Targeted gene mutation approaches to the study of anxiety-like behavior in mice

Targeted gene mutation approaches to the study of anxiety-likebehavior in mice

Andrew Holmes*

Section on Behavioral Pharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, NIH, Bethesda, MD, 20892-1375, USA

Abstract

Studying the behavioral phenotypes of transgenic and gene knockout mice is a powerful means to better understand the pathophysiology of

neuropsychiatric disorders and ultimately improve their treatment. This paper provides an overview of the methods and ®ndings of studies

that have tested for anxiety-related behavioral phenotypes in gene mutant mice. In the context of improving the side effect burden of

benzodiazepines, gene targeting has been valuable for dissociating the functional roles (i.e., anxiolytic, sedative, amnestic) of individual

GABAA receptor subunits. Supporting the link between abnormalities in CRH function and anxiety, CRH overexpressing transgenic mice

and CRH-R2 receptor knockout mutants have displayed signi®cantly increased anxiety-like behavior, while CRH-R1 receptor knockout

mice have shown an anxiolytic-like phenotype. Consistent with an important role for the serotonergic system in anxiety, 5-HT1A receptor

de®cient mice have consistently exhibited heightened anxiety-like behavior, while the evidence from 5-HT1B and 5-HT2C de®cient mice

remains somewhat equivocal. Mutant mice lacking either of the monoamine degrading enzymes, MAOA or COMT, have shown a number of

behavioral and neurological effects, including alterations in anxiety-like behavior. With enhanced spatial and temporal control over gene

mutations, in combination with an improved battery of behavioral tests, gene mutant mice will provide an increasingly valuable tool for

understanding the neural substrates of anxiety. q 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Anxiety; Behavior; Transgenic; Knockout; Gene; Mouse; Genetic background; GABAA receptor; 5-HT receptors; Corticotropin-releasing hormone

1. Introduction

The emergence of molecular techniques that allow the

alteration of genes and gene function in the intact animal is

fashioning a new era in behavioral neuroscience. Protocols

used in the generation of transgenic and gene knockout mice

are now established as routine, and their widespread avail-

ability is producing proliferating numbers of gene mutant

mice with direct relevance to the study of neural processes

[1]. Fortunately for the behavioral neuroscientist, the mouse

represents a good subject for studying the effects of genetic

manipulations on behavior. Once generated, a mutation can

be maintained in numbers of mice large enough to facilitate

sound behavioral studies, with relatively little expense.

Moreover, while there has been a long history of using the

rat in behavioral neuroscience, many laboratories now have

behavioral tests that are speci®cally designed, or can be

successfully adapted, for use with mice.

The technology behind the generation of transgenic and

gene knockout mice has been extensively described else-

where, and the reader is referred to one of many excellent

sources for a fuller discussion [2±4]. A transgenic mouse is

often generated in order to study the functional conse-

quences of gene overexpression. Brie¯y, this is achieved

by microinjecting foreign DNA containing copies of a

given gene into developing mouse embryos, where they

have a chance to integrate into the host genome. The overall

level of transgene expression is largely determined by the

location at which the transgene inserts into the genome,

which is random. However, patterns of expression can be

directed to speci®c areas of the brain by using a region or

cell speci®c promoter. With a gene knockout mouse, one

can study the effects of removing a gene. First a DNA

construct is designed that causes a functional disruption in

the gene of interest. The DNA is then integrated into plur-

ipotent embryonic stem cells (via homologous recombina-

tion) and inserted into foster embryos. Progeny are

examined to see whether they have incorporated the null

mutation (e.g., via coat color), and those chimeric offspring

carrying the mutation in germline cells (thereby allowing

the mutation to be transferred across generations) can be

interbred to produce mice that are heterozygous for the

missing gene. Assuming that the absence of the gene does

not impact prenatal survival, interbreeding heterozygote

offspring will normally give a complement of heterozygous

Neuroscience and Biobehavioral Reviews 25 (2001) 261±273PERGAMON

NEUROSCIENCE AND

BIOBEHAVIORAL

REVIEWS

0149-7634/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.

PII: S0149-7634(01)00012-4

www.elsevier.com/locate/neubiorev

* Tel.: 11-301-496-4838; fax: 11-301-480-1164.

E-mail address: [email protected] (A. Holmes).

null mutant, homozygous null mutant and `wild type' litter-

mate controls, in the ratio of 2:1:1.

Where a genetic alteration does impact prenatal survival,

studying the behavioral consequences of that mutation is, of

course, impossible. The `classical' methods for generating

gene knockout and transgenic mice produce genetic muta-

tions in all cells where the gene is expressed, throughout

ontogeny and adulthood. Therefore, even in viable animals,

the presence of the mutation during development can

complicate interpretation of behavioral phenotypes

(observed or absent) in a mutant mouse due to indirect or

multiple effects of the gene mutation. Thus, additional gene

product in a transgenic mouse may lead to a cascade of

molecular and neurochemical effects, confusing the causal

link between phenotype and targeted gene. Conversely,

where a gene has been inactivated, developmental adapta-

tions may compensate for the deletion, thereby masking that

gene's normal function. On one level, developmental effects

can provide insight into the plasticity of neural systems [5].

Moreover, rendering a genetic alteration that is both global

and chronic is a powerful means to model genetic contribu-

tions to a neurological or a neuropsychiatric disorder, when

that is the goal. However, these same factors become unde-

sirable when chronic, global expression impacts survival, or

when studying the function of a gene in the normal adult

brain [6]. In this context, techniques which allow for greater

control over the temporal and spatial characteristics of a

gene mutation represent an important advance [7].

