen & Williams, 1998).

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37 Early life alterations such as handling and weaning have shown to alter HPA functioning as late as weaning age (Plotsky & Meaney, 1993; Cook, 1999). In Experiment 1, a litter from the non-injected group was prematurely weaned by 2-3 days because the mother died over a weekend due to unknown causes. To discover if this premature weaning had an effect on alcohol intake, each non-injected litter was compared to the other three treatment groups. There were no significant differences between the litter weaned early and the other control litter in 5% intake before puberty. Therefore, their data were retained in the overall analysis. In Experiment 3, three mothers died overnight of unknown causes, about a week after giving birth. Their litters subsequently were euthanized. The deaths happened over three successive days and may have provided a social stressor to the remaining animals in an olfactory form. Odors have been found (Zuri et al., 1998) to have an influence on behavior. It is possible that the mothers dying affected the remaining litters. Unfortunately the effects of these events and the subsequent maternal environment of the remaining litters are unknown. The different times of LPS administration may also play a part in subsequent alcohol intake. Differences in the drinking trends were seen between the L1 and L2 group in Experiments 1, 2 and 3. In each of these experiments, the L2 group differed the most from the other groups, either by drinking significantly more or less than the others. LPS administered in the first week of life may not have been intrinsic in influencing early adult drinking behavior because the effects we saw were bidirectional. In a study by Dent and colleagues (1999), an LPS injection on P 18 showed a higher CORT response after 2 hours than animals injected on P 6. LPS is active mainly during

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38 an acute phase response, which lasts up to 3 h after administration, where cytokines and stress hormones are at their peak (Kakizaki et al., 1999). It is possible that in our study the L2 group experienced a higher CORT response and this manifested as a different effect on alcohol drinking behavior in adulthood. Gender Differences There is some evidence for gender differences in alcohol intake and preference. Lancaster et al. (1996) used a paradigm in which tubes of 5% beer were offered to male and female rats ad libitum, in addition to water and food. In a free access situation, females were found to increase intake and preference of 5% beer around 50 days of age. With the added stressor of handling (Lancaster, 1998), female rats showed no significant increase in 5% beer consumption but a significant increase in water intake around puberty. On the contrary, male rats had an increase in alcohol intake and preference around puberty. Gender differences in mice are not as evident. In a two bottle choice of 8% alcohol and water under no-stress conditions B6 mice were found to show no gender differences in intake and preference (Little et al., 1999). In contrast, female CD-1 mice were found to drink more than males in a two bottle choice between alcohol and water (Naassila et al., 2002). Following LPS as the putative stressor, in our study not all treatment groups showed gender differences in alcohol intake. In Experiment 1, 10% intake before puberty for saline injected mice was higher for females than males. In Experiment 2, saline injected females drank more than males throughout the study. In Experiment 3, L2 females started drinking more than males before puberty during the 10% ethanol phase and continued until the end of the study.

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39 The Effects of Saline Injections Dent and colleagues (1999) have reported that saline injections have an effect on the release of stress hormones. Dent et al. (1999) found evidence of a graded effect by using an early gene marker, c-Fos. Cells in the paraventricular nucleus of the hypothalamus (PVN) were found to be active following administration. The number of fos positive cells were lower in number in saline injected rats, than LPS injected animals but greater than in non-injected controls. In our experiments saline injections proved to have an effect on the animals body weights. In Experiments 1, 2 and 3, the weights of the saline injected group were lower than the non-injected animals during the injection and intake phases. Though saline injections had metabolic effects, in our studies we anticipated there would be a graded effect on alcohol intake, where the intake of alcohol would be contingent on the degree of stress administered. Little et al. (1999) used C57BL/10 mice and screened them in the first three weeks of life for ethanol preference. Mice were then separated into high and low preference groups. The saline injection, a mild stressor, has been implicated in increasing the preference for alcohol. Mice were injected with saline or not injected at all during 7-9 weeks of age and then tested for free choice ethanol consumption. Animals receiving saline preferred significantly more ethanol than non-injected animals (Little et al., 1999). In Experiments 1,2, and 3, saline injected animals showed a preference for ethanol as compared to water during the periods of 5% exposure before and after puberty. Therefore, there may be a possibility that the early life saline injections served as a mild stressor. However, intake of the saline injected group was not always higher than the

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40 non-injected group. Our results along with Little et al. may suggest that the saline injections affect alcohol preference but not necessarily intake. Conclusion This is one of the first studies to look at a long term effect of LPS on drinking behavior. LPS proved to be a reliable stressor in regards to depressed body weight; it produced the predicted decrease in body weight. However, it is doubtful if the administration phases were close enough to the ethanol phases to exert a reliable influence. LPS administered during the stress hyporesponsive period was less effective in increasing intake throughout the ethanol phases. This may be due to the lack of responsivity of the stress axis. LPS administered in after weaning was more effective in producing long lasting effects on the system. Whether this was mediated though immune system or endocrine system cannot be concluded from our experiments. Puberty normally sees a surge in sexual hormones that affect growth and reproduction as well as the ingestion capacity and metabolism of alcohol. Alcohol ingestion has been founded to increase serum ACTH and cortisol levels of human adolescents, similarly to the response seen after encountering stressful stimuli (Frias et al., 2000). In our experiments, puberty may have played a larger role in alcohol intake than the early life LPS injections. Though early life stress has been shown in various protocols to induce behavioral changes in adulthood, the stressor used in this model was not successful. Utilizing the idea of alcohol use being a common coping response in adolescence one could say that the inconsistent intake of the LPS treated groups in the experiments occurred simply because the system was no longer being challenged. Due to the lack of an immediate immune challenge there was no longer a stressful reason to drink.

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41 An attenuation of a fever response occurred in a different protocol, where LPS was administered after two weeks of alcohol exposure (Taylor et al., 2002). It is possible that the exposure to alcohol for our adult mice negated any remaining neuroendocrine effects of early LPS administration. Based on body weight data, the lasting effect of LPS can be assumed. However, the length of the alcohol phases themselves could have worked against bringing out any effects of LPS in relation to alcohol intake. For future directions, we plan on looking at the short term effects of LPS injections on alcohol intake minutes to hours following the stressor. This would better elucidate the time course of the interaction between an immune challenge and alcohol intake or preference. We plan on also testing whether the mice were drinking alcohol to make up for some caloric deficit by offering a three bottle choice, where a nutritive liquid would be added. Experiments 1,2 and 3 did demonstrate that LPS has enduring effects on body weight. Additional investigation needs to be done to determine the relation between early LPS administration and adult alcohol intake.

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BIOGRAPHICAL SKETCH Cheryl Hope Vaughan was born in Clarendon, Jamaica, in 1980. She moved to Miami, FL, at age 8. She completed high school at Chaminade-Madonna College Preparatory in Hollywood, FL. Cheryl received her B.A. in psychology from St. Thomas University in May of 2000. She began graduate school at the University of Florida in September of 2000. She plans to stay after her M.S. in psychology to pursue a docotoral degree in psychology in the behavioral neuroscience area. 48


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NEONATAL ENDOTOXIN EXPOSURE AND ALCOHOL INTAKE OF MICE IN
EARLY ADULTHOOD












By

CHERYL H. VAUGHAN


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003



























Copyright 2003

by

Cheryl H. Vaughan




























The love and support I have received from my parents has made all the difference in my
pursuit of graduate studies. Their unconditional love has made me the person I am today.
This thesis is dedicated to my mother, Dahlia, and my father, Arthur.















ACKNOWLEDGMENTS

I would like to thank Dr. Neil Rowland for his support, patience and encouraging

words throughout the last three years. I would also like to acknowledge my other

committee members for their insights and contributions to my research at the University

of Florida.

I have made some close friends here at UF and their friendship has made the past few

years a stronger learning experience.
















