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The antidepressant drug tianeptine blocks working memory errors

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The antidepressant drug tianeptine blocks working memory errors pharmacological and endocrine manipulations of stress-induced amnesia in rats
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Campbell, Adam Marc
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University of South Florida
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Adrenalectomy
Hippocampus
NMDA
Radial-arm water maze
Corticosterone
Dissertations, Academic -- Psychology -- Doctoral -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: Stress has been shown to influence learning and memory in humans and rats (Diamond et al, 1996; Diamond et al, 1999; Krugers et al, 1997; Kirschbaum et al, 1996; Lupien et al, 1997). The hippocampus and is an area of the brain involved in memory function in humans and rats (Kirschbaum et al, 1996; Lupien et al, 1997) and is highly susceptible to stress (Diamond et al, 1990). Research has indicated that a number of stressors such as exposure to a predator (Diamond et al, 1999) can lead to stress effects. Recently efforts have been made to counteract the effects of stress on brain function and related behavioral performance. The antidepressant drug tianeptine has been used in this setting.Little is known about tianeptine's role in blocking stress effects on behavior and memory performance with regard to interactions with stress hormones, such as corticosterone. Here a set of experiments delineates the role of corticosterone and its link to stress effects on memory as well as an investigation into the actions of tianeptine and ADX in the blockade of stress effects on memory. First, I examined the effects of tianeptine on multi-day RAWM working memory training and a novel one-day learning and memory training task. Second, the effects of propranolol, an anti-anxiety medication, were tested with regard to the alleviation of stress effects on memory, allowing for a comparison between two anti-anxiety drugs, tianeptine and propranolol. Third, adrenalectomy (ADX) and the resultant depletion of adrenal hormones were examined in connection with learning and memory in the one-day learning task. Fourth, the effects and interactions of tianeptine and ADX were examined to see if tianeptine can exert its effects in the absence of adrenal hormones. Tianeptine blocked stress-induced memory errors in two different tasks and under ADX conditions. All effects were independent of corticosterone levels. In contrast, propranolol was ineffective in blocking stress-induced memory changes. The current data may prove useful in the development of antidepressant drugs and further the study of the mechanisms by which stress affects memory.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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by Adam Marc Campbell.
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Includes vita.
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Title from PDF of title page.
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Document formatted into pages; contains 90 pages.

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ABSTRACT: Stress has been shown to influence learning and memory in humans and rats (Diamond et al, 1996; Diamond et al, 1999; Krugers et al, 1997; Kirschbaum et al, 1996; Lupien et al, 1997). The hippocampus and is an area of the brain involved in memory function in humans and rats (Kirschbaum et al, 1996; Lupien et al, 1997) and is highly susceptible to stress (Diamond et al, 1990). Research has indicated that a number of stressors such as exposure to a predator (Diamond et al, 1999) can lead to stress effects. Recently efforts have been made to counteract the effects of stress on brain function and related behavioral performance. The antidepressant drug tianeptine has been used in this setting.Little is known about tianeptine's role in blocking stress effects on behavior and memory performance with regard to interactions with stress hormones, such as corticosterone. Here a set of experiments delineates the role of corticosterone and its link to stress effects on memory as well as an investigation into the actions of tianeptine and ADX in the blockade of stress effects on memory. First, I examined the effects of tianeptine on multi-day RAWM working memory training and a novel one-day learning and memory training task. Second, the effects of propranolol, an anti-anxiety medication, were tested with regard to the alleviation of stress effects on memory, allowing for a comparison between two anti-anxiety drugs, tianeptine and propranolol. Third, adrenalectomy (ADX) and the resultant depletion of adrenal hormones were examined in connection with learning and memory in the one-day learning task. Fourth, the effects and interactions of tianeptine and ADX were examined to see if tianeptine can exert its effects in the absence of adrenal hormones. Tianeptine blocked stress-induced memory errors in two different tasks and under ADX conditions. All effects were independent of corticosterone levels. In contrast, propranolol was ineffective in blocking stress-induced memory changes. The current data may prove useful in the development of antidepressant drugs and further the study of the mechanisms by which stress affects memory.
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The Antidepressant Drug Tianeptine Blocks Working Memory Errors: Pharmacological and Endocrine Manipulations of Stress-Induced Amnesia in Rats by Adam Marc Campbell A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Psychology College of Arts and Sciences University of South Florida Major Professor: David M. Diamond, Ph.D. Paula Bickford, Ph.D. Cynthia Cimino, Ph.D. Cheryl Kirstein, PhD. Toru Shimizu, Ph.D. Date of Approval: March 23, 2004 Keywords: adrenalectomy, hippocampus, NMDA, radial-arm water maze, corticosterone Copyright 2004, Adam Marc Campbell

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Dedication My dissertation is dedicated to the me mory of my grandmother Lillie Campbell and my grandfather Lloyd Van Winkle.

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Acknowledgments I would first like to thank my major professor Dr. David M. Diamond, Ph.D. for his guidance and expertise. I would also lik e to thank the members of my dissertation committee, Dr. Bickford, Dr. Cimino, Dr. Kirs tein, Dr. Shimizu, and Dr. Pierce for taking time out of their busy schedules to assist my project. I would like to thank all of the people I have worked with while in Dr. Diamonds lab, especially Dr. Collin Park. Collin, your help has been extremely valuable, and I thank you again. I would also like to thank Dr. Carmen Munoz at I.R.I.S, Courbe voie, France, for the opportunity to examine the behavioral psychopharmacology of tianeptin e. I also thank Dr. Monika Fleshner and her lab in the Department of Integrative Physiology at the Un iversity of Colorado for her help with the corticos terone sampling and analysis. I w ould also like to thank John Soto at the James A. Haley VA Hospital Animal Research Facility for his endless help. My love goes out to the person who has seen me through this journey and has kept me smiling, Dawn. I love you. I would like to give my love and thanks to my family, my dad Ron, my mom Peggy, my sister Sara my grandfather William, my grandmother Elsie, and my uncle Greg, whose unending l ove and support have gotten me where I am today. Everything I achieve is as much theirs as it is mine. And last but definitely not least, I would like to thank Dr. Walter Isaac. My passion for neuroscience is a direct product of his generosity, wisdom and patience.

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i Table of Contents List of Tables iv List of Figures v Abstract vii Chapter One: Introduction 1 Hippocampus, Neuronal Atrophy and Memory 1 Stress, Corticosterone and Synaptic Plasticity 4 Current Stress and Working Memory Research 6 Tianeptine, Serotonin, NMDA and Memory 8 The Beta-Adrenergic System and Propranolol 9 Adrenalectomy and Memory 12 Current Experiments 16 Chapter Two: Experiment One: Tianeptine Blocks Stress-Induced Memory Errors on the Criterion-Based Multi-Day RAWM Working Memory 18 Task Method 18 Rats and Handling 18 Drug 19 Stress 19 Statistical Procedures 20 Results : Within Trials Analysis 20 Results: Retention Trials Analysis 21 Results: Plasma CORT Analysis for RAWM Criterion Testing 23 Chapter Three: Experiment Two: Tianeptine does not block Stress-Induced Memory Errors Wh en Given After the Stressor on the Multi-day RAWM Task 24 Method 24 Rats and Handling 24 Drug 25 Stress 25 Statistical Procedures 26 Results: Within trials Analysis 26 Results: Retention Trial Analysis 27 Chapter Four: Experiment Three: Tianeptine Blocks Stress-Induced Memory Errors on the Novel One-Day Learning (ODL) Task 29 Method 29 Rats and Handling 29 Drug 30

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ii Statistical Procedures 30 Results: Within Trials Analysis 30 Results: Retention Trials Analysis 32 Results: Plasma CORT Analysis for One-Da y Learning Paradigm 33 Chapter Five: Experiment Four : Propranolol Does Not Block Stress-Induced Memory Erro rs on the One-Day Learning Task 35 Method 35 Rats and Handling 35 Drug 36 Statistical Procedures 36 Results: Within Trials Analysis: 5 mg/kg Propranolol Dose 37 Results: Retention Trial Analysis: 5 mg/ kg Propranolol Dose 38 Results: Within Trials Analysis: 10 mg/ kg Propranolol Dose 39 Results: Retention Trial Analysis: 10 mg /kg Propranolol Dose 41 Independent T-Test Analysis Between 5 and 10 mg/kg Propranolol Doses 41 Results: Plasma CORT Analysis for 5 mg/ kg Propranolol Dose 41 Results: Plasma CORT Analysis for 10 mg /kg Propranolol Dose 41 Results: Independent T-Test Between Stress Groups 42 Chapter Six: Experiment Five: Adrenalectomy, Stress and Memory: Effects of Adrenal Steroids on Stress-Induced Memory Changes 44 Method 44 Rats and Handling Procedure 44 Adrenalectomy (ADX) Procedur es 44 Corticosterone and Sodium Replacement Procedures 45 Behavioral Testing Regime 46 Blood Sampling and Preparation 46 Statistical Procedures 47 Results: Within Blocks Analysis 47 Results: Retention Trial Analysis 48 Results: Blood/CORT Analysis for ADX Experiments 50 Chapter Seven: Discussion Tianeptine Blocks Stress-Induced Memory Errors 52 Serotonergic Mechanism of Tianeptine Action 52 NMDA Receptor Mediated Mechanism of Tianeptine Action 53 Testing the Anti-Anxiety Properties of Tianeptine 54 Stress and Major Depressive Disorder (MDD): Pre-Clinical Applications of Tianeptine 56 Involvement of Corticosterone in the Formation and Blockade Of Stress Effects 57 The Role of Adrenal Hormones on Stress and Memory 60 Summary and Conclusions 61

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iii References 66 About The Author end page

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iv List of Tables Table One: Means and Standard Errors of the Mean for all Groups by Blocks of Two Trials: Adrenalectomy, Tianeptine and One-Day Learning Task. 47

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v List of Figures Figure One : Acquisition Curve and Retention Trial Performance for All Groups by Trial: Tianeptine and Crite rion-Based Task. 21 Figure Two : Mean Errors per Group on the Retention Trial: Tianeptine and Criterion-Based Task. 22 Figure Three : Corticosterone Levels pe r Group: Tianeptine and Criterion-Based Task. 23 Figure Four: Acquisition Curves and Retention Performance for All Groups by Trials: Tianeptine Give n After Stress. 27 Figure Five : Mean Errors by Group for Retention Trial When Tianeptine is Given after Cat Exposure: Tianep tine Given After Stress. 28 Figure Six : Acquisition Curves and Retention Performance for all Groups by Blocks of Trials: tianepti ne and One-Day Learning Task. 32 Figure Seven : Mean Errors per Group on Retention Trial: Tianeptine and One-Day Learning Task. 33 Figure Eight: Mean Plasma CORT Levels by Group for One-Day Learning Paradigm: Tianeptine and One-Day Learning Task. 34 Figure Nine : Acquisition Curves and Rete ntion Performance for all Groups by Blocks of Trials: 5 mg/kg Dose of Propranolol and One-Day Learning Task. 37 Figure Ten : Errors on Retention Trial for all Groups: 5 mg/kg Dose of Propranolol and One-Day Learning Task. 38 Figure Eleven : Acquisition Curves and Retention Performance for all Groups by Blocks of Trials: 10 mg/kg Dose of Propranolol and One-Day L earning. 39 Figure Twelve : Retention Errors for all Groups: 10 mg/kg Dose of Propranolol and One-Day Learning Task. 40 Figure Thirteen : Mean Plasma CORT Levels for All Groups: 5 mg/kg and 10 mg/kg Dose of Propranolol and One-Day Learni ng Task. 42 Figure Fourteen : Errors on Retention Trial for all Groups: Adrenalectomy, Tianeptine and One-Day Learning Task. 49

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vi Figure Fifteen : Mean Plasma CORT Levels for All Groups: Adrenalectomy, Tianeptine and One-Day Learning Task. 51

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vii The Antidepressant Drug Tianeptine Blocks Working Memory Errors: Pharmacological and Endocrine Manipulations of Stress-Induced Amnesia in Rats Adam Marc Campbell ABSTRACT Stress has been shown to influence learning and memory in humans and rats (Diamond et al, 1996; Diamond et al, 1999; Krugers et al, 1997; Kirschbaum et al, 1996; Lupien et al, 1997). The hippocampus a nd is an area of the brain involved in memory function in humans and rats (Kirschbaum et al, 1996; Lupien et al, 1997) and is highly susceptible to stress (Diamond et al, 1990). Research has indicated that a number of stressors such as exposure to a predat or (Diamond et al, 1999) can lead to stress effects. Recently efforts have been made to counteract the effects of stress on brain function and related behavioral performance. The antidepressant dr ug tianeptine has been used in this setting. Little is known about ti aneptines role in bloc king stress effects on behavior and memory performan ce with regard to interactions with stress hormones, such as corticosterone. Here a set of experiments delineates the role of corticosterone and its link to stress effects on memory as well as an investigation into the actions of tianeptine and ADX in the blockade of stress effects on memory. First, I examined the effects of tianeptine on multi-day RAWM working memo ry training and a novel one-day learning and memory training task. Second, the effects of propranolol, an anti-anxiety medication, were tested with regard to the alleviati on of stress effects on memory, allowing for a comparison between two anti-anxiety dr ugs, tianeptine and propranolol. Third, adrenalectomy (ADX) and the resultant deple tion of adrenal hormones were examined in connection with learning and memory in the one-day learning task. Fourth, the effects

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viii and interactions of tianeptine and ADX were examined to see if tianeptine can exert its effects in the absence of adrenal hormones. Tianeptine blocked stress-induced memory errors in two different tasks and under ADX c onditions. All effects were independent of corticosterone levels. In contrast, propranol ol was ineffective in blocking stress-induced memory changes. The current data may prove useful in the development of antidepressant drugs and further the study of the mechanisms by which stress affects memory.

