Experimental Animal Models in Neurobehavioral Research [1 ed.] 9781616680527, 9781606920220

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Experimental Animal Models in Neurobehavioral Research, Nova Science Publishers, Incorporated, 2008. ProQuest Ebook Central,

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EXPERIMENTAL ANIMAL MODELS IN NEUROBEHAVIORAL RESEARCH

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EXPERIMENTAL ANIMAL MODELS IN NEUROBEHAVIORAL RESEARCH

ALLAN V. KALUEFF AND JUSTIN L. LAPORTE Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

EDITORS

Nova Science Publishers, Inc. New York

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All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Available Upon Request

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Evolution and Schizophrenia

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CONTENTS Preface Chapter 1

Chapter 2

Chapter 3

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Chapter 4

ix Methodological and Theoretical Issues in Experimental Modeling of Anxiety in Animals Leandro José Bertoglio and Antônio de Pádua Carobrez A Standardized Behavioral Test Battery to Identify and Validate Targets for Neuropsychiatric Diseases and Pain Vladimir M. Pogorelov, Kevin B. Baker, Murtaza M. Malbari, Thomas H. Lanthorn and Katerina V. Savelieva Behavior Recognition—W5: The Next Generation of Technology for Behavioral Research James H. Thompson, Vikrant Kobla, Xuesheng Bai, Fayin Li, Dongliang Liu, Mu Sun and Yiqing Liang Pharmacological Dissociation of Anxiety State in the Elevated Star Maze in Chicks Elizabeth P. Rainey and Kenneth J. Sufka

Chapter 5

Unpredictable Chronic Mild Stress in Mice Alexandre Surget and Catherine Belzung

Chapter 6

Sleep Structure during Chronic Stress and Anhedonia in the Mouse Model of Depression Tatyana Strekalova, Raymond Cespuglio and Vladimir Kovalzon

Chapter 7

Understanding the Stress Response through Mouse Genetics Amy F. Eisener-Dorman and Valerie J. Bolivar

Index

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17

47

67 79

113 129 167

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PREFACE Behavioral research has been an exciting staple of the neuroscientific community‘s efforts in combating the rising challenges of mental healthcare. They have provided researchers with low-cost high-yield ―faux‖ subjects that have aided in the development of treatments and therapies for brain disorders. Constantly, new innovations are being developed that bring researchers a greater understanding of the pathogenetic mechanisms behind the disorder, as well as clues on how to make progress towards their remedies. As new animal models are being developed on a daily basis through genetic modification or the creative use of a new species, researchers have an unprecedented opportunity to explore these disorders with the help of these new tools. This translational approach is of critical importance for the study of neuropsychiatric disorders. While the differences between the subjects of clinical research and basic research are obvious, there are many neurobiological similarities that allow for the possibility of creating an accurate animal analogue of a human disorder. Although many new treatments have been developed, there is always the need for better and more accurate models. These tools contribute to translational research and are the best hope for countering the challenges facing the field of mental health. This book is another installation in a series of books on animal models. In 2006, the book ―Animal models in biological psychiatry‖ was published, marking the first of the series. The subsequent book ―Behavioral models in stress research‖ provided further important updates on the innovative techniques and models that are on the cutting edge of research in this exciting field. While having similar scope, this book complements the other books of the series by providing chapters on a range of topics in biopsychology and behavioral neuroscience. The present book offers a rich spectrum of both innovative methodological and conceptual advances regarding investigations on sleep, depression, anxiety, as well as how these domains interact. In our opinion, this research on the interaction is particularly important in today‘s psychiatry, given the frequent comorbidity of numerous brain disorders. The chapters presented here are in-depth and diverse. Drs. Bertoglio and Carobrez‘s chapter will cover methodological and theoretical issues in modeling anxiety in animals, and provides a conceptual background for the practice of behavioral phenotyping as applied to the study of effective disorders. The practical application of this theoretical contribution can also be found in Pogorelov et al.,‘s chapter, which is a look into their laboratory‘s methodolological transformation from using the existing behavioral assays into a highthroughoutput phenotyping test battery.

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The tools to create such a laboratory are addressed in Dr. Thompson et al.,‘s chapter that expounds upon the new wave of animal observation technology. Specifically, their chapter will focus on a novel videotracking behavioral recognition system that promises to change marjedly the way that traditional neurobehavioral research is conducted. Another new direction of investigation is explored by Drs. Rainey and Sufka‘s chapter that introduces the reader to a new model of anxiety using a species not often used in behavioral research –the chick – and will address the applicability for this translational research to neuropsychiatric disorders such as depression and anxiety. Another interesting model, the unpredictable chronic mild stress model in mice, will be featured in Drs. Surget and Belzung‘s chapter. The authors review the data on this paradigm of depression and highlights how it induces features that are isomorphic to the human disease and how these alterations are induced by factors involved in the aetiology of major depression in humans. Additional factors that contribute to the pathogenesis of depression, as well as the effect on other body processes will be discussed in Dr. Kovalzon et al.,‘s chapter. These authors review their own research into the links between stress, anhedonia, and alterations in sleeping patterns in the mouse model of depression. Dr. Bolivar‘s chapter will focus on the genetics that underlie the mouse responses to stress, thereby providing a logical summary to the preceding chapters in this book. This topic emphasizes the key role played by the genetic factors in brain disorders and stress responses in general. Importantly, all authors of the book are recognized experts in the field, representing laboratories from all over the world. This makes the book a very international effort that we hope could increase its scientific value. The book is intended for an international audience of basic and clinical neuroscientists who are interested in behavioral neurophenotyping, as well as translational significance of these behaviors from animal to human subjects. The writing will also be accessible to students at the undergraduate level, while still providing an important update to graduate students and professional researchers in the disciplines of psychology, biology, and neuroscience on this rapidly developing field. Finally, we need to acknowledge the role of NARSAD (National Alliance for Research on Schizophrenia and Depression) in this book. NARSAD, the world‘s leading charity dedicated to mental health research, has an established history of supporting research on neuropsychiatric disorders. YI Award (2007-2009) from NARSAD has been pivotal to the creation of this book, and this project could not have been completed without NARSAD‘s support. The Editors take this opportunity to thank NARSAD for their support, and hope that this multidisciplinary book will be yet another contribution to advancing mental health research.

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In: Experimental Animal Models in Neurobehavioral Research ISBN 978-1-60692-022-0 Editors: A. V. Kalueff and J. L. LaPorte © 2009 Nova Science Publishers, Inc.

Chapter 1

METHODOLOGICAL AND THEORETICAL ISSUES IN EXPERIMENTAL MODELING OF ANXIETY IN ANIMALS Leandro José Bertoglio and Antônio de Pádua Carobrez Department of Pharmacology, Biological Sciences Center, Federal University of Santa Catarina, Florianópolis, SC, 88049-900, Brazil

ABSTRACT

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An animal model of anxiety is an alternative approach you can use instead of direct testing this complex emotion in humans. It is useful not only to drug discovery/development, but also to investigate the neurobiological mechanisms underlying anxiety. The assessment of anxiety-related behavior in genetically-modified animals is also conducted by means of them. Depending on the case, however, it has some drawbacks. The objective of this chapter is to review general concepts related to animal models of anxiety, and how their validity, reliability and relevance can be estimated. Rodent defensive behaviors associated with this emotion are also concisely described. Moreover, to estimate whether the current use of the animal model of anxiety matches the ideal panorama (i.e., takes the above aspects into account), we conducted a literature survey using the elevated plus-maze test as an example. Results showed that the introduction of experimental refinements have been rather slow to occur. For instance, although risk assessment measures have proven to be more sensitive to anxiety modulating drugs than traditional measures, only a quarter of studies have adopted them. It is hoped that this chapter can provide insights into pertinent methodological and conceptual issues, allowing thus a more rational use of animal tests for anxiety.



Corresponding author. Fax: +55 48 3337-5479; E-mail address: [email protected] (L. J. Bertoglio).

