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Section C

In vivo/Integrative

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25Behavioural Methodology

Emma RobinsonPhysiology & Pharmacology, University of Bristol, UK

25.1 Basic ‘how-to-do’ and ‘why-do’ sectionBehavioural methodologies are used to quantify specific behavioural outputs. The

measurement of behaviour is a hugely diverse area, encompassing studies such as, the

assessment of basic sensory or motor function, complex cognitive behaviours such as

learning andmemory, animalmodels of disease andphenotypinggeneticallymodified

animals. In this brief overview of behavioural methodology, issues relating to the

following are all considered, and specific examples are used to illustrate key points:

� The choice and validity of a method.

� Animal models of disease.

� Experiment design.

� Data interpretation.

A detailed review of the different methods available is not provided. Further

information relating to individual methodologies can be sourced by referring to

literature databases or relevant textbooks and protocol journals (e.g. Nature

Protocols). Examples of different behavioural methods and a brief synopsis are

given in Tables 25.1 and 25.2 at the end of this primer.

25.2 Animal models and behavioural testingIn biomedical ‘behavioural’ research, an animal model is a term used to describe

experimental studies, using animals, that provide insight into human health and

disease. In many cases, the animal models and clinical tests in humans measure very

Essential Guide to Reading Biomedical Papers: Recognising and Interpreting Best Practice, First Edition.

Edited by Phil Langton.

� 2013 by John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd.

CH25 09/26/2012 12:17:19 Page 232

similar processes (e.g. detection of a noxious heat stimulus). In other cases, themodel

has beenvalidated (see below) in terms of its ability to replicate some aspect of human

disease symptom(s) and/or to predict drugs that have efficacy in treating disease. For

example, the forced swim test is an animal model of depression which can predict

drugs with antidepressant efficacy; however, this model does not directly mirror any

aspect of the human depression, nor the methods used to quantify mood in humans.

Table 25.1 Examples of different behavioural methods used to assess specific aspects ofsensory or motor function.

Method Primary application Summary

Locomotoractivity

Drug-induced sedation

Control for methods

which depend of

motor function

Animals are placed in a test arena, wheretheir total activity is quantified. Thiscan be carried out using an automatedsystem, with infrared detectors used toquantify both horizontal and verticalmovements, or by manual counting.

Rotorod Motor coordination/ataxia

This uses a customised piece of equipmentwhere animals are placed onto a rod,which is then turned at an increasingspeed until the animal falls.Impairments in motor coordination andsedation are detected by a reduction intime on the apparatus.

Acoustic startle

Pre-pulse inhibition

(PPI)

Auditory response andsensory-motorgating

Animals are placed in an apparatus whichdetects vibration. A loud tone is playedand the subsequent startle response isquantified. When combined with a pre-pulse (preceding low volume tone),normal animals show a reduced startleresponse. This PPI tests sensory motorgating.

Tail flick/hot platetest

Sensitivity to noxious

stimuli

Pain research

Animals are exposed to a non-damagingnoxious stimuli and their time torespond to the stimulus is quantified.Impaired sensory function andantinociceptive treatments increase thetime to detect and respond to thestimulus.

SHIRPA (SmithKlineBeecham, Harwell,Imperial College,Royal LondonHospital,phenotypeassessment)

Series of testsdesigned to assessthephenotype ofgeneticallymodified mice

This is a standardized set of proceduresused test muscle function, cerebellarfunction, sensory function and basicneuropsychiatric function in geneticallymodified mice. Relevant to assessingphenotype, as well as determiningpotential confounds when using otherbehavioural methods.

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Table

25.2

Exam

plesofdifferentbehaviouralmodelsassociated

withspecificCN

Sdiseases.

Method

Primaryapplication

Brief

description

Elevated

plusmaze

Anxietyresearch

Methodusesrodents,naturalaversionto

heightandopen

spaces.Rodentstendto

spend

more

timeexploringthesafe

‘closed’arm

s,butanxiolytictreatm

entsincrease

timespent

inthemore

aversive

‘open’arm

s.