TABLE OF CONTENTS


A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ......................................... ............... vii

LIST OF FIGURES ............................................... viii

A B S T R A C T ....................................................................................................................... ix

INTRODUCTION .................................. .. .......... .............. ............... 1

A alcohol Intake in H um ans .... ................................................................ .. .................. 1
Proposed Reasons behind Alcoholism................................................................... 2
Genetic Basis .............................................................................. . .. .............. 3
Trauma and Stress .......................... .. ........... .............................. 5
Animal Models of Alcohol Intake ........................................................ .............. 6
Mouse Models .......................................................................................................... 7
Stress Induced D drinking .................................................................... .............. 9
E arly L ife Stress and D drinking ...................................... ........................ ................. 11
Stress H yporesponsive P eriod........................................ .................... ................. 11
Animal Models of Early Life Stress and Drinking .......................................... 12
A im o f S tu d y ................ ............................................................................................ .. 1 3
Lipopolysaccharide as a Stressor ..................................................................... 13
H y p o th e sis ............................................................................................................. 14

MATERIALS AND METHODS.................................................................................15

A n im als ......................................................................................................... ............ 1 5
P ro c e d u re ...................................................................................................................... 1 5
E x p e rim e n ts ................................................................................................................ .. 1 6
S statistical A n aly sis ........................................................................................................ 17

R E S U L T S .......................................................................................................... ........ .. 18

E x p e rim e n t 1 ............................................................................................................... .. 1 8
B o dy W eig h ts ......................................................................................................... 18
E th an o l In tak e ........................................................................................................ 18
E x p e rim e n t 2 ............................................................................................................... .. 1 9
B o dy W eig h ts ......................................................................................................... 19
A alcohol Intake ............................................................................................. 20


v









E xperim ent 3 ........................................................................................................ 21
B ody W eights ......................................................................................... 2 1
A alcohol Intake ......................................................................................... 2 1

D ISCU SSION ............................................................................................... .............. 34
The Effects of LPS Injections on Body W eights ........................................ .............. 34
The Effects of LPS Injections on Alcohol Intake...................................... .............. 35
A alternate Influences of A alcohol Intake..................................................... .............. 36
G ender D differences .. .... ........ ......... .................................................. ..... ........ 38
The E effects of Saline Injections...................................... ....................... .............. 39
C o n c lu sio n .................................................................................................................... 4 0

L IST O F R E FE R E N C E S ... ........................................................................ ................ 42

BIOGRAPHICAL SKETCH ...................................................... 48






















LIST OF TABLES


Table page

1 Group weights (g) during two injection phases......................................................23

2 Group w eights (g) during ethanol phases ............................................... ................ 24

3 Number of mice showing a net preference (>50%) for ethanol over water.............. 25

4 Mean water intake for treatment groups during ethanol phases ..............................26

5 Ethanol intake before and after puberty,m l ............................................ ................ 27















LIST OF FIGURES


Figure page

1. Experiment 1. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, before puberty in CD-1 mice ............................... ................ 28

2. Experiment 1. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, after puberty in CD-1 mice...... .............................. ................ 29

3. Experiment 2. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, before puberty in CD-1 mice..........................30

4. Experiment 2. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, after puberty in CD-1 mice...........................31

5. Experiment 3. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, before puberty in CD-1 mice.... ........... ...........32

6. Experiment 3. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in
choice with water, after puberty in CD-1 m ice ....................................... ................ 33















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

NEONATAL ENDOTOXIN EXPOSURE AND ALCOHOL INTAKE OF MICE IN
EARLY ADULTHOOD

By

Cheryl H. Vaughan

May 2003

Chair: Neil E. Rowland
Major Department: Psychology

Hormonal manipulation during the perinatal period in rodents has been shown to result

in permanent effects on brain organization and function. In the present study, we examine

whether a physiological stress during early life will affect spontaneous alcohol intake in

early adulthood in mice. Hsd:ICR (CD-1) mice were injected intraperitoneally (i.p.) with

a dose of .05 mg/kg lipopolysaccharide (LPS) either in the first postnatal week, postnatal

day (P) 3-7, or after weaning (P21-25) to induce acute immunological stress. Two other

groups received either saline injections or no injection, serving as controls. Subsequent

alcohol intake for the four groups was recorded at two intervals, before puberty (P30-44)

and after puberty (P50-64). During these two intervals, a two-bottle choice was offered

between water and beer containing either a 5% or 10% concentration of alcohol. The

acute anorectic effects of LPS administration and chronic effects on growth were

monitored by recording body weights throughout the experiment. The physiological

stress induced by LPS administration during either the first week of life or after weaning









was found to reduce body weight gain but to have an inconsistent effect on the preference

for alcoholic beer during early adulthood.















INTRODUCTION

Alcohol Intake in Humans

The etiology of alcohol intake and abuse has been of importance in the past decades

because of the possibility of identifying reliable predictors of the behavior. In a recent

Gallup Poll (Gallup Organization, 2002) 64% of both males and females self reported

consuming alcohol. In 12 -17 year olds, the reported alcohol use is 38% (National

Household Survey on Drug Abuse [NHSDA], 1999). In a survey of eighth graders, 13%

report using alcohol five times or more in the past two weeks (National Institute of Drug

Abuse [NIDA], 2001). The incidence of alcohol intake then doubles in the next 6 years

of life. By ages 18-25, 84% of the population has reported alcohol use in his or her

lifetime (NHSDA, 1999). Among high school seniors, 30% report using alcohol five

times or more in the past two weeks (NIDA, 2001). Thus, there is roughly a two-fold

increase from the transition from late adolescence into early adulthood. The later life

increase raises questions as to why this occurs. With more than half the population trying

alcohol by adulthood coupled with an increased consumption, the onset of alcohol intake

appears to be linked to further alcohol use

Several studies have examined the factors that contribute to adolescent drinking.

Adolescence is of physiological meaning due to the documented hormonal alterations

that help in developing secondary sexual characteristics (Ducharme and Collu, 1982).

Frias and colleagues (2000) conducted a study on the effects of acute alcohol intoxication

on the hypothalamic pituitary-gonadal (HPG) axis and hypothalamic pituitary adrenal









(HPA) axis hormones. HPG axis hormones include testosterone, prolactin, luteinizing

hormone and follicle stimulating hormone. One of the HPA axis hormones,

adrenocorticotrophic hormone (ACTH), was included as well during assessment to

determine stress levels of the adolescents.

The adolescents studied (N =27; 11 males, 16 females) were admitted to the

emergency department with acute intoxication. Intoxication was behaviorally defined as

slurred speech and unstable walking. Twenty-one adolescents (10 males, 11 females)

served as controls. Controls were admitted to the emergency room for mild traumas, like

contusions or sprains. Both groups' age ranged from 13-17 years.

Using ACTH levels of the controls as a comparison, adolescents with acute

alcohol intoxication were found to display neuroendocrine function similar to levels

found in the adolescents that were not intoxicated that solely encountered the stress of the

emergency room. Serum ACTH levels were higher in the intoxicated subjects in

comparison to the controls in both sexes. On average, the increase was about 9-fold

higher in females and 5-fold higher in males. Serum testosterone was decreased in

intoxicated males and increased in intoxicated females, as compared to the controls.

Serum prolactin levels were higher, as well, in both sexes. In females it increased 5-fold

and in males, 2.6-fold in females. In this example, acute alcohol intoxication affected

hormones that perform necessary modifications that are vital to a growing adolescent.

Proposed Reasons behind Alcoholism

Many reasons have been proposed to explain how moderate alcohol use can change

into abuse. Exacerbating features usually include existing disorders that complicate

effective coping strategies. Male alcoholics have been shown to have a concurrence of

alcoholism with childhood behavioral disorders; primarily, attention deficit disorder and









conduct disorder. Females usually experience alcoholism with affective disorders. A

study by Glenn and Nixon (1991) studied early symptom onset (ESO; alcoholic

symptoms occurring before 25), and late symptom onset (LSO; alcoholic symptoms

occurring after the age of 25) of alcoholism. ESO subjects were more anxious and felt a

lesser capability to interact in social situations than LSO subjects. Reportedly, female

alcoholics experience more mood changes, anxiety and trouble with concentration,

compared to alcoholic men. This suggests a lack of sufficient resources is a component

of dealing ineffectively with psychological problems (Nixon & Glenn, 1995).

Genetic Basis

One argument behind the development of alcoholism is whether the problem is either

learned or inherited. In trying to decipher which alternative is most accurate, findings

suggest both may play a role in determining alcohol intake. There is genetic evidence in

the literature of a link to alcoholism. Chromosomal linkage studies have implicated

chromosomes 1, 2, 4, 7, and 11 (Enoch & Goldman, 1999). There is also evidence of

heredity playing a part in alcoholism. Family histories are one way to study the influence

of both the nature and nurture aspects of alcohol intake and abuse.