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1 Chapter One Introduction Hippocampus, Neuronal Atrophy and Memory Stress has been shown to influence lear ning and memory in humans and rats (Diamond et al, 1996; Diamond et al, 1999; Krugers et al, 1997; Kirschbaum et al, 1996; Lupien et al, 1997). The hippocampu s is an area of the brain that is highly susceptible to stress (Diamond et al, 1990; Diamond et al 1994; Diamond et al 1996; Joels et al, 2001; Kim et al, 1996). The hippocampus is involved with declarative memory in humans (Kirschbaum et al, 1996; Lupien et al, 1997) and spatial and working memory in rats (de Quervain et al, 1998; Diamond et al, 1996). Re search has indicated that a number of stressors including restraint (K uroda et al, 1998; Magarinos et al, 1995), exposure to a novel environment (Diamond et al, 1994) and exposure to a predator such as a cat (Diamond et al, 1999) can lead to behavi oral and physiological stress effects. Chronic stress or chronically elevated levels of stress hormones such as corticosterone in rats or cortisol in humans can lead to neural atrophy and cell death in the hippocampus (Luine et al, 1993; Arbel et al, 1994; Magarinos et al, 1995; Conrad et al, 1999; Uno et al, 1989). Luine et al ( 1993) found that 21 days of corticosterone treatment or 21 days of 6-hour restraint stress per day in rats caused atrophy of apical dendrites of pyramidal neurons in the CA3 region of the hippocampus. Furthermore, the atrophy was restricted to the apical dendrites of the CA3c pyramidal cell population in the hippocampus. No atrophy was present in other hippocampal sub-regions such as dentate gyrus, the CA1 and CA2 pyramidal cells or the basal dendri tic tree of the CA3 pyramidal cell population. Although the atrophy was restricted to a specific sub-region of

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2 hippocampal cells, Luine et al (1995) showed that chronic stress or corticosterone treatment and the subsequent dendritic atrophy was associated with impairment of initial learning of a hippocampal-dependent radi al arm spatial learning task. Work by Magarinos et al (1995) again s howed that 21 days of stress or corticosterone application resulted in apical dendritic atrophy of CA3c pyramidal cells in the hippocampus. Magarinos et al (1995) reported that rats treated with th e steroid synthesis blocker cyanoketone showed an impaired secretion of corticosterone in response to stress while maintaining basal corticosterone levels. Al so, cyanoketone treated rats exhibited no apical dendritic atrophy suggesting that the atrop hy was, in part, caused by the actions of stress levels of corticoste rone on the CA3c hippocampal excitatory amino acid (EAA) receptor sites. Two EAA receptors found pl entifully in the hippocampus are the Nmethyl-D-aspartate (NMDA) receptor and the AMPA receptor. Magarinos et al (1995) also showed that application of CGP 43487, a competitive NMDA receptor antagonist, blocked stress-induced dendritic atrophy, wh ile the AMPA receptor antagonist NBQX did not block the atrophy. This suggests that the occurrence of dendritic atrophy is specific to an NMDA receptor mechanism in th e CA3c region. Similar research with tree shrews has suggested that a chronic psychos ocial subordination stre ssor, as opposed to a restraint stressor, cause s CA3 dendrites to atrophy (Magar inos et al, 1996). Subsequent work indicates that dendritic atrophy induced by stress or corticosterone administration could be prevented by phenytoin (Dilantin) an anti-epileptic drug (Watanabe et al, 1992c). The fact that phenytoin is an anti-epileptic drug and, in effect, reduces the release of the excitatory amino acid (EAA) glutam ate, suggests that the mechanism of the dendritic atrophy is based upon the release of excitatory amino acids, and the EAAs

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3 actions on the corresponding receptor. In eff ect, the receptor, due to the increase in glutamate release becomes overexcited, a condi tion that can lead to neuronal atrophy or cell death. As stated above, the presence of large numbers of NMDA receptors makes the hippocampus extremely vulnerable to the effect s of EAAs such as glutamate when they are released in increased quantities. In fact, in concert with the glutamate action theory is the idea that glutamate is released in the hippocampus during stress (Joels and DeKloet, 1993; Reineld et al, 1984), thus linking the stress release of glutamate to the EAA mechanism of dendritic atrophy. Other resear ch has found that the antidepressant drug tianeptine blocks the effect s of stress on hippocampal cell morphology (Watanabe et al, 1992b). Watanabe et al (1992b) found that 21 days of restraint stress or corticosterone treatment led to dendritic atr ophy in the CA3c region of the hippocampus. Watanabe et al (1992b) also reported that daily treatment with the drug tianeptine (15 mg/kg) given concurrently with the chroni c stressor prevented the apical dendrite atrophy. Give the evidence that tianeptine has effects on th e serotonin system and the NMDA receptor system, Watanabe et al (1992b) and others (Kole et al, 2002) suggests that hippocampal atrophy processes may be influenced by se rotonin as well as corticosterone or glutamatergic processes. Other recent rese arch (Conrad et al, 1999) also found that tianeptine treatment (10 mg/kg) given duri ng the three-week chronic restraint stress regimen prevented CA3 atrophy. Conrad et al (1999) also found that 10 days after the cessation of the stressor the dendritic atrophy re versed to pre-stress le vels, indicating that the chronic stressor did not produce permanen t cell damage. Conrad et al (1999) also reported that there was a pres ervation of hippocampal-depend ent behaviors (freezing to

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4 context) in both tianeptine injected and non-in jected rats, suggesting that chronic restraint stress does not effect tasks specific to fear conditioning. Stress, Corticosterone, and Synaptic Plasticity Stress or corticosterone treatment has b een found to inhibit the induction of longterm potentiation (LTP), a form of syna ptic plasticity found among hippocampal cell groups (Foy et al, 1987; Shors et al, 1989, Kim et al, 2002). L ong-term potentiation is an enhancement in synaptic efficacy following elec trical stimulation of an afferent pathway and is considered a physiological model of memory formation. Foy et al (1987) stressed rats by giving them one tail shock a minute for thirty minutes. After the tail shocks were implemented, the rats hippocampi were removed and the hippocampal sections were tested for the occurrence of long-term pot entiation. Foy et al (1987) found that the stressful event given in vivo blocked the occurrence of long-term potentiation in vitro. Similar research by Shors et al (1989) al so found that inescapable shock blocked hippocampal LTP. A group of rats was trained to escape low-intensity shock in a shuttlebox test, while another group of yoked controls could not escape but was exposed to the same amount and regime of shock. After 1 week of training, long-term potentiation (LTP) was measured in vitro in hippocampal slices. Expos ure to uncontrollable shock massively impaired LTP relative to exposure to the same amount of controllable shock. Extending the idea of stress blocking LTP Diamond et al (1992) found that the magnitude of LTP was related to the stress hormone corticosterone in an inverted-U manner. That is, at low and high stress leve ls of corticosterone, LTP was reduced, and there was induction of LTP at an optimal mode rate level of corticosterone. Diamond et al (1994) also found that when rats were expos ed to an unfamiliar environment, primed-

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5 burst potentiation (PBP), a physiologically rele vant form of LTP, was inhibited in the hippocampus. In a more recent study (Mesches et al, 1999), PBP was blocked in hippocampal slices obtained from rats expos ed to a cat. The study performed by Mesches et al (1999) evaluated the eff ects of acute psychological st ress (cat exposure) in adult male rats on synaptic plasticity assessed in vitro in hippocampal slices. Two physiological models of memory were studied in CA1 in each recording session: first, primed burst potentiation (PBP), a low-thresh old form of plasticity produced by a total of five physiologically patterned pulses; and s econd, long-term potentiation (LTP), a suprathreshold form of plasticity produced by a trai n of 100 pulses. Three groups of rats were studied: (1) undisturbed rats in their home cage (home cage); (2 ) rats placed in a chamber for 75 min (chamber); and (3) rats placed in a chamber for 75 min in close proximity to a cat (chamber/stress). At the end of the chamber exposure period, blood samples were obtained, and the hippocampus was prepared for in vitro recordings. Only the chamber/stress group had elevated stress levels of corticosterone. The major finding was that PBP, but not LTP, was blocked in the ch amber/stress group. Thus, the psychological stress experienced by the rats in response to cat exposure resulted in an inhibition of hippocampal plasticity. It may be deduced that since stress or high levels of corticosterone inhibit hippocampal synaptic plasticity that stress may also interfere with performance on behavioral tasks dependent on the hippocampus. In fact, stress does exert effects on behavioral measures of learning and memory, especially those testing hippocampaldependent memory types such as spatial and working memory (de Quervain, et al, 1998; Diamond et al, 1996; Diamond et al, 1998; Di amond et al, 1999; Krugers et al, 1997;

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6 Luine et al, 1994). Diamond et al (1996) showed that rats placed in a novel environment showed impairments on working memory function but not reference memory function. Reference memory function is hippocampal-i ndependent and can be defined as the longterm memory of events that do not change fr om day to day, or trial to trial. The Diamond et al (1996) work suggests that stress can inhibit wo rking memory (hippocampaldependent) memory function while leaving reference memory (hippocampalindependent) memory intact. Furthermore, this finding suggests that stress impairs hippocampal functioning, as measured by be havioral testing while also impairing physiological measures of memory function, such as PBP and LTP. Current Stress and Working Memory Research More recent behavioral data also support the idea that stress impairs hippocampaltype memory. Diamond et al (1999) showed that rats exposed to a cat exhibited errors on a radial arm water maze (RAWM) task. Rats were trained to locate a hidden submerged platform in one of six arms in the RAWM. The rats were given 4 trials per day to learn where the submerged platform was located. Th e platform was in the same arm for every trial on that day while the platform loca tion was different across days. Changing the platform location required the rat to learn a new location each day constituting the use of working memory. After the fourth trial, rats were stressed 30 minutes with the cat after learning the platform location for a particular day. After the 30 minutes, the rats were given a retention trial to evalua te if they had remembered the platform location. Rats that were stressed committed significantly more errors in the RAWM. That is, they entered arms not containing the platform more often than rats that were not stressed. As the

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7 RAWM is a working memory task, it is implied that the stress interfered with hippocampal functioning. More recently, work from Diamonds group has suggested that rats that have artificially elevated levels of corticos terone (CORT) are not impaired on working memory tasks (Park, et al, 2001). Rats were trained on a spatial working memory task (the radial arm water maze) and then were given a retention trial 30 minutes later. The object was to find the arm containing the hidde n platform on that day in the retention trial. As before, rats that were not exposed to the cat had good memory and rats that were exposed to the cat exhibited memory impairment, and elevated levels of endogenous CORT. However, rats that were injected with stress levels of CORT but were not placed with the cat, did not show memory impairment Since these rats were not stressed, this would suggest that elevated levels of CORT alone, are not suffici ent to cause spatial working memory errors. In related work, Woods on et al (2003) found that rats that were stressed with a cat and those that were given access to an estrous female both exhibited high levels of CORT. But whereas the cat exposed rats showed an increase in errors the rats given access to the female did not show an increase in error rate. The findings of Woodson et al (2003) are consis tent with the Park et al (2001) findings because certain groups of rats in both studies exhibited high levels of CORT while not showing spatial memory impairments. These data again suggest that high levels of CORT alone are not sufficient to produce memory impairment. F ear provoking stimuli such as the cat, which are known to activate the amygdala, may inte ract with corticosterone to impair hippocampal-dependent memory. Based on these findings, the theory that stress effects

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8 on learning and memory are dependent on hi gh corticosterone levels needs to be examined further. Tianeptine, Serotonin, NMDA and Memory In recent years efforts have been made to counteract the effe cts of stress on brain function and related behavioral performan ce. A drug known as tianeptine 7-[(chloro6,11-dihydro-5,5-dioxo-6-methyldibenzo[c,f][1,2] thiazepin-11-y1 amino] heptanoic acid, sodium salt, has been developed and used in this setting. Origin ally developed as an antidepressant medication (Lab rid, 1992), tianeptine has be en shown to have varied effects regarding the physiology, neurochemist ry and behavioral measures associated with learning and memory. Neurochemica lly, tianeptine has been shown to reduce extracellular serotonin (5-HT) levels by increa sing 5-HT reuptake in the rat brain and in rat and human platelets ex vivo (Mennini et al, 1987, Mocaer et al, 1988, de Simoni et al, 1992). Extensive research has since focused on the connection between the decrease in extracellular 5-HT and tianeptines ability to block effects of stress on learning and memory (Conrad et al, 1996; Luine et al, 1994). The theory that tian eptine has its effects via a decrease in 5-HT comes from the suggest ion that stress increases 5-HT levels in the brain (Matsuo et al, 1996; Yosh ioka et al, 1995), and that 5HT has been associated with blockage of primed-burst potentiation (Corradetti et al, 1992). Thus a threefold hypothesis linking stress, 5-HT, and memory can be formed. That is, stress increases 5HT levels, 5-HT has been suggested to impair learning and memory functioning, and tianeptine, which lowers extracellular 5-HT le vels, has been shown to block stress effects on memory and LTP (Shakesby et al, 2002).