One technique which produces regional restriction of a gene

deletion works by ¯anking the gene of interest with loxP

sequences that act as recognition sites for the bacteriophage

enzyme Cre recombinase [8]. Intercrossing animals carrying

the loxP ¯anking regions with Cre-expressing transgenic mice

will result in excision of the `¯oxed' site containing the target

gene. Therefore, when Cre is driven by a promoter that is

speci®c for a given region, inactivation of the gene is restricted

to that region. In order to add temporal control over the genetic

mutation, Cre expression can be linked to a regulating system,

such as tetracycline or interferon [9]. The ability to determine

where and when a gene is inactivated or overexpressed is

already providing important insights into the link between

genes and learning and memory [10,11], and it is hoped that

it will have a similar impact for studying anxiety-related

processes. Of course, understanding the effects of increasingly

sophisticated genetic manipulations necessitates sounds meth-

ods for behavioral phenotyping. The next section provides and

introduction and overview of some of the behavioral methods

used to assess anxiety-related phenotypes in gene mutant

mice.

2. Tests for anxiety-like behavior in mice

2.1. Exploration-based tests

Numerous tests have been designed to test anxiety-like

behavior in rodents [12,13]. The majority of these beha-

vioral paradigms were developed and pharmacologically-

validated in rats and some valuable tests have not readily

transferred for use in mice, e.g., the social interaction test

[14]. Nonetheless, there remains a wide choice of estab-

lished behavioral tests with which to test for anxiety-related

phenotypes in a transgenic or gene knockout mouse. Many

of the behavioral assays that are currently popular for testing

anxiety-related behavior circumvent the need to train condi-

tioned responses and instead exploit the natural exploratory

drive in mice [15]. Probably because of their rapidity,

simplicity of design and uncomplicated face validity,

these ethological exploration-based paradigms have far

and away been the tests of choice in studies which have

tested for anxiety-related phenotypes in mutant mice

(Table 1).

Common to the exploration-based tests for anxiety-like

behavior is the basic premise that the innate tendency to

explore a novel place will be inhibited by increasing the

aversive nature of the environment, thereby producing a

con¯ict between approach and avoidance. Put simply,

high levels of exploration in an aversive environment are

interpreted as low levels of anxiety-like behavior. Exposure

to a novel and well-lit open ®eld represents the experimental

manifestation of this concept in its simplest form [16]. In

this test, increased anxiety-like behavior is primarily asso-

ciated with avoidance of the central, exposed part of the

open ®eld, as ànxious' mice tend to show more defensive

thigmotactic behavior along the walls of the apparatus [17±

19]. While measures of defecation have traditionally been

an index of anxiety-related behavior in the open ®eld [16],

possible abnormalities in gastrointestinal function in a

mutant mouse should be ruled out before drawing inferences

about anxiety-related behavior from such data. Taking over-

all levels of open ®eld ambulatory activity (i.e., line cross-

ings or photocell beam breaks) as measures of anxiety-

related behavior can be even more problematic. Ambulatory

activity in an open ®eld is likely to be the result of both

anxiety-related approach/avoid con¯ict and levels of basal

locomotor activity. Indeed, the open ®eld is commonly used

as a test for locomotor activity phenotypes in mice with

mutations in systems related to locomotor initiation and

control [20]. Thus, reduced open ®eld activity in a trans-

genic or knockout mouse could either re¯ect a con¯ict-

related reduction in exploration [21,22], or a reduced level

of basal activity that is unrelated to anxiety. As such, in the

absence of additional evidence, a pro®le of reduced open

®eld activity in a mutant mouse cannot unequivocally be

interpreted as indicating either hypoactivity or heightened

anxiety-like behavior [23,24].

The light$ dark exploration test has been used as a

screen for pharmacological compounds impacting anxi-

ety-like behavior for over 20 years [25,26]. The test

apparatus comprises two inter-connecting chambers; one

large, open and brightly-lit, the other smaller, covered

and unlit. In most untreated mouse strains, an aversion

A. Holmes / Neuroscience and Biobehavioral Reviews 25 (2001) 261±273262

to the brightly-lit compartment produces a clear prefer-

ence for the dark compartment, and/or more time spent

in the light compartment. Treatment with anxiolytics

generally results in an increased number of exploratory

transitions between light and dark compartments, and/or

more time spent in the light compartment [27±29]. How-

ever, psychomotor stimulants such as amphetamine can

produce false positive anxiolytic-like pro®les in the

light$ dark exploration test [15]. In a similarly way, a

phenotype of locomotor hyperactivity in a gene mutant

mouse [20] could manifest as signi®cantly increased

light$ dark transitions, leading to a false positive inter-

pretation of reduced anxiety-like behavior. Conversely,

retarded locomotor activity in a gene mutant could

present as heightened anxiety-like behavior in the light$dark exploration test. As with the open ®eld test, this

raises the dif®cult issue of dissociating changes in

exploratory behaviors in a test for anxiety-like behavior

from abnormal basal locomotor activity. To tackle this

issue in pharmacological studies, some investigators have

A. Holmes / Neuroscience and Biobehavioral Reviews 25 (2001) 261±273 263

Table 1

Examples of anxiety-related phenotypes in transgenic and gene knockout mice.

