Animal models of anxiety

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Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA

Nervous system disorders

Animal models of anxiety Mathias V. Schmidt*, Marianne B. Mu¨ller Max Planck Institute of Psychiatry, Munich, Germany

Anxiety-related disorders are a leading cause for decreased quality of life worldwide. Treatment options are sparse and the molecular mechanism of anxiety is

Section Editor: Wolfgang Wurst – Institute of Developmental Genetics, GSF Research Centre, Neuherberg, Germany

poorly understood, underlying the need for good animal models of anxiety. Here a concise overview about different approaches is given, including genetic manipulation, strain differences, bidirectional breeding and environmental interventions. Limitations of the different approaches and their best use in anxiety research will be discussed. Introduction Pathological anxiety is one of the key symptoms of human affective disorders, that is, depressive and anxiety disorders [1]. As anxiety is a highly conserved behaviour among all kind of species, testing for anxiety can be reliably achieved in specific and standardized behavioural paradigms in rodents to learn more about the neurobiological mechanisms underlying pathological anxiety in humans [2]. Ideally, an animal model should mimic the human condition of interest with respect to its aetiology, symptomatology and treatment (construct, face and predictive validity) [3]. However, fully meeting such requirements is infeasible in the context of complex psychiatric disorders, where the presence of some of the cardinal features (e.g. anticipatory anxiety, suicidal ideation, feelings of worthlessness and guilt) is defined by a subjective verbal report. Testing for anxiety as a key symptom of affective disorders in animals, therefore, is based on recent neuroscientific approaches which rely on mimicking specific key signs or symptoms rather than mimicking an entire syndrome [4]. *Corresponding author: M.V. Schmidt ([email protected]) URL: http://www.mpipsykl.mpg.de/ 1740-6757/$ ß 2006 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmod.2006.11.002

Anxiety characterizes an emotion, which is only described in higher mammals and humans. As a consequence there are no in vitro or in silico anxiety models available. In animal research, rodents are the most widely used animals of choice. It is therefore logical that most animal models of anxiety have been developed with the use of rats or mice. In this review we will give an overview of the different models of anxiety. Models of genetic manipulation, strain differences, bidirectional breeding and environmental interventions will be addressed, highlighting some prominent examples and their implications for anxiety research. Furthermore, the limitations of the different approaches and their best use will be discussed, to guide readers in the selection of an animal model for a specific research propose.

Genetically engineered mice Mice with a genetic modification are in general used to study the function of a specific gene of interest in the context of anxiety. The interest in the gene is often based on previous knowledge, gathered, for example by pharmacological studies or results obtained in humans. In other cases alterations of anxiety-related behaviour is a more or less incidental co-finding and was neither intended nor expected. In the literature there are numerous reports of genetically modified mouse lines with reported differences in anxiety [5]. Although these mouse lines are valuable in the understanding of molecular mechanisms of anxiety, many need to be more extensively validated before they could be treated as a general model for anxiety. Here we will discuss mouse models with a genetic modification of the 369

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CRH system or the serotoninergic system, which both have been extensively studied and validated as anxiety models.

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that low levels of this receptor may represent a risk factor for anxiety-related disorders.

Future directions of genetic manipulation CRH and anxiety In addition to its well-known endocrine effects, CRH (GenBank accession no. BC119036) has been shown to act as a neurotransmitter or neuromodulator in the brain, orchestrating the response of bodies to stress on various levels [6]. In particular, CRH has been implicated in the control of anxietyrelated behaviour [7]. Two mouse lines lacking the main receptor for CRH in the brain, the CRH receptor 1 (CRHR1; GenBank accession no. BC103675), have been independently generated and tested [8,9]. Both lines displayed a reduced anxiety in several anxiety tests, such as the dark-light box and the elevated plus maze. Further studies with these lines also supported and extended the initial findings and underlined the importance of the CRH–CRHR1 system in anxiety [10– 12]. As conventional gene knockouts may be confounded and possibly misinterpreted by compensatory effects more recent strategies of genetic manipulations apply conditional gene deletions, which are time and region specific. Consequently, forebrain and limbic system specific CRHR1 deficient mice have been developed by using the well-known Cre-lox system [13]. Interestingly, these mice display essentially the same reduced anxiety phenotype as conventional CRHR1 knockouts, giving further support for the important function of central limbic CRHR1 in mediating anxiety-related behaviour. In contrast to the robust behavioural alterations of the various CRHR1 deficient mouse lines, so far no significant differences were found in the autonomous anxiety response in these animals [14].