Forced

swim

test

(tailsuspensiontest)

Depression

Animalsarenorm

allyexposedto

thetestenvironment(inescapablesw

imorsuspensionby

thetail),then

re-tested24hourslater.Animals’norm

alresponse

isto

showreducedescape

behaviouronre-exposure,butthisispreventedifpre-treated

withan

antidepressant.

Novelobject

preference

test

Recognitionmem

ory

Methodto

studyshort-termandlong-termrecognitionlearningandmem

ory.Relieson

rodents’naturalpredispositionto

explore

novelobjects.Followingexposureto

twoormore

objects,animalsarere-testedafteragiven

periodoftime,withorwithouttreatm

ents,and

mem

ory

forthefamiliarversusnovelobject

assessed

usingaratioofexplorationtimeat

each

object.

Morriswater

maze

Spatialworkingmem

ory

Methodto

studylearningandmem

ory

inaspatialtask.Animalsarerequired

tolearnthe

locationofaplatform

hidden

justunderwater.Thetimeto

learnthelocationandabilityto

remem

ber

thelocationarethen

tested.

Conditioned

place

preference

Addiction

Methodusedto

studydrugswithinherentlyrewardingproperties.Associationbetweenan

environmentanddrugorcontroltreatm

entsaremade,andthen

animalspreference-tested

usingachoicetestwhereboth

environmentsaremadeavailable.Drugswhichare

reinforcinginduce

preference

forthedrug-pairedenvironment.Thismethodhas

also

been

usedto

lookat

conditioned

aversion.

Pavlovian

conditioning/

autoshaping

Associativelearning

Based

ontheinitialworkofPavlov,thismethodisusedto

investigatetheform

ationofan

associativemem

ory

followingpairingofastimulus(e.g.lightortone),withan

outcome

(e.g.foodrewardorfootshock).Followingexposure

tothestimulus,animalslearnthe

association,whichisquantified

bymeasuringtheirapproachoravoidance

responses.

Rotationalbehaviour

Basalgangliamotor

function(Parkinson’s

disease)

Thismethodcombines

aneurotoxiclesion,6-hydroxydopam

ine,andbehaviouralmeasure,to

investigatemotorfunctioninvolvingthebasalganglia.Followingaunilaterallesionofthe

dopam

inenigrostriatalpathway,animalsexhibitrotationalbehaviours,particularlywhen

anexogenousdopam

inestimulusisused.Drugswithefficacy

inPDattenuatethis

rotationalbehaviour.

25.2 ANIMAL MODELS AND BEHAVIOURAL TESTING 233

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Other behavioural methods are considered to be tests of a specific aspect of

physiology (e.g. motor function, sensory processing). These tests are designed to

assess the functional consequences of a given manipulation in terms of a behav-

ioural endpoint. This is likely to involve the integration of a number of different

systems of the body, and it takes great skill to ensure that the choice of method and

the interpretation of the data are appropriate. While in vivo behavioural studies

provide essential information about how different systems of the body integrate to

mediate that response, non-specific effects can readily confound the results. This is

particularly the case when looking at genetically modified animals, where a full

behavioural screen is essential before any specific animal model of disease is used.

The vast majority of behavioural tests and animal models rely on an animal’s

movement to provide the quantifiable measure. If the animal has impaired sensory

or motor function, these are likely to confound the results.

Example 1 The results shown in Figure 25.1 illustrate data obtained from an

experiment using the elevated plus maze, an animal model of anxiety. In this model,

non-specific effects associated with both strain differences in baseline behaviour

and locomotor effects are illustrated.

Example 2 If a genetically modified mouse has a locomotor impairment, it may

appear to have impaired abilities in the Morris water maze test of spatial learning,

but in fact it may just be unable to swim properly.

Example 3 If a genetically modified mouse has impaired hearing, it may appear to

show attenuated learning in conditioned fear paradigm, but in fact it may simple be

unable to hear the tone which predicts footshock and so fails to respond in the

manner typical of an animal with normal hearing.