Penick et al. (1978) performed an experiment with 155 male veterans using previous

family history as a predictor of abuse and a possible treatment approach. The average

age for the men was 45 years old. The men volunteered to participate in the study and

agreed to return monthly for a year for evaluation at an outpatient clinic. At the

beginning of the experiment a two-hour interview was conducted to determine the state

of the men's family history.

The men were subsequently categorized into three groups. Group I (n=61) had at least

one grandparent/parent that abused alcohol. Group II (n=57) had a relative other than









grandparent/parent abusing alcohol. A common "other" relative was reportedly brothers

and uncles. Group III (n=37) had no blood relative with a drinking problem. During the

monthly visits for observation, the patients participated in a variety of measures to assess

mood states,1 personality,2 and psychiatric behavior.3 In total, 39 variables from the

different measures were studied. These variables included basic sociodemographic

information and alcohol related happenings.

There was a significant correlation between the onset age of drinking behavior and

having an alcoholic grandparent/parent. Thirty-three percent of the men in Group I

started drinking regularly at age 15 or younger. These results suggest exposure to an

alcoholic family member results in a higher likelihood the subject inadvertently learns the

behavior as normal and accepted. The results demonstrate that early societal and familial

influences can affect drinking in adulthood.

Comparable conclusions were seen in a similar study using women. Using women

with driving under the influence offenses, Lex et al. (1991) found that 75% of the women

had a family history of alcoholism. A majority of the women started drinking at an early

age and reported failure to abstain from drinking. These studies also show that family

history and early forming drinking habits have a role in the severity of alcohol's effects

on everyday functioning.




1 Profile of Mood States (POMS: Tension, Depression, Anger, Vigor, Fatigue and
Confusion)
2 Eysenck Personality Inventory (EPI: Neuroticism, Extraversion, Lie)

3 Symptom Checklist-90 (SCL: Somatization, Obsessive-Compulsive, Interpersonal
Sensitivity, Depression, Anxiety, Anger-Hostility, Phobic Anxiety, Paranoid Ideation,
Psychoticism, Additional)









Trauma and Stress

Though trauma isn't a direct cause for alcohol abuse, there have been connections

between those suffering from posttraumatic stress syndrome (PTSD) and alcoholism. In

fact, 25% to 75% of survivors of abusive or violent trauma report problematic alcohol

abuse. Having PTSD does increase the risk of abuse even if it doesn't have a causal

relationship. In a population of Vietnam veterans diagnosed with PTSD, 60%-80% have

alcohol abuse disorders as well. PTSD symptoms are often times worsened by substance

abuse. About 10%-50% of adults with PTSD also have other serious disorders such as

mood, anxiety, addictive and behavioral disorders (National Center for PTSD, 2000).

Increases in stress have commonly been reported concurrently with increases in

alcohol intake. In a population of middle-aged women, individuals whose coping abilities

were classified as low problem focused consumed more alcohol per occasion than those

that with better problem focused abilities in stressful circumstances (Breslin et al., 1995).

This and other studies utilizing humans have uncovered some of the causes behind stress

motivated drinking. Recent studies with male and female adolescents and young adults

have found that one of the most common reasons for drinking is its use as a coping

strategy to combat stress (Hansell et al., 1999; Laurent et al., 1997). Alcohol drinking in

young adulthood has been attributed to stressful episodes and was found to decrease after

college as stressors diminished and life became more stable (Perkins, 1999).

Stress and the HPA axis

The hypothalamic-pituitary-adrenal axis (HPA) axis is considered to be the primary

system responsible for physiological responses to stress. The HPA axis consists of a

negative feedback system that is triggered by a cascade of hormones. Both corticotrophic









releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from

parvocellular neurons in the paraventricular nucleus of the hypothalamus (PVN).

Normally AVP alone is a weak secretagogue of ACTH, however when secreted in

conjunction with CRH it works synergistically to boost the release of ACTH from

corticotrope cells in the anterior pituitary. AVP works at the molecular level on cells of

the anterior pituitary. Once ACTH is released it regulates the release of glucocorticoids

from the adrenal cortex. Corticosterone (CORT) in rodents and cortisol in humans acts

via the circulatory system, providing negative feedback at the level of the hypothalamus

and the pituitary to modify further secretion of hormones (Whitnall, 1993).

Animal Models of Alcohol Intake

Alcohol intake in rats is generally tested using a two bottle choice procedure. When

given the choice between alcohol and water, the net choice for alcohol is regarded as

preference for the fluid. Exposure to alcohol is presented in incrementing concentrations

so a range of preference can be established. For instance, the range can be used to

characterize a particular line of subjects. Further ways to explore voluntary alcohol

intake are conducting self-administration and fluid discrimination studies.

Rhesus monkeys can be trained to self-administer alcohol through an intravenous

catheter via a lever press. Deneau and colleagues (1969) demonstrated that rhesus

monkeys would self administer to the point of intoxication. During intoxication,

monkeys exhibited poor motor coordination and stupor. Monkeys reduced food intake

and would self-administer alcohol throughout the day for consecutive days.

Animal models utilizing selective breeding and genetic manipulations are beneficial to

the study of alcohol intake. Alcohol preferring (P) and alcohol non-preferring (NP) rats

are one selectively bred strain used to study the physiology and behavior of alcohol









seeking behavior. P rats voluntarily consume alcohol in concentrations of 10%-30%.

They acquire neural tolerance to and physical dependence of alcohol. NP rats avoid

alcohol and show no physical tolerance or dependence (Li et al., 1987).

McMillan and Li (2001) reported that another selectively bred line, high-alcohol

drinking (HAD) rats can be trained to discriminate between three different fluids in a

three lever choice procedure. Rats were trained to lever press for food then began drug

discrimination training. Before discrimination training sessions, rats received saline, a

low dose (0.75 g/kg) of ethanol or a high dose (1.5 g/kg) of ethanol. HAD rats were able

to discriminate between ethanol and saline and responded at high rates for the highest

ethanol dose administered. The high response rate reflects the control of the high dose of

ethanol on behavior.

Mouse Models

One of the results of the completion of the mouse genome, is the ability to use mice in

alcohol studies to test the genetic basis for alcoholism. Quantitative trait locus (QTL)

mapping has been helpful in locating specific genes that may contribute to physiological

disorders. QTLs for ethanol consumption have been identified. To locate a QTL, firstly,

strains of mice with largely different phenotypes are inbred. Secondly, the offspring or

F2 generation, are phenotyped and then genotyped using genetic markers. Once the

identity of the F2 generation is known, behavioral tests can be performed and a

correlation between the behavior and genes can be established (Crabbe et al., 1999).

Mice created by transgenic procedures have been used to study the role of specific

hormones or neuropeptides that may be involved in mediating alcohol intake. Recently,

the involvement of neuropeptide Y (NPY) in alcohol consumption has been reported.









NPY is usually associated with the control of food intake because of its excitatory effect

on the PVN resulting in an increase in food intake (Levine & Morley, 1984).

The NPY subtype 1 receptor (Y1R), in particular is involved in the response of NPY

to ethanol (Thiele et al., 2002). In alcohol tests Y1R knockouts (Y1 -/-) drank more of

3%, 6% and 10% ethanol than wild type mice (Y1 +/+). Additional experiments were

done to rule out alcohol drinking due to caloric and taste influences. The drinking was

not a result of high ethanol metabolism because Y1R knockouts had no significant

differences in plasma ethanol concentrations than wild type mice. Therefore, Y1

receptors are suggested to be involved in voluntary ethanol consumption (Thiele et al.,

2002).

C57BL/6 (B6) and DBA/2J (D2) are two strains of mice used commonly in alcohol

studies due to their opposing profiles for alcohol preference and intake. In a test of taste

preferences, B6 mice have high preferences for ethanol at 5%, 10%, 15%, and 20%

concentrations, while D2s show low preferences in this range. In B6 mice, the peak of

preference occurs at 10% (Bachmanov et al., 1996). Extracellular recordings from brain

slices of the ventral tegmental area were taken from both D2 and B6 mice. Results

indicate that D2 mouse neurons were more responsive to excitation by ethanol. One of

the reasons for the difference in intake in these two strains is proposed to be difference in

the effects of ethanol in the brain. This heightened sensitivity could be why D2 mice will

drink less during alcohol intake studies (Brodie & Appel, 2000).

In a separate set of studies, B6 and D2 mice were crossed to produce B6D2 F2 mice.