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9 Although a large amount of research has been conducted linking memory impairment to serotonin and alleviation of stress effects on memory by tianeptine, tianeptine has recently been shown to exhibi t effects on the NMDA re ceptor (Kole et al, 2002). NMDA receptors became more excitatory when tianeptine was applied in vitro Tianeptine increased excitato ry post-synaptic currents (EPSC s) in the hippocampal slices, indicating that tianeptine app lication is effective in making the NMDA receptor site more efficacious. The Kole et al (2002) study suggests that be sides having 5-HT effects, tianeptine may also have effects linked dire ctly to NMDA activation and enhancement. The NMDA receptor plays an important ro le in the generation of LTP, and NMDA antagonism has been shown to cause disturba nces in learning and memory performance (Castellano et al, 2001; Kawabe et al, 1998). This is an important note considering recent research (Shakesby et al, 2002) found that tianeptine blocked the stress induced inhibition of LTP. Thus, recent data theorizes that th e NMDA receptor may play an important role in tianeptines ability to bl ock stress effects on synaptic pl asticity and memory function. The Beta-Adrenergic System and Propranolol In contrast to the NMDA receptor role in memory function, a second mechanism is involved in the emotional modulation of learning and memory: the beta-adrenergic system. Enhanced memory associated with arousing emotional experiences involves activation of the beta-adrenergic system (Cahill et al, 1995; Cahill and McGaugh, 1996a, 1996b; McGaugh and Cahill, 1997; McGaugh, 2000). A large amount of research has suggested that the amygdala, and more specif ically the basolateral amygdala (BLA), is central to the beta-adrenergic memory system (Hamann et al, 1999; Canli et al, 2000). The noradrenergic system, especially within the amygdala, is a central mechanism in the

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10 modulation of memory consolidation. Curre nt research indicate s that the adrenal hormone epinephrine and the stress hormone co rticosterone act in interactive ways to affect memory consolid ation (McGaugh et al, 2000; Roozendaal, 2000). Epinephrine is also involved in the modulation of memory processes (Gold et al, 2001; Gamaro et al, 1997). Gold et al (1975) first found that systemic injections of epinephrine after inhibitory avoidance traini ng enhanced long-term re tention of the task. Subsequent studies have shown that epine phrine activates beta-a drenoceptors on vagal afferents terminating in the nucleus of the solitary tract (NTS). Memory enhancement is also achieved by electrical stim ulation of the ascending vagus ne rve, similar to that seen with epinephrine injection (Clark et al, 1998). The NTS innervates the amygdala via noradrenergic projections. The amygdala innerv ates several forebrain structures including the hippocampus via the locus coeruleus (van Bockstaele et al, 1998). Inhibition of the NTS with lidocaine blocked the memory enhancing effects of epinephrine (Williams et al, 1993), while the beta-adren ergic agonist clenbuterol infused into the NTS induced memory enhancement (Williams et al, 2000). The NTS-amygdala-hippocampal pathway is thus seen as fundamental to the enhancement of memo ry consolidation. Glucocorticoids (corticoster one in rats, cortisol in humans) also enhance longterm memory consolidation, similar to that seen with epinephr ine (Roozendaal, 2000; deKloet et al, 1999). Blockade of corticostero ne synthesis with the synthesis inhibitor metyrapone prevents enhancement of retention on the inhibitory avoidance (IA) task by epinephrine. The idea that metyrapone can bl ock epinephrines ability to enhancement retention on the IA task indicat es that there is an adrenergic-glucocorticoid interaction influencing memory consolidation.

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11 Previous research indicating epinephrine and glucocorticoids enhance memory consolidation used the inhibitory avoidance task (Lia ng et al, 1986, Roozendaal, 2000). Even though the IA task is stressful, an enhancing effect was seen upon hormone administration. The memory being enhanced wa s that of the stressf ul experience itself, which is the context in which the shock that was to be avoided was given. Subsequent epinephrine or glucocorticoid administration sought to strengthen the memory of the stressful experience. Further research has i ndicated that the beta-adrenergic antagonist propranolol impairs the enhancement of me mory for emotional experiences in rats (Roozendaal et al, 1999). Memo ry consolidation of emotiona l experiences has also been shown to be impaired by propranolol in hum ans (Cahill et al, 1994; van Stegeren et al, 1998). That is, the memory of the stressful experience was not as strong after administration of the antagonist. In the presen t experiments predator (cat) exposure is the source of stress. In theory, a pplication of epinephrine or gl ucocorticoids such as CORT would enhance the memory of the cat and inhi bit the memory of events peripheral to the stressful experience; in the case of the curr ent experiments the peripheral memory event would be the memory of the platform location (see Methods below). Based on the findings of previous research (Roozendaal et al, 1999), the beta-blocker propranolol can be given in an effort to reduce or eliminat e the enhancement of the memory of the cat exposure. The reduction of the memory for the cat would be beneficial for the memory of the platform location. Thus, propranolol woul d be expected to preserve the platform memory by inhibiting the memory of the cat.

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12 Adrenalectomy and Memory When discussing the idea of stress it is important to note that stress has two separate components: the physical and the psychological. The curren t set of experiments has focused on both of these components. The e xposure of the rat to the cat predator is both a psychological and physical stressor. The physical component of a stressful event was further investigated in the current experi ment by examining the levels of the stress hormone corticosterone in the blood str eam. The physical component of the stress reaction focuses, in part, on the activation of the endocrine syst em. Upon the introduction of a stressor the hypothalamic-p ituitary-adrenal (HPA) axis is activated. A neurochemical cascade occurs within the HPA-axis eventually leading to the rel ease of epinephrine, norepinephrine and corticosterone into th e bloodstream by the adrenal glands. The chemical cascade begins with the release of corticotropin-releasing hormone (CRH) by the hypothalamus. The release of CRH then causes the pituitary gland to release adrenocorticotrophic hormone (ACTH), which in turn triggers the adrenal glands to release corticosterone. The adrenal gland is considered the major gland in reacting to stress. The adrenal gland is made up of two intera cting bodies; the adrenal medulla (central adrenal) and the adrenal cortex (outer adrenal). The adrena l medulla is controlled by the sympathetic division of the autonomic nervous system which is the division activated under conditions of sudden stress. Motor axons fr om the autonomic nervous system synapse upon specialized cells in the adrenal medulla cal led chromaffin cells. It is the chromaffin cells that are responsible for the release of epinephrine and nore pinephrine into the bloodstream shortly after the stre ssful event has occurred. It is the release of epinephrine

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13 and norepinephrine that leads to physical manifestations of stress like increased heart rate, increased blood pressure and metabolic changes to name a few. While the adrenal medulla is integral to the release of epine phrine and norepinephrine, the endocrine gland cells of the adrenal cortex are responsi ble for the release of the stress hormone corticosterone and the regulatory horm one aldosterone by the adrenal glands. Aldosterone is important for the regulation of sodium, potassium and chloride within the body. The increase of the adrenal neurochemicals and hormones under conditions of acute stress are highly adaptive and return to basal levels shortly after the event. For example, corticosterone levels in the blood ha ve been shown to peak 20-30 minutes poststress. Chronic release of these substances under conditions of chronic stress becomes maladaptive and injurious. The current experi ments incorporated acute stress situations (exposure to the cat for thirty minutes) in all instances. The removal of the adrenal glands elim inates the production of corticosterone, epinephrine, norepinephrine and aldosterone. A large body of research has focused on the effects of adrenalectomy (ADX), that is, the removal of the adrenal glands on behavior and the brain. One line of research has fo cused on ADX and its effects on hippocampal morphology. Granule cells within the dentate gyrus of the hippocampus have a high rate of turnover and new cells are continuous ly formed (Gage et al, 1998; Gould and Cameron, 1996; Gould and McEwen, 1993). Previous research found that adrenalectomized rats exhibited selective loss of granule cells in the dentate gyrus of the hippocampus (Sloviter et al, 1989; Conrad et al, 1993), whic h suggests that ADX accelerates both the neurogenesis and apoptos is of granule cells (Cameron and Gould, 1994; Gould et al, 1990; Hornsby et al, 1996; Hu et al, 1997; Jaarsma et al, 1992;

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14 Sapolsky et al, 1991). In additi on, the replenishing of granule cells occurs with a longer delay than under non-ADX conditions (Cameron and Gould, 1996). The accelerated granule cell turnover caused by ADX is preven ted by corticosterone replacement at low doses (Reul and DeKloet, 1985). Degeneration of granule cells is evid ent as early as two to three days after ADX surgery (Gould et al, 1990; Jaarsma et al, 1992; Hu et al, 1997). Adrenal steroids such as corticosterone cont rol the rate of cell tu rnover in the dentate gyrus and may also control behavioral meas ures associated with hippocampal activity such as spatial memory tasks. Vaher et al (1994) showed that rats that had been adrenalectomized exhibited neuronal degenera tion and cell loss in the dentate gyrus as well as deficits in memory performance on a spatially oriented eight-arm radial maze. Vaher et al (1994) found that corticosterone le vels were lower in ADX rats than in sham control animals, and that corticosterone levels were negativel y correlated with maze performance. That is, the rats with lower corticosterone levels (the ADX rats) made a greater number of errors on the memory test. Th e idea that lower corticosterone levels are associated with a deficit in memory perfor mance suggests that adrenal hormones play an important role in the physical maintenance of hippocampal cells, most notably dentate granule cells, and also an important role in the performance of hippocampal-dependent spatial memory tasks. Adrenalectomy has also been found to ha ve detrimental effects on measures of electrophysiology within the hippocampus (Stienstra et al, 1997; Stienstra et al, 2000; Joels et al, 2001). Stienstra et al (1997) found that thr ee days after adrenalectomy orthodromic field responses, an electrophysiologi cal measure of synaptic plasticity in the dentate gyrus were reduced in amplitude in vitro While the adrenalectomized rats

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15 showed a marked decrease in field respons es, sham-operated controls exhibited normal synaptic plasticity. Stienstra et al (1997) also showed that ad renalectomized rats that were treated with corticosterone during the three days post-ADX di d not exhibit a reduction in cell signal amplitude. Stienstra et als ( 1997) results indicate that measures of hippocampal electrophysiology, in this cas e the dentate gyrus, are disrupted in vitro by adrenalectomy three days prior. Stienstr a and Joels (2000) found that post-synaptic potentials in dentate gyrus cells were bl ocked by ADX, and that treatment with corticosterone in vitro increased the EPSP slopes 2.5-3 hours after treatment in ADX rats. These findings indicate that de layed corticosterone effects in vitro are sufficient to normalize synaptic transmission in the dentate gyrus of ADX rats, even in the presence of apoptotic cells three days after ADX. Thus, th e impairment of synaptic transmission after ADX may not be due to cell loss but rather due to a reduction of adre nal steroids and its actions on the NMDA receptor. The aforementioned research has indicated that corticosterone treatment prevents cell loss in the hippocampus due to ADX (Reul and DeKloet, 1985) and that corticosterone treatment in vitro reverses the deleterious effects of ADX on synaptic transmission (Stienstra and Joels, 2000). In the current e xperiment rats received a low dose of corticosterone replacement after ADX, thus preventing rapid cell loss within the hippocampus. The corticosterone manipulation after ADX allows for the study of the effects of the elimination of adrenal steroids on memory while keeping the hippocampus intact. Thus, the current experiment studied the effect of ADX, per se, on memory performance rather than ADX-induced hippoc ampal damage. While previous research has shown that ADX leads to spatial memory impairment, the current study hypothesized

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16 that spatial memory will be unaffected by ADX due to our use of corticosterone replacement. Also, based on pilot data which showed that tianeptine was effective in blocking a stress effect in the presence of stress levels of corticosterone, we hypothesized that stress would still cause impa irment in the presence of ADX. Current Experiments Although a large amount of research exists, little is known about tianeptines role in blocking stress effects on behavior and memory performance with regard to interactions with stress hormones, such as co rticosterone. What was needed was a set of experiments to delineate the role of corticos terone and its link to stress effects on memory as well as an investigation into the actions of tianeptine and ADX in the blockade of stress effects on learning and memory. What follows is an examination of the effects of stress on memory in general, and how tianept ine, propranolol and adrenalectomy interact with stress and memory. First, the current se ries examined the effects of tianeptine on standard multi-day RAWM working memory training. Also, tianeptines effects on a novel one-day learning and memory training task were examined. Specifically, the following experiments were conducted to de termine if tianeptine could block stress effects on memory in each of the two types of memory testing, bringing the level of prior experience in the maze for the rats into cons ideration. Second, the effect of propranolol was tested with regard to the alleviation of stress effects on memory, allowing for a direct comparison between tianeptine and propranolol in their memory modulating capacity. It was hypothesized that propranolol would block the enhanced memory of the cat stress experience and reduce the cat stress effects on the peripheral (non-stress) memory of the platform location. Third, adrenalectomy (ADX) and the resultant depletion of adrenal

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17 hormones were examined in connection w ith learning and memory in the one-day learning task, allowing the further study of th e role of corticosterone on learning and memory. Fourth, the effects and interactions of tianeptine and ADX were examined to see if tianeptine can exert its effects in the absence of adrenal hormones.