25.2.1 Validity

Behavioural tests are required to be ‘valid’ (from the French ‘valide’, meaning

‘strong’). The validity of an animal model is tested against a number of criteria:

� How well the animal model reflects the symptoms in man is referred to as face

validity.

� How well the animal model replicates underlying biology is referred to as

construct validity.

� How well the animal model predicts treatments which will be effective in the

human condition is referred to as predictive validity.

� How readily data obtained from the animal model can be translated to a benefit

in the clinic is referred to translational validity.

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It is rare that an animal model can achieve all three levels of validity, particularly

in relation to CNS diseases. Recently, emphasis has been put on translational

validity, reflecting the commercial interest in biomedical research and drug

development. Whilst designing an animal model which can achieve validity across

all these areas is difficult, a lack of validity in one or more area does not necessarily

restrict the use of the model, but it should be a key factor when deciding which

Figure 25.1 Schematic representation of anxiety testing apparatus and data. The elevated plusmaze is one of the most widely used methods to study anxiety related behaviour in rodents. Itutilizes their natural aversion to height and open spaces, and provides a number of differentmeasures of anxiety-related behaviour and general activity. Panel A illustrates the basicapparatus. Two arms are open, while the other two arms are enclosed. The animal is usuallyplaced in the centre and behaviour is recorded and analyzed over a period of time (�10minutes).A key measure of anxiolytic behaviour is an increase in the time spent in the open arms. Panel Billustrates the type of data whichmay be obtained in this model. Note that there is a difference inbaseline behaviour between the strains, and the response to the drug treatment differs. It is wellknown that different strains exhibit very different behavioural profiles, often exhibiting effectsgreater than the drug treatments. A common mistake when looking at EPM data is a failure toconsider non-specific effects such as differences in motor function. The behaviour of the animalin this task is dependent on exploration, so an apparent decrease in time on the open arms (panelC) may appear to show an anxiogenic effect. However, analyzing these data as a relative value(i.e. percentage time in the open arm (panel D) reveals no overall difference. When the overallactivity of the animals is analyzed (panel E), it is clear that the effects in panel D are due to anoverall increase in locomotor activity. (�indicates a significant difference from control treatedanimals). Courtesy of Dr. Emma Robinson.

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model is most appropriate to the hypotheses being tested and how the data obtained

are interpreted.

25.3 Required controls (and issues ofexperimental design)

The principle of the 3Rs and animal welfare

Behavioural studies depend on the use of a conscious living animal, so therefore

there is potential for an animal to experience pain suffering and lasting harm. In

the UK, these procedures will most likely require a Home Office licence, with

other countries having similar national or local requirements. The experiment

design should consider the ‘3Rs’ – reduce, refine and replace – and good animal

welfare and handling techniques are essential to achieving high quality,

reproducible data. Stress responses in animals can have major effects on their

behaviour, and these may even be greater than the effect of the manipulation

which is being investigated. It is also worth considering that a stressed animal

will exhibit physiological, endocrine and neurochemical changes, which will

interact with the treatment administered.

This section is structured to provide some general information relating to the use

of behavioural methodology. Some points may not be relevant or practical when

considering a specific method. It may prove useful to review Primer 2 (on

experimental design) and Primer 4 (statistics).

25.3.1 Behavioural testing equipment

The majority of behavioural testing carried out as part of drug development uses

automated equipment.This reduces the time taken to complete a study, allows formore

animals to be tested at a given time and reduces experimenter error and bias. Purpose-

built automated equipment is expensive, so therefore is not necessarily realistic for

smaller academic research groups. Not all methods can, or have been, automated, and

many research groups still carry out non-automated behavioural testing.