The new recombinant strain was tested for free choice ethanol consumption. Genomic

DNA was collected from mouse spleens at the termination of ethanol testing. Genetic









markers corresponding to high ethanol drinkers were located on loci on both

chromosomes 2 and 9 (Phillips et al., 1998). Identification of these functional genes

helps to answer some of the questions of the influence of the environment on behavior.

Marking the location additionally will aid in finding genes and gene products and their

homologs in humans.

Stress Induced Drinking

One factor that has been confirmed in both human and animal data to have an effect

on alcohol drinking is stress. Young adult college graduates reported consuming more

alcohol during college to relieve stress in both social and academic situations. After

college, subjects reported a decrease in stress encounters and this highly correlated with

the decrease reported in alcohol intake (Perkins, 1999). Rats stressed with either

immobilization or isolation in adulthood show significantly higher ethanol intake than

non-stressed controls. Alcohol intake was increased three weeks after cessation of the

stressors, showing that stress can have a lasting effect on inducing coping behavior (Nash

& Maickel, 1985).

Genetic models have also been used to test stress induced alcohol intake. CRH is one

of the hormones involved in the cascade of activation during stress induced HPA axis

activation. CRH binds to two types of receptors in the anterior pituitary, CRH1 and

CRH2. The CRH1 receptor (CRH1R) is highly expressed in the anterior pituitary, cortex,

hippocampus, amygdala and cerebellum (Potter et al., 1994). Chronic psychosocial stress

significantly reduces the binding sites of CRH in anterior pituitary, dentate gyrus, and

hippocampus (Fuchs & Flugge, 1995). Due to the importance of CRH, its receptor also

has a large role in regulating the body's response to stress. Mice CRH1R knockouts

have an impaired stress response and an increased ACTH/CORT response after stress









(Timpl et al. 1998). Therefore the binding abilities of CRH IRs are important in stress

and can be indicators of the stress response in the presence of alcohol.

Mice lacking functional CRH1IRs were used in an experiment by Sillaber et al. (2002)

to test their importance to stress induced drinking. CRH1R knockouts and wild type

mice were offered a choice between increasing concentrations of ethanol and water.

Baseline intake was recorded for eight weeks. Baseline intake of alcohol did not differ

significantly for the two groups of mice. After the eight weeks, mice were exposed to

social defeat stress for three consecutive days.

During and immediately following the stressor, there was no difference in alcohol

intake. Three weeks after the repeated social defeat exposure, CRH1R knockout mice

showed an increase in alcohol intake compared to baseline. This increase was not seen in

the wild type mice. Following the alcohol testing, CRH1R knockout mice were then

exposed to a forced swim test for three consecutive days. There were no significant

differences in alcohol intake during the stress days. Three weeks after the forced swim

alcohol intake the intake of the knockout mice rose significantly higher than the wild type

mice. The post stress response of CRH1R knockout mice lasted six months after the

second stressor, the forced swim.

Alcohol metabolism did not play a role in the difference in alcohol intake between

wild type and CRH1R knockout mice. Blood alcohol concentrations did not differ

significantly between the two groups. The prolonged drinking of alcohol in CRH1R

knockout mice may be due to an increase in NR2B subunit of the NMDA receptor in the

nucleus accumbens and hippocampus. Neurons with mostly NMDA receptors have been









implicated in the reinforcing effects of alcohol and the increased expression of the NR2B

subunits have been linked to a stressful experience (Bartanusz et al., 1995).

Early Life Stress and Drinking

Stress experienced at specific times during development has been shown to have long

lasting effects on the responsivity of the HPA axis in adult rodents. Thus, prenatal (Reul

et al., 1994) and postnatal studies (Shanks et al., 1995) have shown that immunological

stress during critical periods of development produces increased HPA responsiveness.

Early maternal separation and handling are examples of processive stressors that have

been also been reported to play a part in a heightened stress response.

Rats that have experienced maternal separation once a day during days 2 to 14 of life

demonstrate an altered HPA axis in adulthood. The result of this manipulation is an adult

rat that is characterized by increased CRF mRNA and plasma corticosterone levels

(Plotsky & Meaney, 1993). Levine (1967) was one of the first to report that rat pups that

were reared by non-handled dams exhibited a high adrenocortical response. Early

experience in the dam can affect the offspring's response to a novel stimulus. This

evidence illustrates there is an important influence of early maternal interaction between

pups and dams. The results of early life stress affecting adult behavior have been

replicated in other studies using other stressors indicating that the effect can be

generalized (i.e., Weinberg, 1987).

Stress Hyporesponsive Period

The behavioral response to early life stress is pertinent because during the first two

weeks of life the stress axis activation is thought to be hyporesponsive. Beginning on

postnatal day 2, rat pups would fail to respond or do so weakly if challenged with a

stressor. Circulating basal levels of plasma CORT are low for the first two weeks of life.









The levels start to appear around postnatal day (P) 7 and reach adult levels by P15. The

CORT response to stress does not develop fully until after P16.

The hyporesponsive period, between P 2-14, has an adaptive function because if

excessive glucocorticoids were released during this time, they would promote decreased

cell division, protein synthesis and uptake of amino acids. All of these actions are vital

for growth and development (Sapolsky & Meaney, 1986). The responsivity of the HPA

axis during this period indicates that there are key points in development. Butte and

colleagues (1973) tested for the specificity and implemented a stressor (subcutaneous

injection of histamine) at various times throughout the stress hyporesponsive period.

There were significant increases in plasma CORT secretion at days 1, 2, 3, 16 and 21.

Subsequent studies have also verified the idea of critical periods. Using this information,

administration of a stressor in early life can result in adult behavioral and neuroendocrine

reactivity to stress.

Animal Models of Early Life Stress and Drinking

In animal models, physical (i.e. early handling & immobilization) and psychological

stressors have been employed to examine the subsequent effects on alcohol intake

(Weinberg, 1987). Early handling in BALB/cJ mice performed during the first three

weeks of life increased a preference for alcohol as compared to non-handled mice of the

same strain (Jones et al., 1985). Lancaster (1998) found that handling as well increased

a preference for alcohol in rats, specifically during 32-45 days of age. This evidence

suggests this period is influential in preparing the individual's level of stress

responsiveness. This work and the large literature on the neuroendocrine response of

stress itself suggest that there is a possible interaction between stress and increased









alcohol preference. Weaning age has also been shown to affect HPA axis activity in

response to stress during adulthood (Cook, 1999).

Aim of Study

This study asks whether perinatal stress of another sort, an immunological challenge,

would affect alcohol intake in young adult mice. Mice have been widely used and studied

as a model for alcohol preference (Belknap et al., 1993) and have previously been

founded to exhibit an increased preference following of an early life stressor (Jones et al.,

1985). Hsd:ICR (CD-1) mice were used in this experiment because this outbred strain

does not consume large amounts of ethanol when presented with a free choice situation

(McMillen & Williams, 1998).

Lipopolysaccharide as a Stressor

To induce the immunological stress, lipopolysaccharide (LPS) was chosen because it

has been used previously and reliably as an agent to modify the HPA axis (Reul et al.,

1994) and it can be administered in controlled doses. LPS is a bacterial endotoxin that

produces an elevation in temperature signaling the release of cytokines. Abdominal

temperature starts to rise 1 hour after injection and peaks at 2-12 hours post injection

(Konsman et al., 1999). LPS exposure proves to be a potent stressor because it increases

CRH, ACTH, AVP and plasma CORT (Shanks et al., 1995, 2000; Laugero & Moberg,

2000a). Dent et al. (1999) found that LPS begins to exert these effects 2 hours after

injection. Plasma ACTH and CORT were increased significantly in rats aged 6, 12 and

18 days old.

LPS affects metabolism and it is known to depress energy deposition. Both factors are

important in maintaining the energy requirement of young adult mice. LPS significantly

reduced energetic efficiency, the ability of the animal to allocate energy into tissues from









an equal unit of energy intake. These changes are indicative of alterations in growth and

deposition of lean and fat tissues. The decreased energy intake helps to explain the

depression in weight that occurs after LPS injection. After an immune challenge LPS

increases IL-13 (Laugero & Moberg, 2000a). IL-1f3 increases energy expenditure and

lean and fat tissue catabolism resulting in anorexia (Benson et al., 1993)

LPS activation is found in the PVN as shown by significant c-fos, an early gene

marker, expression in neurons linked to the induction of the neuroendocrine cascade of

the HPA axis. In addition to altering HPA activity, LPS administered to mice in the first

week of life was found to increase social reactivity in adulthood (Granger et al., 1996).