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INTRODUCTION In behavioral neurosciences, animal models enable investigation of brain-behavior relations, with the aim of gaining insight into human behavior and its underlying processes. Beyond doubt, the most significant information derives from the study of humans, but this is not always possible due to ethical, methodological and/or economical constraints. Alternatively, a comparative approach that relies on animal models could be used to reach these goals. This approach is based on the evolutionary theory proposing that fundamental aspects of the behavior of humans are shared with other animals [46,51]. The ground for this proposal was established by Charles Darwin in his book entitled The Expression of Emotions in Man and Animals [21]. According to van der Staay [67], an animal model is a living organism used to study brain-behavior relations under controlled conditions, with the final goal to gain insight into, and to enable predictions about these relations in humans. An additional implicit purpose is that animal models can provide a simplification of complex phenomena. Various experimental preparations have been developed to assess behavioral parameters indicating anxiety, especially in rodents such as rats and mice. They may be useful in investigating the fundamental neuronal mechanisms underlying anxiety, and may contribute to the development of new medications. The evaluation of anxiety-related behavior in animals submitted to biochemical and/or gene targeting manipulations is also conducted by means of them [19,29,35,39]. Before describing how to test/model anxiety in animals, some aspects have to be remembered. Firstly, the use of animal models stands on the assumption that anxiety in animals is comparable to anxiety in humans. Although it cannot be proven that an animal experiences anxiety in the same way as a human being, it is well accepted that some behavioral patterns in rodents indicate anxiety [47]. Secondly, in both humans and laboratory animals this emotional state is not a unitary phenomenon as it includes innate (trait) anxiety, which is considered to be an enduring feature of an individual, and situation-evoked (state) or experience-related anxiety [67]. Thirdly, modeling anxiety in animals is critically dependent on the test systems used. As the behavior of a species has been shaped during evolution, a valid test for anxiety should respect and maximize the animal natural defensive behavior. Although this may sound trivial, the ethological relevance of behavioral tasks has long been ignored in basic research [19,56]. In view of these facts, one should always consider that data derived from animal models are of value only to the extent that the models are valid, and that the severity of the disorder evoked in animals may not be the level of human disorder modeled [27,35]. One of the current challenges is therefore to optimize existing neurobiological approaches to anxiety. The present chapter considers important methodological and theoretical issues which may improve the validity, and thus provide a more rational use, of animal tests for anxiety.

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Table 1. Some frequently used animal tests for anxiety1 Name Elevated plus-maze Open-field (arena) Social interaction Exposure to predator odors (e.g. cat) Light/dark exploration Elevated T-maze Vogel conflict test Mouse defense test battery Ultrasonic (or stress-induced) vocalization Defensive burying

Reference for reviewing details [19] [53] [24] [2] [17] [29] [45] [15] [61] [22]

Table 2. Principal behavioral profiles in experimental tests for anxiety (adapted from references [35] and [49]) Avoidance Exploration Risk assessment Self-grooming Defecation, urination Aggression Others (e.g. defensive burying)

   or  (depending on the model)  or  (frequency; depending on model)   

Legend:  = increase;  = decrease.

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GENERAL CONCEPTS The behavioral repertoire of animals has long been employed to detect effects on, and impact of anxiety [15,49,54,55,64]. A number of models based on animal emotional reactivity have been designed and proven to be bidirectionally sensitive to stressful manipulations (Table 1). Many of these tests involve simple, rapid and inexpensive ways of evaluating an animal‘s condition (Table 2). The classification of experimental tests for anxiety can be based on the nature and the type of stressors employed. They can either induce (by drugs, targeted gene mutations or brain lesions/stimulations or external stressful factors) or measure anxiety, in terms of behavioral and physiological reactions [8,50,66,67]. With regard to the eliciting stimulus, experimental preparations can be categorized as being "natural" or "artificial". Whereas the former is outlined to maximize the naturally occurring defensive behavior in response to aversive stimuli with ecological meaning to the species (e.g. predator odor and open spaces), the latter employs strong and often painful stressors (e.g. shock) to elicit behaviors not normally seen in natural conditions [33]. Natural animal models allow for a reliable evaluation of a number of external factors including pharmacological agents [29,50]. Such 1

Based on ethopharmacological and neurophysiological evidence, it has been proposed that anxiety and fear are categorically distinct entities [43,44]. Further details about defensive behaviors thought to reflect fear (e.g. freezing and escape) and relevant experimental tests to study them in animals can be found elsewhere [e.g. references 13-15,29].

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ethologically based paradigms are more sensitive to stress when compared to artificial ones [55,58]. Clearly, the stressfulness of the test has to be taken into account when analyzing the behavior, as it may significantly affect behavioral performance. The present chapter will focus on the first group of anxiety tests.

BEHAVIORAL DIMENSIONS RELATED TO ANXIETY The expression of some species-specific behaviors has been shown to be related to anxiety in rodents. In this section several of them will be concisely described, and their use to increase the reliability and sensitivity of tests for anxiety will be discussed.

Avoidance It is well known that rodents tend to avoid the unprotected area of a novel environment when first entering it [7]. In an experimental setup, usually represented by a defined area, rodents will typically start to explore the environment along the walls while avoiding the open (i.e., unprotected) area. Its aversive characteristic can be modulated by illumination levels, by the height of the apparatus, and by enabling the animal to see the edge. The expression of avoidance behavior also depends on the sensorial capabilities of the animal and can further be influenced by its locomotor activity, motivational factors, and by its exploration strategy as well [47]. Besides, this defensive response is sensitive to compounds, such as benzodiazepines, that show an anxiolytic-like activity in humans and animals [2,19, 29].

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Risk Assessment When confronted with a threatening stimulus, rodents display particular patterns of response such as stretched-attend posture, which occurs when the animal stretches forward (Figure 1C) and then retracts to its original position, and is categorized as risk assessment behavior [15,58]. The biological function of these behaviors is to gather information about the potential threat by cautiously approaching the threatening stimulus or by scanning the surrounding area. Risk assessment behavior is thought to be an active defense pattern closely related to anxiety [15,19,30]. As rodents may display an increase in stretched-attend postures even when no longer avoiding an unprotected area, for instance, it has been advocated that risk assessment, thus representing the most enduring behavioral expression of anxiety, may even be more sensitive to anxiety modulating drugs than avoidance behavior [58,59,64].

Exploration Being confronted with novelty, behavior in rodents is determined by the conflict between the drive to explore the unknown area/object and the motivation to avoid potential danger. The exploratory behavior in rodents summarizes a broad spectrum of behavioral patterns such as risk assessment behavior, walking, rearing, climbing, sniffing, and manipulating objects

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[4]. Exploration is gradually inhibited by anxiety, thereby representing an indirect measurement of anxiety [9]. Notably, primary alterations in exploratory motivation may confound measures of anxiety [6], which has to be considered when behaviorally phenotyping rodents [39].

Cognition Cognitive and emotional domains interact in a fundamental manner [36,47]. As a result, learning and memory processes should be controlled especially when characterizing (genetically modified) animal models for anxiety or anxiety-modulating effects of drugs [34,36-38,47,48]. So far, however, this is not routinely done, and when it takes place, it has been limited to selected ―reference‖ memory tests [36]. This is of concern because the anxiety-memory interplay is complex and nonlinear, implying that not always altered anxiety is seen together with altered memory, as vise versa [34,47]. One promising strategy to overcome this situation may be to apply more extensively the experimental models that simultaneously profile anxiety and memory functions. This is the case of test/retest in the elevated plus-maze. As later discussed, its usefulness rests on the fact that a non-selective and/or false-positive anxiolytic-like effect of drugs can be detected [19].

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VALIDITY AND RELIABILITY ASPECTS Validity can be defined as the agreement between a test score and the quality that it measures [40]. It represents the process by which the reliability and the relevance of a method are established for specific purposes [35]. Reliability is characterized by the reproducibility of a test within and between laboratories and over time. Since numerous differences exist between laboratories, good reproducibility at least within the same laboratory has to be established [60]. Relevance reflects the meaningfulness and usefulness of results obtained with a particular animal test [67]. It has been affirmed that anxiety animal models should possess face, predictive, and construct validity [62]. This order reflects the hierarchy of the categories of validity, where the construct validity is the highest one [44]. To be a good model, it should fulfill all these three criteria at the same time. This situation, however, is not seen in animal modeling very often [20]. An animal model possesses face validity if there are resemblances between the test and the situation to be modeled. Several authors, however, consider face validity to be of limited value [40,62]. One of the main reasons is that animals have their own species-specific behavioral repertoire to survive in their habitat, and thus, there may be little resemblance between their behavior and that of humans even though similar underlying processes may guide these behaviors [44]. In relation to the predictive validity, it takes place when the result obtained in the test has some value to the process to be modeled, i.e., if it allows extrapolation of the effect of a particular experimental manipulation from one condition to others (e.g. laboratory to real world). In psychopharmacology, predictive validity usually refers to the ability of a drug screening or an animal model to correctly identify the efficacy of a putative therapeutic approach [67]. Finally, it is assumed that a certain animal model possesses construct validity if its procedures provide a sound theoretical framework [41]. In this respect,

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Darwin [21] has advocated that behavioral characteristics are acquired as a result of selective pressure exerted by evolution. As several environmental constraints are similar, many adaptations are general to species, among which stand basic emotions such as anxiety. This view therefore justifies the use of animals other than humans.