25.3.2 Choice of method

It is essential that the experiment design is based around a central hypothesis. The

behavioural method or methods are then chosen, based on the purpose of the

experiment. Consideration should also be given to the specificity of the test

to the behaviour of interest and whether additional, control tests should be included

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(e.g. assessment of motor function). Having chosen the method(s), consideration

should be given to the limitations of the method and their impact on the interpreta-

tion of any data. A common problem with behavioural studies is the over-

interpretation of a specific behavioural deficit – particularly one that has been

observed but which is not consistent with the hypothesis.

25.3.3 Experiment design

The design of any experiment is vital to ensuring the reliability of the results

obtained. With behavioural methods, this is particularly important, as many factors

can lead to experiment design bias or experimenter bias (see Primers 2 and 3). Key

design issues are:

� Avoiding experimenter bias. It is essential that any non-automated behavioural

testing is carried out with the experimenter blind to treatment, otherwise

experimenter bias will undoubtedly influence results (this is probably true of

any experiment, but behavioural studies largely follow this procedure).

� Counter-balancing study design. Behavioural experiments can use either

between-subject designs (animal only receives one treatment and observations

are regarded to be unpaired) orwithin-subject designs (each animal receives all

treatments and observations are regarded to be paired). A between-subject

design avoids adaptation to repeated testing in the apparatus (e.g. elevated plus

maze), but introduces a larger scatter (variance) within the data, which can

require larger numbers of animals in each group.Within-subject studies tend to

reduce variance and the numbers of animals needed, but are only suitable

where animals are unlikely to adapt to the test or where the animal has been

trained to a stable level of performance. Within-subject studies should provide

statistical justification for this. Whichever type of experiment design is used,

treatments must be fully counterbalanced to avoid bias through experiment

design. This is normally done using a technique known as a ‘fully randomized

Latin Square’ design. This means that on any given day, all treatments are

represented and factors such as the time of day are balanced within the study.

� Species, strain and genetic modification. Different species offer advantages

and disadvantages, depending on the method and experimental objectives.

Choosing the right species and strain for the behavioural method can greatly

increase the validity of the data obtained. For example, the forced swim test

(FST) was originally designed for rats, which are a species known to swim

within their natural environment. In contrast, mice are highly averse to water

and experience a significant stress response when exposed to the forced swim

test, which can confound the results. As an alternative, the mouse tail

suspension test was developed to apply the same principles as the FST, but

using a more species-relevant procedure.

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� Different strains of animal also show a high degree of variability in terms of their

behavioural responses, and this can be both an advantage and a disadvantage. For

example, some strains show higher baseline anxiety and locomotor activity in a

novel environment. These strain differences can been used to facilitate detection

of specific treatment effects, but can also result in false positives. For example, if

the aim is to study the efficacy of an anxiolytic drug, selecting a strain of animal

with high baseline anxiety will increase the chances of detecting an effect

(see Figure 25.1, panel B). In contrast, strain differences in baseline performance

in a number of behavioural tasks have led to over-interpretation of data from

genetically modified mice where strain-matched controls were not used.

� Control experiments for non-specific effects. The majority of behavioural

methods depend on animals using some sensory and/or motor processes to

perform the test. If the manipulation used causes a generalized impairment in

sensory or motor function, this may lead to an apparent deficit in the test when,

in fact, it is a non-specific effect. In order to control for non-specific effects, the

inclusion of additional behavioural tests may be necessary. This is particularly

relevant when looking at the behavioural consequence of genetic modification.

It is also important when assessing the effects of a novel pharmacological agent

where effects on locomotor function, for example, have not been established.

As most behavioural methods depend on motor function, tests of motor

coordination and sedative effects are useful. If the test utilizes a specific

sensory domain (e.g. visual processing), some assessment of normal function

should be included when using genetically modified animals. For most

laboratory strains, information about sensory functions is already known.

� Combining behavioural and pharmacological methods. The most common

types of experiments performed using behaviouralmethods are pharmacological

studies, where animals are treated with doses of a drug and the behavioural

effects are quantified and compared to vehicle controls. Antagonist experiments

are also performed using behavioural methods in order to assess the receptor(s)

involved in mediating the response elicited by a given agonist. A typical agonist

dose response experiment and antagonist study is illustrated in Figure 25.2. In

this example, animals receive different doses of the agonists in order to establish

the dose response relationship and EC50. These experiments often generate bell-

shaped dose-response curves, where the quantified behaviour initially increases

(or decreases), and then non-specific effects start to counteract the effects or

inhibit the animals’ ability to express the behaviour (e.g. induction of sedation).