Hypothesis

We hypothesize that receiving this immunological stress in either the first week of life

or after the weaning period will increase alcohol preference in adulthood. Recording the

intake of alcohol in adolescence will be a reflection of the physiological changes taking

place during puberty and may also suggest a coping response, which has been the

common explanation for alcohol intake in human adolescents.















MATERIALS AND METHODS

Animals

Untimed pregnant female Hsd:ICR mice were purchased from Harlan Labs

(Indianapolis, IN). After the dams gave birth, litters were culled to eight (approximately

4 of each sex) and left with the mother. Each litter was randomly assigned to

experimental groups by order of birth date. In these experimental groups mice received

no injection, saline injections, LPS injections in the first postnatal week or LPS injections

after weaning. Litters were weaned at 21 days of age and were then group housed until

the beginning the alcohol intake testing at postnatal day (P) 30.

Animals were housed in shoebox cages with Sani-Chips bedding. Food (PMI

Rodent Chow) was available ad libitum throughout the experiment and tap water was

available ad libitum prior to and after ethanol testing. Animal rooms were kept at 22 + 2

C and had a 12 hr light/dark cycle with lights off at 1900 h.

Procedure

The LPS and saline groups were injected intraperitoneally (i.p.) with 5[l of LPS

or saline during the light cycle. LPS injected groups received 0.05 mg/kg Salmonella

entiritidis, (Sigma Chemical Co.) either on P 3 & 5 or P 21 & 23. Mice in the saline

injected group received equivalent saline injections on P 3, 5, 21 & 23. Weights were

recorded for all groups on P 3, 5, & 7. Injections were given immediately after weighing

to monitor each group's body weight. Non-injected control mice were only handled for

weighing. Animals were returned to their mothers immediately after injections.









Weights were recorded again on P 21, 23 & 25 during the second round of

injections. Once animals reached P 30, all were housed singly and given a choice

between 5% or 10% ethanol (ethanol + non-alcoholic beer) and water. These fluids were

presented in 20 ml pipettes, fitted with metal drinking spouts and rubber stoppers.

Pipettes were mounted to home cages by wedging them firmly into the bars of the cage

lid. In the first week of testing (P 30-37) mice had a choice between water and 5%

ethanol. In week two (P 38-44) of testing mice had a choice between water and 10%

ethanol. The same sequence was repeated at P 50-57 and P 58-64, respectively. Daily

intake was recorded and data are presented as mean ml + SE per week for each ethanol

concentration.

Experiments

All three experiments followed the same procedure. Experiment one consisted of

2 control litters (9 female, 7 male), 3 saline injected control litters (11 female, 13 male), 3

litters injected with LPS in the first postnatal week (11 female, 12 male) and 3 litters

injected with LPS after weaning (12 female, 10 male). All animals in experiment one

were weaned at P 21, with the exception of one litter in the non-injected group that was

weaned 2-3 days early.

Experiment two and three were conducted in an attempt to replicate experiment

one. Experiment two consisted of 4 non-injected control litters (11 female, 21 male), 4

saline injected control litters (14 female, 17 male), 4 litters injected with LPS in the first

postnatal week (15 female, 16 male) and 4 litters injected with LPS after weaning (12

female, 17 male). Experiment three consisted of 3 non-injected control litters (14 female,

10 male), 4 saline injected control litters (14 female, 16 male), 3 litters injected with LPS









in the first postnatal week (6 female, 18 male) and 3 litters injected with LPS after

weaning (11 female, 13 male).

Statistical Analysis

One-way ANOVA (Sigma Stat 1.0, SPSS) was used to test the relationship

between treatment and mean intake and Bonferroni's method was used for multiple

comparisons between groups. One-way repeated measures ANOVA was used to compare

before puberty drinking to after puberty drinking. Preference ratios were derived from

dividing the total alcohol consumed by total fluid consumed (ml alcohol/ml water + ml

alcohol). A chi-squared test was used to compare preference ratios. A t-test was used to

compare male and female alcohol intake. A p-value of < 0.05 was considered significant















RESULTS

Experiment 1

Body Weights

The body weights are shown in the top panels of Table 1. The non-injected (NON)

mice weighed consistently more than the other groups throughout the study. Early and

late treatment with LPS (LI and L2) decreased significantly the weights during P3-P7

and P21-25. By the weaning period (P21-25), groups LI and L2 weighed less than the

saline injected mice. Non injected animals weighed more than group L2 during 5% and

10% alcohol phases (see Table 2). The other LPS injected group (LI) only showed

significant differences at P 30 and P38.

Ethanol Intake

In intake tests before puberty for 5% and 10% EtOH, groups LI and L2 drank a

significantly greater mean volume of ethanol than non-injected animals (see Figure 1).

During the 5% alcohol phase, L2 drank more ethanol than saline injected animals. All

animals decreased total volume intake during the 10% alcohol phase, however both LPS

injected groups drank more than the SAL and NON groups (p< .05). Figure 2a shows 5%

ethanol intake after puberty. Groups LI and L2 drank more 5% alcohol than NON

animals. Group L2 was the only LPS injected group that drank significantly more than

the SAL group (p < .05).

Table 3 shows the ethanol preference by group, expressed as the number of animals

showing a net preference (>50%) for alcohol. There were significant differences between









groups during both the prepubertal (P30-37) and postpubertal (P50-57) periods. The

water intakes during the two-bottle choice are shown in Table 4 and were significantly

higher for the NON group than other treatment groups during intervals P30-37 and P 50-

57.

Ethanol intakes before (P30-44) and after (P50-64) puberty are compared in Table 5.

The non-injected control group showed no difference in either 5% or 10% ethanol intake

between the two phases of testing, and the saline-injected controls showed a modest

increase in only 5% intake after puberty compared with before puberty. In contrast, both

LPS groups had significantly lower 10% ethanol intakes after puberty than before,

although no such change was evident for the 5% ethanol.

The results suggest that the LPS injections did have an effect not only on the animals'

weights but also on the preference and intake of alcohol in adulthood. Experiment 2

attempts to build on these findings by replicating the study with a larger set of animals.

Experiment 2

Body Weights

The middle panel of Table 1 shows group weights during the two injection phases. In

the first week of life, group LI weighed significantly less than NON and SAL groups.

By P7, contrary to experiment one, the SAL group weighed significantly more than the

other three groups. This result shows there was no suppressant effect of the saline

injection on body weight gain. After injections in the weaning period, group LI weighed

significantly less than the NON and SAL groups. An effect of the second LPS injection

was seen only on P25, when LI weighed significantly less than the NON group (p <

.05).









Table 2 shows weights of mice during alcohol testing done before and after puberty.

SAL and NON groups weighed significantly more than LI before the 5% alcohol phase.

The NON group weighed more than LI after 10% alcohol phase. The NON group

weighed significantly more than LI throughout the second phase of 5% and 10% alcohol

testing (p < .05).

Alcohol Intake

Figure 3 shows 5% and 10% intake for all treatment groups before puberty. Both non-

injected and saline injected groups of mice drank significantly more 5% alcohol than

group L2 during P30-37. For 10% alcohol consumption, only the CON group drank

significantly more than L2 (p < .05). Table 3 shows the number of animals that preferred

alcohol to water. LI, SAL and NON mice showed a net preference (>50%) for 5%

alcohol over water both before and after puberty. The NON group had the largest

percentage of animals that showed a net preference for 5% alcohol at 23 out of 32 (72%)

mice. Comparison of preference ratios during the four intervals of ethanol exposure,

revealed a significant difference in P 30-37 (p < .005) and P 38-44 (p < .005).

Figure 4 shows alcohol intake after puberty. During 10% alcohol testing, group L2

drank less than the non-injected group (p < .05) (see Figure 4b). About 1/3 of mice in

groups LI, SAL and CON prefer 10% alcohol to water after puberty (see Table 3). The

water intake values during the two-bottle choice are shown in Table 4. During P50-57,

L2 mice drank more water than NON and LI mice (p<.05). Table 5 contains the

comparison of alcohol testing before (P30-44) and after (P50-64) puberty. There were

significant differences for both LPS groups. LI and L2 mice consumed the 5% alcohol

during P50-57 as compared to P30-37 (p<.02).