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18 Chapter Two Experiment One: Tianeptine blocks stres s-induced memory errors on the criterionbased multi-day working memory task Method Rats and Handling Fifty-six male Sprague-Dawley rats ( 175-200 grams) were given a two-week habituation period within the vivarium upon a rrival. Rats were housed two to a cage in standard Plexiglas cages and given food and water ad libitum All rats were handled on Days 10, 12 and 14 of the two-week habitu ation period. During the handling procedure rats were taken in their cages, four cages at a time, to the behavioral testing room and placed on a table along one wall. Rats were then gently handled one at a time for approximately 2 minutes each. On Day 14 of th e habituation period rats were handled in addition to receiving numerical tail markings with a permanent marker. At the end of each handling session rats were taken back to the vivarium. All handling sessions were given in the morning, the same time as subsequent behavioral testing. Rats were run in the radial six-arm water maze (RAWM) using a criterion-based working memory task (as previously desc ribed, Diamond et al, 1999). The RAWM was a black galvanized round tank (168 cm diameter 56 cm height, 43 cm deep) filled with clear water (23 -24 C). The tank was divided into six arms radiating from a central area using 6 V-shaped stainless steel walls (54 cm height, 56 cm length). A plastic platform (12 cm diameter) was hidden from view 1 cm below the surface of the water at the end of one of the arms.

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19 Rats were trained to find the submerged platform placed at the end of one of the six arms. Rats were given four acquisition tria ls per day in which they started in one of the non-goal arms. The platform remained in the same arm within each day, but the platform location was changed between days, giving the rats a new location to learn each day. After the four acquisition trials the rats were given a 30-minute period in the home cage. After the 30 minutes the rats were given a retention probe trial to the platform. All rats were run until they met the criterion of no more than one error on the retention trial over three consecutive training days. Once a rat met the criterion it was placed into one of four groups: Home Cage/Saline (n=15), Home Cage/Tianeptine (n=14), Stress/Saline (n=15), Stress/Tianeptine (n =12). After meeting the cr iterion and placed into a manipulation group, rats were run for two days post-criterion using the same within-day training regimen. During these two post-criter ion days drug manipulations and stress manipulations were performed. After the se cond post-criterion day blood samples were taken within two minutes after the retention trial. Blood samples we re centrifuged and the plasma collected. The plasma samples were th en assayed for levels of corticosterone. Drug Tianeptine (10 mg/kg) or saline-vehicle (0.9 % NaCl in di stilled water) was administered 30 minutes before the first acq uisition trial on the two post-criterion days. All injections were given interperitoneally (IP). Stress During the 30-minute manipulation peri od on the two post-criterion days, rats were either 1) put back into the home cage for 30 minutes, or 2) placed in a small Plexiglas box and transported to a separate room containing a larger sound attenuating

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20 chamber box (750 cm x 570 cm x 580 cm). The rats were placed in the large chamber containing the cat for 20 minutes and then placed into the home cage for 10 minutes. While in the chamber with the cat the rats we re housed in the sma ll Plexiglas boxes with small holes in the top, allowing them to s ee and smell the cat but not allowing the cat access to the rat. After the 30-minute period th e rats were given the retention trial. Statistical Procedures A repeated-measures ANOVA was performed on the mean errors on all groups by trial for the two days post-criterion comb ined. A one-way ANOVA was performed on the means for all groups on the retention trial. A one-way ANOVA was also performed on the means for the corticosterone assays. Results: Within Trials Analysis Means and standard errors of the mean were obtained for all four groups (HC/ Vehicle, HC-TIA, Stress/Vehicle, Stress/TIA) by trial (T1-RT). A within trial test of linearity showed that there was a significan t linear trend for all groups combined by trial (F [1, 51] = 103.737, p < 0.0001), indicating sign ificant acquisition performance across the four learning trials (T 1-T4) (see Figure One). A repeated-measures ANOVA indicated that there was a significant TRIALS effect for all groups combined (F [4, 204] = 43.932, p 0.0001). All other within subject effects and interactions were not signifi cant. The repeated measures ANOVA also found that there was a significant between subjects STRESS main effect among the groups (F [1, 51) = 5.412, p < 0.024). All other between su bject effects and interactions by trial were not significant (see Figure One).

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0123456T1T2T3T4RTTrialMean Errors HC-VEH HC-TIA STR-VEH STR-TIA Figure One: Acquisition Curve and Retention Trial Performance for All Groups by Trial: Tianeptine and Criterion-Based Task. Results: Retention Trial Analysis Mean errors for the two-day post-criterion days combined are as follows: Home Cage/Vehicle = 0.433 0.118, Home Cage/TIA = 0.578 0.198, Stress/Vehicle = 2.133 0.291, Stress/Tianeptine = 0.708 0.224. Again, all variances are calculated as Standard Errors of the Mean (SEM). There was a significant STRESS main effect (F [1, 52] = 22.704, p < 0.0001) with stress animals making significantly more errors than control animals overall. There was also a significant DRUG main effect (F [1, 52] = 11.144, p < 0.002) indicating that Vehicle groups made significantly more errors than the tianeptine groups overall. There was also a STRESS x DRUG interaction effect (F [1, 52] = 16.440, p < .0001) showing 21

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that the Stress/Vehicle group made significantly more errors than the other groups. The Stress/Tianeptine group was significantly different from the Stress/Vehicle group, but not significantly different from the two control groups (see Figure Two). These data indicate that stress significantly impaired spatial learning in the Stress/Vehicle group on the task compared to both home cage groups. Cat exposure led to an increase in errors on the retention trial in the Stress/Vehicle group. The data also indicate that tianeptine was effective in blocking the stress-induced memory deficit. That is, the mean error rate was significantly reduced in the Stress/Tianeptine group compared to the Stress/Vehicle group. Also, tianeptine injection did not increase error rates in the Home Cage/Tianeptine group, eliminating the idea that IP injection alone could increase stress effects on spatial memory. 00.511.522.53HC-SALHC-TIASTR-SALSTR-TIAGroupMean Errors per Group Figure Two: Mean Errors per Group on the Retention Trial: Tianeptine and Criterion-Based Task. 22

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Results: Plasma CORT Analysis for RAWM Criterion Testing The mean corticosterone values (g/dl) obtained from plasma samples are as follows: Home Cage/Vehicle = 11.880 1.351, Home Cage/TIA = 8.773 1.591, Stress/Vehicle = 41.093 3.044, Stress/TIA = 39.623 2.868. There was a significant STRESS main effect for corticosterone levels (F [3, 25] = 47.928, p < .001). (see Figure Three). All other effects were not significant. Data indicate that both stress groups had significantly higher CORT levels than those of the home cage groups independent of drug manipulation. Tianeptine alone did not raise CORT levels in the HC-TIA group nor did tianeptine lower CORT levels significantly in the STR-TIA group. 05101520253035404550HC-SALHC-TIASTR-SALSTR-TIAGroupPlasma CORT levels (ug/dl) Figure Three: Corticosterone Levels per Group: Tianeptine and Criterion-Based Task. 23

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24 Chapter Three Experiment Two: Tianeptine does not bl ock stress-induced me mory errors when given after the stressor on the multi-day RAWM task Method Rats and Handling Thirty-two male Sprague-Dawley Rats, (175-200 grams) were given a two-week habituation period within the vivarium upon a rrival. Rats were housed two to a cage in standard Plexiglas cages and given food and water ad libitum All rats were handled on Days 10, 12 and 14 of the two-week habitu ation period. During the handling procedure rats were taken in their cages, four cages at a time, to the behavioral testing room and placed on a table along one wall. Rats were then gently handled one at a time for approximately 2 minutes each. On Day 14 of th e habituation period rats were handled in addition to receiving numerical tail markings with a permanent marker. At the end of each handling session rats were taken back to the vivarium. All handling sessions were given in the morning, the same time as subsequent behavioral testing. Rats were run in the same radial sixarm water maze (RAWM) as in Experiment One using the same criterion-based working memory task (as previously described, Diamond et al, 1999). The RAWM was a black galvanized round tank (168 cm diameter, 56 cm height, 43 cm deep) filled with clear water (23 -24 C). The tank was divided into six arms radiating from a central area by us ing 6 V-shaped stainless steel walls (54 cm height, 56 cm length) (Figure 1). A plastic platform (12 cm diameter) was hidden from view 1 cm below the surface of the wate r at the end of one of the arms.

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25 Rats were trained to find the hidden plat form placed at the end of one of the six arms. Rats were given four acquisition trials per day in which they started in one of the non-goal arms. The platform remained in the same arm within each day, but the platform location was changed between days, giving th e rats a new location to learn each day. After the four acquisition trials the rats were given a 90-minute period in the home cage. After the 90 minutes the rats were given a rete ntion probe trial to th e platform. All rats were run until they met the cr iterion of no more than one retention error over three consecutive training days. Once a rat met the criterion it was placed into one of four groups: Home Cage/Vehicle (n=6), Home Cage /Tianeptine (n=6), Stress/Vehicle (n=7), Stress/Tianeptine (n=7). After meeting th e criterion and placed into a manipulation group, rats were run for two da ys post-criterion to the platform. During these two postcriterion days drug manipulations and st ress manipulations were performed. Drug Tianeptine (10 mg/kg) or saline vehicl e (0.9 % NaCl in distilled water) was administered immediately after the rats were taken from the cat or 30 minutes after they were placed back into the home cage, depe nding on group. The rats were then placed in the home cage for sixty minutes to simulate th e time course of the drug in Experiment One. After the sixty minutes in the home cage, the rats ran the retention trial. Stress During the 90-minute manipulation peri od on the two post-criterion days, rats were either put back into the home cag e for 90 minutes or placed in a large box containing a cat for 30 minutes and then pl aced into the home cage for 60 minutes. While in the cat box the rats were housed in sma ll Plexiglas boxes, allowing them to see and

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26 smell the cat but not allowing the cat access to the rat. After the 90-minute period the rats were given the retention trial. Statistical Procedures A repeated measures ANOVA was perf ormed to analyze the significant differences among all groups by trial. A one-way ANOVA was also performed on the mean errors on the retention trial for the two days post-criterion. Results: Within Trials Analysis Means and standard errors of the mean (SEM) were calculated for all groups by trial. A test of linearity among the groups by tr ials indicated that th ere was a significant linear trend across the trials (F [1, 22) = 89.306, p < 0.0001) (see Figure Four). The significant linear trend among the groups indica ted that there was a significant acquisition of the task or reduction in errors across the learning trials (T1-T4). The repeated measures ANOVA revealed that there was a significant within-subject TRIALS effect (F [4, 88] = 42.001, p < 0.0001). There was also a significant TRIALS x STRESS interaction (F [4, 88] = 4.462, p < 0.002). The between-subjects analys is indicated that there was a significant STRESS effect among all groups combined across trials (F [1,22] = 5.573, p < 0.028). The DRUG main effect a nd the STRESS x DRUG interaction was non-significant (see Figure Four).