5-HT and anxiety Among the various described 5-HT receptor subtypes the 5-HT1A receptor (GenBank accession no. NM008308) has been the main serotonin receptor implicated in fear and anxiety. Based on the earlier findings that partial as well as total agonists of the 5-HT1A receptor have anxiolytic properties, in 1998 three independent groups published the generation of 5-HT1A knockout mice [15–17]. Even though the mutant mouse lines were generated on different genetic backgrounds, all three lines displayed an increased anxietyrelated behaviour in several standard anxiety tests. Importantly, subsequent tests with these mice also revealed increased vegetative responses to mild stressors, such as enhanced heart rate and blood pressure, indicating that the increased behavioural anxiety in these animals is paralleled by changes in autonomic activity [18,19]. Taken together with reports of a 5-HT1A receptor deficit in several different affective disorders, among which post-traumatic stress disorder, panic disorder and major depression [20–23], the data obtained in 5-HT1A deficient mice indicate 370

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Many of the currently available genetic animal models of anxiety deactivate or over-express the gene of interest in the whole body or at least in a large number of functionally different tissues or structures. As a consequence, the data obtained with these models can be flawed owing to compensatory mechanisms, which occur over time or between different regions. Novel technical advances such as RNA interference hold the possibility to modulate gene expression in the very time and region specific manner, thereby minimizing side effects.

Strain differences These approaches for models of anxiety utilize the vast amount of different rat and especially mouse lines that are available for research purposes. Some of the more generally used strains in anxiety research include C57/Bl6, BALBc, 129S and DBA/2, although many more strains (inbred and outbred) have been tested for anxiety-related behaviour. Several studies have described pronounced behavioural and autonomic differences of anxiety responses in the various mouse strains, including inbred strains (e.g. C57/Bl6, A/J, BALB/c, DBA/2, C3H or 129) as well as some outbred strains (e.g. Swiss Webster or NMRI) [24–29]. Although there are some inconsistencies regarding the reported anxiety levels of some strains, several more prominent strains have been extensively validated as high anxiety (e.g. DBA/2) or low anxiety (e.g. C57/Bl6) strains [30]. However, even with those well validated strains variable results regarding anxietyrelated behaviour can be obtained, depending on confounding factors such as the sub-strain, the litter experience of their mother, the animal provider, transport conditions to the laboratory, time of recovery before the test or the specific test conditions (e.g. illumination). Nonetheless inbred mouse strains have been widely used in anxiety research, predominantly for the validation of anxiolytic or anxiogenic compounds. In addition, recent approaches using either chromosome substitution strains (CSS) or recombinant inbred strains (RIS) have opened up new possibilities to study complex genetic traits such as anxiety [31–33], which will surely advance our understanding of the genetic factors modulating anxiety in the coming years.

Bidirectional breeding of phenotype extremes The strategy of bidirectional breeding is to select genetic factors influencing anxiety-related behaviour in two extreme phenotype lines. This approach is based on the genetic variability of outbred mouse or rat strains and uses a specific selection criterion. Numerous of these genetic models have been described in the literature over the past years, some of which

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have specifically focused on an anxiety-related selection criterion [34–36]. The observed anxiety differences in these models is thus not based on an external stimulus, but represents an innate predisposition to express high or low levels of anxiety (trait). The high anxiety behaviour (HAB) and low anxiety behaviour (LAB) rat lines are a prominent example, which has been extensively validated as a genetic animal model of anxiety [37]. Here the time spent in the open arm of an elevated plus maze is taken as a selection criterion. After a few generations, an average of less than 5% of the test time on the open arms of the elevated plus maze was spent on breeding HAB animals, whereas for LAB animals the open arm time averaged around 50%. The anxiety phenotype of these two rat lines was further validated by several behavioural and pharmacological approaches [38]. Recently, the single nucleotide polymorphism (SNP) in the promoter of the vasopressin gene (GenBank accession no. BC051997) could be shown to be an underlying genetic basis of the difference in anxiety-related behaviour between the two rat lines [39,40]. This example demonstrates that bidirectional breeding approaches offer the possibility to identify novel anxietyrelated target genes, which can later be extensively studied by using, for example genetic manipulations.

Environmental or pharmacological intervention Various paradigms and treatments (perinatal and adult) have been shown to alter anxiety-related behaviour (acutely or persistently). These models are fundamentally different from the above-mentioned approaches, as they are based on state anxiety rather than trait anxiety. Most of the used paradigms increase fear or anxiety in the animals, whereas few are able to decrease anxiety-related behaviour. Several prominent examples will now be discussed.