The weight loss of the LPS injected groups was a trend that was evident in

experiments one and two. However, the increased alcohol intake of groups LI and L2 in

experiment one was not replicated in experiment two. A third replication was done in

hopes of a definitive answer to whether an immune challenge would increase alcohol

intake in adulthood.

Experiment 3

Body Weights

The bottom panel of Table 1 shows the weights for the third set of mice during the two

injection phases. In the first week of life the NON group weighed more than all three

other groups. This trend was significant on P 3 and 5. After the second round of

injections group L2 weighed significantly less than the other three groups (p<.05). In the

period before puberty, the weights of L2 remained lower than NON and LI mice (see

Table 2). This trend occurred until the end of the 10% alcohol phase; the L2 group

weighed significantly less than LI mice.

Alcohol Intake

Figure 5 shows alcohol intake for all treatment groups before puberty. Although there

were no significant differences between the groups, Table 3 shows that during the 5%

alcohol phase more than half the animals preferred alcohol to water. The number of

alcohol preferring animals decreased considerably during the 10% phase. Alcohol intake

after puberty shows was significantly different during the 10% alcohol phase (see Figure

6). The L2 group drank significantly more 10% alcohol than LI and SAL group, and

75% of these mice preferred the 10% alcohol to water (see Table 3).

A comparison of preference ratios during the four ethanol phases revealed a

significant difference from chance ratios for consumption of 5% and 10% alcohol after






22


puberty. The water intakes during the two-bottle choice are shown in Table 4; there were

no significant differences. Table 5 contains the comparison of alcohol testing before

puberty (P30-44) and after puberty (P50-64). All four groups of mice drank significantly

more 10% the second time it was offered; this was not evident in the other two

experiments.










Table 1. Group weights (g) during the two injection periods.


First week of life


Weaning period


Experiment one


+ .1a
+ .1 a
+ .1
+ .1


P5
4.3 .1a
4.2 .1a
4.6 .1a
5.1 .1


P5
ab 3.2 .1 ab
3.6 .1
3.8 .1
3.6 .1


L1
L2
SAL
NON


L1
L2
SAL
NON


L1
L2
SAL
NON


P5
3.4 .1 a
3.4+ .1
3.4 .1
3.7 .1


5.7 +.1a
5.6 + .la
5.9 + .Ia
6.5 +.1


14.1 .4 ab
13.4 + .3 ab
15.9 + .3 a
17.6 + .6


Experiment two
P7 P21
4.2 .1 ab 8.9 .3 ab
4.4 .1 b 10.5+ .5
4.8 .1 10.7 .1
4.5 .1 b 11.1 .3
Experiment three
P7 P21
4.6 .1 a 11.0 .2
4.5 .1 a 9.5 .3 abc
4.4 .l a 11.5 .2
5.0 .1 11.7+ .4


P23
16.7 .5 ab
15.4 + .4 ab
18.9 + .4
19.2+ .4

P23
11.0 .3 ab
12.5 + .6
13.0+ .2
13.8 + .4

P23
13.1 + .3
11.4 + .3 abc
13.1 + .3
13.7+ .5


P25
19.1 .5 ab
17.7 + .4 ab
21.6+ .4
22.2 + .6

P25
13.2 .4 ab
14.7 .6 a
15.3 + .2
16.6 + .4

P25
15.6+ .3
13.5 .3abc
15.1 + .3
16.5 + .6


L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline
injected group on P3, 5, 21 &23; NON= non injected group
a Significantly less than non-injected group
b Significantly less than saline group
' Significantly less than LPS group injected on P3 & 5
Significance level at p<.05; numbers expressed are mean SE grams


+ 1 a
.+ 1a
a+ 1a
+ .1









Group weights (g) during ethanol phases


Before puberty


After puberty


5% ethanol
P30
24.5 + .7 ab
23.2 + .6 ab
27.2 + .5
27.6 + .9

5% ethanol
P30
19.5 + .5 ab
21.0 + .9
21.7 + .4
23.0 + .5

5% ethanol
P30
22.4 + .5
19.4 + .4 a c
21.1 + .4
22.3 + .7


P38
27.5 + .7 a
26.9 + .6 a
29.2 + .6
30.2 + .9


L1
L2
SAL
NON



L1
L2
SAL
NON



L1
L2
SAL
NON


Experiment one
10% ethanol
P44
29.9 .8 31.2
28.8 + .7 a 30.0


31.4
32.5


5% ethanol
P50
.8
.8 a


32.6+ .8
34.0 + 1.1


Experiment two
10% ethanol 5% ethanol
P44 P50
26.7 .8 a 28.1 .8 a
29.0 + .8 30.4 + .8
29.3 + .8 30.7 + .8
30.2+ .6 31.6+ .6
Experiment three
10% ethanol 5% ethanol
P44 P50


a c


30.0+ .7
26.5 + .7 c
27.3 + .6
29.0 + 1.0


31.5 + .7
27.5 + .8 c
28.3 + .6 c
30.2 + 1.0


I


10% ethanol
P64
34.4 + .9
33.7 + .9 a
36.5 + .8
37.5 + 1.3

10% ethanol
P64
30.8 .9 a
33.4 + .9
33.0 + .9
34.7 + .7

10% ethanol
P64
34.0 + .8
29.6 + .9
30.5 + .7 c
33.0 + 1.1


L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline
injected group on P3, 5, 21 &23; NON= non injected group
a Significantly less than non-injected group
b Significantly less than saline injected group
' Significantly less than LPS group injected on P3 & 5
Significance level at p<.05; numbers expressed are mean SE in grams


P38
25.0 + .6
26.7 + .7
26.1 + .9
27.4 + .5


P38
27.0 + .6
24.2 + .5
25.2 + .4
26.7 + .8


Table 2


P58
33.5 + .9
32.1 + .9 a
34.8 + .7
36.0 + 1.2


P58
29.2 + .9 a
31.7 + .9
31.6 + .9
33.3 + .6


P58
32.6 + .8
28.7+ .8 c
29.9 + .6
31.6 1.1









Number of mice showing a net preference (>50%) for ethanol over water


Ethanol concentration
5%
10%
5%
10%

Ethanol concentration
5%
10%
5%
10%

Ethanol concentration
5%
10%
5%
10%


P
30-37 a
38-44
50-57 b
58-64

P
30-37 c
38-44 d
50-57
58-64

P
30-37
38-44
50-57 e
58-64 f


Experiment one
L1 (n=23) L2 (n =22)
20 19
12 16
18 18
10 11
Experiment two
L1 (n=31) L2 (n =29)
20 8
5 1
24 18
10 4
Experiment three
L1 (n=24) L2 (n = 24)
19 15


SAL (n=
19
12
20
11

SAL (n=
16
6
21
11

SAL (n=
20
5
21
9


NON (n= 16)
8
5
6
4

NON (n=32)
23
7
21
10

NON (n=24)
15
5
11
6


L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline
injected group on P3, 5, 21 &23; NON= non injected group
a Significant difference from chance during this period (2 = 8.73, p< .05)
b Significant difference from chance during this period (X2 = 12.83, p<.01)
c Significant difference from chance during this period (2 = 13.2, p< .005)
d Significant difference from chance during this period (X2 = 19.4, p<.005)
e Significant difference from chance during this period (2 = 10.4, p<.025)
f Significant difference from chance during this period (X2 = 15.3, p<.005)


Table 3









Mean water intake for treatment groups during ethanol phases, ml


P
30-37
38-44
50-57
58-64

P
30-37
38-44
50-57
58-64

P
30-37
38-44
50-57
58-64


L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline
injected group on P3, 5, 21 &23; NON= non injected group
a Significantly more than LPS group injected on P3 & 5
b Significantly more than LPS group injected on P21 & 23
c Significantly more than saline injected group
d Significantly more than non injected group


L1
1.9+ .3
3.5 + .4
2.2+ .2
3.3 + .3

L1
3.9+ .4
4.6+ .3
2.4+ .3
3.7+ .3

L1
2.7+ .3
5.3 + .3
3.2+ .3
4.1 .3


Experiment one
L2 SAL
1.9 .3 2.3 .3
2.6 .4 3.4 .3
2.1 .5 2.0 .3
3.3 .3 2.9 .3
Experiment two
L2 SAL
4.8 .4 4.3 .4
5.8 .3 5.5 .3
3.6 .4 ad 2.6 .3
4.7 .3 3.9 .3
Experiment three
L2 SAL
3.2 .4 3.2
5.2 .4 4.8 .3
3.0 .4 3.2 .2
3.3 .4 4.1 .3