Genetic mutation Anxiety-related phenotype Behavioral test [Ref]

GABAA a1 subunit mutant No anxiety-related phenotype elevated plus-maze [103]

light$ dark exploration [103]

GABAA d subunit knockout No anxiety-related phenotype elevated plus-maze [104]

GABAA y2 subunit heterozygous

knockout

Increased anxiety-like behavior elevated plus-maze [50]

Light$ dark exploration [50]

Free exploratory paradigm [50]

GABAA y2 subunit (long-variant)

knockout

Increased anxiety-like behavior elevated plus-maze [105]

Glutamate decarboxylase enzyme

(GAD65) knockout

Increased anxiety-like behavior elevated zero-maze [111]

open ®eld center time [111]

Monoamine oxidase A knockout Decreased anxiety-like behavior open ®eld center time [58]

Monoamine oxidase B knockout No anxiety-related phenotype elevated plus-maze [114]


Catechol-O-methyltransferase

knockout

Increased anxiety-like behavior Light$ dark exploration [114]

Dopamine D3R knockout Decreased anxiety-like behavior elevated plus-maze [24]

No anxiety-related phenotype elevated plus-maze [117]

Dopamine D4R knockout Increased anxiety-like behavior open ®eld center time [52]

emergence test [52]

novel object exploration [52]

5-HT1A receptor knockout Increased anxiety-like behavior elevated plus-maze [126]

open ®eld center time [46,126±128]

elevated zero-maze [46]

novel object exploration [46]

5-HT1B receptor knockout Decreased anxiety-like behavior elevated plus-maze [45]

No anxiety-related phenotype open ®eld center time [127,131]

ultrasonic vocalizations in pups [45]

No anxiety-related phenotype elevated plus-maze [131]



5-HT2c receptor knockout Increased anxiety-like behavior emergence test [124]

5-HT5A receptor knockout No anxiety-related phenotype elevated plus-maze [73]


defensive burying [73]

Corticotropin-releasing hormone Increased anxiety-like behavior elevated plus-maze [135]

(CRH) overexpressing transgenic open ®eld center time [135]


CRH knockout No anxiety-related phenotype elevated plus-maze [138]

CRH R1 receptor knockout Decreased anxiety-like behavior elevated plus-maze [51,140]

emergence test [51]

light$ dark exploration [140,141]

CRH R2 receptor knockout Increased anxiety-like behavior elevated plus-maze [145,146]


emergence test [146]

Decreased anxiety-like behavior open ®eld center time [146]

No anxiety-related phenotype light$ dark exploration [145]

sought to identify behavioral indices within a given test that

measure general activity per se. This approach has arguably

achieved some success in the elevated plus-maze test.

As a screen for novel anxiolytics, the elevated plus-maze

has become commonplace in behavioral laboratories [30]

and is currently the most popular test for anxiety-like beha-

vior. The conceptual basis for this test was derived from the

innate preference shown by rats for (elevated) enclosed

alleys over (elevated) open alleys [31]. This observation

led directly to the familiar design of the elevated plus-

maze; two open arms perpendicular to two enclosed arms

(i.e., walled), interconnected by a single central platform,

and elevated approximately 0.5±1m above ¯oor level [32].

Baseline behavior in most mouse strains is characterized by

an avoidance of the open arms (in favor of the protection

afforded by the enclosed parts). In both rats [33] and mice

[34], open arm avoidance is exaggerated by drugs with pro-

anxiety effects and reversed by treatment with standard

anxiolytics. Locomotor activity in the elevated plus-maze

has traditionally been measured by total number of arm

entries; i.e., a selective anxiety-related effect was inferred

from changes in open arm exploration in the absence of

concomitant changes in total entries. However, using factor

analysis to examine commonalities between individual plus-

maze measures, a number of studies have indicated that total

arm entries co-load across factors relating to `locomotor

activity' and ànxiety-like behavior' [35±37]. This pattern

is not surprising given that total arm entries is a composite

measure of closed and (anxiety-related) open entries. In

contrast to this ambiguous pattern of loadings, closed arm

entries often load exclusively on the `locomotor activity'

factor, suggesting that closed arm entries is a more accurate

index of locomotor activity in the elevated plus-maze.

Further re®nement of the elevated plus-maze has origi-

nated from a greater understanding of the form and function

of rodent defensive behavior [38,39]. Vigilance and `risk

assessment' behaviors are related to information gathering

about potential threat [40±42], with parallels drawn with the

hypervigilance for threat cues evident in some anxiety disor-

ders [41]. It has been suggested that the incorporation of risk

assessment behaviors into plus-maze ethograms can

increase the test's sensitivity to anxiolytic and anxiogenic

compounds [43]. Furthermore, it is interesting that

`stretched approach/stretch attend' postures in the plus-

maze show higher correlations with hypothalamic±pitui-

tary±adrenal axis-mediated stress responses than the more

traditional spatio-temporal exploratory indices [44].

However, to date only a small number of research groups

have included risk assessment behaviors in tests for anxiety-

like behavior in mutant mice [24,45,46]. A more wholesale

change to the elevated plus-maze led to the design of the

elevated zero-maze, which consists of a single annular plat-

form divided into two open and two closed quadrants (i.e.,

no central platform) [47]. While the elevated zero-maze has

not been fully examined for its sensitivity to known anxio-

lytics, it has been able to identify behavioral pro®les of

mutant mice that parallel anxiety-related phenotypes

evident in pharmacologically-validated behavioral tests

[46,48].

Some authors have argued that providing a safe area from

which to explore an aversive environment presents the

animal with a less anxiety-provoking test situation, and

one which may more closely measure trait anxiety than

state anxiety [49,50]. The free exploration test described

by Griebel and colleagues [49] entails housing animals in

one compartment for 24 h prior to giving the animal a free

choice between remaining in the familiar compartment or

exploring a novel area. Based on a similar concept, the

emergence test has been used in a number of studies to

test anxiety-like behavior in mutant mice [51,52]. The emer-

gence test involves placing the mouse inside an opaque

object, such as a cylinder, contained within a larger open

®eld. While the primary measure of anxiety-like behavior is

taken to be the latency to emerge into the novel arena,

auxiliary markers of anxiety-like behavior may include

the number of emergences and the time spent out of the

cylinder during a 5 min test session. Indeed, Dulawa et al.

