Tests of unconditioned anxiety — Pitfalls and disappointments

PHB-10447; No of Pages 17 Physiology & Behavior xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Physiology & Behavior journal homepage: www.elsevier.com/locate/phb

Review

2

Unconditioned tests of anxiety — Pitfalls and disappointments

3Q1

A. Ennaceur ⁎ University of Sunderland, Department of Pharmacy, Wharncliffe Street, Sunderland SR1 3SD, UK

6

H I G H L I G H T S

1 6 1 5 17 18 19 20 21

a r t i c l e

22 23 24 25 26 27 44 28 29 30 31 32 33

Keywords: Fear Stress Avoidance Novelty Preference Grooming Rearing GABAA Anxiolytics Mice Rats

Contents 1. 2. 3. 4. 5. 6.

R O

R

R

E

C

T

E

The plus-maze, the light–dark box and the open-field are the main current unconditioned tests of anxiety for mice and rats. Despite their disappointing achievements, they remain as popular as ever and seem to play an important role in an ever-growing demand for behavioral phenotyping and drug screening. Numerous reviews have repeatedly reported their lack of consistency and reliability but they failed to address the core question of whether these tests do provide unequivocal measures of fear-induced anxiety, that these measurements are not confused with measures of fear-induced avoidance or natural preference responses — i.e. discriminant validity. In the present report, I examined numerous issues that undermine the validity of the current tests, and I highlighted various flaws in the aspects of the tests and the methodologies pursued. This report concludes that the evidence in support of the validity of the plus-maze, the light/dark box and the open-field as anxiety tests is poor and methodologically questionable. © 2014 Published by Elsevier Inc.

N C O

47

51 52 53 54 55 56 57 58 59 60 61 62

a b s t r a c t

Article history: Received 23 December 2013 Received in revised form 21 April 2014 Accepted 28 May 2014 Available online xxxx

48 46 45

50 49

i n f o

P

Review of the discriminant validity of the current unconditioned tests of anxiety Issue with translation of the operational definition of anxiety into behavior tests There is no concordance between spatiotemporal and ethological parameters Pharmacology is not sufficient or necessary in the validation of behavioral tests Novel open space anxiety tests are proposed as alternatives to the current one.

D

• • • • •

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Definitions of fear and anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Issue with aversion, natural preference, conflict and security . . . . . . . . . . . . . . . . . Issue with avoidance of aversive stimuli and pending threat . . . . . . . . . . . . . . . . . Issue with experience of stress and anxiety in the protected/lit space vs. unprotected/unlit space Issue with sensitivity, state and trait anxiety . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Sensitivity to drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.1. GABAA subtype receptors . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.2. Other neurochemical targets . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Sensitivity to strain difference . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Sensitivity depending on anxiety types . . . . . . . . . . . . . . . . . . . . . . . 6.4. Sensitivity depending on state and trait anxiety . . . . . . . . . . . . . . . . . . .

U

7 8 9 10 11 12 13 14

O

4 5

F

1

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

⁎ Tel.: +44 773 249 0550. E-mail address: [email protected].

http://dx.doi.org/10.1016/j.physbeh.2014.05.032 0031-9384/© 2014 Published by Elsevier Inc.

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

0 0 0 0 0 0 0 0 0 0 0 0

34 35 36 37 38 39 40 41 42 43

7.

Issue with pharmacological validation . . . . . 7.1. Animal models of human behavior . . . 7.2. Animal models of human pathology . . . 8. Issue with stress-induced potentiation of anxiety 9. Ethological parameters . . . . . . . . . . . . 9.1. Self-grooming and rearing behavior . . . 9.1.1. Self-grooming . . . . . . . . 9.1.2. Rearing behavior . . . . . . . 9.1.3. Grooming vs. rearing . . . . . 9.2. Stretch attend posture and head-dipping . 9.2.1. Stretch-attend posture (SAP) . . 9.2.2. Head-dipping . . . . . . . . . 10. Alternatives to the current tests of anxiety . . . 11. Conclusion . . . . . . . . . . . . . . . . . . 12. Uncited reference . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