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0123456T1T2T3T4RTTrialMean Errors HC-SAL HC-TIA STR-SAL STR-TIA Figure Four: Acquisition Curves and Retention Performance for All Groups by Trials: Tianeptine Given After Stress. Results: Retention Trial Analysis The mean number of errors on the retention trial by group was as follows: Home Cage/Vehicle = 0.33 0.105; Home Cage/TIA = 0.333 0.105; Stress/Vehicle = 1.786 0.565; Stress/TIA = 2.071 0.456 (see Figure Five). A one-way ANOVA was performed on the retention trial errors. The ANOVA indicated that there was a significant STRESS main effect (F [1, 22] = 18.639, p < .0001) between groups. The ANOVA also showed that there was no significant main effect of DRUG (F [1, 22] = .279, p < .602) and no significant STRESS x DRUG interaction (F [1,22] = .279, P < .602). Stress impaired spatial learning as evidenced by an increase in errors compared to home cage groups. A significant stress effect was found in both the STR-SAL and STR-TIA groups compared to home cage groups even after a 60 minute delay between the 27

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stress exposure and the retention trial. The increase in the delay period, extended to keep the time course of tianeptine consistent among experiments, did not hinder the stress exposures ability to create a stress-induced memory deficit. Finally, data indicate that tianeptine given after the stressor did not significantly reduce the stress-induced memory impairment. 00.511.522.53HC-SALHC-TIASTR-SALSTR-TIA GroupMean Errors per Group Figure Five: Mean Errors by Group for Retention Trial When Tianeptine is Given after Cat Exposure: Tianeptine Given After Stress. 28

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29 Chapter Four Experiment Three: Tianeptine reduces stress-induced memory errors on the novel One-Day Learning (ODL) task Method Rats and Handling Thirty-two male Sprague-Dawley Rats, ( 175-200 grams) were given a two-week habituation period within the vivarium upon a rrival. Rats were housed two to a cage in standard Plexiglas cages and given food and water ad libitum All rats were handled on Days 10, 12 and 14 of the two-week habitu ation period. During the handling procedure rats were taken in their cages, four cages at a time, to the behavioral testing room and placed on a table along one wall. Rats were then gently handled one at a time for approximately 2 minutes each. On Day 14 of th e habituation period rats were handled in addition to receiving numerical tail markings with a permanent marker. At the end of each handling session rats were taken back to the vivarium. All handling sessions were given in the morning, the same time as subs equent behavioral testing. The rats were assigned to the following groups after handling: Home Cage/Vehicle (n=8); Home Cage/Tianeptine (n=8); Stress/Vehicle (n=8); Stress/Tianeptine (n=8). Rats were run in the radial six-arm water maze (RAWM) using a one-day learning task. The RAWM was a black galvanized r ound tank (168 cm diameter, 56 cm height, 43 cm deep) filled with clear water (23 -24 C). The tank was divided into six arms radiating from a central area by using 6 V-shaped st ainless steel walls (54 cm height, 56 cm length). A plastic platform (12 cm diamet er) was hidden from view 1 cm below the surface of the water at the end of one of the arms.

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30 Each rat was run only one day, with each day containing an in itial acquisition of twelve trials in succession. The twelve trial acquisition sequence was then followed by a thirty-minute manipulation period in which the rat was either returned to the home cage or given cat exposure. After the thirty-minut e manipulation period the rats were given a single retention trial. After the retention trial tail blood was taken from each rat and frozen for future analysis of plasma corticosterone levels. Drug Drug and saline vehicle injections were given thirty minutes prior to Trial One. Both drug (Tianeptine, 10 mg/kg) and vehi cle (0.9 % NaCl in distilled water) were administered interperitoneally. Statistical Procedures A repeated measures ANOVA was used to analyze the significance among all groups by blocks of two trials (6 blocks of 2 trials + RT = 13 tota l trials). A one-way ANOVA was performed on the mean errors per group on the retention tr ial (Trial 13). A one-way ANOVA was also performed on th e mean results per group for plasma corticosterone levels. Results: Within Trials Analysis Means and standard errors of the mean (SEM) were calculated for all groups by block of trials. A test of lin earity among the groups revealed that there was a significant linear trend among all groups (F [1, 28] = 58.474, p < 0.0001) (see Figure Six). The test for linearity also showed a significant BL OCKS x STRESS linear trend (F [1, 28] = 10.208, p < 0.003). A significant linear trend wa s also found for the BLOCKS x DRUG interaction (F [1, 28] = 4.839, p < 0.036.

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31 The repeated measures ANOVA revealed that there was a significant BLOCKS effect (F [6,168] = 20.636, p < 0.0001). Th ere was also a BLOCKS x STRESS interaction effect shown by the ANOVA (F [6, 168] = 3.862, p < 0.001). A significant BLOCKS x DRUG effect was also revealed (F [6, 168] = 2.902, P < 0.01). The repeated measures ANOVA also indicated a signifi cant BLOCKS x STRESS x DRUG interaction (F [6, 168] = 2.377, p < 0.031). The between su bjects component of the ANOVA showed a significant STRESS x DRUG interaction (F [1, 28] = 12.501, p < 0.001). The two main effects, STRESS and DRUG were not significant (see Figure Six).

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00.511.522.533.544.55B1B2B3B4B5B6RTBlocks of TrialsMean Errors HC-SAL HC-TIA STR-SAL STR-TIA Figure Six: Acquisition Curves and Retention Performance for all Groups by Blocks of Trials: Tianeptine and One-Day Learning Task. Results: Retention Trial Analysis Means for each group were as follows: Home Cage/Vehicle = 0.25 0.164; Home Cage/Tianeptine = 0.375 0.263; Stress/Vehicle = 4.00 0.707; Stress/Tianeptine = 0.625 0.324 (see Figure Seven). 32

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012345HC-SALHC-TIASTR-SALSTR-TIAGroupMean Errors Figure Seven: Mean Errors per Group on Retention Trial: Tianeptine and One-Day Learning Task. The ANOVA indicated that there was a significant STRESS effect (F [1, 28] = 22.828, p < .0001). The ANOVA also showed that there was a significant DRUG effect (F [1, 28] = 15.070, p < 0.001). Lastly, the ANOVA indicated that there was a significant STRESS x DRUG effect (F [1, 28] = 17.478, p < .0001). The data show that there was a stress-induced memory impairment in the STR-SAL group, and this impairment was blocked by tianeptine treatment (STR-TIA). The Experiment Three data are analogous to the Experiment One data in that tianeptine was effective in blocking the memory deficit due to cat exposure. Results: Plasma CORT Analysis for One-Day Learning Paradigm Means were calculated and a one-way ANOVA was performed on plasma corticosterone levels for all groups. The means were as follows: Home Cage/Vehicle = 33

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13.269 1.16; Home Cage/TIA = 18.94 2.70; Stress/Vehicle = 54.174 3.20; Stress/TIA = 55.297 3.27 (see Figure Eight). The ANOVA revealed a significant STRESS effect (F [1, 36] = 202.103, p < 0.0001). The DRUG main effect and the STRESS x DRUG interaction were not significant (p < 0.219 and 0.408, respectively). As in Experiment One, Experiment Three CORT data indicate that CORT levels were significantly elevated in both stress groups compared to home cage groups. Again, tianeptine injection did not independently raise CORT levels nor did they reduce CORT levels in the STR-TIA group. 0.0010.0020.0030.0040.0050.0060.0070.00HC-SALHC-TIASTR-SALSTR-TIAGroupMean CORT Levels per Group Figure Eight: Mean Plasma CORT Levels by Group for One-Day Learning Paradigm: Tianeptine and One-Day Learning Task. 34

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35 Chapter Five Experiment Four: Propranolol does not bl ock stress-induced memo ry errors on the One-Day Learning Task Method Rats and Handling Fifty-six male Sprague-Dawley Rats, (175-200 grams) were given a two-week habituation period within the vivarium upon a rrival. Rats were housed two to a cage in standard Plexiglas cages and given food and water ad libitum All rats were handled on Days 10, 12 and 14 of the two-week habitu ation period. During the handling procedure rats were taken in their cages, four cages at a time, to the behavioral testing room and placed on a table along one wall. Rats were then gently handled one at a time for approximately 2 minutes each. On Day 14 of th e habituation period rats were handled in addition to receiving numerical tail markings with a permanent marker. At the end of each handling session rats were taken back to the vivarium. All handling sessions were given in the morning, the same time as subs equent behavioral testing. The rats were assigned to the following groups after handling: Home Cage/Vehicle (n=8); Home Cage/5 mg PROP (n=8); Stress/Vehicle (n=8 ); Stress/5 mg PROP (n=8), Home Cage/10 mg PROP (n = 8); Stress/10 mg PROP (n = 8). Rats were run in the radial six-arm water maze (RAWM) using the one-day learning working memory task (as previously described). To review, the RAWM was a black galvanized round tank (168 cm diameter 56 cm height, 43 cm deep) filled with clear water (23 -24 C). The tank was divided into six arms radiating from a central area by using 6 V-shaped stainless st eel walls (54 cm height, 56 cm length). A plastic platform

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36 (12 cm diameter) was hidden from view 1 cm below the surface of the water at the end of one of the arms. Each rat was run only one day, with each day containing an in itial acquisition of twelve trials in succession. The twelve trial acquisition sequence was then followed by a thirty-minute manipulation period in which the rat was either returned to the home cage or given cat exposure. After the thirty-minut e manipulation period the rats were given a single retention trial. After the retention trial tail blood wa s taken from each rat within two minutes of the retent ion trial and frozen for future an alysis of plasma corticosterone levels. Drug Drug and vehicle injections were given thirty minutes prior to Trial One (T1). Both drug (Propranolol, 5mg/kg and 10 mg/kg) and vehicle (0.9 % NaCl in distilled water) were administered interperitoneally. Statistical Procedures A repeated measures ANOVA was perfor med on all groups by six blocks of two trials. An ANOVA was performed on the mean errors per group on the retention trial (Trial 13). As well, an ANOVA was also performed on the mean results per group for plasma corticosterone levels. Each of the ANOVA procedures was performed individually on the 5 and 10 mg/kg PROP groups. Independent t-tests were then performed to compare the means across th e two STRESS, and STRESS x DRUG groups for each dose, to preserve statistical power.

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Results: Within Subject Analysis: 5 mg/kg Propranolol Dose Means and standard errors of the mean were calculated for all groups by blocks of trials. The repeated measures ANOVA showed that there was a significant BLOCKS effect for all groups combined (F [6, 168) = 6.623, p < 0.0001). There was also a significant BLOCKS x STRESS interaction (F [6, 168] = 5.373, p < 0.0001). A test of linearity indicated that there was a significant linear trend among the groups on all acquisition blocks (F [1, 28) = 11.765, p < 0.002). The test of linearity also indicated a significant linear trend for the BLOCKS x STRESS interaction (F [1, 28) = 4.235, p < 0.049). The between subjects analysis indicated that there was a significant DRUG main effect among groups by blocks of trials (F [1, 28] = 10.358, p < 0.003) (see Figure Nine). 00.511.522.533.544.55B1B2B3B4B5B6RTBlocks of TrialsMean Errors HC-SAL STR-SAL HC-5 STR-5 Figure Nine: Acquisition Curves and Retention Performance for all Groups by Blocks of Trials: 5 mg/kg Dose of Propranolol and One-Day Learning Task. 37

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Results: Retention Trial Analysis: 5 mg/kg Propranolol Dose The between subjects ANOVA performed on retention trial means indicated that there was a significant STRESS main effect (F [1, 44] = 30.188, p < 0.0001). The ANOVA revealed that both the DRUG and STRESS x DRUG interaction were not significant, suggesting the ineffectiveness of the 5 mg/kg dose of propranolol (PROP) on stress-induced memory changes (see Figure Ten). The data showed that rats given the 5 mg/kg dose of PROP and were placed in the home cage during the delay period did not exhibit more spatial memory errors on the retention trial. Also, the 5 mg/kg dose of PROP was not effective in blocking the stress effect on spatial memory retention performance. 00.511.522.533.544.55HC-SALSTR-SALHC-5 STR-5 GroupMean Errors Figure Ten: Errors on Retention Trial for all Groups: 5 mg/kg Dose of Propranolol and One-Day Learning Task. 38

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Results: Within Subject Analysis: 10 mg/kg Propranolol Dose Means and SEMs for all groups by blocks of two trials were calculated. The repeated measure ANOVA showed that there was significant BLOCKS effect (F [6, 168) = 6.817, p < 0.0001). There was also a significant BLOCKS x STRESS interaction (F [6, 168] = 5.317, p < 0.0001). A test of linearity indicated that there was a significant linear trend for the BLOCKS effect (F [1, 28] = 10.125, p < 0.004). There was also a significant BLOCKS x STRESS linear trend (F [1, 28] = 6.596, p < 0.016). The between subjects analysis revealed that there was a significant STRESS main effect (F [1, 28] = 10.248, p < 0.003). There was also a significant DRUG effect (F [1, 28] = 27.179, p < 0.0001). The STRESS x DRUG interaction was not significant (see Figure Eleven). All groups exhibited a significant learning curve over the six blocks of two trials. 0123456B1B2B3B4B5B6RTBlocks of TrialsMean Errors HC-SAL STR-SAL HC-10 STR-10 Figure Eleven: Acquisition Curves and Retention Performance for all Groups by Blocks of Trials: 10 mg/kg Dose of Propranolol and One-Day Learning. 39