Figure 1. The (rat) elevated plus-maze is consisted of two opposite open-arms (surrounded by a small ledge), and two enclosed-arms, about 50 cm above the ground (A). Using the elevated plus-maze to measure anxiety is relatively simple: one may score the number of entries and the time spent on the open-arms (B). In addition to these spatiotemporal measures, there are more subtle postures associated with anxiety such as the stretched-attend (C). They are collectively referred to as risk assessment behaviors. An ―anxious‖ animal is one that displays risk assessment behavior very often, and rarely ventures out on the open-arms. In general, whereas anxiolytics (e.g. diazepam) increase open-arm exploration and reduced stretched-attend postures, anxiogenic drugs (e.g. pentylenetetrazole) produce the opposite effect. One possible complication that an animal might not come out because it is inherently inactive, rather than anxious, can be dealt with by scoring the number of enclosed-arms entries, an index of general exploratory activity in this test (D).

Table 3. Variables proven to influence both behavioral responses and pharmacological effects in the elevated plus-maze test (adapted from references [19], [57] and [69]) Type Organismic Procedural

Examples Species; strain; gender (estrous cycle/lactation); age. Housing condition; circadian rhythm/light cycle; prior handling and injection experience; prior stress; apparatus construction (e.g. floor surface, walls/arms color, arm width, open-arm ledges); illumination level; prior test experience; behavioral measures scored: conventional, ethological; definition/validation of measures (e.g. arm entry); method of scoring (i.e., live, manual/automated, videotape).

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PANORAMA OF CURRENT USE OF ANIMAL TESTS: DIFFERENCES FROM THE IDEAL

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The preceding sections reviewed defensive behaviors associated with anxiety, general concepts related to animal models of this emotion, as well as how their validity, reliability and relevance can be evaluated. Using the elevated plus-maze (EPM) test as an example, this section further stresses the importance of (1) identifying and controlling the major sources of variability; (2) introducing the measurement of more subtle defensive behaviors such as risk assessment; and (3) adopting other methodological refinements such as min-by-min scoring and use of a test/retest protocol. The EPM (Figure 1) stands as one of the most popular in vivo animal tests currently in use. Its popularity, with around 3400 published papers so far [71], is likely due to its obvious and numerous advantages, namely: economy, rapidity, simplicity of design and bidirectional drug sensitivity, coupled with the fact that it does not require lengthy training procedures or the use of food/water deprivation or electric shock [57]. This implies that the popularity of the EPM owes more to practical than theoretical considerations. This assumption is also perhaps valid to some extent to other experimental tests for anxiety [19,56]. The main sources of inter-laboratory variability in the use of the EPM are summarized in Table 3. Based on the fact that behavioral responses and pharmacological effects observed in the EPM are under the influence of these variables, it would be imperative that laboratories using, or planning to use, this test dedicate time and effort in order to define the optimal experimental conditions before starting their respective studies. From such a perspective, the next paragraphs focuses on the advantages of applying both ethological measures (i.e., score more subtle behaviors such as risk assessment instead of conventional only), and detailed temporal analysis (i.e., min-by-min scoring) of the rodents' behavior in the EPM. It also deals with the importance of the EPM test/retest protocol.

Refining the Information Gathered The primary indexes of EPM anxiety comprise spatiotemporal measures of open-arm avoidance (percentage of entries and of time spent in). Risk assessment is a significant behavioral dimension closely related to anxiety that has been almost entirely ignored not only in the EPM but also in other animal tests [56,58]. As previously mentioned, the biological function of these acts and postures is to inform behavioral strategies in potentially dangerous situations, therefore strengthening their measurement as a valuable tool to measure more precisely emotional reactivity in rodents [59]. For instance, while doses of 2.5-5.0 mg/kg of the serotonin 1A receptor partial agonist buspirone induces an anxiolytic-like effect on spatiotemporal measures in the EPM, coupled with a profound suppression of most active behaviors, at lower doses significant anxiolysis was observed in several ethological measures, including reductions in risk assessment behaviors such as stretched-attend postures [30]. Moreover, the utility of risk assessment measures in discriminating between anxiogenesis and sedation has also been demonstrated [72]. The anxiogenic drug 1-(3-chlorophenyl)piperazine (mCPP) reduced the time spent in the open areas but increased the occurrence of stretched-attend

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postures, supporting the conclusion that these effects of mCPP are due to anxiogenesis rather than sedation or locomotor impairment [63]. Despite the mounting evidence, a panorama of the recent literature (Table 4) shows that adoption of this methodological refinement has been rather slow to occur. The recording of risk assessment behaviors in the EPM test was introduced almost 15 years ago [19,57], but even now only a quarter of current studies have adopted this approach. One might explain this by the fact that it demands a substantially greater investment of time than conventional scoring. This argument, however, has proven to be unfounded [58,63]. In fact, it is likely that neither the min-by-min scoring nor the adoption of a test/retest protocol significantly increases the time spent on data collection. Whatever additional time investment were required for these analyses, it would be compensated for by their numerous advantages, such as the enhancement of sensitivity (and specificity) to (anxioselective) drug action and the improved distinction between general activity and emotional reactivity [11,12,30,58,59]. An interesting feature of the EPM concerns the effect of prior test experience, which usually lasts 5 min, on subsequent behavioral responses. There is growing evidence that it increases open-arm avoidance during Trial 2. This pattern of response appears to be acquired early on Trial 1, as revealed by min-by-min scoring in both mice and rats [19,32]. More specifically, up to the second min of Trial 1, roughly equal open and enclosed-arm exploration is observed, suggesting novelty/curiosity as the main motivational stimulus during this phase. As the session continues, however, rodents display a clear enclosed-arm preference, which remains in Trial 2 as well. Both risk assessment behavior and general exploratory activity, represented by stretched-attend postures and enclosed-arms entries, respectively, remained stable throughout Trial 1, suggesting that the pattern of change in open-arm exploration cannot be attributed to a general behavioral suppression. In Trial 2, the first minutes are characterized by higher levels of these risk assessment and general exploratory activity measures when compared to the end of Trial 1. This profile seems to reflect an initial and rapid re-familiarization with the EPM apparatus before resorting to the typical open-arm avoidance [19]. Besides risk assessment analysis and min-by-min scoring of the rodents' behavior in the EPM task, several laboratories worldwide have also employed a test/retest protocol. This approach has proven that prior EPM experience not only produces enduring changes in behavioral responses, but also strongly affects future drug responsiveness in this test (Table 5). The usefulness of the test-retest protocol also rests on the fact that a non-selective and/or false-positive anxiolytic-like effect of a given drug can be detected in the EPM. For example, considering that there was an increase in open-arm exploration after the systemic administration of midazolam and scopolamine in Trial 1, one could advocate that both drugs have an anxiolytic-like property. Nevertheless, while midazolam undoubtedly possesses an anxiolytic-like action [e.g. 10], scopolamine-induced learning acquisition deficit has been systematically reported using several animal models of learning and memory [e.g. 23,68]. Evidence that scopolamine given prior to Trial 1 disrupted open-arm avoidance, the usual behavioral strategy adopted throughout Trial 2 performance, is given by the min-by-min analysis. Whereas the group treated with midazolam prior to Trial 1 displayed a percentage open-arm time score similar to controls in Trial 2, the group receiving scopolamine before Trial 1 performed differently [12].

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Table 4. Behavioral measures scored during elevated plus-maze testing based on a survey a including 350 studies (25 per year/species) published since 2001 Year 2001 2002 2003 2004 2005 2006 2007 Average

Rat 85 80 60 76 84 76 72 76

Conventional onlyb Mouse 80 68 77 74 88 68 80 76

Conventional + Ethologicalc Rat Mouse 15 20 20 32 40 23 24 26 16 12 24 32 28 20 24 24

Legend: a = performed on the PubMed site (www.pubmed.com) using the entering expression ―elevated plus-maze‖. Within each year/species, the studies were randomly chosen; b = It comprises the percentage of entries and of the time spent on the open-arms; c = essentially comprising risk assessment behavior such as stretched attend postures.