79

2. Definitions of fear and anxiety

110 111

Fear and anxiety are “overlapping, aversive, activated states centered on threat” [296]. The distinction between the two has been difficult and controversial [80,427] due to their overlapping nature. Fear and anxiety have been considered to refer to the same [256,370,428]

102 103 104 105 106 107

112 113

t1:1 t1:2

C

E

R

R

100 101

O

98 99

C

96 97

N

94 95 Q2

U

92 93

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

or different constructs [23,67,152,253,305,319,323] or regarded as parts of the same continuum [128,232,288,354]. Generally, fear is defined as a negative emotional state associated with the perception of imminent or present threat to wellbeing or survival. It is a defensive reaction that motivates and/or facilitates the detection, escape, and avoidance of impending identifiable danger. Anxiety, on the other hand, is defined as a negative emotional state associated with the perception of potential or ambiguous threat. Like fear, it is a defensive reaction, but is characterized by a feeling of apprehension, uncertainty, worries, uneasiness or tension stemming from the anticipation of potential threat or negative outcomes [23,98,152,295,296]. Hence, in fear conditions, humans and animals face an unambiguous situation; they can avoid the threatening stimulus or escape to safety. The aversive stimulus does not carry any positive incentive that diminishes or moderates the need to avoid or escape. However, in anxiety conditions, humans and animals face an ambiguous situation. They are unable to avoid/escape or approach the perceived threat stimulus [but see, Sections 4 and 11]. They experience a high level of uncertainty and unpredictability as the threat stimulus appears to be associated with both positive and negative outcomes [248,342].

114

3. Issue with aversion, natural preference, conflict and security

134

The EPM consists of four arms radiating from a central platform forming a plus sign shape; it is elevated from the ground with two opposed walled arms and two opposed open arms [168]. The EZM is a modification of the EPM. It consists of a circular runway divided in two enclosed quadrants opposite to two open quadrants [359,409]. The light–dark box consists of two chambers one lit and the other dark connected through a small opening or a tunnel [19,87,175]. The OF consists of either a cylindrical, rectangular or a square box with open top [54,163,412]. In all these three tests, animals seem to avoid the open and/or lit space of the open arms of the EPM, the lit chamber of the light–dark box and the central area of the OF. This avoidance response or “natural aversion” [242] is used as an indicator of anxiety in animals. It is based on the assumption that anxiety involves a conflict between the drive to avoid and the drive to explore a perceived threatening stimulus (i.e. an open space and/or a lit area of a test apparatus), and that the current tests set into play these conflicting drives [46,90, 169,281,346]. However, one can also view that animals demonstrate a “natural preference” for dark and/or protected spaces [177,261,272, 279,355,389,413], or that such preference optimizes safety and security [12,272,290,414]. Animals are offered a choice between aversive and non-aversive stimuli, and they choose the latter; they are not compelled to venture into the open and/or lit space. Animal scientists appear to hold a paradoxical attitude. They recognize that only the open and/or lit space is anxiogenic while at the same time attributing anxiety to

135

R O

109

90 91

. . . . . . . . . . . . . . . .

P

108

The elevated plus-maze (EPM) or zero-maze (EZM), the light–dark box (LDB) and the open-field (OF) are the main current unconditioned tests of anxiety for mice and rats. They are all intensively used, particularly the EPM, in the study of the neurobiological basis of anxiety and in screening for novel targets and anxiolytic compounds. The validity of these tests has been questioned in numerous reports [29,45,51,64,86, 101,183,314,329,321,390]. However, these tests, in particular the EPM, are considered very popular and elected as the reference standard for their sensitivity to benzodiazepines. Hence, introduction of a novel methodology and approach is systematically rejected if it does not include one of these tests for comparisons. But, do these tests really measure the construct of anxiety, and not something else? What standard of reference status do they provide for enforcing their comparisons with novel alternative tests? Does pharmacological validity have any relevance to the construct validity of a behavioral test? (See Table 1.) In this review, I will examine various aspects of the current unconditioned tests of anxiety and highlight the numerous issues that in my view undermine the validity of these tests. The review is divided in two major parts; one concerns the spatio-temporal parameters of these tests and the second part concerns the ethological parameters. The lack of concordance between these two and the ambiguity in the interpretations of the observed animal responses is discussed. I hope that this critical assessment will initiate a constructive debate about the process of validation of behavioral tasks in animal studies. I would like to emphasize here that my concern is not whether or not animals experience anxiety in the EPM, the light–dark box and the OF. It is likely that they do. My concern is whether these tests provide unequivocal measures of anxiety.