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Results: Retention Trial Analysis: 10 mg/kg Propranolol Dose The between subjects ANOVA performed on the retention trial means indicated that there was a significant STRESS main effect (F [1, 44] = 30.188, p < 0.0001). The ANOVA showed that the DRUG and STRESS x DRUG interaction was not significant (see Figure Twelve). As with the 5 mg/kg dose of PROP, the 10 mg/kg dose of PROP did not significantly raise error rates in the HC-10 mg/kg PROP group. Also, the 10 mg/kg dose of PROP was ineffective in reducing retention errors in the STR-PROP group. 0123456HC-SALSTR-SALHC-10STR-10 GroupMean Errors Figure Twelve: Retention Errors for all Groups: 10 mg/kg Dose of Propranolol and One-Day Learning Task. Independent T-test Analysis Between 5 mg and 10 mg Propranolol Doses An independent two-tailed t-test revealed that there was no significant difference between the STRESS-5 mg/kg PROP group and the STRESS-10 mg/kg group (t = 0.615). An independent t-test also showed that there was no difference between the HC-5 mg/kg PROP group and the HC-10 mg/kg PROP group (t = 0.794). The independent t40

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41 test shows that there was no difference among the groups when comparing the groups across dosage. Results: Plasma CORT Analysis for 5 mg/kg Propranolol Dose Means were calculated and a one-w ay ANOVA was performed among groups. The following are the means and standard er rors of the mean for all groups: Home Cage/Vehicle = 13.26 1.16; Home Cage /PROP = 62.595 4.041; Stress/Vehicle = 55.174 3.27; Stress/PROP = 60.393 3.036 (see Figure Thirteen). The ANOVA showed that there was a signi ficant STRESS main effect (F [1, 32] = 43.123, p < 0.0001). There was also a significa nt DRUG main effect (F [1, 32] = 88.821, p < 0.0001). The STRESS x DRUG interacti on was also signific ant (F [1, 32] = 53.494, p < 0.0001). The 5 mg/kg dose of PROP significantly raise CORT levels in rats compared to the HC-SAL group. The 5 mg/kg dose of PROP, however, did not significantly lower high stress levels of CORT in the STR-PROP group. Results: Plasma CORT Analysis for 10 mg/kg Propranolol Dose Means were calculated and a one-way ANOVA performed on data for all groups. The following are the means for all groups : Home Cage/Saline = 13.26 1.16; Home Cage/PROP = 60.951 3.985; Stress/Salin e = 55.174 3.27; Stress/PROP = 84.095 10.56 (see Figure Thirteen). An ANOVA indicated that there was a significant STRESS main effect between groups (F [1, 29] = 54.473, p < 0.0001). There was also a significant DRUG main effect (F [1, 29] = 79.966, p < 0.0001). The STRESS x DR UG interaction was also significant (F [1, 29] = 4.189, p < 0.05). The 10 mg/kg dose of PROP significantly raise CORT

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levels in rats compared to the HC-SAL group. The 10 mg/kg dose of PROP, however, did not significantly lower high stress levels of CORT in the STR-PROP group. 0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00100.00HC-SALSTR-SALHC-5mg/kgHC-10mg/kgSTR-5mg/kgSTR-10/mg/kgGroupMean CORT per Group Figure Thirteen: Mean Plasma CORT Levels for All Groups: 5 mg/kg and 10 mg/kg Dose of Propranolol and One-Day Learning Task. Results: Independent T-Test Between Stress Groups An independent t-test was performed to analyze the difference between the Stress/5 mg/kg PROP and Stress/10 mg/kg group. The t-test was not significant (t = 0.02). This indicated that the 10 mg/kg dose did not lead to significantly more errors in the stress groups than did the 5 mg/kg dose. In summary, Experiment Four found that propranolol was not effective in blocking stress-induced memory errors in the RAWM. All three stress groups, Stress/Saline, Stress-5 mg/kg of propranolol and Stress10 mg/kg of propranolol 42

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43 exhibited statistically significant elevations in mean error rate compared to Home Cage groups. Also there was no statistically significant difference between the 5 mg/kg and the 10 mg/kg dosages of propranolol and its eff ects on memory. Both doses of propranolol did raise CORT levels in both Home Cage/P ropranolol groups, but error rates in both groups remained at control levels.

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44 Chapter Six Experiment Five: Adrenalectomy, Stress and Memory: Effects of adrenal steroids on stress-induced memory changes Method Rats and Handling Procedure Seventy-one rats (Harlan La boratories) were used in th is experiment. The rats per group were as follows: Sham/Vehicle (n = 12 ), ADX/Vehicle (n = 12), Sham/Stress (n = 9), ADX/Stress (n = 10), Sham/TIA (n = 6), ADX/TIA (n = 6), Sham/Stress/ TIA (n = 8), ADX/Stress/TIA (n = 8). Rats were appr oximately 250 grams upon arrival and had an approximate weight of 350 grams at the beginn ing of surgery and te sting. All rats were housed two to a cage in standard Plexiglas ra t cages with wire tops. Rats were given a habituation period of two week s. During the two-week habitu ation period all rats were given water and standard rat chow ad libitum Also during the two-week habituation period the rats received the handli ng regime as described earlier. Adrenalectomy (ADX) Procedures The day after the two-week habituation period had ended all rats were taken from the vivarium in their home cage by cart to the surgery room. Rats were given one hour to acclimate to the surgery room surroundings. During the one-hour pre-surgery period all rats were weighed in a standard electronic scale and placed back into the home cage. Separate cages were used on a far ta ble to house the rats post-surgery. At the beginning of the surgical proce dure individual rats were placed upon the surgery table and administered the anesthe tic halothane with an oxygen mixture via a nose cone. During the surgery the rats were gi ven continuous maintenance applications of

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45 the anesthesia through the nos e cone. Upon proper anesthesia the rat was then shaven bilaterally posterior to the rig cage, an area directly a bove the visceral location of the adrenal gland/kidneys. After sh aving the rat was swabbed with an antiseptic solution and placed on a heating pad on the surgery table. A one-inch incision was then made through the skin and muscle. After the incision th e adrenal gland was carefully located and removed using a circle-tipped forceps and sm all surgical scissors. The rat was then swabbed internally and monitored for exce ss bleeding. After any possible bleeding had stopped the muscle and skin was sutured usi ng Ethicon Coated vicryl 18-inch suture thread (muscle) and silk braided 18-inch suture thread (skin). After one side was complete the rat was then carefully turn ed and the procedure was repeated on the remaining side. All surgeries were done left side first on all rats for continuity. After the final suturing rats were treated with an an tiseptic solution and placed in a holding cage for post-surgery evaluation. Respiration and he artbeat were monitore d for all rats. Shamcontrol rats received all procedures consiste nt with adrenalectomy except for the removal of the glands. When rats were successfully reco vered they were returned to their original home cage. After all rats were done they were returned to the viva rium and were closely monitored for regular home-cage activitie s such as eating, drinking and grooming. Corticosterone and Sodium Replacement Procedures Sham-control rats were given norma l drinking water upon returning to the vivarium after surgery. ADX rats were gi ven a special saline water solution to compensate for the loss of corticosterone (C ORT) and aldosterone. Corticosterone (100 mg) was dissolved in 8 ml of ethyl alcohol (Et OH) and then added to four liters of saline

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46 solution (20 g of NaCl per 4 liters of distilled water w ith CORT replacement) All ADX rats remained on the CORT replacement salin e solution for the entirety of the study. Behavioral Testing Regime Rats were given seven days to recove r from ADX surgery. After the seven day period rats were run using the One-Day Lear ning (ODL) radial arm water maze (RAWM) task. The exact procedures for the ODL task were stated previously. In short all rats were given twelve trials (six visibl e, six hidden/submerged) to fi nd the escape platform. After the twelve acquisition trials were given the ra ts were either placed back in the home cage or with the cat for thirty minutes depending on group. After the thirty minutes rats were given a one-trial retention test to the previous platform location. The thirteen total trials for each individual rat were consistent, but the platform location was randomized among the rats so no two consecutive rats went to the same platform location. Blood Sampling and Preparation After the final retention tr ial the rat was quickly taken to an adjacent room for blood sampling. Each rat was placed in a wire re straint after each rats tail was soaked in warm water for thirty seconds to aid blood flow from the tail. After the tail soaking a 1mm cut was made at the end of the tail. The tail was then gently massaged from base to tip to take the blood. Blood was collected in to plastic centrifuge tubes and placed under a blanket out of the light and allowed to settle. After all blood samples were taken the blood was centrifuged and the resulting cleare d plasma was pipetted into a new plastic tube. All blood samples were then placed into a deep freezer and frozen at degrees centigrade and awaited radioimm unoassay (RIA) manipulation.

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47 Statistical Procedures Means and standards errors of the mean (SEM) were calculated for all groups on all individual trials. A univariate ANOVA was performed on bl ocks of two trials for the twelve acquisition trials (six blocks of two trials). An ANOVA was also performed on the means of the one-trial retenti on test (Trial 13). Means and SEMs were also calculated for serum CORT levels, and an ANOVA was performed on serum CORT levels for differences among the various groups. Results: Within Blocks Analysis The following table contains the means a nd standard errors of the mean for all groups by blocks of two trials (see Table One). ADXSAL SHAM-SAL ADXSTR SHAM-STR Mean SEM Mean SEM Mean SEM Mean SEM B1 2.667 0.386 2 0.511 3.8 0.309 2.611 0.415 B2 1.625 0.283 2 0.364 2.25 0.454 1.5 0.363 B3 1.291 0.339 1.542 0.217 1.3 0.249 1.278 0.237 B4 0.917 0.192 0.875 0.262 0.95 0.240 0.778 0.169 B5 0.833 0.112 0.958 0.199 0.5 0.129 0.889 0.320 B6 0.375 0.175 0.458 0.114 0.75 0.200 0.389 0.111 RT 0.25 0.179 0.333 0.188 3.5 0.619 2.444 0.669 ADXTIA SHAM-TIA ADX-STR-TIA SHAM-STR-TIA Mean SEM Mean SEM Mean SEM Mean SEM B1 2.667 0.615 3.583 0.5833 2.688 0.582 3.125 0.440 B2 2.083 0.810 1.667 0.279 2.063 0.678 2.563 0.417 B3 2.5 0.695 1.667 0.667 0.875 0.263 1.625 0.245 B4 1.25 0.309 1.333 0.210 0.75 0.25 1.125 0.363 B5 1.083 0.238 0.833 0.210 0.688 0.230 1.125 0.295 B6 0.917 0.300 0.833 0.307 0.438 0.147 0.563 0.290 RT 0.667 0.422 0.667 0.333 0.625 0.375 0.875 0.295 Table One: Means and Standard E rrors of the Mean for all Grou ps by Blocks of Two Trials: Adrenalectomy, Tianeptine and One-Day Learning Task.

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48 The repeated measures ANOVA showed that there was a significant BLOCKS effect (F [6, 378] = 39.314, p < 0.0001). There was also a significant BLOCKS x STRESS interaction effect (F [6, 378] = 5.869, p < 0.0001). The repeated measures ANOVA also revealed a BLOCKS x DRUG interaction effect (F [6, 378] = 3.060, p < 0.006). Also, a significant BLOCKS x STRESS x DRUG interaction effect was shown (F [6, 378] = 3.833, p < 0.001. A test of linearity among all groups by block revealed a significant linear trend for BLOCKS (F [1, 63] = 127.877, p < 0.0001). A significant linear trend was also evident within the BL OCKS x DRUG effect (F [1, 63] = 6.276, p < 0.015). The between subjects analysis showed significant STRESS x DRUG interaction effect (F [1, 63] = 9.098, p < 0.004), and a significant SURGERY x STRESS x DRUG three-way interaction (F [ 1, 63] = 4.760, p < 0.033). All groups showed a significant learning curve across the six tw o-block acquisition trials. Results: Retention Trial Analysis The means and Standard Error of the Mean (SEM) for Retention Trial (RT) performance for all groups are as follows: Sham/Saline = 0.333 0.188; ADX/Saline = 0.25 0.179; Sham/Stress = 2.444 0.669; ADX/Stress = 3.5 0.619; Sham/TIA = 0.667 0.333; ADX/TIA = 0.667 0.422; Sham/Stress/TIA = 0.875 0.295; ADX/Stress/TIA = 0.625 0.375. The analysis of variance (ANOVA) perf ormed on the RT error rates between groups indicated that there was a signifi cant STRESS main effect, (F [1, 63] = 20.637, p < 0.0001). The ANOVA also showed that there was a significant DR UG main effect by the tianeptine, (F [1, 63] = 9.218, p < 0.003) Conversely, the ANOVA found that there was not a significant main effect of ADX (F [1, 63) = 0.352, p < 0.555). In addition, the

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ANOVA indicated that there was a significant STRESS x DRUG two-way effect, (F [1, 63] = 18.223, p < 0.0001). The remaining two-way effects, ADX x STRESS (F [1, 63] = 0.534, p < 0.468) and ADX x DRUG (F [1,63} = 1.009, p < 0.319) were not significant. The three way interaction ADX x STRESS x DRUG was not significant (F [1.63) = 1.303, p < 0.258 (see Figure Fourteen). The cat exposure significantly increased errors in the ADX-STR and Sha-STR groups. These data indicate that ADX did not block the stress effect on memory. Also, the data indicate that in the presence of ADX and sham surgery tianeptine significantly lowered memory errors in the ADX-STR-TIA and Sha-STR-TIA groups. 00.511.522.533.544.5ADX-VEHSha-VEHADX-STRSha-STRADX-TIASha-TIAADX-STR-TIASha-STR-TIAGroupMean Errors Figure Fourteen: Errors on Retention Trial for all Groups: Adrenalectomy, Tianeptine and One-Day Learning Task. 49