NON
3.5 + .6 ab
4.3 + .6
3.9 + .5 abc
4.3 + .5

NON
3.5 + .3
6.3 + .7
2.4 + .3
3.7 + .3

NON
3.1 + .4
5.6 + .5
3.7 + .4
4.5 + .4


Table 4









Ethanol intake before and after puberty, ml


Concentration


5%

10%


Concentration


5%

10%


Concentration

5%

10%


Stage


P30-37
P50-57
P38-44
P58-64

Stage

P30-37
P50-57
P38-44
P58-64

Stage
P30-37
P50-57
P38-44
P58-64


Experiment one
L1


L2 SAL


5.8 .4 6.1 .4
5.5 .4 6.5 .4
4.0 .2 a 4.3 .4 a
3.4 .2 a 3.5 .3 a
Experiment two
L1 L2

4.4 .2 a 3.5 .3 a
5.1 .3 a 4.4 .3 a
2.7 .2 2.1 .2
2.9 .2 2.3 .2
Experiment three
L1 L2
5.2 .3 a 5.3 .4
4.5 .3 a 5.5 .5
2.1 .2 a 2.9 .3 a
3.3 .2 a 4.6 .3 a


4.4 .3 a
4.9 + .3 a
2.9+ .2
3.1 +.2

SAL

4.7+ .3
5.2+ .4
2.5 + .2
2.8 .3

SAL
4.2+ .3
4.0+ .3
2.2+ .2 a
2.8 + .2 a


L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23; SAL= saline
injected group on P3, 5, 21 &23; NON= non injected group
a Significant difference between before puberty and after puberty intake
Significance level at p<.05; Expressed as mean SE ml/day


NON


3.7
3.7
2.3
2.6


NON

5.2 + .3
5.3 + .4
3.1 +.2
3.4+ .3

NON
5.4+ .4
4.7+ .6
2.4+ .4 a
3.6+ .4 a


Table 5












(a) 5% EtOH
7 L1
6 SAL
6 NON
5 L






(b) 10% EtOH


















week period. LI and L2 consumed more alcohol than NON mice at both
SSASignificantly greater than SAL.
E 677Z NON
5 -









Figure 1. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, before puberty in CD-P3 & mice. The four treatment groups received
each concentration for o week each. Intake is shown as the average over the I
week period. L1 and L2 consumed more alcohol than NON mice at both
concentrations. Fluid intakes are expressed as mean + SE ml/day
Significantly greater than NON.
4 Significantly greater than SAL.
LI=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group













(a) 5% EtO H
8 -


Figure 2. Mean ( SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, after puberty in CD-1 mice. The four treatment groups received
each concentration for 1 week each. Intake is shown as the average over the 1
week period. LI and L2 consumed more ethanol than NON mice during 5%
exposure. No significant differences for 10% ethanol intake. Fluid intakes are
expressed as mean + SE ml/day
Significantly greater than NON.
# Significantly greater than SAL.
L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group

















(a) 5% EtOH


(b) 10% ETOH
8 -


- L1
7ZZZ L2
- SAL
Z2 NON


Figure 3. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, before puberty in CD-1 mice. The four treatment groups received
each concentration for 1 week each. Intake is shown as the average over the 1
week period. L2 mice drank less than NON mice at both concentrations and
less than SAL mice at the 5% concentration. Fluid intakes are expressed as
mean + SE ml/day
Significantly less than NON.
# Significantly less than SAL.
L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group













(a) 5% ETOH


Figure 4. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, after puberty in CD-1 mice. The four treatment groups received
each concentration for 1 week each. Intake is shown as the average over the 1
week period. No significant differences occurred for 5% ethanol intake. L2
intake for 10% alcohol was less than NON mice. Fluid intakes are expressed
as mean + SE ml/day
Significantly less than NON.
L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group













(a) 5% EtOH


(b) 10% ETOH
8 ------


- L1
ZZ1 L2
- SAL
ZZZ] NON


Figure 5. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, before puberty in CD-1 mice. The four treatment groups received
each concentration for 1 week each. Intake is shown as the average over the 1
week period. No significant differences occurred for 5% and 10% ethanol
intake. Fluid intakes are expressed as mean + SE ml/day
L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group





















(a) 5% ETOH


E

(b) 10% ETOH

m L1
-a 7 ZZ2 L2
m SAL
6777 NON
6

5

4

3

2


0




Figure 6. Mean (+ SE) intakes of (a) 5% ethanol and (b) 10% ethanol, given in choice
with water, after puberty in CD-1 mice. The four treatment groups received
each concentration for 1 week each. Intake is shown as the average over the 1
week period. No significant differences occurred for 5% ethanol intake. L2
drank significantly more 10% alcohol than LI and SAL. Fluid intakes are
expressed as mean + SE ml/day
Significantly less than L2.
L1=LPS group injected on P3 & 5; L2= LPS group injected on P21 & 23;
SAL= saline injected group on P3, 5, 21 &23; NON= non injected group
















DISCUSSION

LPS is an endotoxin that produces an immune and HPA axis response. It is believed

to function as a stressor because of its activation of the HPA axis (Reul et al., 1994;

Granger et al., 1996). The first week of life and the week during weaning have been

reported to be critical periods in development (Dent et al., 1999). There is evidence of

manipulations that were done during these critical periods that have affected behavior in

adulthood. We chose LPS injections as a stressful manipulation and alcohol intake as our

behavioral measure in adulthood. Alcohol intake is a problem in adult youth and one

hypothesized reason as to why that is occurring could be an early life immune challenge.

In our experiments we saw differing results after testing this hypothesis.

The Effects of LPS Injections on Body Weights

As previously reported, LPS did produce characteristic lowering of body weight in all

three experiments. In experiments one and three, the L2 group remained at a significantly

lower weight than controls until the end of the study (P64). In experiment two, the LI

group was the LPS injected group that showed suppressed body weight gain until the end

of the study.

This suppression of body weight gain is due to the short and long-term metabolic

effect of LPS. After acute injection of LPS, body weight gain and energy deposition

decrease in the first 24 hours (Laugero & Moberg, 2000). Short term effects last up to 48

hours, during which the endotoxin functionally activates the HPA axis (Shanks et al.,









1995; Konsman et al., 1999). Other short-term effects of the endotoxin include the

increase of central production of stress hormones, abdominal temperature and brain IL-13

(Shanks et al., 1995; Konsman et al., 1999). LPS increases circulating CORT, which also

contributes to decreased weight gain via its catabolic effects and inhibition of caloric

efficiency (Laugero & Moberg, 2000a).

The slowed weight gain, that can last indefinitely, seems to occur as a result of the

release of interleukin-1 3 (IL-1 3) that happens when LPS triggers an inflammatory

response to sickness (Inui, 2001). There is an endogenous IL-1 receptor antagonist (IL-

Ira) that inhibits the effects of IL-1 on cells, therefore decreasing a full immune response

(Arend, 1993). In mice lacking a naturally occurring IL-Ira, Inui (2001) showed that

there was decreased weight of the mice with a deficient IL-13 receptor antagonist in

comparison to control littermates. The weight difference was apparent at six weeks of age

and continued until 13 weeks of age. This lasting weight difference suggests that IL-1 J3

has a large part in mediating the long term effects of LPS. Although there was a reliable

weight loss observed following LPS exposure in Experiments 1,2, and 3, the degree of

weight loss did not serve as a reliable predictor of alcohol intake in adulthood in our

studies.

The Effects of LPS Injections on Alcohol Intake

In Experiment 1, LPS administered in the first week of life and around weaning

influenced an increased preference for ethanol before puberty (P 30-44) and through a

portion of early adulthood (P 50-57). LPS injections, as seen in prior studies, contributed

to a modified stress response later in life (Granger et al., 1996; Laugero & Moberg,

2000b; Plotsky and Meaney, 1993; Shanks et al., 1995, 2000). The results of experiments









2 and 3 did not however confirm this influence of LPS. Though methodologically all

three experiments were the same, the results were inconsistent. Physiological changes,

environmental changes and taste properties of the alcohol itself could be possible

contributors to why the results did not consistently support our hypothesis.