88 89

. . . . . . . . . . . . . . . .

D

81

86 87

. . . . . . . . . . . . . . . .

E

1. Introduction

84 85

. . . . . . . . . . . . . . . .

T

80

82 83

. . . . . . . . . . . . . . . .

O

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

F

2

Table 1 Number of publications reports. *any suffix, •April 13, 2013.

t1:3

PubMed search terms

Published reports•

t1:4 t1:5 t1:6 t1:7

(plus maze) AND anxi* AND (rats OR mice) (zero maze) AND anxi* AND (rats OR mice) (open field) AND anxi* AND (rats OR mice) (light dark box) AND anxi* AND (rats or mice)

4523 192 2455 398

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198

202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222

C

178 179

E

176 177

R

223 224 225 226 227 228 229 230 231

5. Issue with experience of stress and anxiety in the protected/lit 232 space vs. unprotected/unlit space 233 It proved difficult to determine whether animals do or do not experience anxiety when their exploration is mostly, if not totally, limited to the protected/unlit space. There are some physiological studies based on measures of the concentration of plasma corticosterone, which indicate increased level of stress in animal exposed to the anxiety tests. However, most of these studies failed to indicate whether this level of stress is directly linked to animals' experience of the open/unprotected space. A number of studies reported an increase in plasma level of corticosterone and/or adrenocorticotropic hormone (ACTH) in animals exposed to the EPM [11,165,189,230,239,249,333], the light–dark box [189,200,258] and the OF [144,164,167] compared to the home cage. Other studies reported no differences in plasma corticosterone between anxious and less anxious animals in the EPM [192,195,358] and in the OF [8,405] and no effect of corticosterone injection on anxiety measures in any of these test [61,153,206,289]. In addition, mice overexpressing glucocorticoid receptor were found to display increased anxiety in the EPM compared to wild type mice without changes in plasma ACTH and corticosterone levels [408]. All these studies did not discriminate between the behavior of animals restricted to parts of the anxiety test apparatus that are considered anxiogenic and those parts that are non-anxiogenic. Hence, they do not indicate whether animals experience a high or low level of stress in the protected and/or unlit space. Currently, high level of anxiety is attributed to animals that show a preference for the protected and/or unlit space. Some studies reported that high anxiety rats confined to the open arms displayed high plasma ACTH and corticosterone compared to low anxiety rats [230,344]. Unfortunately, in these studies the level of these hormones was not examined in rats confined to the enclosed arms for comparison. The differences between the two substrains could have been present in both conditions. A previous study by Liebsch et al. [239] did not report any significant difference in plasma ACTH and corticosterone between high and low anxiety rats exposed to the EPM and no significant correlations between these two hormones and open arm entries. Rodgers et al. [333] also did not find significant correlations between plasma corticosterone and open arm entries in both rats and mice. They found instead, that plasma corticosterone was highly correlated with measures of risk assessment. This was confirmed in a subsequent study by Mikics et al. [275] who reported that measures of anxiety were not affected by metyrapone, a glucocorticoid synthesis blocker, or corticosterone treatment, and that plasma corticosterone levels correlated positively with measures of risk assessment in the EPM and OF tests. Another study [11] also confirmed that metyrapone did affect risk assessment but had no effects on anxiety measures in the EPM. Since risk assessment is directed towards the open arms, one can assume that animals express high level of stress towards these arms in the central area of the maze and that they experience less stress in the enclosed arms [see Section 10.2.]. A recent study by Mendes-Gomes et al. [270] assessed the level of corticosterone in mice exposed to an EPM with two open and two enclosed arms, an EPM with all four arms open or an EPM with all four arms enclosed. They reported that the plasma corticosterone level was increased in all the three maze types. These results confirm earlier