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CONCLUDING REMARKS As noted by Rodgers [55], many of the inconsistencies in this area might be solved if the emphasis were directed not only to pharmacological but also to behavioral validation of animal models. Additionally, since not all robust behavioral changes seen in the experiments represent meaningful parameters for evaluation of animal anxiety, there is a need for clear-cut measures, resistant to experimental conditions or apparatus design of particular laboratories, showing reliable and predictable changes following experimental manipulations affecting anxiety [47,70]. In its normal form, anxiety can be divided into two categories: state anxiety, a measure of the immediate or acute level of anxiety, and trait anxiety, which reflects the long-term tendency of an individual to show an increased anxiety response. In its pathological form, anxiety can severely interfere with normal life. In humans, this latter has been split into six disorders, namely: generalized-anxiety, social anxiety, phobia, panic, posttraumatic stress and obsession-compulsion [1]. It seems therefore indispensable to increase the range of behavioral tests used, including animal models of state and trait anxiety, when assessing behaviors related to this emotion in rodents. Nevertheless, nearly all of the animal models of anxiety currently known rely on normal laboratory animals in acute experimental procedures. This situation favors anxioselective drug treatment predictions rather than the study of the brain mechanisms underlying anxiety disorders. A related question is whether mice with biochemical and/or gene targeting manipulations are animal models of pathological anxiety per se, because they display high level of defensive behaviors associated with anxiety in classical tests [25,26], or merely animal models of a single gene dysfunction [8,34,39]. It is still a matter of investigation (and debate) whether the use of inbred anxious strains (e.g. [28]) and/or selected lines (e.g. [31]) of rodents to provide models of anxiety that have greater validity than state ( Table 1) or single-gene deletion models of anxiety [39,49,67]. There is, therefore, an urgent need for systematic approaches to define and characterize pathological anxiety in animals [49].

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Table 5. Experimental evidence showing that prior test experience interferes with both behavioral responses and pharmacological effects of drugs in the elevated plus-maze. As observed in the test session, open-arm exploration and midazolam anxiolytic-like effect depend on the maze type used during pre-test (adapted from references [9] and [10]) Maze type used in the pre-test (5 min duration)

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1. None

Legend:

Test session in the elevated plus-maze (5 min duration) Open-arm exploration Effect of midazolam Normal range (20-30 %)

Anxiolytic

2. Elevated plus-maze

Decreased

None

3. Elevated T-maze

Decreased

Untested

4. Elevated L-maze

Decreased

Untested

5. Open-arm confinement

Unchanged

Anxiolytic

6. Enclosed-arm confinement

Unchanged

Anxiolytic

7. Open-arm + central platform confinement

Unchanged

Untested

= Open-arm;

= Enclosed-arm;

= Blocked-arm.

The full range of what can be defined as anxiety, encompass in one side, normal defensive responses to potential threat (physical or psychological), which can be analogous or homologous among species, including humans. On the other side is the atypical or exaggerated defensive behavior frequently expressed in normal unthreatened situations exhibited by subject that clearly possess some degree of genetic, developmental and/or environmental vulnerability. Thus, an improvement in test validity could be achieved when the complete defensive repertoire of animals is considered [55]. An important caveat is the fact that the defensive repertoire of animals will seldom match a specific anxiety disorder in humans. For example, when analyzing the behavioral symptoms of generalized-anxiety disorder, avoidance and risk assessment appear to be predominant, but escape reaction may also occur. Conversely, risk assessment and avoidance behaviors might arise as a consequence of panic disorder as well. Further, considering that symptoms of psychiatric disorders are often being revised and their pathogenesis revisited [16,62], additional caution is needed before claiming or using an animal model of anxiety. In view of this, one should always keep in mind that generating the perfect animal model does not represent a separate

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goal of research. Rather, the model, and its constant evolution, represents an integral part of neuropsychobiology [42]. As various forms of psychopathologies in animals and humans can be characterized as context-regulation disorders, subjects may sometimes produce normal behavior in inappropriate contexts. Thus, special analysis of behavioral contexts may be needed in the field of animal anxiety. It is also worth mentioning that animal emotional behavior is not just plus or minus, but has several dimensions including anxiety, exploration, locomotion, general arousal and coping [60]. These dimensions interact with each other as well as with cognitive functions, giving a complex mosaic picture of behavior. The traditional quantitative behavioral methods (i.e., latency, frequency and duration parameters and their spatial, temporal or sequential patterns) to study animal anxiety should therefore be combined with sophisticated analysis of "not just the presence or absence of these behaviors, but also whether or not the acts, postures and gestures are fully developed in intensity, latency and patterning" [5]. Although depression and anxiety are considered to be separate entities according to current diagnostic classifications, in clinical practice these two conditions often co-exist [52]. Whether or not this comorbidity also occurs in laboratory animals awaits further investigation [37]. The development of experimental approaches allowing an integrated measurement of behaviors related to anxiety and depression in animals may represent an important direction for future studies [36-39]. As a final point, an alternative perspective views anxiety and depression along a temporal continuum [3,18]. This implies we should also attempt to model the inter-relatedness of these clinical states in a preclinical setting. In this regard, it is noteworthy that promising experimental approaches [e.g. 65] has been developed to test this assumption.

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AKNOWLEDGEMENTS Leandro José Bertoglio and Antônio de Pádua Carobrez are supported by fellowships from CNPq, Brazil.

REFERENCES [1] [2]

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Leandro José Bertoglio and Antônio de Pádua Carobrez Belzung C (1999): Measuring rodent exploratory behavior. In: Cruzio WE, Gerlai TT (Eds.). Handbook of Molecular Genetics for Brain and Behavior Research. Elsevier, New York, pp. 77-99. Belzung C, Le Pape G (1994): Comparison of different behavioral test situations used in psychopharmacology for measurements of anxiety. Physiol Behav 56:623-628. Belzung C, Griebel G (2001): Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav Brain Res 125:141-149. Bertoglio LJ, Carobrez AP (2000): Previous maze experience required to increase open arm avoidance in rats submitted to the elevated plus-maze model of anxiety. Behav Brain Res 108:197-203. Bertoglio LJ, Carobrez AP (2002): Prior maze experience required to alter midazolam effects in rats submitted to the elevated plus-maze. Pharmacol Biochem Behav 72:449455. Bertoglio LJ, Carobrez AP (2003): Anxiolytic-like effects of NMDA/glycine-B receptor ligands are abolished during the elevated plus-maze trial 2 in rats. Psychopharmacology 170:335-342. Bertoglio LJ, Carobrez AP (2004): Scopolamine given pre-Trial 1 prevents the onetrial tolerance phenomenon in the elevated plus-maze Trial 2. Behav Pharmacol 15:4554. Bittencourt AS, Nakamura-Palacios EM, Mauad H, Tufik S, Schenberg LC (2005): Organization of electrically and chemically evoked defensive behaviors within the deeper collicular layers as compared to the periaqueductal gray matter of the rat. Neuroscience 133:873-892. Blanchard RJ, Blanchard DC (1989): Anti-predator defence behaviors in a visible burrow system. J Comp Psychol 103:70-82. Blanchard DC, Griebel G, Blanchard RJ (2003): The Mouse Defense Test Battery: pharmacological and behavioral assays for anxiety and panic. Eur J Pharmacol 463:97116. Borsini F, Podhorna J, Marazziti D (2002): Do animal models of anxiety predict anxiolytic-like effects of antidepressants? Psychopharmacology 163:121-131. Bourin M, Hascoet M (2003): The mouse light/dark box test. Eur J Pharmacol 463:5565. Boyer P (2000): Do anxiety and depression have a common pathophysiological mechanism? Acta Psychiatr Scand Suppl 406:24-29. Carobrez AP, Bertoglio LJ (2005): Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 29:11931205. Clement EY, Calatayd F, Belzung C (2002): Genetic basis of anxiety-like behaviour: a critical review. Brain Res Bull 57:57-71. Darwin C (1872): The Expression of Emotions in Man and Animals. Philosophical Library, New York. De Boer SF, Koolhaas JM (2003): Defensive burying in rodents: ethology, neurobiology and psychopharmacology. Eur J Pharmacol 463:145-161. De-Mello N, Carobrez AP (2002): Elevated T-maze as an animal model of memory: effects of scopolamine. Behav Pharmacol 13:139-148.