(1999) have recently suggested that the latency to emerge

measure may, in fact, be more sensitive to genotype differ-

ences in basal locomotor activity than in anxiety-related

behavior [52].

2.2. Issues of methodology

Behavior in rodent tests for anxiety is sensitive to a host

of organismic and procedural variables that vary across

experiments and from laboratory to laboratory [30,53]. In

addition to the design and material construction of the appa-

ratus itself [54,55], subject gender, age, housing, handling,

and husbandry can all contribute to behavioral pro®les in

tests for anxiety-related behavior [39]. Given the in¯uence

of social experience on anxiety-related behaviors in mice

[56,57], anxiety-related behavior in a mutant mouse should

be considered in the context of any parallel phenotypes in

maternal, social or aggressive behavior [45,58]. Prior expo-

sure to certain anxiety tests can carry over and in¯uence

future test pro®les. For example, exposure to the elevated

plus-maze for as little as 2 min has been found to alter

behavioral baselines and/or responsivity to anxiolytics on

subsequent plus-maze trials [59,60]. Clearly, great care

should be taken when repeatedly testing individuals in the

same test. Similarly, when using multiple behavioral para-

digms to test for an anxiety-related phenotype in the same

subject, test-order and the interval between tests should be

carefully considered and always clearly outlined in the

Methods. The sensitivity of anxiety-like behavior to extra-

neous variables was recently underscored in a study which

attempted to control for all aspects of laboratory environ-

ment, animal husbandry, and experimental protocol [61]. In

spite of their efforts to harmonize testing for anxiety-like

behavior in three separate laboratories, Crabbe and collea-

gues [61] found that the observation of genotype differences


in anxiety-related behavior across eight inbred mouse

strains was dictated by the laboratory in which testing was

conducted. These ®ndings further highlight the importance

of controlling procedural variables in studies of anxiety-

related behavior in gene mutant mice.

Even ostensibly simple behavioral tasks draw upon a

complex of neurological, sensory and motor abilities. For

example, performance in the elevated plus-maze and zero-

maze will be determined by visual (depth) perception and

tactile sensitivity to the thigmotactic support in the enclosed

parts of the apparatus. Similarly, suf®cient vision is needed

in order to discriminate levels of illumination which under-

pin the open ®eld, light$ dark exploration, and emergence

tests. In terms of motor capabilities, a mutant mouse that is

unable to maneuver around the arms of the elevated plus-

maze could spend an exaggerated amount of the test session

trying to turn at the distal ends of the open arms, thus arti®-

cially biasing results to show high open arm time. Altered

nociceptive responses will in¯uence behavior in the Vogel

water lick and defensive-burying of a shock probe tests

among others, while adequate vision/hearing is integral to

performance in the light-enhanced startle paradigm.

More general de®cits in physical health and well-being

can manifest in any behavioral test, probably as inhibited

performance. As such, it is important to examine a mutant

mouse for any gross abnormalities in neurological, sensory

and motoric function that may comprise performance (and

interpretation) of behavior in paradigms designed to test

anxiety-like behavior [62]. One approach is to systemati-

cally examine mutant mice using a screen of simple physical

checks, neurological re¯exes, and sensory capabilities

[63,64]. A less direct alternative is to infer intact function

from performance in other test situations. Thus, good vision

can be con®dently inferred from an ability to ®nd a visually-

cued platform in the Morris water maze. Valuable informa-

tion on hearing and somatosensory capacities can be

obtained by measuring startle amplitude to auditory and

tactile startle stimuli respectively. Monitoring the ability

to remain on an accelerating rotarod is a proven way to

assess balance and motor coordination in a mutant mouse

[65,66].

It has already been noted that, for anxiety tests that

are based upon exploratory responses, a hyperactive or

hypoactive phenotype in a mutant mouse can confound

interpretation of tests for anxiety-like behavior. Perhaps

the surest way to test for a locomotor activity pheno-

type that is uncontaminated by the response to an aver-

sive environment is to record activity of single animals

within the home cage over a number of days. In cases

where a gene mutation does impact locomotor activity

in the home cage, tests that are less reliant on explora-

tory responses may be more suitable for measuring

anxiety-like behavior; e.g., stressor-induced ultrasonic

vocalizations in pups and adults [45,67,68], fear/light-

enhanced startle paradigm [69,70], shock-probe defen-

sive burying test [71±73], mouse defense test battery

[74,75], acoustic startle response [73,76]. Of course,

while some of these tests may be less in¯uenced by

abnormal locomotor activity, they come with their

own complexities and potential confounds (e.g., altered

sensitivity to pain).

2.3. Genetic background

A prominent issue for behavioral studies with mutant

mice concerns the in¯uence of genetic background on the

detection of mutation-induced phenotypes [5,77±81]. Any

behavioral phenotype observed in a gene mutant mouse

will be the product of a complex, epistatic interaction

between the mutation and the genetic background on

which it is placed. This fact acquires further signi®cance

in view of the extensive genetic heterogeneity that exists

across different mouse strains. In terms of anxiety-like

behavior, it is well known that mouse strains exhibit

widely different pro®les in tests including the elevated

plus-maze [76,82±84], light$ dark exploration test

[28,84,85], free-exploratory paradigm [86], and open

®eld [76], as well as in certain neurotransmitter systems

related to anxiety, e.g., GABAA receptor function

[28,83,84,87]. For transgenic and gene knockout studies,

DNA constructs and embryonic stem cells are invariably

derived from 129 substrains (e.g., 129/SvJ, 129/SvEv,

129/Ola), while a separate inbred strain (often C57BL/

6) is used for the purpose of blastocyst donation and

breeding. Therefore, understanding the anxiety-related

behavior of these particular inbred strains can provide

important insight into the in¯uence of genetic back-

ground in a mutant mouse.