F

174 175

R

172 173

N C O

170 171

U

168 169

O

Exposure to an aversive stimulus does not lead automatically to the experience of anxiety, particularly when escape or avoidance responses are possible. Animals that cannot avoid or escape an aversive stimulus are likely to express more fear than animals that can avoid or escape [388]; escape or avoidance can lead to termination of the aversive stimulus and reduction of fear and anxiety [see, 246,429]. It can be argued that in both humans and animals avoidance responses were shown to indicate anxiety. In this case, one would question when fear-induced avoidance or escape is not fear-induced anxiety [23,99,377]. There are a number of situations from everyday life were avoidance is not accompanied with fear and/or anxiety despite the threatening or aversive nature of these situations. Furthermore, fear and anxiety experienced from exposures to a threatening object or a place are terminated by avoiding that object or a place. Avoidance instates or reinstates a sense of security; successful avoidance of these negative stimuli can act as a reward [112,210,316,343,352]. However, this is true only if the possibility to avoid these stimuli remains available, and that there is no pending threat from these stimuli in the near future. In humans, a pending threat from an aversive stimulus is a source of worry and apprehension. Humans anticipate and ruminate possible negative outcomes whether they are in or away from anxiogenic situations [35,43,336,374]. In this case, escape and avoidance would be considered

166 167

R O

201

165

P

4. Issue with avoidance of aversive stimuli and pending threat

163 164

D

200

161 162

as coping responses, which do not lead to the termination of the aversive stimulus. In the current tests of anxiety in animals, escape to or avoidance from the protected and/or unlit space is not associated with any pending threat; animals do not expect to be forced to move towards the aversive stimuli or that the aversive stimulus will be brought to them. Uncertainty and unpredictability can be a major source of fear and anxiety in animals exposed for the first time to a novel environment, but how one will measure anxiety in animals that freely choose safety and security over risk-taking? (see Section 11).

T

199

animals that naturally prefer and chose the protected and/or unlit space. The present paradox arises from a confusion between a conflict that emerges from a visual contrast formed by two physical entities presented opposite or side by side (i.e. open vs. enclosed, light vs. dark, white vs. black) and a conflict that results from the action of two opposite drives (i.e. approach vs. escape or avoidance). In the current unconditioned tests of anxiety, there is no evidence of conflicting drives, neither in the case of entry into the protected/unlit space nor in the case of avoidance of the unprotected/lit space. For a conflict to occur, each available choice option needs to be associated with both positive and negative outcomes, hence contributing to a feeling of loss of control and predictability [244,429]. It has been suggested that withholding entrance (passive avoidance) is indicative of anxiety [266,267]. In a passive avoidance task, rats or mice are exposed to a test chamber divided into a black chamber, for which they show innate preference, and a brightly lit chamber, for which they show an innate aversion. Administration of a mild electric shock after an entry into the dark chamber is followed on subsequent exposure to the test by avoidance of this chamber. Animals are considered to experience anxiety as they found themselves in the brightly lit chamber withholding entrance into the dark chamber which is their naturally preferred area. This passive avoidance does not compare to that observed in the EPM or EZM, the light–dark box and the OF. In these tests, animals show preference for the protected and/or unlit space, and stay there most of the duration of the test session. It is not clear how anxiety is determined in these tests when animals are considered as withholding entrance into a protected and/or unlit space for which they have a natural aversion. Furthermore, McNaughton and Corr [268] stated that “one only approaches a threat if there is some positive, conflicting, reason that makes avoidance inappropriate”. Is there any positive than can be sought by rats or mice in the open and/or lit space of the current unconditioned tests of anxiety, and why would avoidance of this space be inappropriate? In addition to the above, there is a question mark regarding avoidance responses of open/unprotected spaces where there is nothing to explore. We observed in an OF and an elevated platform that rats and mice did cross into and spent time in the central area when it was occupied by an object than when there was no object [3,120,122]. Hence, it is possible that animals do not venture into the central area of an OF not because of fear but because there is nothing there to stop at and explore.