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[24] File SE, Seth P (2003): A review of 25 years of the social interaction test. Eur J Pharmacol 463:35-53. [25] Finn DA, Rutledge-Gorman MT, Crabbe JC (2003): Genetic animal models of anxiety. Neurogenetics 4:109-135. [26] Flint J (2003): Animal models of anxiety and their molecular dissection. Sem Cell Devel Biol 14:37-42. [27] Fuchs E, Flïugge G (2006): Experimental animal models for the simulation of depression and anxiety. Dialogues Clin Neurosci 8:323-333. [28] Gaalen MM van, Steckler T (2000): Behavioural analysis of four mouse strains in an anxiety test battery. Behav Brain Res 115:95-106. [29] Graeff FG, Zangrossi HJr (2002): Animal models of anxiety disorders. In: D'haenen H, Den Boer JA, Westenberg H, Willner P (Eds.). Textbook of biological psychiatry. Wiley, London, pp. 879-893. [30] Griebel G, Rodgers RJ, Perrault G, Sanger DJ (1997): Risk assessment behaviour: evaluation of utility in the study of 5-HT-related drugs in the rat elevated plus-maze test. Pharmacol Biochem Behav 57:817-827. [31] Hinojosa FR, Spricigo L Jr, Izídio GS, Brüske GR, Lopes DM, Ramos A (2006): Evaluation of two genetic animal models in behavioral tests of anxiety and depression. Behav Brain Res 168:127-136. [32] Holmes A, Rodgers RJ (1998): Responses of Swiss-Webster mice to repeated plusmaze experience: further evidence for qualitative shift in emotional state? Pharmacol Biochem Behav 60:473-488. [33] Kalueff AV (2003): Today and tomorrow of anxiety research. Stress Behav 8:145-147. [34] Kalueff AV (2007): Neurobiology of memory and anxiety: from genes to behavior. Neural Plast, article ID 78171. [35] Kalueff AV, Tuohimaa P (2004): Experimental modeling of anxiety and depression. Acta Neurobiol Exp 64:439-448. [36] Kalueff AV, Murphy DL (2007): The importance of cognitive phenotypes in experimental modeling of animal anxiety and depression. Neural Plast, article ID 52087. [37] Kalueff AV, Nutt DJ (2007): Role of GABA in anxiety and depression. Depress Anxiety 24:495-517. [38] Kalueff AV, Ren-Patterson RF, LaPorte JL, Murphy DL (in press): Domain interplay concept in animal models of neuropsychiatric disorders: a new strategy for highthroughput neurophenotyping research. Behav Brain Res. [39] Kalueff AV, Wheaton M, Murphy DL (2007): What's wrong with my mouse model? Advances and strategies in animal modeling of anxiety and depression. Behav Brain Res 179:1-18. [40] Kaplan RM, Saccuzzo DP (1997): Psychological Testing. Principles, Applications, and Issues. Brooks-Cole Publishing Company, Pacific Grove. [41] McGuire M, Troisi A (1998): Darwinian Psychiatry. Oxford University Press, Oxford. [42] McKinney WT (2001): Overview of the past contributions in animal models and their changing place in psychiatry. Sem Clin Psychiatry 6:68-78. [43] McNaughton N, Corr PJ (2004): A two-dimensional neuropsychology of defense: fear/anxiety and defensive distance. Neurosci Biobehav Rev 28:285-305.

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[44] McNaughton N, Zangrossi HJr (in press): Theoretical approaches to the modeling of anxiety in animals. In: Blanchard RJ, Blanchard DC, Griebel G, and Nutt DJ (eds). Handbook of fear and anxiety. Elsevier. [45] Millan MJ, Brocco M (2003): The Vogel conflict test: procedural aspects, gammaaminobutyric acid, glutamate and monoamines. Eur J Pharmacol 463:67-96. [46] Nesse RM (1999): Proximate and evolutionary studies of anxiety, stress and depression: synergy at the interface. Neurosci Biobehav Rev 23:895-903. [47] Ohl F (2003): Testing for anxiety. Clin Neurosci Res 3:233-238. [48] Ohl F, Roedel A, Storch C, Holsboer F, Landgraf R (2002): Cognitive performance in rats differing in their inborn anxiety. Behav Neurosci 116:464-471. [49] Ohl F, Arndt SS, van der Staay FJ (in press): Pathological anxiety in animals. Vet J. [50] Overall KL (2000): Natural animal models of human psychiatry conditions: assessment of mechanisms and validity. Prog Neuropsychopharm Biol Psychiatry 24:727-776. [51] Panksepp J, Moskal JR, Panksepp JB, Kroes RA (2002): Comparative approaches in evolutionary psychology: molecular neuroscience meets the mind. Neuro-Endocrinol Lett 23:105-115. [52] Pollack MH (2005): Comorbid anxiety and depression. J Clin Psychiatry 66:22-29. [53] Prut L, Belzung C (2003): The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3-33. [54] Ribeiro-Barbosa ER, Canteras NS, Cezário AF, Blanchard RJ, Blanchard DC (2005): An alternative experimental procedure for studying predator-related defensive responses. Neurosci Biobehav Rev 29:1255-1263. [55] Rodgers RJ (1997): Animal models of ‘anxiety‘: where next? Behav Pharmacol 8:477496. [56] Rodgers RJ (2007): More haste, considerably less speed. J Psychopharmacol 21:141143. [57] Rodgers RJ, Cole JC (1994): The elevated plus maze: pharmacology, methodology and ethology. In: Cooper SJ, Hendrie CA (Eds.). Ethological Pharmacology. John Willey and Sons, New York, pp. 56-67. [58] Rodgers RJ, Cao BJ, Dalvi A, Holmes A (1997): Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 30:289-304. [59] Roy V, Chapillon P (2004): Further evidences that risk assessment and object exploration behaviours are useful to evaluate emotional reactivity in rodents. Behav Brain Res 154:439-448. [60] Salome N, Viltart O, Darnaudery M (2002): Reliability of high and low anxiety-related behaviour: influence of laboratory environment and multifactorial analysis. Behav Brain Res 136:227-237. [61] Sanchez C (2003): Stress-induced vocalisation in adult animals. A valid model of anxiety? Eur J Pharmacol 463:133-143. [62] Sarter M, Bruno JP (2002): Animal models in biological psychiatry. In: D‘haenen H, Den Boer JA, Willner P (Eds.). Biological Psychiatry. John Willey and Sons, New York, pp. 47-79. [63] Shephard JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT (1994): Behavioral and pharmacological characterization of the elevated ‗zero-maze‘ as an animal model of anxiety. J Psychopharmacol 116:56-64.

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[64] Shuhama R, Del-Ben CM, Loureiro SR, Graeff FG (2007): Animal defense strategies and anxiety disorders. An Acad Bras Cienc 79:97-109. [65] Sufka KJ, Feltenstein MW, Warnick JE, Acevedo EO, Webb HE, Cartwright CM (2006): Modeling the anxiety-depression continuum hypothesis in domestic fowl chicks. Behav Pharmacol 17:681-689. [66] Uys JD, Stein DJ, Daniels WM, Harvey BH (2003): Animal models of anxiety disorders. Curr Psychiatry Rep 5:274-281. [67] van der Staay FJ (2006): Animal models of behavioral dysfunctions: Basic concepts and classifications, and an evaluation strategy. Brain Res Brain Res Rev 52:131-159. [68] Zanotti A, Valzelli L, Toffano G (1986): Reversal of scopolamine induced amnesia by phosphatidylserine in rats. Psychopharmacology 90:274-275. [69] Walf AA, Frye CA (2007): The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nat Protoc 2:322-328. [70] Wall PM, Messier C (2001): Methodological and conceptual issues in the use of the elevated plus-maze as a psychological measurement instrument of animal. Neurosci Biobehav Rev 25:275-286. [71] Web of Science (2007): According to the search performed in December 2007, using the entering expression ―elevated plus-maze‖ at the site http://isi9.isiknowledge.com. [72] Weiss SM, Wadsworth G, Fletcher A, Dourish CT (1998): Utility of ethological analysis to overcome locomotor confounds in elevated maze models of anxiety. Neurosci Biobehav Rev 23:265-271.

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In: Experimental Animal Models in Neurobehavioral Research ISBN 978-1-60692-022-0 Editors: A. V. Kalueff and J. L. LaPorte © 2009 Nova Science Publishers, Inc.

Chapter 2

A STANDARDIZED BEHAVIORAL TEST BATTERY TO IDENTIFY AND VALIDATE TARGETS FOR NEUROPSYCHIATRIC DISEASES AND PAIN Vladimir M. Pogorelov, Kevin B. Baker, Murtaza M. Malbari, Thomas H. Lanthorn, and Katerina V. Savelieva* Lexicon Pharmaceuticals, Inc., Department of Neuroscience, 8800 Technology Forest Place, The Woodlands, TX 77381, USA

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ABSTRACT A standard battery of behavior tests for mice designed for high-throughput phenotypic screening is described. In addition to pain models, the tests cover four endophenotypes relevant to psychiatric disorders: anxiety, depression, memory impairment, and psychosis/schizophrenia. The pain models, hot plate and formalininduced flinching, were validated with the reference analgesics morphine, gabapentin, and ketorolac. All psychiatric models were validated with clinically effective drugs. In order to increase throughput, some tests were optimized to shorten their duration without compromising their predictive validity. This chapter documents our success in adapting existing behavioral assays for high-throughput capacity. Our experience may be a useful guide for other investigators setting up their own test batteries, and also serve as a point of comparison between assay variants.