Perinatal manipulations Pre- or postnatal paradigms involving either a pharmacological treatment, some kind of stressor or a disturbance of mother–pup interaction have been described to permanently alter anxiety-related behaviour [41–45]. Unfortunately the vast amount of methodologically and also conceptually different approaches makes the interpretation of the sometimes conflicting data rather difficult [46]. One of the oldest and perhaps most reliable procedures, which alter anxiety-related behaviour during adulthood, is the early handling paradigm. Here, rat or mouse pups are separated from their mothers for 15 min per day over the course of their first three weeks of life whereas control animals remain undisturbed [47]. This procedure has been consistently shown to decrease anxiety-like behaviour in several different test situations [48–52]. However, the molecular mechanism underlying this persistent effect on anxiety is still unclear. Differences in maternal care, which also result in differences of anxiety during adulthood,

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have recently been suggested to be conveyed by epigenetic processes [53].

Acute or chronic stress paradigms Aversive stressful events have been shown to result in increases in anxiety and fear [54–56]. The number of paradigms used is again unmanageable and confusing. A good example for acute stress are conditioned fear paradigms, although there is again a large variation of methodology. Anxiety models of conditioned fear examine freezing behaviour that is provoked by stimuli (e.g. the environment or a tone) associated with an aversive situation, for example an electric foot shock. This fear potentiated freezing response is sensitive to several anxiolytic drugs, such as benzodiazepine agonists [57]. Most prominently variations of this paradigm have been used to model aspects of panic disorder and PTSD [58,59]. In addition to the more anxious phenotype, many of these paradigms have been used to model certain aspects of major depression. Although anxiety disorders and mood disorders have been classified as two separate types of disorders there is a high degree of symptom overlap and comorbidity [60]. In addition, some antidepressants have been successfully used to treat anxiety disorders, although some anxiolytics have also been shown to be effective in the treatment of depressive symptoms. Thus, animal models of depression often use an increased anxiety-related behaviour as key symptom, whereas animal models of anxiety often include symptoms of depression (e.g. differences in the forced swim test). Similar to the situation with perinatal approaches the molecular mechanisms by which acute or chronic stress paradigms alter anxiety-related behaviour is not clearly understood. This is mainly based on the fact that stress alters bodily functions on such a large variety of different levels, that it is nearly impossible to distinguish between them. Glucocorticoids, acting via transcriptional or non-transcriptional mechanisms, exert a vast amount of different effects in the brain and the periphery, which are cell, region and time specific [61]. In addition, stress activates the peripheral sympathetic nervous system and affects many central neurotransmitter systems, which can modulate anxiety-related behaviour. Thus, to unravel the molecular mechanisms by which stress affects anxiety-related behaviour a combination of these models with, for example genetic models seems most promising.

Model comparison Of the discussed animal models of anxiety there is no ideal one, which would generally be better suited for anxiety research. Each model has its own advantages and disadvantages and should therefore be used accordingly (see Table 1). Genetic models bear the advantage that the function of a specific gene in respect to anxiety can be studied. Although www.drugdiscoverytoday.com

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Table 1. In vivo models Genetic manipulations

Inbred strains

Selection lines

Environmental/ pharmacological intervention

Pros

Tests involvement of a specific gene in anxiety

 Easily available  High number of different inbred strains

 High specificity to the selection criterion  Possibility of identification of genetic vulnerability markers

High specificity of environmental risk factor

Cons

Genetic manipulation may result in unwanted and hard to interpret compensatory effects (can be minimized by using time and region specific somatic mutations)

 Often unstable results between laboratories  Genetic or environmental cause of differential anxiety not clear

 High operating expense due to the need of continuous selection testing  Co-selection of anxiety-independent genetic markers

Large methodological variation, often with discrepancies in the reported effects on anxiety

Best use

Hypothesis driven approach with a specific gene of interest

Pharmacological screening

Identification of novel, anxiety-related genes

In combination with any of the genetically based models

Access

 Collaboration  Commercial animal suppliers  New initiative

Commercial animal suppliers

 Collaboration  New initiative

Easy access through published methods descriptions

References

[12,66]

[24,25,28]

[40,67]