E

159 160

3

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 Q3 282 283 284 285 286

310 311

A number of studies have examined the sensitivity of the current tests of anxiety to the difference between strains of animals [48,211, 278,317,334,358,402], and to the effect of drugs [94,159,235,314,335], lesions [309,379,391] and genetic manipulations [91,297,410]. The inconsistent and contradictory results of these studies have been discussed and debated in numerous publications [48,51,64,183,243,294, 331,335,390,402,403] but these remain unsatisfactory as they did not address correctly the core question I raised earlier concerning whether they provide unequivocal measures of the construct (i.e. anxiety) that they are meant to measure.

312

6.1. Sensitivity to drugs

313 314

The validity of the current tests of anxiety relies heavily on their sensitivity to drugs that have shown anxiolytic properties in the clinic. Benzodiazepines form the basis of their validity; hence, they are considered a gold standard of reference for any novel potential anxiolytic compounds. A lack of sensitivity to this class of drugs seems to invalidate the construct validity of any novel behavioral test. However, with the introduction in the clinic of buspirone, a 5-HT-1A partial agonist and a first non-benzodiazepine anxiolytic, the current tests of anxiety proved unable to accommodate this novel drug [16,63,77,100,157,171, 191,259,314]. Inconsistent and contradictory results are exemplified with serotonin-selective reuptake inhibitor (SSRI), fluoxetine, which was reported to exert anxiolytic as well as anxiogenic effects with both acute and chronic treatment [114,116,158,293,325,361,362]. This issue of sensitivity was further exacerbated by the observation that the current tests were unable to distinguish psychostimulant from anxiolytic effects [100,101,409]. Their insensitivity to non-benzodiazepine anxiolytics and their sensitivity to psychostimulants raised questions about their validity. Have it not been to clinical evidence, buspirone and other 5-HT compounds would have been most likely discarded in preclinical studies. Unfortunately, despite these major drawbacks, the EPM, the LDB and the OF remained and are still as popular as ever.

319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347

6.1.2. Other neurochemical targets Over the years it emerged that various drugs acting at receptor targets other than GABAA receptors can also reduce anxiety [see, 209]. These include not only 5-HT [9,149,410], dopamine [104,109,233], noradrenaline [172,193], glutamate [94,174,240,371], histamine [324,423,426], neuropeptides [159,188,300,375,386], cannabinoids [180,282,301], and acetylcholine [55,105,237] but also GABAB [143,194] and GABAC [95] receptor subtypes. This diversity and ever increasing number of neurotransmitter receptor targets suggest that anxiety is spread over various neurochemical systems though a drug action at any one system is shown to be sufficient to elicit anxiolysis. It is not clear how drugs acting by a vast number of different mechanisms converge to produce the same output. Attempts to accommodate interactions between systems have been very limited [2,162,373,399,415]. There are a number of reports where the effects of different drugs acting by different mechanisms is accounted for by the presence of different forms of anxiety [27,138,190, 184,227,318]. However, there are also numerous studies where the anxiolytic effect of the same drug is reported for the same behavioral tests that have been associated with these different forms of anxiety [see Section 4.3].