Keywords: Knockout mice, behavior test battery, predictive validity, novel drug targets

*

Corresponding author: Katerina V. Savelieva, Tel.: 1-281-863-3603; Fax: 1-281-863-8098; E-mail address: [email protected]

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1. INTRODUCTION We have undertaken a genome-wide effort to identify, evaluate, and prioritize genes for neuroscience drug discovery. Our strategy is to use genetic inhibition of potential drug targets in knockout (KO) mice to model the action of pharmacological antagonists. By identifying therapeutic phenotypes revealed by genetic analysis we can select targets for drug discovery whose modulation is highly likely to result in safe and efficacious agents so long as potent and specific modulators can be identified (West et al., 2000; Walke et al., 2001; Abuin et al., 2002; Friddle et al., 2003; Zambrowicz et al., 2003a; Austin et al., 2004). The KO mice are derived from embryonic stem cells mutated either by retroviral insertion or homologous recombination and then screened for phenotypes indicative of potential for drug development. We have designed a standard battery of tests for an initial high-throughput phenotypic screen that incorporates assays selected for their ability, as a set, to probe a wide swath of animal behavior that has been associated with CNS disorders and their treatment by pharmaceuticals. Additionally, the individual assays and the collection of tests as a whole have been selected, and modified where necessary, for their short duration and equipment requirements to allow assessment of large numbers of mice with relatively modest effort. The tests are also carefully sequenced to maximize the information that can be gained from a given cohort of mice without introducing inter-test influences. The battery covers general behavior and health as well as behaviors related to anxiety, depression, schizophrenia, learning and memory, nociception, and sleep disorders. If a knockout line scores with a potentially therapeutic phenotype in the first pass test battery, the finding is followed up by application of more time-consuming secondary assays as well as repetition of the original assay on larger cohorts of animals in order to confirm and expand upon the initial observation. A behavioral test as an animal model of a human condition must show adherence to a criterion called validity (Willner, 1984). It may be symptomatic similarity between the model and the illness (face validity), similarity in pharmacological responses to pharmacological therapies (predictive validity) or similarity in the etiology and disease mechanism (construct validity). Accordingly, animal models are ―validated‖ against any of these criteria, which may be independent of each other. Since the mechanism of psychopathology is often not known, construct validity is frequently inaccessible in behavioral models. This is especially true for diseases with complex genetics such as depression, schizophrenia, anxiety disorders, etc. On the other hand, psychiatric disorders are classified as clusters of symptoms with alterations in mental processes that are unique attributes of humans. Species differences in the manifestation of a particular internal state also contribute to the complexity of the issue. These considerations have led to the concept of the endophenotype in psychiatry and human genetics in order to facilitate investigation of psychiatric disorders (Gottesman and Gould, 2003; Gould and Gottesman, 2006; Cryan and Slattery, 2007; Einat, 2007). Endophenotype refers to a set of behavioral or physiological characteristics that constitute a core process or function that is abnormal in a clinical population rather than a specific set of symptoms that are part of the clinical diagnosis. The endophenotype approach has two clear advantages over previous attempts to encompass ―disease states‖. First, it allows for incorporating species-specific manifestations of the core process being modeled. Second, it makes screening for genetic abnormalities more comprehensive because the genetic factors

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associated with the discrete process are much more limited compared to those associated with the whole syndrome. In this chapter we describe our experience working with animal models targeting certain endophenotypes in psychiatric disorders and our attempts to adapt existing behavioral assays for high throughput capacity. In particular, we will describe assays for endophenotypes related to the following disease indications:

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   

Anxiety – increased inhibition as reaction to threat, both diffuse and cued, Depression – adoption of immobility in an inescapable situation, Dementia – impairment in reference memory, Schizophrenia/psychosis– impairment in sensorimotor gating.

With respect to anxiety, one of the approaches to modeling anxiety-like behaviors involves the cross-species generality of defensive reactions to threat. In rodents, anxiety states cause an arrest of ongoing activity resulting in ―freezing‖ behavior or immobility. In this context, the light-dark test takes advantage of inhibition of normal exploration of a brightly-lit area. Conflict tests like Vogel‘s suppressed drinking also make use of interruption of reinforced behavior by aversive stimulation. By contrast, fear potentiated startle is based on unconditioned reflex potentiation by a fear conditioned stimulus. Startle as well as freezing response is well-conserved across species. Depression is a heterogenic, multifaceted disorder with symptoms manifested at the psychological, behavioral, and physiological levels (American Psychiatric Association, 1994). This is why it is often difficult to assign animal models to the human condition of depression. For instance, immobility in a swimming tank was initially conceived of as ―behavioural despair‖ by Porsolt et al., (1978). Other views were that immobility is an attempt at conserving energy (Nishimura et al., 1988) or an evolutionary preserved coping strategy where immobility represent the psychological concept of ―entrapment‖ described in clinical populations (Thierry et al., 1984). Rodent models of depression are mainly based on predictive validity, objectivity of measured response and their high reliability and reproducibility within and between laboratories. Validation with clinically proven antidepressants provides a compelling means for vetting these models and provides way to gauge measured responses. Sensorimotor gaiting deficits are a marker for schizophrenia-spectrum disorders in humans (Braff et al., 2001) and have construct validity. Prepulse inhibition (PPI) is widely used as an animal model for sensory gaiting abnormalities and refers to normal reduction in the magnitude of the startle response to an intense stimulus when that stimulus is preceded by a prepulse. Sensorimotor gating is one mechanism by which an organism filters information from its surroundings. Schizophrenic, schizotypal, and obsessive-compulsive disorder patients have lower PPI than healthy control subjects. Disruption of PPI can be caused by psychostimulant drugs like amphetamine or psychotomimetics like non-competitive NMDA antagonist PCP that elicit psychotic-like symptoms in normal volunteers or exacerbate psychosis in schizophrenic patients. The Morris water maze (WM) is a test for spatial learning and memory. Rodents must have abilities for encoding, storage and retrieval of spatial information to successfully navigate in a pool. In the cued version of WM an animal learns to escape from the water by

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Vladimir M. Pogorelov, Kevin B. Baker, Murtaza M. Malbari et al.,

locating a platform that is visible. The swimming pool place task requires the animal to learn the location of a platform relative extramaze cues in the room and form a spatial map of the location (O‘Keefe and Nadel, 1978). It is thought that performance in these two versions reflects the same motor, motivational, and sensory processes; but differs in the nature of associative or memory processes. Morris et al., 1982 showed that place navigation task but not cued learning is disrupted by hippocampal damage. There has been a tendency to extend this relation to components of human memory system taxonomy that has been linked to hippocampus through clinical work. For instance, place versus cued goal learning could map onto episodic versus semantic types of memory. Unfortunately, a supporting set of results, methods and memory task analyses does not exist for rodents. Nevertheless, controlling noncognitive behaviors like thygmotaxis or spiral search strategies makes the test an elegant tool to assess learning and memory. Thus, transgenic mice expressing beta-amyloid precursor protein exhibit age-dependent deficits in place learning and in probe trials (without platform) but not in visible platform (cue) learning (Moran et al., 1995). Some aspects of the declarative memory that are severely compromised in patients with temporal lobe lesions or early-stage Alzheimer‘s disease (Squire, 1987) may, therefore, tap into spatial learning/memory measurements in the water maze. Because of global impairment and neurochemical correspondence with Alzheimer‘s disease scopolamine administration may be an attractive model of cognitive decline. Scopolamine was reported to produce acquisition and performance impairments in the hidden platform task (Buresova et al., 1986). Finally, the hot plate test for thermal sensitivity and the formalin test for tonic pain were included in the battery. The formalin test is automated and the hot plate test has proved to be quick and reliable in our hands. Both tests are easy to perform by trained personal and suitable for high-throughput screening. As we have established the assays mentioned above in our laboratories we have been mindful that differences in behavior are often observed by different groups at different sites (Crabbe et al., 1999). While one approach to addressing this issue is to standardize the assays used, this is not always successful. Therefore pharmacological validation of behavioral tests in benchmarking experiments using known drugs is required when establishing a behavioral model assay. Here we present pharmacological validation results for assays commonly used in our test battery.