[41,42,54]

the comparison of different inbred strains can predominantly be used for pharmacological validation, the comparison of selective breeding lines may result in the identification of novel anxiety-related genes. The strength and power of these approaches would certainly increase if different lines with dissimilar selection criteria would be used for comparison. Identified genes can then be tested by creating mouse lines with a specific genetic manipulation. The timing and location of the genetic manipulation is thereby crucial and should be chosen as precise as possible. A nice example is the study of Gross et al., suggesting that the expression of the 5HT1A receptor during infancy is responsible for an altered anxiety-related behaviour during adulthood [62]. With the current technological advancement, for example using in vivo RNA interference, it is now possible to alter gene expression in the very time and location specific manner, which will help to further unravel the genetic basis of anxiety [63]. Current problems including possible alterations of maternal behaviour or differences due to the genetic background can also be avoided by using a more specific approach. Anxiety models using environmental or pharmacological interventions build on the assumption that long-term alterations in state anxiety have a similar molecular and physiological basis as pathological alterations of anxiety in humans. As environmental interventions often alter a variety of physiological and behavioural endpoints, it remains unclear whether differences in anxiety-related behaviour are causally connected with the treatment or a secondary consequence. To test the usefulness of these models they should be combined with genetic anxiety models to study the interaction of environment and genetic vulnerability. Epigenetic processes, which might be involved in the long372

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term alteration of anxiety due to certain environmental influences, should also be included in future studies.

Model translation to humans Investigating anxiety in rodent models aims at identifying the neurobiological mechanisms underlying normal and pathological anxiety in humans. Keeping the limitations and difficulties in modelling complex psychiatric diseases in mind, however, identifying key disease symptoms amenable to study reliably in animals seems to be a feasible approach to increase our knowledge about the neurobiology of normal and pathological emotional states and to open up new avenues for the development of novel and innovative treatment strategies. The finding of CRH-elicited anxiety in experimental animals, mediated predominantly via the CRHR1 [9,13], and the subsequent development and clinical testing of specific CRHR1-antagonists [64,65] is just one example of this powerful strategy.

Conclusions In this review we have described several good and reliable animal models, which can be used to study the underlying mechanisms involved in anxiety-related disorders. One has to keep in mind although that each animal model of anxiety stands and falls with the chosen anxiety readout (see Box 1). Currently used anxiety tests have a lot of pitfalls. An over-interpretation and unwarranted extrapolation of results obtained with one or two anxiety tests is certainly a risky approach, which should be discouraged. This problem can be tackled by standardizing procedures for each anxiety tests, which would make the comparison of results between laboratories a lot easier. In addition,

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Box 1. Anxiety tests All animal models of anxiety are critically dependent on the anxiety behavioural tests, which are used to define the anxious phenotype. The most widely used behavioural anxiety tests include the elevated plus maze (Fig. I), the dark-light box, the open field test or the conditioned fear approach. Several other valuable test, like the novelty induced suppression of feeding test or the modified hole board, are used far less often. Many of the currently available anxiety tests were developed and validated based on their effectiveness with classical anxiolytics. They are therefore suited to test compounds with similar mechanism of action, but may be disadvantageous when novel treatment strategies are to be discovered. Anxiety in rodents is a complex phenotype, which is represented in a large number of different facets. For instance, an enhanced social anxiety does not necessarily result in an enhanced explorative anxiety. Furthermore, a behaviourally more anxious phenotype might not be linked to an enhanced somatic fear response and vice versa. Thus, when defining an animal model of anxiety it is important to combine a variety of different anxiety tests (behavioural as well as somatic measurements), which can help to define the different aspects of anxiety altered in the particular model. This is of specific importance when models for specific anxiety disorders (e.g. PTSD, panic disorder among others) are developed and characterized. Explicit care should be taken to use models where the animals can display their natural anxiety-related behaviours. In addition, the assessment of subtle behavioural alterations in the different tests (e.g. risk assessment behaviours) may add important information to the described phenotype. Thus, instead of measuring only time in the centre or periphery of the open field, researchers should also include measures such as locomotor adaptation, stretched attends or rearings. A main hindrance in anxiety research seems to be the misinterpretation of test results. This is largely due to the neglect of confounding factors, which can alter the test outcome without necessarily being directly linked to anxiety. For instance, it is often difficult to distinguish between differences in motivation and differences in anxiety in tests, which assume an equal motivation for food intake or exploration. In addition, animals may use a different exploratory strategy, which can easily be misinterpreted as increased anxiety. Other confounding factors include variations in basal locomotor activity, drug metabolism or cognition. Again, the best way to minimize these confounding factors is the use of several principally different anxiety tests and measurements.

claims of differences in anxiety in a given animal model should always be supported by several different anxiety tests, including also the investigation of vegetative symptoms of anxiety.

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Figure I. The elevated plus maze (EPM). Anxiety is measured by the time the animals spend in the open, unprotected arms of the maze and by the number of entries to theses arms. Additional parameters, as the number of stretch attends or the number of head dips are also indices for anxietyrelated behaviour.

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