In order to test whether this effect is mediating by a specific receptor, a second

experiment is carried out, where a selective antagonist for the hypothesized

receptor is used. In this second study, an EC50 dose of the agonist is normally

used, and this is ideally tested against two doses of the antagonist. Although not

always feasible, the doses of antagonist should not affect the behaviour when

administered alone, and specifically and dose-dependently attenuate the agonist-

induced behavioural response.

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25.4 Pitfalls in execution or interpretationThe points below can be summarized as issues that require consideration when

interpreting behavioural data and translating the findings to human disease:

1. How relevant is the behaviour that has been measured in the animal to the

human symptom or disease?

2. Is the deficit or improvement observed specific? Have control behavioural tests

been included or referenced?

3. Is the experiment design unbiased?

Figure 25.2 In vivo dose response data. These two figures provide an example of the type ofdata which may be obtained from a behavioural experiment looking at locomotor activity. Theresults in panel (a) show the dose dependent effects of an agonist. There is an increase inlocomotor activity exhibited over a relatively small dose range (1 log unit), with an overall bell-shaped dose response effect observed. In many cases, the behavioural method is only sensitiveto effects over a narrow range because low doses are sub-threshold and higher doses induceother, non-specific effects, which themselves affect behaviour. For example, the drug used inthis experiment may activate receptors which increase locomotor activity at low doses (specificeffect) but, at higher doses, other receptors are activated or inhibited, leading to non-specificeffects. The data shown in panel (b) illustrate a well-designed experiment to investigate thereceptor involved in mediating a behavioural response. An antagonist selective for thehypothesized receptor is used in the study. A total of six treatment groups are included toprovide controls. The vehicle group is used to control for the procedure and vehicle used todissolve the drug. The agonist-alone group is a positive control to ensure the effect is present.The antagonist-alone groups are included to test whether the antagonist has any effects whengiven alone at the doses to be tested in combination with the agonist. The agonist is then alsotested in the presence of two antagonist concentrations. Ideally (although often not realistic),the antagonist alone has no effect and a dose-dependent attenuation of the agonist effect isobserved. (�indicates a significant difference from control treated animals, #indicates asignificant difference from agonist alone treated animals). Courtesy of Dr. Emma Robinson.

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4. What is the specificity of the drug(s) used in the experiment at the doses

administered?

5. Are the doses of drug used relevant to clinical doses? How does the dose used

compare to the occupancy of the receptors in vivo? (This is not always easy to

find out, but very relevant; different species metabolize drugs at different rates,

so comparing receptor occupancy is the most reliable way of knowing that the

dose is relevant.)

6. What is the time course of the drug effects and are these relevant to the

experiment design? For example, targeted brain infusions may reach receptors

after five minutes, while oral drug dosing may require one or two hours for

peak plasma concentration to be reached. When using an antagonist, this is

ideally administered first, and at a time which allows it to equilibrate at the

receptor before the agonist is administered.

Further reading and resourcesCrawley, J.N. (1999). Behavioral phenotyping of transgenic and knockout mice: exper-

imental design and evaluation of general health, sensory functions, motor abilities, and

specific behavioral tests. Brain Research 835(1), 18–26.

Hatcher, J.P., Jones, D.N., Rogers, D.C., Hatcher, P.D., Reavill, C., Hagan, J.J. & Hunter, A.J.

(2001). Development of SHIRPA to characterise the phenotype of gene-targeted mice.

Behavioural Brain Research 125(1–2), 43–47.

Nestler, E.J. & Hyman, S.E. (2010) Animal models of neuropsychiatric disorders. Nature

Neuroscience 13, 1161–1169.

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