2. MATERIAL AND METHODS Male and female littermates bred in a mixed (albino) C57BL/6J-Tyrc-Brd x 129S5/SvEvBrd genetic background were used for all behavioral studies unless otherwise noted (Zheng et al., 1999, Zambrowicz et al., 2003b). C57BL/6J is a standard and well studied mouse line, perhaps one of the most employed inbred strains available; therefore, its genetics are supported by numerous studies (e.g., Tucker et al., 1992; Slingsby et al., 1996; MGI, 2007). C57 Black mice are suitable for many laboratory procedures and reproduce reliably (Morse, 1979). The albino variant (Zheng et al., 1999) allows easy recognition of chimeras because they will have dark pigment in certain areas (contributed by stem cells from 129S5/SvEvBrd). Such visual screening provides an easy and reliable means to identify mice that need to be tested for use in transmitting the introduced mutation to a new generation, and the eventual creation of animals that are homozygous for the null mutation.

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129S5/SvEvBrd was used as a source of embryonic stem cells because of its reliability in the production of tractable embryonic stem cells (Simpson et al., 1997). The earliest studies of knockout mice employed similar inbred strains (Koller et al., 1989), making the choice of ES cell donor, and recipient, obvious for a high throughput operation. Moreover, the work by Simpson et al., (1997) provided a clear understanding of the various 129 strains, as well as a foundation for intelligent selection of the 129S5/SvEvBrd substrain. Mice were maintained under a standard light/dark cycle from 7 am to 7 pm. They were housed in groups of five in 30x20x20 cm acrylic cages with food and water freely available. All mice were 9-12 weeks old and weighed 25-30 g at the time of testing. After being transferred from vivarium facility mice were always acclimated to a holding room for 1 hour before testing. All animal studies were approved by The Lexicon Pharmaceuticals Institutional Animal Care and Use Committee. Primary screening assays in our test battery include Circadian activity evaluation, Open Field Activity, Stress-Induced Hyperthermia assay, Inverted Screen, Functional Observational Battery (FOB), Hot Plate, Prepulse Inhibition of the Acoustic Startle Response (PPI), Tail Suspension (TS), Trace Fear Conditioning and Formalin paw test. Since 2000, our research group has looked at behaviors of more than 4000 different strains of genetically-engineered mice by using our primary behavioral test battery (Table 1).

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Table 1. Comprehensive behavioral test battery (Primary Screen) Test order

Measurement

(1) Circadian home cage monitoring

24 h activity in home cage, circadian rhythm

(2) Stress-induced hyperthermia

Rectal temperature, anxiety-like behavior

(3) Open field Activity

Locomotor activity in the novel environment; anxiety-like behavior

(3) Inverted Screen

Muscle strength and coordination

(4) Marble burying

Anxiety-like and depression-like behavior

(5) Functional observational battery/FOB

Body weight, whisker, coat, simple reflexes

(5) Hot Plate

Nociception (acute pain)

(6) Prepulse inhibition/startle response

Sensory-motor gating, hearing, startle

(7) Tail suspension test

Depression-like behavior

(8) Trace fear conditioning

Learning and memory

(9) Formalin paw

Nociception (acute and tonic pain)

We conduct the primary screen tests in the order listed, using 8 mutant mice and 8 wild-type littermates for control (wild type population pooled data is also used in determining significance of effects from primary screen results). Each experiment is done on separate days (indicated in brackets before each assay), except that Open field test is followed by Inverted screen assay; and FOB is followed by Hot Plate test. Circadian testing duration in primary screen is three light-dark cycles and carried out only in female mice since males cannot be placed back together to original group housing after 3-days separation period.

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Selection of follow-up assays is based on the specific indication determined during the primary test battery, and a new cohort of naïve mice is tested in the selected assays (Table 2). Follow-up assays include Forced Swim Test (FST) for depression, Platform (Light/Dark) Test, Fear Potentiated Startle Assay (FPS) and Vogel Test for anxiety, Delay Fear Conditioning, Novel object recognition, social recognition and the Morris Water Maze (WM) for learning and memory, Latent Inhibition (LI) and Conditioned Avoidance response (CAR) for psychosis, and the social interaction test for diseases that have social deficits as one of the symptoms (i.e., autism spectrum disorders and schizophrenia). Only assays for which we present pharmacological validation results in this chapter are described below in detail. While pharmacological validations of each assay were run on separate cohorts of mice the order and choice of the tests for screening purposes is usually from less to more stressful and according to the primary phenotype. For example, for follow-up testing of an anxiety phenotype, the platform test is run first, followed by open field, FPS or Vogel test. The open field test is a prerequisite for testing memory-associated phenotypes, psychotic-like or depressive-like behaviors, as these involve a motor component. The WM is run 2 weeks after trace fear conditioning experiment if there are significant alterations in freezing to context or tone. We did explore the effects of a specific test order to make sure that tests applied at the beginning of the battery do not confound the tests that follow up. For example we found that the order in which tail suspension and forced swim tests are applied do not affect results (data not shown). Finally, the pain assays if applicable are usually left to the end of a battery. Table 2.Follow-up assays are selected based on the therapeutic indication. See text for further details

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Secondary Behavioral Screening Battery

Cognition

Depression

Anxiety

Trace Fear Conditioning

Tail Suspension

Platform Light/Dark Test

Prepulse Inhibition

Hot Plate

Fear Potentiated Startle

Latent Inhibition

Formalin

Porsolt Forced Swim Test Novel Object Recognition

Morris Water Maze

Vogel test

Schizophrenia

CAR

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Pain

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2.1. Nociception Assessment Formalin Test The mice were tested for nociception with Automatic Nociception Analyzers (purchased from the Ozaki lab at University of California, San Diego). A metal band was placed around the left hind paw of each mouse with superglue 30 minutes prior to testing. After the 30minute acclimation period, 20 µl of 5% formalin was subcutaneously injected in the dorsal surface of the left hind paw. Mice were individually housed in cylindrical chambers for 45 minutes. A computer software recorded flinches per minute, total flinches for Phase I (acute phase = first 8 minutes), and total flinches for Phase II (tonic phase between 20 - 40 minutes) through an electromagnetic field (for details see Yaksh et al., 2001). Morphine was injected at 5 and 10 mg/kg, gabapentin was injected at 50 and 200 mg/kg, and ketorolac was injected at 10 and 50 mg/kg 30 min prior to formalin injection. Hot Plate Assay The Hot Plate Assay was performed using a Columbus Instruments Hotplate Analgesia meter (model 1440). The hot plate was set to 55 C and controlled to stay within 0.1 C. The size of the heated surface area was 10‖ x 10‖ x 0.75‖. The response time for each animal (hind-paw flinching or licking) was automatically recorded by a computer when an experimenter blind to genotype of the animals being tested pressed the stop button on the chamber. Morphine was injected at 5 and 10 mg/kg, and gabapentin was injected at 10 and 50 mg/kg 30 min prior to testing.

2.2. Anxiety Assessment

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Platform Test (an analog of the Light/Dark test) is described in Pogorelov et al., 2007.

Fear Potentiated Startle Assay (FPS) Fear potentiated startle is assessed using acoustic startle chambers (SR-LAB, San Diego Instruments, San Diego, CA, USA) for mice as described in the PPI section below. Highfrequency speakers mounted above the cylinders produced all acoustic stimuli. Scrambled constant-current foot shocks were delivered through a cradle-shaped grid mounted on the floor of the cylinders. The protocol consisted of two phases. During the training a mouse was placed in the cylinder for 5 min of acclimation. Following acclimation, mice received 30 training trials with an average ITI of 110 sec (range: 90-130 sec). These trials consisted of a 30 sec 85 db tone co-terminating with a 0.5 sec 0.2 mA foot shock. During training the chamber lights were on. The test session began 24 hours after training. Mice were placed in the cylinder for a 5 min acclimation period followed by delivery of 20 startle pulses (105 db white noise bursts 40 msec in duration), with half of the pulses presented during the 30 sec 85 db tone cue and the other half without the cue present (no tone). The trials were presented in a pseudorandom order at an average ITI of 110 sec. During the test the chamber lights were off to reduce effect of the training context on FPS expression. Percentage of FPS was calculated for each animal using the following formula: %FPS = [(mean cue trial startle response – mean

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no-cue trial startle response)/(mean no-cue trial startle response)] x100. Chlordiazepoxide was injected at 6 and 12 mg/kg 30 min prior to the test session.