Alternate Influences of Alcohol Intake

In Experiment 1, both LPS injected groups demonstrated significantly high ethanol

intake until P 57. A lack of an increase in alcohol intake was observed in Experiments 2

and 3. This lack of an effect could be due to hormonal changes that occur around

puberty. In Experiment 3, during P 58-64, the L2 group 10% intake was significantly

higher than the LI and SAL groups.

Lancaster et al. (1996) found similar results in rats. Under no-stress conditions,

increased voluntary ethanol intake was observed around P 52. In our experiment, it is

possible that changes in circulating pubertal hormones affected high ethanol intake in

adulthood. Ethanol intake, whether high or low, could be due to these hormonal changes

independent of LPS administration at critical periods in development.

The preference for 5% alcohol over 10% alcohol is in agreement with a study done by

McMillen & Williams (1998). McMillen and Williams found CD-1 mice to prefer

ethanol at a 6.8% concentration in a free choice situation with no physiological

manipulation in comparison to the commonly high alcohol preferring B6 strain. The

inconsistent alcohol intake results of alcohol in Experiments 1, 2, and 3 cannot be readily

attributed to low taste reactivity. CD-1 mice can learn a LiCl induced taste aversion to

alcohol as well as prefer water to a bitter quinine solution in a two bottle choice test

(McMillen & Williams, 1998).









Early life alterations such as handling and weaning have shown to alter HPA

functioning as late as weaning age (Plotsky & Meaney, 1993; Cook, 1999). In

Experiment 1, a litter from the non-injected group was prematurely weaned by 2-3 days

because the mother died over a weekend due to unknown causes. To discover if this

premature weaning had an effect on alcohol intake, each non-injected litter was compared

to the other three treatment groups. There were no significant differences between the

litter weaned early and the other control litter in 5% intake before puberty. Therefore,

their data were retained in the overall analysis.

In Experiment 3, three mothers died overnight of unknown causes, about a week after

giving birth. Their litters subsequently were euthanized. The deaths happened over three

successive days and may have provided a social stressor to the remaining animals in an

olfactory form. Odors have been found (Zuri et al., 1998) to have an influence on

behavior. It is possible that the mothers dying affected the remaining litters.

Unfortunately the effects of these events and the subsequent maternal environment of the

remaining litters are unknown.

The different times of LPS administration may also play a part in subsequent alcohol

intake. Differences in the drinking trends were seen between the LI and L2 group in

Experiments 1, 2 and 3. In each of these experiments, the L2 group differed the most

from the other groups, either by drinking significantly more or less than the others. LPS

administered in the first week of life may not have been intrinsic in influencing early

adult drinking behavior because the effects we saw were bidirectional.

In a study by Dent and colleagues (1999), an LPS injection on P 18 showed a higher

CORT response after 2 hours than animals injected on P 6. LPS is active mainly during









an acute phase response, which lasts up to 3 h after administration, where cytokines and

stress hormones are at their peak (Kakizaki et al., 1999). It is possible that in our study

the L2 group experienced a higher CORT response and this manifested as a different

effect on alcohol drinking behavior in adulthood.

Gender Differences

There is some evidence for gender differences in alcohol intake and preference.

Lancaster et al. (1996) used a paradigm in which tubes of 5% beer were offered to male

and female rats ad libitum, in addition to water and food. In a free access situation,

females were found to increase intake and preference of 5% beer around 50 days of age.

With the added stressor of handling (Lancaster, 1998), female rats showed no significant

increase in 5% beer consumption but a significant increase in water intake around

puberty. On the contrary, male rats had an increase in alcohol intake and preference

around puberty.

Gender differences in mice are not as evident. In a two bottle choice of 8% alcohol

and water under no-stress conditions B6 mice were found to show no gender differences

in intake and preference (Little et al., 1999). In contrast, female CD-1 mice were found

to drink more than males in a two bottle choice between alcohol and water (Naassila et

al., 2002). Following LPS as the putative stressor, in our study not all treatment groups

showed gender differences in alcohol intake. In Experiment 1, 10% intake before

puberty for saline injected mice was higher for females than males. In Experiment 2,

saline injected females drank more than males throughout the study. In Experiment 3, L2

females started drinking more than males before puberty during the 10% ethanol phase

and continued until the end of the study.









The Effects of Saline Injections

Dent and colleagues (1999) have reported that saline injections have an effect on the

release of stress hormones. Dent et al. (1999) found evidence of a graded effect by using

an early gene marker, c-Fos. Cells in the paraventricular nucleus of the hypothalamus

(PVN) were found to be active following administration. The number of fos positive cells

were lower in number in saline injected rats, than LPS injected animals but greater than

in non-injected controls.

In our experiments saline injections proved to have an effect on the animals' body

weights. In Experiments 1, 2 and 3, the weights of the saline injected group were lower

than the non-injected animals during the injection and intake phases. Though saline

injections had metabolic effects, in our studies we anticipated there would be a graded

effect on alcohol intake, where the intake of alcohol would be contingent on the degree of

stress administered.

Little et al. (1999) used C57BL/10 mice and screened them in the first three weeks of

life for ethanol preference. Mice were then separated into "high" and "low" preference

groups. The saline injection, a mild stressor, has been implicated in increasing the

preference for alcohol. Mice were injected with saline or not injected at all during 7-9

weeks of age and then tested for free choice ethanol consumption. Animals receiving

saline preferred significantly more ethanol than non-injected animals (Little et al., 1999).

In Experiments 1,2, and 3, saline injected animals showed a preference for ethanol as

compared to water during the periods of 5% exposure before and after puberty.

Therefore, there may be a possibility that the early life saline injections served as a mild

stressor. However, intake of the saline injected group was not always higher than the









non-injected group. Our results along with Little et al. may suggest that the saline

injections affect alcohol preference but not necessarily intake.

Conclusion

This is one of the first studies to look at a long term effect of LPS on drinking

behavior. LPS proved to be a reliable stressor in regards to depressed body weight; it

produced the predicted decrease in body weight. However, it is doubtful if the

administration phases were close enough to the ethanol phases to exert a reliable

influence. LPS administered during the stress hyporesponsive period was less effective

in increasing intake throughout the ethanol phases. This may be due to the lack of

responsivity of the stress axis. LPS administered in after weaning was more effective in

producing long lasting effects on the system. Whether this was mediated though immune

system or endocrine system cannot be concluded from our experiments.

Puberty normally sees a surge in sexual hormones that affect growth and reproduction

as well as the ingestion capacity and metabolism of alcohol. Alcohol ingestion has been

founded to increase serum ACTH and cortisol levels of human adolescents, similarly to

the response seen after encountering stressful stimuli (Frias et al., 2000). In our

experiments, puberty may have played a larger role in alcohol intake than the early life

LPS injections.

Though early life stress has been shown in various protocols to induce behavioral

changes in adulthood, the stressor used in this model was not successful. Utilizing the

idea of alcohol use being a common coping response in adolescence one could say that

the inconsistent intake of the LPS treated groups in the experiments occurred simply

because the system was no longer being challenged. Due to the lack of an immediate

immune challenge there was no longer a stressful reason to drink.









An attenuation of a fever response occurred in a different protocol, where LPS was

administered after two weeks of alcohol exposure (Taylor et al., 2002). It is possible that

the exposure to alcohol for our adult mice negated any remaining neuroendocrine effects

of early LPS administration. Based on body weight data, the lasting effect of LPS can be

assumed. However, the length of the alcohol phases themselves could have worked

against bringing out any effects of LPS in relation to alcohol intake.

For future directions, we plan on looking at the short term effects of LPS injections on

alcohol intake minutes to hours following the stressor. This would better elucidate the

time course of the interaction between an immune challenge and alcohol intake or

preference. We plan on also testing whether the mice were drinking alcohol to make up

for some caloric deficit by offering a three bottle choice, where a nutritive liquid would

be added. Experiments 1,2 and 3 did demonstrate that LPS has enduring effects on body

weight. Additional investigation needs to be done to determine the relation between early

LPS administration and adult alcohol intake.















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BIOGRAPHICAL SKETCH

Cheryl Hope Vaughan was born in Clarendon, Jamaica, in 1980. She moved to

Miami, FL, at age 8. She completed high school at Chaminade-Madonna College

Preparatory in Hollywood, FL. Cheryl received her B.A. in psychology from St. Thomas

University in May of 2000. She began graduate school at the University of Florida in

September of 2000. She plans to stay after her M.S. in psychology to pursue a docotoral

degree in psychology in the behavioral neuroscience area.