In this context, there are marked differences in anxiety-

related behaviors between C57BL/6 and 129 substrains, as

well as in learning and memory [88,89], sensory functions

[90±92] and motor behaviors [93]. More speci®cally,

Montkowski et al. [94] have found that 129/SvJ and 129/

Sv-ter substrains showed less anxiety-like behavior than

C57BL/6 mice in the open ®eld test and/or elevated plus-

maze. Similarly, Rogers and coworkers [81] have reported

lower anxiety levels in the 129/SvHsd strain, as compared to

C57BL/6, tested in the elevated plus-maze. In contrast,

Homanics and colleagues [95] have found that 129/SvJ

mice exhibit higher, rather than lower, levels of anxiety-

like behavior in the elevated plus-maze as compared to

C57BL/6J mice, while no differences were evident for

open ®eld center time. The 129/SvIJ substrain has also

shown higher levels of anxiety-like behavior than C57BL/

6J mice in the elevated zero-maze [96]. Taken together

these ®ndings indicate that the detection of differences

between C57BL/6 and 129 strains is dependent upon

which 129 substrain is tested and which behavioral assay

is performed.

Genetic and behavioral variability between parental

strains leads to greater variability in genetic background.

This increased ªnoiseº could mask a mild anxiety-related


phenotype in a gene mutant mouse. In order to dilute the

in¯uence of 2 sets of parental genes, a mutation can be

repeatedly backcrossed onto a given strain, often C57BL/

6, to ultimately produce a congenic mutant strain with a near

pure genetic background. However, even with a congenic

mutant mouse, it is possible that an anxiety-related pheno-

type will actually be the result of parental genes that are

linked to the chromosomal locus of the mutant gene, rather

than due to the mutated gene per se [78]. Thus, cases where

altered anxiety-like behavior is evident in multiple congenic

mutant lines (e.g., heterozygous GABAA g2 knockout mice

[50]), provide the most compelling evidence that the pheno-

type is a direct result of the mutation, and not an artifact

related to genetic background. The issue of genetic back-

ground and other problems more associated with the beha-

vioral methodology highlight some of the caveats that are

essential to consider when studying anxiety-like behavior in

transgenic and knockout mice. The following section

provides a brief overview of some of the anxiety-related

phenotypes reported in gene mutant mice to date, many of

which have been studied with due consideration of the wider

methodology.

3. Anxiety-related phenotypes in mutant mice

Historically, understanding the neural bases of anxiety-

related behavior in rodents has been mainly driven by study-

ing the effects of exogenous manipulations (pharmacologi-

cal, electrical), and the consequences of neural ablations.

Perhaps the principal disadvantage of these approaches is

that they can lack selectivity or reproducibility. With the

molecular approach, brain manipulations can potentially

be rendered at the genetic level with both precision and

reliability. The literature on the effects of genetic mutations

on anxiety-like behaviors is ever growing and the number of

reports of unexpected anxiety-related phenotypes in mutant

mice perhaps presages some novel and serendipitous ®nd-

ings using this approach [97,98]. A review of all relevant

®ndings to-date is clearly beyond the scope of the present

paper. Instead, the following section will focus primarily on

gene mutations that relate to pharmacological treatments for

anxiety disorders. A summary of the ®ndings is provided in

Table 1.

3.1. Manipulations of GABAergic transmission and

benzodiazepine receptor function

The effects of pharmacological compounds acting at

benzodiazepine receptors in the brain are intimately linked

to anxiety. Benzodiazepine receptor agonists such as diaze-

pam have been hugely effective therapies for anxiety and,

despite concerns about their safety and long-term use,

remain the treatment of choice for anxiety disorders [99].

These drugs, along with certain other compounds with

known anti-anxiety effects such as barbiturates, ethanol

and neuroactive steroids, exert their anxiolytic effects via

modulation of GABAergic transmission at GABAA recep-

tors [100,101]. GABAA receptors are made up some 20

protein subunits [102], the composition of which determines

the function of the receptor complex. However, while it is

known that the distribution of these complexes varies

throughout the brain, their respective function roles are

poorly understood. Thus, a better understanding the func-

tional signi®cance of GABAA heterogeneity could help

dissociate the anti-anxiety effects of benzodiazepine

agonists from their unwanted side effects (i.e., amnesia,

sedation, ataxia, dependence). In this context, rendering

genetic alterations at the level of individual GABAA subu-

nits represents a very powerful means for studying their

respective roles in mediating anxiety-related behavior.

Rudolph and colleagues [103] have recently reported

that mice with mutation of the a1 GABA subunit are

insensitive to the sedative, amnestic, and anticonvulsant

effects of diazepam, but remain responsive to the drug's

anxiolytic effects (probably via action at intact a2 and a3

subunits). In a separate study, Mihalek et al. [104] have

found that inactivating the d subunit renders mice insen-

sitive to the anxiolytic effects of neuroactive steroids in

the elevated plus-maze. In combination with a and bsubunits, the g2 subunit is essential for the expression

of the benzodiazepine site on the GABAA receptor

complex. Homanics et al. [105] have deleted the long

variant of the g2 subunit gene and found that this did

not alter total g2 subunit expression, thereby indicating a

compensatory increase in the short variant of the g2

subunit. However, even with this ontological compensa-

tion, g2L subunit knockout mice exhibited heightened

anxiety-like behavior in the elevated plus-maze.