Vogel Test The testing apparatus consists of Coulbourn Instruments test chambers. Each chamber is equipped with a metal grid floor and a waterspout protruding from one of the walls. The water bottle in the chamber is filled with fresh water. The spout is connected to a drinkometer while the grid floor is connected to a shock generator. A computer program counts the number of licks the mouse makes. During the test session the subject gets a mild shock (0.2 mA for 0.1 sec) every time it completes a specific number of licks. Mice are water deprived 24 h before the start of experiment. On Day 1 mice are placed into experimental chambers for 30 min with water available from a drinkometer. One hour after the session mice receive water in the home cages for 60 min. The next day (Day 2) the mice are exposed to the chambers with drinking tubes for 40 min to provide additional habituation. Animals that score less than 100 licks on Day 2 are excluded from further testing. On this day mice do not receive water in the home cages after the experiment. The conflict procedure is performed on Day 3. Mice are allowed to make 20 unpunished licks to start the session. If a mouse does not score 20 licks within the first 10 min of the test session it is excluded from further testing. After the initial 20 licks are made a mouse receives a mild shock (0.2 mA for 0.10 sec) through the grid. Shocks are delivered every 20 licks afterwards, and the number of licks is recorded. The session is terminated 20 minutes after the first 20 licks are completed. Chlordiazepoxide was injected at 12 mg/kg 30 min prior to testing in the conflict procedure.

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2.3. Depression Assessment Forced Swim Test (FST) Three glass cylinders (30 cm in height, 18 cm in diameter) were filled with 25 cm of room temperature water. An automated VideoTrack system for Forced Swim (ViewPoint Life Sciences, Inc., Montreal, Canada) was used to track animals‘ movements in the water. The thresholds for the tracking software were set by the experimenters based on the following definitions for behavior parameters. Immobility was defined as when a mouse was only making movements required to keep its head above the water. Struggling was defined as vigorous movements with all 4 limbs breaking the water, usually against the walls. On the first day mice were individually placed in the cylinders for a 15 min period (pre-test phase) and behaviors were recorded for the first 5 min. At the end of this phase each mouse was removed from the water, dried with a paper towel and placed back into the home cage. The next day the mice were exposed to the same experimental conditions for a 5 min period (test phase). Water was changed between animals. Fluoxetine or imipramine was injected 3 times: right after the first 15 min session, 6 h later, and 1 h before the testing on day 2. Tail Suspension Test (TS) The tail suspension test for depression-related behavior was conducted using chambers from Med Associates (PHM-300TSS Mouse Tail Suspension System, Med Associates, Georgia, VT). The test cubicle is made of ½‖ white PVC with inside dimensions of 13‖ x 13‖

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x 12.5‖. The mouse was securely fastened with medical adhesive tape to a metal hook by the tip (~1.0-1.5 cm) of the tail and suspended above the floor in a visually isolated cubicle. Mobility was recorded by a precision linear load cell, load cell amplifier, and filter with the gain set at 4. Immobility was defined as the area under the curve using an immobility threshold of 2. The total duration of immobility over a single 6 min session was recorded for each mouse. Animals that climbed their tails during testing were excluded from data analysis. Fluoxetine or imipramine was injected 1 h before testing.

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2.4. Psychosis and Schizophrenia Assessment Acoustic Startle and Prepulse Inhibition of the Startle Response The apparatus consisted of acoustic startle chambers for mice (SR-LAB, San Diego Instruments, San Diego, CA, USA). The SR-LAB cylindrical animal enclosure monitors animal movements with an ultra-stable, hermetically sealed motion sensor using full 12 bit resolution (a range from zero to over 5000 millivolts) for accurately measuring the wide range of startle responses. The tubular design of the animal enclosure ensures that the animal remains centered over the sensor for consistently reliable results. Animals were individually placed into enclosures inside the sound-attenuated startle chambers and acclimated for 3 min. PPI was assessed in a test session lasting for 15 min, in which the subjects were presented with a series of discrete trials comprising a mixture of six types of trials: startle pulse (120 dB, for 40 ms), no stimulus (70 dB background noise), or prepulse stimulus of one of four sound levels (74, 78, 82 or 90 dB) for 20 ms, followed 100 ms later by an acoustic startle (120 dB for 40 ms). A total of 6 trials under each condition were delivered in a random sequence and all trials were separated by a variable inter-trial interval of 10–30 s. The maximum response to the stimulus (V max) was averaged for each trial type. In the startle alone trials, the basic auditory startle (startle response) was measured. Animals with an average startle response value equal to or below 100 were excluded from PPI analysis. In ―no stimulus‖ trials a baseline measure was taken to assess movement in the enclosure under no stimulation. In the prepulse plus startle trials, PPI was calculated as a percentage score for each acoustic prepulse trial type using the formula: % PPI = 100−[((startle response for prepulse + pulse)/(startle response for pulse alone))] x100. Phencyclidine (PCP) and amphetamine were injected 15 min prior to the experiment and risperidone pretreatment was 30 min before testing.

2.5. Dementia Assessment Morris Water Maze The set up is a circular pool that is 2 meters in diameter and 40 cm in depth (Accuscan Instruments, Inc., Columbus, OH) and a WaterMaze Video Tracking System (Actimetrics, Inc.,Wilmette, IL) with a camera suspended from the ceiling. The pool was filled to a depth of 30 cm with water at a temperature of about 24 – 26 degrees Celsius. In order to hide the visibility of the escape platform, the water was made opaque by the addition of non-toxic water based paint. The escape platform was then placed about 1 cm below the water surface

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in the middle of one of the pool quadrants (N, S, E, or W), designated as the test quadrant. There were 2 learning phases and 1 probe trial. During the first phase, also known as the visible phase, the platform was made visible with a conical tube placed into a cylinder which was placed on the platform. The maze was surrounded with curtains in order to hide all external cues. The mouse was released into the pool facing the wall of one of the quadrants adjacent to the platform. The trial ended as soon as the mouse climbed onto the platform, and/or 90 seconds had elapsed. If the mouse did not reach the platform in the 90 seconds, the experimenter guided it to the platform. At the end of each trial, the mouse was left to rest on the platform for 10 seconds. This phase had 2 trials per day for 3 days. The inter-trial interval was 10 to 15 minutes. During the inter-trial interval the mouse was placed into the holding cage under a heat lamp until its next trial to dry and to prevent hypothermia. During the hidden platform phase the platform was no longer marked and remained hidden below the water surface, the curtains were removed and extra-maze cues (rectangular pieces of paper with striped patterns) were fixed to the walls. The same procedure was followed on each trial as stated for the visible phase. This phase had 2 trials per day for 5 days. The probe trial occurred 72 hours after the last hidden training trial. During the probe trial, the platform was removed from the pool, and the mouse was placed in the pool facing the wall in the quadrant opposite to the training quadrant. The duration of the probe trial was 60 seconds and the percentage of time spent in each quadrant was recorded. Latency time to reach the platform, swim paths and velocity were calculated by the WaterMaze image software (Actimetrics, Inc.).

Drugs Morphine hydrochloride, Gabapentin, Ketorolac (tris salt), Chlordiazepoxide hydrochloride, Fluoxetine hydrochloride, Imipramine hydrochloride, D-amphetamine sulfate, phencyclidine (PCP) hydrochloride, and Scopolamine hydrochloride (all from Sigma, St. Louis, MO) were dissolved in sterile water. Risperidone (Sigma) was first diluted in a drop of 50% HCl, and then in PBS. All drugs were administered I.P. in a volume of 10 ml/kg. All drug doses were chosen based on published literature. Fresh 5 % formalin solution was prepared by diluting formaldehyde (Formalde-fresh 20%, Fisher Scientific, Fair Lawn, NJ) with distilled water. Statistical Analysis The STATISTICA 7.0 software package (StatSoft, Inc., Tulsa, OK, USA) was used to determine significant differences between groups. Repeated measures ANOVA or one-way ANOVA with drug dose as a main effect were performed on each data set followed by posthoc tests when significant effects of dose or dose x repeated measures interaction were determined.

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Standardized Behavioral Test Battery to Identify and Validate Figure 1 Targets … Formalin paw test, Morphine

Sum Paw Flinches

A

550 500 450 400 350 300 250 200 150 100 50 0

Control (n=9) 5 mg/kg (n=10) 10 mg/kg (n=10)

*

**

Phase 1

B

Sum Paw Flinches

Formalin paw test, Gabapentin 550 500 450 400 350 300 250 200 150 100 50 0

Control (n=16) 50 mg/kg (n=16) 200 mg/kg (n=15)

**

* **

Phase 1

**

Phase 2

Formalin paw test, Ketorolac

C

Sum Paw Flinches

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Phase 2

550 500 450 400 350 300 250 200 150 100 50 0

Control (n=10) 10 mg/kg (n=8)

*

Phase 1

Phase 2

Figure 1. Effects of systemically delivered morphine (A), gabapentin (B) and ketorolac (C) on paw flinching in phase 1 and phase 2 of the formalin test. * - p