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50 Results: Blood/CORT Anal ysis for ADX Experiments Means were calculated and a one-way ANOVA was performed for all groups. The following are the means and standard erro rs of the mean for all groups: Sham = 13.45 0.76; ADX = 3.092 0.45; ADX/Stress = 3.792 0.442; Sham/Stress = 43.55 5.323; ADX/TIA = 4.273 = 0.385; Sham/TIA = 14.159 1.588; ADX/Stress/TIA = 4.738 0.826; Sham/Stress/TIA = 44.97 6.164 (see Figure Fifteen). The ANOVA revealed that there was a si gnificant ADX main effect (F [1, 41] = 129.714, p < 0.0001). There was also a significant STRESS main effect (F [1, 41] = 49.749, p < 0.0001). There was also a significant ADX x STRESS interaction effect (F [1, 41] = 46.083, p < 0.0001). All other effects a nd interactions were not significant. The data show that stress significantly increased CORT levels in Sham-STR and the ShamSTR-TIA groups. In summary, Experiment Five showed that ADX alone did not impair spatial memory performance in rats. The ADX/Vehicle and Sham/Vehicle groups were not significantly different and both exhibited mean error rates significantly low than both ADX/Stress and Sham/Stress groups. Experime nt Five showed that stress-induced memory errors were produced in the pr esence of ADX. ADX/Stress and Sham/Stress groups showed a significant incr ease in mean error rate and both groups error rates were significantly higher than all other groups. Tian eptine was shown to maintain efficacy in reducing stress-induced memory errors even in the presence of ADX. ADX/Stress/TIA and Sham/Stress/TIA groups were both significantly lower than ADX/Stress and Sham/Stress groups, and were significantly sim ilar to all Home Cage groups. Experiment Five also showed that the ADX/STR group exhi bited significantly hi gher error rates and

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the groups CORT levels were at low ADX levels. These data suggest that high stress levels of CORT are not necessary for stress-induced errors to occur. 0.0010.0020.0030.0040.0050.0060.00ShamADXADX-STRSham-STRADX-TIASham-TIAADX-STR-TIASham-STR-TIAGroupMean CORT Levels per Group Figure Fifteen: Mean Plasma CORT Levels for All Groups: Adrenalectomy, Tianeptine and One-Day Learning Task. 51

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52 Chapter Seven Discussion Tianeptine Blocks Stress-Induced Memory Errors The first four experiments in the current series examined the effects of the antidepressant tianeptine on stress-induced me mory errors in rats. Tianeptine has been shown to reverse the stress-i nduced blockade of synaptic-p lasticity (Kole et al, 2002; Shakesby et al, 2002) and has re versed spatial memory defic its in rats (Conrad et al, 1996; Luine et al, 1994). The current rese arch found that acute administration of tianeptine was effective in blocking stress -induced memory errors in two different training paradigms within the radial-arm water maze (RAWM). In both the criterionbased training regimen (Experiment One) a nd the one-day learning regimen (Experiment Three), tianeptine blocked the stress-induced memory deficit on the post-stress retention trial. In both experiments tia neptine was given thirty minutes before the first acquisition or learning trial. Tianeptine administration blocked stress-induced memory errors on the retention trial, but did not a ffect acquisition. Both vehicle and tianeptine groups showed a significant learning curve over th e acquisition trials prior to any stress during the thirty minute manipulation period. Serotonergic Mechanism of Tianeptine Action Research suggests that ti aneptine exerts its effects through a serotonergic mechanism (Mennini et al, 1987, Mocaer et al 1988, de Simoni et al, 1992), namely increasing the reuptake of serotonin from th e synapse, in turn reducing the amount of synaptic serotonin. The serotonergic theory is attractive due to the idea that stress increases serotonin levels in the synapse (Matsuo et al, 1996; Yo shioka et al, 1995).

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53 Tianeptine would then be hypot hesized to alleviate the st ress effects by reducing the serotonin due to the stress. NMDA Receptor Mediated Mechanism of Tianeptine Action While the serotonin hypothesis is still a viable possibility, recent research has shown that tianeptine may also act through an NMDA receptor mechanism, buffering the NMDA receptor from the effects of stress (Kole et al, 2002; Shakesby et al, 2002). The glutamatergic NMDA receptor is a logical mechanism for stress-induced effects on memory and the blockade of these effects by tianeptine based on the idea that the NMDA receptor has been shown to mediate cellular and functional effects of stress. Following a stressful event, receptor binding and recep tor subunit expression for hippocampal NMDA receptors are enhanced (Bartanusz et al, 1995; Krugers et al, 1993). Also, stress lowers the threshold for long-term depression (LTD), a form of hippocampal synaptic plasticity, via NMDA receptor activation (Kim et al 1996). The administration of an NMDA receptor antagonist prevents stress-induced dendritic remodeling of CA3 pyramidal neurons (Magarinos and McEwen, 1995). De ndritic remodeling is known to be a consequence of chronic stress. Other research indicates that the dimi nishing of NMDA receptor function may be linked to the activity of antidepressant drugs (Skolnick et al, 1999; Petrie et al, 2000; Krystal et al, 2002). For instance, the expression of hippocampal NMDA receptor subunits is reduced after chronic administrati on of antidepressants (S kolnick et al, 1999). Recent research reviewed by Petrie et al ( 1999), indicates that in animal models of depression NMDA antagonists exhibit similar potencies as antidepressant medications. Also, recent work by Berman et al (2000) showed that ketamine, an NMDA-antagonist,

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54 produced a transient improvement in the mood state of patients with major depression. As seen here, both preclinical and clinical wo rk has shown that the NMDA receptor may be heavily linked to stress effects and disorders closely associated to stress such as major depressive disorder (MDD). A further discussion of MDD and stress will follow below. Another line of research involving hippocam pal synaptic plasticity has indicated that stress blocks long-term potentiation (LTP ) in the hippocampus and this blockage is reversed by tianeptine (Shakesby et al, 1999). The work with tianep tine and LTP suggests that tianeptine may work via NMDA receptors by increasing the amount of glutamate available to the receptor, t hus buffering it from the effects of stress. Tianeptine would then be seen as a drug that w ould protect the receptor from th e effects of stress while also setting a set of chemical pr econditions, through increased rele ase of glutamate that would make the receptor less susceptible to the effects of stress. Testing the Anti-Anxiety Properties of Tianeptine In Experiment Two, tianeptine was given af ter the stressful experience within the criterion-based regimen. Tianep tine was not effective in re ducing stress-induced memory errors on the retention trial when administered after the stressful event. Thus tianeptine was effective in reducing the effects of st ress on memory only when administered in a proactive manner. Tianeptine seems to set a series of chemical and receptor-based conditions that make the cell less susceptible to stress. This is the case mentioned above in Experiments One and Three when tianeptine was given thirty minutes before the first training trial. Tianeptine is also thought to exhibit anti -anxiety properties as well as antidepressant properties (Wilde et al, 1995; R odgers et al, 1997; Dr obizhev et al, 2000; Lepine et al, 2001; Rumiantsev a et al, 2003). The effects of tianeptine given after the

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55 stressful experience are useful in gauging the anti-anxiety properties of the drug. It would be possible that the errors exhibited by th e stress/vehicle groups could be caused by increases in anxiety levels. An increase in an xiety levels could aff ect performance of the rat in the maze by increasing the overall speed of motor function (i.e. swimming). If this was the case, giving the tianeptine after the stressful event should have reduced anxiety levels in the rat, thus reducing the error rate on the retention trial. The fact that errors were not reduced in stressed rats given tiane ptine after the stressful event suggests that the reduction in stress-induced errors was due to stress-related factors other than increased levels of anxiety and related cha nges in motor function. However, we did not measure anxiety behavior, per se, in the rats Therefore, whether tianeptine blocked postcat exposure anxiety can not yet be determined. Experiment Four also examined the role of anxiety and performance by administering the beta-blocker drug propranol ol. Two doses of propranolol (5 mg/kg and 10 mg/kg) were ineffective in reducing stress -induced errors on the retention trial on the one-day learning task. The ineffectiveness of propranolol on bl ocking stress-induced memory errors on the one-day learning task su ggests that the beta-adrenergic system is not a viable mechanism for stress-induced memory change under the current RAWM-cat conditions. The idea that propr anolol did not block stress effects on memory is not consistent with previous research which s howed that propranolol blocked the memory enhancing effects of glucocorticoids and epinephrine. Previous research showed that glucocorticoid injection enhanced memory for an inhibitory avoidance task and that this enhancement was blocked by propranolol (Rooz endaal et al, 1999). The blockade of enhancement of the stressful memory woul d be analogous to the enhancement of the

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56 stressful memory of the cat in the current studies. The enhancement of the cat stress memory would cause the stre ss induced impairment of the peripheral memory, the memory of the platform location. However, in the current studies propranolol was ineffective in blocking stress-induced memo ry impairment of the peripheral memory. This suggests that the blockade of beta-adren ergic activity at the time of stress is not effective in the predator exposure stress situation. Stress and Major Depressive Disorder (MDD): Pre-Clinical Applications of Tianeptine The effects of tianeptine detailed here ar e important to the investigation of the pathophysiology and treatment of major depres sive disorder (MDD). There are similar neurological effects seen in stressed subjects and those with MDD. Namely in both instances, stress and MDD, hippocampal and prefrontal cortical functioning is impaired whereas amygdaloid functioning is enhanced (Burghardt et al, 2003; Vouimba et al, 2003). Also certain biomarkers including neurotransmitter abnormalities and hormone levels, including the stress hormone cort icosterone, and brain structure morphology (Hindmarch et al, 2001; Ph illips et al, 2003; Sapolsky, 2000) have commonalities among stress and MDD. It is known that during a stress response and with MDD corticosterone levels in rats or cortisol levels in hum ans are elevated (Boyer et al, 2000; Moghaddam, 2002, Parker et al, 2003). This commonality between stress and MDD makes stress a prime area of study to better unde rstand the etiology and treatment of MDD. It is also intriguing to note that severe life stressors can sometime s lead to the occurrence of clinical depression. The study of stress and it s neurological and chemical substrates will lead to better understanding of how stressful life experien ces can lead to depressive

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57 disorders. The study of stress can also l ead to a better understanding of effective treatments for depressive disorders. Also it must be taken into account the different mechanisms by which tianeptine and other antidepressants such as selective se rotonin reuptake inhi bitors (SSRIs) work. SSRIs are thought to alleviate symptoms of depression over time by increasing the amount of serotonin available in the synapse. Increases in serotonin are achieved by the inhibition of reuptake of serotonin back into the pre-synaptic cell. Conversely, tianeptine has been shown to be a serotonin reuptake e nhancer in rats and in humans (deSimoni et al, 1992; Wilde et al, 1995). After treatment s ranging in duration from 4 weeks to 3 months at the time of testi ng, tianeptine was seen to have the same efficacy as amitriptyline, imipramine, and fluoxetine (Wilde et al, 1995). As stated above, tianeptine enhances the reuptake of serotonin into the pr e-synaptic cell. The pr e-synaptic cell would then have an increase in the amount of serotonin that could possibl y be released. Over time, it is possible that SSRIs and tianeptin e may indeed have the same mechanism of action, increased amounts of serotonin in th e synapse. SSRIs accomplish this by blocking reuptake, whereas tianeptine may accomplish this by having more serotonin to release from the pre-synaptic cell due to the enhancem ent of reuptake. It is important to keep in mind the possible long-term pharmacological e ffects of drugs with seemingly different mechanisms of action in the treatment of depression. Involvement of Corticosterone In the Formation and Blockade of Stress Effects One area of interest in th e current series of experiments was the actions of corticosterone and its effects on stress and st ress-induced memory change. In Experiment One it was found that rats that were stressed and received a vehicle treatment exhibited

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58 elevated levels of CORT. The finding of elevat ed CORT levels is consistent with other data indicating raised levels in response to cat exposure (Diamond et al, 1999). In addition, rats that were stressed and receive d tianeptine thirty minutes prior to training also exhibited statistically si gnificant elevated levels of CO RT, similar to the levels found in stressed rats that were administered ve hicle treatment. The fact that CORT was elevated in the STRESS/TIA rats suggests that tianeptine exerts its effects independent of modifying circulating blood CORT levels. That is, the blocking of stress-induced memory errors in Experiment One within the criterion-based multi-day training regimen was not due to a reduction in circulating blood levels of CORT. In Experiment Three, similar elevated CORT levels were found in stressed rats trained on the one-day learning regimen. Thus, tianeptine did not lower CORT levels in either training regimen, the multi-day training nor the one-day learning task. The current series of experiments showed that stress-induced memory deficits can be alleviated by tianeptine in the presence of high CORT or in the absence of CORT via ADX. The current data lend to the discussion of the interaction betw een CORT levels and memory performance. Extensive research ha s shown that stimuli that are considered arousing can enhance memory (Cahill, 2000; LeDoux, 2000; McGaugh, 2000). Conversely, stimuli that increase the emotiona lity of the subject, such as stress also impair memory (Diamond and Park, 2000; Kim and Diamond, 2002) and cause amnesia (Loftus and Kaufman, 1992; Joseph, 1999). An apparent paradox exists therefore between the type of arousing stimulus and its effects on memory and behavior. While arousing stimuli such as predator exposure cause amnesic effects on a spatial memory task in rats (Diamond et al, 1996, 1999b) other arousing stimuli that do not contain a fear