GuÈnther and co-workers [106] have reported that

complete genetic deletion of the g2 subunit drastically

reduced both the number of benzodiazepine sites in the

brain and the perinatal survival of mice carrying the muta-

tion. Most mice homozygous for the mutation died within

days of birth, while those surviving a little longer exhibited

severe sensorimotor de®cits. Mice heterozygous for the g2

subunit had a less marked reduction in benzodiazepine sites

and remained viable and healthy, allowing an examination

of their behavior as adults. Adult heterozygous g2 subunit

knockout mice exhibit increased anxiety-like behavior in

the elevated plus-maze, light$ dark exploration test, and

free-exploratory paradigm [50]. In addition to clear

evidence of spontaneous anxiety-like behavior, these

mutant mice also showed evidence of greater learned fear

responses to a partial, ambiguous stimulus in a cued and

contextual fear conditioning paradigm. Crestani et al. [50]

suggest that this reduced discrimination among learned

threat cues may relate to the bias for attributing danger to

neutral stimuli seen in individuals with anxiety disorders. In

this context, it will be important to assess whether a facil-

itation of sensory processing per se could underpin the beha-

vioral phenotype of these mutant mice. Notwithstanding,

increased anxiety-like behavior in mutant mice lacking


benzodiazepine binding sites is signi®cant in the light of

evidence from pharmacological challenge and neuroima-

ging studies showing that anxiety disorders are associated

with altered benzodiazepine receptor binding [107,108].

Evidence for the role of GABA receptor function in anxi-

ety states from gene knockout mice has also derived from

engineering alterations in GABA levels. Reducing GABA

levels by deleting the synthesizing-enzyme glutamate

decarboxylase, GAD67, produces a prenatal lethal pheno-

type [109,110]. Genetic inactivation of the GAD65 synthe-

sizing-enzyme produces a less marked reduction in GABA

levels and a corresponding increase in the survival of mutant

mice [111]. GAD65 mutant mice showed increased anxiety-

like behavior as measured by the elevated zero-maze and

center time in a novel open ®eld [111], a phenotype the

authors directly attribute to a reduced ability to synthesize

GABA rather than a change in postsynaptic GABAA recep-

tor density in these mice. Radioligand receptor binding

results supported this interpretation, as did the ®ndings

that muscimol (which acts directly on postsynaptic recep-

tors) retained its sedative action in mutant mice. Moreover,

the locomotor activating effect of diazepam and the sedative

effect of pentobarbital were found to be either diminished or

lost in mutant mice.

3.2. Mutations of monoamine function

Monoamine oxidase A (MAO-A) inhibitors, such as

phenelzine, which act by blocking the metabolism of dopa-

mine, norepinephrine and 5-HT, remain valuable treatments

for anxiety (particularly panic disorder) [112]. Cases et al.

[58] have studied mutant mice lacking the MAO-A gene,

which show elevated brain levels of 5-HT and norepinephr-

ine. In addition to a signi®cant aggressive phenotype, MAO-

A-de®cient mice showed evidence of reduced anxiety-like

behavior, spending more time in the center of an open ®eld

than wild type controls. These mutant mice also showed

evidence of enhanced fear conditioning in a standard cued

and contextual fear conditioning paradigm (albeit in

comparison to very low freezing scores in wild type

controls) [113]. These emotion-related phenotypes were

paralleled by neurological abnormalities in the somatosen-

sory cortex and a hypersensitivity to sensory stimuli

[58,113]. Therefore, it is possible that a more general altera-

tion in sensory ability might have contributed to the pheno-

types observed in anxiety-related tests. In contrast to the

major phenotype of MAO-A knockouts, mice lacking the

MAO-B gene did not show any signi®cant alterations in

brain monoamine concentrations or any alterations in anxi-

ety-like behavior in either the elevated plus-maze or the

open ®eld center time assay [114].

In addition to the monoamine oxidases, the catechola-

mines are also degraded by catechol-O-methyltransferase

(COMT). Gogos and colleagues [115] have found that

male COMT-de®cient mice exhibited substantially elevated

levels of dopamine (but not norepinephrine or 5-HT) in the

frontal cortex, paralleled with a heightened level of aggres-

sion. In contrast, female COMT-de®cient mice did not show

any signi®cant neurochemical or aggressive phenotype and,

instead, demonstrated increased anxiety-like behavior in a

modi®ed version of the light$ dark exploration test. Gogos

et al. [115] suggest that differences between male and

female mutant mice may re¯ect hormonal control over

COMT activity and speculate that the COMT gene may

contribute to sex differences in the prevalence of emotional

disorders.

There are now a number of studies that have involved a

more speci®c targeting of the dopaminergic system by way

of deleting dopamine receptor subtypes, such as the dopa-

mine D3 receptor (D3R), which is highly expressed in the

limbic system [116]. Independently generated lines of D3R-

de®cient mice have exhibited inconsistent behavioral

phenotypes in terms of both locomotor activity

[23,117,118] and anxiety-like behavior. Thus, while Steiner

et al. [24] report a reduced level of anxiety-like behavior in

the elevated plus-maze, Xu et al. [117] report no genotype

differences using the same test. In a study of dopamine

D4R-de®cient mice, Dulawa et al. [52] have found a more

consistent anxiety-like phenotype across multiple tests; i.e.,

less center exploration in a novel open ®eld, reduced novel

object exploration, and greater preference for the home-base

in the emergence test than wild type controls. Finally,

Campbell and colleagues [119] have reported that a trans-

genic stimulation of a restricted subset of dopamine D1

receptors on neurons in the amygdala and cortex produced

a striking phenotype characterized by perseverative beha-

viors, such as leaping and an intense biting/grooming of

cage mates. Given there was no concomitant evidence of

increased aggression when mutant mice were tested in a

standard resident-intruder paradigm, Campbell et al. [119]

propose the phenotype may model compulsive symptoms in

obsessive compulsive disorder.

3.3. 5-HT receptor knockouts

The hypothesized involvement of serotonergic mechan-

isms in the mediation of anxiety is long-standing [120] and

the use of drugs that preferentially impact 5-HT function,

such as the SSRIs and buspirone, now rival the benzodiaze-

pines as ®rst line treatments for anxiety disorders

[112,121,122]. Identifying the relative roles of each of the

14 1 5-HT receptor subtypes has been hampered by the

dif®culty in obtaining pharmacological ligands that are

suf®ciently selective for individual receptors. Given the

utility of transgenic and gene knockout mice for deleting

receptor subtypes (especially if the mutation can be induced

in the intact, adult animal), gene targeting offers signi®cant

advantages over current pharmacological methods of 5-HT

receptor function [123]. Preliminary evidence of an anxiety-

like phenotype in 5-HT2C receptor knockout mice stemmed

from the ®nding that mutant mice showed extended laten-

cies to emerge in the emergence test [124]. However, given


that emergence latencies can be in¯uenced by alterations in

spontaneous locomotor activity [52], and 5-HT2C mice

exhibit a complex locomotor phenotype [125], the relevance

of these ®ndings to anxiety-like behavior per se remains

unclear until these mice are subject to further tests. In a

study employing multiple behavioral tests, Grailhe et al.

[73] have recently found that inactivation of the 5-HT5A

receptor subtype had no impact on behavior in the elevated

plus-maze, defensive burying test, or open ®eld center time

assay.

In view of the fact that buspirone, a partial agonist at the

5-HT1A receptor, is a clinically effective anxiolytic, the

anxiety-related phenotype of mice lacking the 5-HT1A

receptor gene has been a major discovery. A line of 5-

HT1A receptor knockout mice generated by Hen and collea-

gues have consistently displayed heightened anxiety-like

behavior in various tests, e.g., the elevated plus-maze

[126] and center time in an open ®eld assays [126,127]. It

is interesting to note that anxiety-like phenotype was espe-

cially strong in male 5-HT1A mutant mice [126]. Evidence

of increased anxiety-like behavior in 5-HT1A knockout mice

has been con®rmed in an independently-generated line

extensively tested in the elevated zero-maze, center time

open ®eld assay, and novel object exploration test [46],

and in a third line that has been tested in the center time

open ®eld assay [128].

Given the absence of 5-HT1A and the concomitant

removal of autoreceptor inhibition of 5-HT release, one

could predict an increased level of extracellular 5-HT in

these mutant mice. However, neither Heisler et al. [46],

nor Ramboz et al. [126] have observed gross alterations in

5-HT tissue concentrations in 5-HT1A mutant mice. A

compensatory alteration, such as an up-regulation of 5-

HT1B receptors in 5-HT1A receptor-de®cient mice would

explain why this neurochemical phenotype was not

observed [128]. The recent ®nding that 5-HT1A knockout

mice are insensitive to the anxiolytic effects of diazepam

also implicates changes at the level of the GABAA-benzo-

diazepine receptor in the anxiety-like phenotype of 5-HT1A

mutant mice. Sibille and colleagues [129] have found

reduced benzodiazepine binding in the amygdala and cortex

in 5-HT1A mutant mice, coupled with an insensitivity to

anxiolytic doses of diazepam in the elevated plus-maze

and open ®eld center time assay. The authors speculate

that these changes are due to a loss of 5-HT1A-mediated

control over expression of a1 and a2 GABAA subunits in

the amygdala in mutants. In the context of these ®ndings, an

inducible gene knockout strategy to delete the 5-HT1A

receptor in the adult brain could prove to be valuable for

studying the functional roles and inter-relationships of the

receptor in anxiety-related behavior.

In contrast to the robust anxiety-like phenotype seen in 5-

HT1A mutant mice, 5-HT1B receptor knockout mice have

displayed unaltered or decreased anxiety-like behavior.

Ramboz et al. [130] found no evidence of anxiety-like beha-

vior in 5-HT1B receptor-de®cient mice tested in the light$

dark exploration test nor for open ®eld center time, while

Zhuang et al. [127] reported an anxiolytic-like phenotype in

terms of increased open ®eld center time in the same line of

mutants. Further evidence of attenuated anxiety-like beha-

vior in 5-HT1B mice comes from a study by Malleret et al.

[131] in which mutants showed no phenotype in the

elevated plus-maze, but increased center time in an open

®eld. Brunner et al. [45] also recently reported an anxioly-

tic-like phenotype in 5-HT1B mice by measuring separation-

induced ultrasonic vocalization in mutant mouse pups, but

again failed to see altered responses in adult mice tested in

the elevated plus-maze. The reason for the inconsistency of

anxiety-related behavior in 5-HT1B mice is unclear but may

re¯ect compensatory alterations in other 5-HT receptors or,

alternatively, a minor role for this receptor in the anxiety-

related processes.