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59 element such as giving a male rat access to an estrous female rat, do not disrupt spatial memory (Woodson et al, 2003). Woodson et al (2003) found that rats that were stressed with a cat and those that we re given access to an estrous female both exhibited high levels of CORT. But whereas the cat exposed ra ts showed an increase in errors the rats given access to the female did not show an increase in error rate. The findings of Woodson et al (2003) are consistent with th e current findings because certain groups of rats in both studies exhibite d high levels of CORT while not showing spatial memory impairments. Other research (Park et al, 2001) found that rats that were not stressed with the cat but were injected with stress le vels of exogenous CORT showed no spatial memory impairment when tested in the RAWM, suggesting that CORT alone does not cause spatial memory impairment. This hypot hesis suggests that the nature of the arousing or stressful stimulus must be taken into account much like that in the Woodson et al (2003) findings. Past studies have shown that ce rtain stressors including shock, restraint and exposure to a novel environmen t have resulted in spatial (hippocampaldependent) memory impairment (Diamond et al 1994; Kuroda et al, 1998; Magarinos et al, 1995). And impairment of spatial memory (Kim and Diamond, 2002) and the impairment of synaptic plasticity (Foy et al 1987; Shors et al, 1989; Kim et al, 2002) in the hippocampus have been associated with high stress levels of CORT. The current experiments and the work of Woodson et al (2003) extend thes e findings by showing memory impairment is not always tied to the presence of high CORT. The current data elucidates the nature of the interaction between elevated CORT levels and memory impairment by showing that high CORT al one does not lead to spatial memory impairment. Experiment Five in the current se ries also finds that stress effects and the

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60 alleviation of stress-induced memory deficits by tianeptine occur in the absence of CORT via ADX. As discussed below, the findings of Experiment Five are consistent with the idea that elevated CORT is not the sole cause of spatial memory impairment. The Role of Adrenal Hormones on Stress and Memory Experiment Five further examined th e effects of CORT and other adrenal hormones on stress and its effects on memor y. Adrenalectomized (ADX) rats that were not stressed showed acquisition of the one-day learning task in a manner statistically equal to that of sham operated controls. Previous research has shown the ADX rats exhibited impaired learning (Vaher et al, 1994). The difference between previous research and the current experiment is that ADX rats in the current experiment received CORT replacement in their drinking wate r. The amount of CORT given in the replacement therapy was designed to maintain the integrity of hippocampal cells that would normally be at risk under ADX conditio ns, namely granule dentate gyrus cells within the hippocampus. Previous research showing memory im pairment did not wish to maintain hippocampal cell integrity thus impairments were seen. It was imperative that the hippocampal cells of Experiment Five rats remain intact, allowing the effects of stress and tianeptine to be studied independent of the effects of hippocampal cell atrophy processes. Data from Experiment Five also showed that ADX rats that were exposed to the cat displayed stress-induced memory errors on the retention trial. The stress effect in ADX rats implies that adrenal hormones, most notably CORT, are not necessary for a stress-induced memory impairment to occur. Also in Experiment Five, tianeptine exerted its effects on stressed rats in both sham and ADX conditions. As in the results from Experiments One and Three, in which tianeptin e was effective in reducing stress-induced

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61 memory impairments in the presence of elevated levels of CORT, Experiment Five showed that tianeptine was effec tive in reducing errors in the absence of adrenal hormones, including CORT. The evidence given here puts into debate the necessity of CORT to the production of a stress effect. In the current experiments stress effects were found in the absence of circulating CORT. The current research showing the induction of a stress effect in the absence of CORT lead s to the analysis of the reactions of the hypothalamic-pituitary-adrenal (H PA) axis during a stressful event. The HPA axis and hippocampus act as a negative feedback mech anism for circulating CORT levels. If CORT levels are elevated, as in a stressful situation, the CORT-receptor rich hippocampus will signal the HPA-axis to reduc e the amount of stress hormones that are being released. If the feedback mechanism is disrupted as in the case of ADX the actions of the hypothalamus and the pituitary gland remain unchecked. In this case, the release of corticothophin-releasing hormone (CRH) by th e hypothalamus and andrenocorticotrophic hormone (ACTH) by the pituitary gland con tinue to be released in stress-induced quantities. Research has shown that admini stration of CRH and ACTH can lead to the production of stress effects similar to those of CORT (Wang et al, 1998). Summary and Conclusions The current set of experiments investig ated tianeptines ability to block stressinduced memory errors. Experiment One found that tianeptine blocked stress-induced errors on a criterion-based memory task. The ra ts were very well trained in the RAWM at the point of tianeptine administ ration. The criterion-based task dictates that the rat be trained for a period of approximately two week s. In Experiment Three tianeptine blocked stress-induced memory deficits on a one-day le arning task. On the one-day learning task,

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62 the rats were not as well trained to the task as in Experiment One. Taken together, the results of Experiments One and Three suggest that the level of training on the task, extended criterion-based vs. one-day traini ng, did not alter the efficacy of tianeptine administration. Experiment Two indicated th at when tianeptine was given after the stressful event, stress-induced memory errors were not blocked. The idea that tianeptine given after the stressful event did not block errors suggests that tianeptine acted in a proactive manner, in essence se tting a set of chemical and/or electrical conditions before the stress occurs. The preconditions set by the administration of tianeptine may serve to strengthen the memory of the hidden plat form location making the hidden platform memory less susceptible to stress. If, as in Experiment Two, the tianeptine was given after the stressor, there was no opportunity for the setting of preconditions and the strengthening of the platform memory vi a NMDA receptor activation. Experiment Two also elucidated the idea that tianeptine was not merely reducing the level of anxiety in response to the cat. If this were so giving ti aneptine after the stress ful event should lower the anxiety level and, in turn, promote the re trieval of the hidden platform memory. Also, propranolol, at tw o doses, was not effective in reducing errors on the retention trial. It is also not ed that the amygdala has a larg e population of beta-adrenergic receptors, and even though the propranolol was administered globally, in theory the blockage of beta-adrenergic receptors by pr opranolol should compromise the functioning of the amygdala. In fact, Experiment Four s howed that rats given propranolol exhibited good retention memory. Thus, the blockade of beta-adrenergic receptors did not disrupt spatial memory function, suggesting the amygda la may not play a role in tianeptines blockade of stress-induced me mory effects. Also, Roozendaal et al (1999) found that

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63 propranolol blocked the gluc ocorticoid-mediated enhancement of memory in the inhibitory avoidance task. The glucocortico id enhanced the memo ry of the stressful event, the context in which the shock was administered. In Experiment Four, it was hypothesized that the beta-adrenergic antagonist would have similar effects in the cat exposed rats. That is, the propranolol would bl ock the fear-intensified memory of the cat experience, and, in turn, not cau se an impairment of the memo ry peripheral to the stress memory, the memory of the platform locati on. In Experiment Four, neither the 5 mg/kg nor the 10 mg/kg dose of propranolol blocked stress-induced errors. Since the peripheral memory was impaired, it can be stated that propranolol was not effective in reducing the effect of the memory of the cat on the memory of the location of the platform. Propranolol also exhi bits anxiolytic effects by acti ng on serotonin receptors. More specifically, propranol ol acts on the 5-HT 1 receptor. Graeff et al (1990) showed that propranolol exhibited anxiolytic effect s on elevated plus-maze performance when administered into the midbrain central gray re gion. Nevertheless, rats in Experiment Four when administered propranol ol at both 5 mg/kg and 10 mg /kg doses did not show a blockage of stress-induced memory errors. It is interesting to note that in Experiment Five, tia neptine blocked stress effects on memory. Tianeptine blocked the effects of stress in the absence of adrenal hormones including corticosterone and pe ripheral epinephrine. The idea that stress effects occurred in the absence of epinephrine suggests that stress effects in the current series may not depend on the actions of either corticosterone or adrenergic (or noradrenergic) substrates. The CORT data submitted in the current expe riments illuminates the idea that CORT is not necessary for a stress effect to occur with respect to memory. In Experiments One and

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64 Three CORT levels were elevated in both st ress groups, Stress/Saline and Stress/TIA. But it was shown that tianeptine reduced errors in the Stress/TIA group. Thus, tianeptine blocked stress-induced working memory errors in the presence of stress levels of CORT. CORT data in Experiment Three shows a simila r effect in the one-day learning task. That is, tianeptine blocked stre ss-induced memory errors in the presence of stress levels of CORT. The idea that errors were reduced in th e presence of stress-induced elevations in CORT suggests that CORT alone is not res ponsible for the induction of working memory errors. Interestingly, in Experiment Four, Home Cage rats that received propranolol showed elevated levels of CORT. Neverthele ss, the Home Cage/PROP rats (at both 5 and 10 mg/kg doses) exhibited control levels of memory errors, again showing that high levels of CORT alone do not directly i nduce working memory errors. To compound the idea that CORT may not be th e causal influence for memory errors, Experiment Five tested rats in the presence of adrenalectomy, essentially elim inating circulating levels of CORT. In Experiment Five, stress effects occurred in the absence of CORT and other adrenal hormones, and tianeptine blocked stress-induced memory errors in the absence of adrenal hormones including CORT. Taken toge ther, the CORT data indicate that CORT is not essential to the production a nd alleviation of a stress effect. In summary, the current set of experiments identified that tianeptine was effective in blocking stress-induced memory errors in two different working memory training tasks. Also, the current experiments found that the reduction in errors was not due to the lowering of anxiety levels, but rather a possi ble strengthening of NMDA receptors within the hippocampus. The experiments also found that the formation of a stress effect is not dependent on CORT or adrenal hormones. Thes e experimental results will lead to further

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65 investigation to examine the possible mechanis ms of tianeptines actions as well as lead to examination of the non-CORT/non-adrenal theory on the formation of a stress effect on working memory. The current set of e xperiments may also lead to a better understanding and future research on the mechanism of action and the efficacy of tianeptine as a treatment for Major Depressive Disorder (MDD).

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71 Jaarsma, D., Postema, F., and Korf, J. (1992). Time course and distribution of neuronal degeneration in the dentate gyrus of rat after adrenalectomy: a silver impregnation study. Hippocampus, 2, 143-150. Joels, M., and deKloet, E.R. (1993). Corticosterone actions on amino acidmediated transmission in rat CA1 hippocampal cells. J. Neurosci., 13, 4082-4090. Joels, M. (2001). Corticosteroid actions in the hippocampus. J. of Neuroendocrinology, 13, 657-669. Joseph, R. (1999). The neurology of di ssociative amnesia: Commentary and literature review. Child A buse and neglect, 23, 715-727. Kawabe, K., Ichitani, Y., and Iwasaki, T. (1998). Effects of intrahippocampal AP5 treatment on radial-arm maze performa nce in rats. Brain Res., Jun 19, 781(1-2), 300-6. Kim, J.J., Foy, M.R., and Thompson, R.F. (1996). Behavioral stress modifies hippocampal plasticity through N-methyl-D-asp artate receptor activ ation. PNAS USA, 93, 4750-4753. Kim, J.J. and Diamond, D.M. (2002). The stressed hippocampus, synaptic plasticity and lost memories. Nat. Rev. Neurosci., 3, 453-462. Kirschbaum, C., Wolf, O.T., May, M., Wippich, W., and Hellhammer, D.H. (1996). Stressand treatment-induced elevati ons of cortisol leve ls associated with impaired declarative memory in healthy adults. Life Sciences, 58, 1475-1483. Kole, M.H., Swan, L., and Fuchs, E. (2002). The antidepressant tianeptine persistently modulates glutamate receptor currents of the hippocampal CA3 commissural

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About the Author Adam M. Campbell received his Bachelors degree in psychology from the University of Cincinnati in 1994. In 1997 he received a Masters degree in Experimental Psychology from East Tennessee State University. At ETSU Adam served as vice-president of Psi Chi, and was accep ted into the Gamma Beta Phi and Phi Kappa Phi Honor Societies. Adam began teach ing psychology courses while at ETSU and continued teaching for four y ears after enrolling in the C ognitive and Neural Sciences program in the Department of Psychology at the University of South Florida in 1997. While at USF, Adam continued his doctoral research at the James A. Haley VA Hospital in Tampa, and was awarded the Outstanding Research Poster at the 1 st Annual College of Arts and Sciences Graduate Research Sym posium in 2002. Adam has also made several presentations at the annual Society for Neuroscience meetings.