3.4. Corticotropin-releasing hormone mutations and CRH-

receptor knockouts

Corticotropin-releasing hormone (CRH) has been

described as the master molecule of the hypothalamic-

pituitary-adrenal (HPA) axis, triggering the cascade of

hormonal, behavioral, and neurochemical events mediated

by the HPA in response to stress. CRH also acts as a

neurotransmitter. Dysfunction in mechanisms of the HPA

axis and CRH function have also been implicated in the

etiology and symptomatology of anxiety disorders [132].

This link is supported by animals studies which have

shown how central administration of CRH produces

alterations in anxiety-related behavior in rats [133,134].

In full agreement with these effects of exogenous CRH,

Stenzel-Poore et al. [135] have reported that transgenic

mice overexpressing CRH exhibit heightened anxiety-like

behavior as measured by the elevated plus-maze and

center time in a novel open ®eld. Further work has indi-

cated that the anxiety-like behavior in CRH transgenic

mice is centrally mediated, since intracerebroventricular

administration of a CRH antagonist reversed the pheno-

type [135], while adrenalectomy did not [136]. Interest-

ingly, two recent studies have found that mice in which

the CRH gene has been inactivated do not show reduced

anxiety-like behavior [136,137].

In agreement with the observation that antisense knock-

down of the CRH-R1 receptor leads to reductions in anxi-

ety-like behavior in rats [139], targeted deletion of CRH-R1

receptors produces alterations in anxiety-related behavior in

mice. To date, two groups have reported on mutant mice

lacking the CRH-R1 receptor, which has a widespread

distribution in the brain. Koob and colleagues have reported

dramatically reduced anxiety-like behavior in three separate

behavioral paradigms; elevated plus-maze, light$ dark

exploration test, and emergence test [51,140]. In an inde-

pendent line of CRH-R1 knockout mice, Timpl and cowor-

kers [141] have also seen evidence of an anxiolytic-like

phenotype in the light$ dark exploration test. These data


further indicate a role for central CRH function in the

mediation of anxiety-like behavior, and advocate the possi-

ble clinical utility of non-peptide CRH-R1 receptor antago-

nists.

The CRH-R2 receptor shows a pattern of distribution that

is primarily restricted in the lateral septum and ventromedial

hypothalamus, but also evident in the amygdala, anterior

and lateral hypothalamus, rapheÂ nuclei, bed nucleus of the

stria terminalis, and hippocampus [142]. Studies examining

the effects of CRH-R2 antisense to block receptor function

in rats have suggested that there was not a signi®cant role

for the CRH-R2 receptor in anxiety-like behavior [143,144].

However, there are two recent reports of altered anxiety-

related behavior in mice lacking the CRH-R2 receptor. Bale

et al. [145] report that CRH-R2 receptor knockout mice

display increased anxiety-like behavior in the elevated

plus-maze and open ®eld center time assay, but not the

light$ dark exploration test. In agreement with these ®nd-

ings, an independently generated line of CRH-R2 receptor

mutant mice have shown heightened anxiety-like behavior

in the elevated plus-maze and the emergence test (but a

pro®le more consistent with a reduced level of anxiety in

the open ®eld) [146]. However, while Bale et al. [145] found

that anxiety-like behavior was not speci®c to any one

gender, Kishimoto and colleagues found that increased

anxiety-like behavior was restricted to female mice [146].

In the absence of further evidence, these sex difference in

different lines of CRH-R2 receptor mice are dif®cult to

reconcile, but likely relate to differences in genetic back-

grounds, experimental procedures and/or behavioral tests.

4. Concluding remarks

Studies in transgenic and gene knockout mice have

already produced some signi®cant insights into the neural

mechanisms underlying anxiety. Evidence that mice with

either loss-of-function or gain-of-function mutations in a

speci®c gene exhibit signi®cant alterations in anxiety-

related behavior can have direct implications for the discov-

ery of novel drug therapies for anxiety disorders. In this

context, the more recent clinical classi®cations have

increasingly considered anxiety as a heterogeneous diagnos-

tic and biological disorder [147,148, but see 149]). In paral-

lel, converging evidence from the laboratory supports the

view that different tests for anxiety-like behavior in rodents

may be measuring different forms of behavior [12,13,35±

37,150,151]. This begs the question of whether particular

behavioral tests might more or less relate to speci®c anxiety

disorders.

In terms of predicting anxiolytic drug action, much of the

validity of the exploration-based tests as models of anxiety-

like behavior has rested upon their acute and reliable sensi-

tivity to low potency benzodiazepines, leading to the

suggestion that they best model benzodiazepine-sensitive

forms of anxiety, such as generalized anxiety disorder

[152]. Indeed, tests such as the elevated plus-maze are

less reliable in predicting anxiolytic activity of drugs used

to treat panic disorder, obsessive compulsive disorder, or

social anxiety disorder, which largely act via monoaminer-

gic mechanisms, rather than the GABA/benzodiazepine

system [13]. Therefore, there is a danger that an over reli-

ance on a small number of existing behavioral assays that

may be good tests for one or more speci®c forms of anxiety

(e.g., generalized anxiety disorder), may be too narrow a

strategy for discovering novel therapeutic targets for other

forms of anxiety (e.g., panic disorder). In this context, the

continued success of the gene mutant approach to ®nding

genes relevant to anxiety-related behavior will partly be

dependent on the development of a broad battery of beha-

vioral tests for anxiety.

Acknowledgements

I would like to thank Dr Jacqueline Crawley, Dr Dennis

Murphy and Professor John Rodgers for their critical read-

ing of a draft of this manuscript, and Sara Kinsley for her

help in constructing the References Section.

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Targeted gene mutation approaches to the study of anxiety-like behavior in mice