392 393

T

C

317 318

E

315 316

R

308 309

R

306 307

O

304 305

C

298 299

N

296 297

U

294 295

F

302 303

293

O

6. Issue with sensitivity, state and trait anxiety

291 292

348 349

R O

301

289 290

sedative effects [41,223,265], and GABAA receptors containing α5subunits have been implicated in mediating their effects on cognition [78,92,315]. This hypothesis has yet to be subjected to thorough investigations as access to mutant mice and to specific α2/α3 and α5 ligands has yet to become widely available. However, the accumulated evidence so far seems to undermine the acclaimed dissociation of GABAA subunit functions. Kralic et al. [223] reported that the sedative/hypnotic effects of non-selective benzodiazepines involve action at receptor subtypes other than α1-containing receptors. This was supported by subsequent reports, which suggest that sedation may be partly dependent on activity mediated by α5- [347,397] and α3- [139] containing GABAA receptors. In addition, α1 was reported to mediate ataxia while α5 mediate muscle relaxation [276] and analgesia [214,287], and both α1 and α5 were reported to mediate the stimulant or reinforcing effects of alcohol [173,198,199,222,339] and the impairing effects of benzodiazepines in learning and memory [32,348,349,416]. The specific function of α1-GABAA receptors in mediating sedation is further compromised by the introduction of a novel drug with high selectivity at α1. This drug is reported to reduce anxiety in mice without myorelaxant or amnesic effects [356]. A recent report [366] implicates the effect of benzodiazepines in reducing conditioned fear to their specific action on α1-GABAA receptors, and in reducing anxiety to their specific action on α2-GABAA receptors alone. The specific function of the other subunits-containing GABAA receptors has also been compromised. It has been reported that loss of righting reflex is dependent on the activation of α2-GABAA receptors by benzodiazepines [382], that these receptors control the rate and regularity of sustained feeding [85], modulate theta activity in REM sleep [219], and contribute to the reinforcing effects of drugs of abuse [113,284], that α3-GABAA receptors mediate benzodiazepines-induced hyperphagia [285] and that both α2- and α3-containing GABAA receptors mediate the rewarding [322] and analgesic [107,182,214,215,287] but not the anxiolytic [182] effects of benzodiazepines. In addition, TPA023, a GABAA α2,3 subtype-selective partial agonist that have no efficacy at the α1- or α5-containing GABAA receptors, was reported to reverse ketamine-induced impairments of a spatial delayed response in Rhesus monkeys [66]. Another drug with selective activity at GABAA receptors containing α2 or α3 subunits, MK-0777, was reported to improve cognitive functions in individuals with schizophrenia [238] but this was not confirmed in a subsequent report [60]. While it was predicted that α2- and/or α3-containing GABAA receptors would be the specific mediators of the anxiolytic effects of benzodiazepines [92,108,247], it appears now that this may not be the case. Here once again, the current tests of anxiety demonstrate no specificity and no predictive validity.

D

300

report by Copland and Balfour [82], which demonstrated that rats subjected to acute exposure to a fully enclosed or fully open X-maze produced comparable increase in plasma corticosterone. However, Mendes-Gomes et al. [270] also reported that the plasma corticosterone level was significantly low in animals exposed to the EPM with all arms enclosed compared to animals exposed to the other mazes. These results confirm an earlier study [304], which demonstrated in rats that the level of corticosterone was significantly more elevated in animals confined to the open than to the enclosed space of the EPM. Hence, both studies suggest that the level of stress experienced in the protected space is significantly lower than that experienced in the unprotected space. Animals continued to prefer the safety of the enclosed arms of the EPM after a previous confinement experience of the open arms [389].

E

287 288

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

P

4

6.1.1. GABAA subtype receptors Since the discovery of the effectiveness of benzodiazepines in reducing anxiety in humans, pharmacological interventions and later genetic manipulations have been conducted in animals to determine the receptor targets of these drugs. These approaches were mainly driven by the need to develop novel anxiolytics without the undesirable effects of the benzodiazepines. The current tests of anxiety played a major role in this venture. They were used to determine whether a particular drug reduces anxiety, and whether such effect is not a consequence of non-specific effects of a drug. GABAA receptors containing α2- and α3-subunits have been implicated in mediating the anxiolytic effects of benzodiazepines [108,139,247,265,283,341] while GABAA receptors containing the α1-subunit have been implicated in mediating their

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391

394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 Q4

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

445 446

Another suggestion made by Rogers et al. [1997] is that some behavioral tests tap into one aspect or type of anxiety and should be limited to benzodiazepines or to particular anxiolytic drugs that they proved sensitive to. However, they do not indicate what aspects of the existing tests contribute to drug specificity [see Section 4.4]. In addition, this proposition seems to ignore a number of reports where the anxiolytic effect of the same class of drugs has been demonstrated in different behavioral tests. Diazepam and chlordiazepoxide were shown to increase the number of entries and/or time spent in the open-arms of the EPM [72,359], in the lit compartment of the LDB [72,88], and in the center of the OF [74,314]. Similarly, the anxiolytic effect of drugs acting by different mechanisms has been demonstrated in more than one anxiety tests that proved sensitive to benzodiazepines [149,159,292,380].

447

6.4. Sensitivity depending on state and trait anxiety

448 449

Lister [243] suggested that attention needs to be paid to the distinction between state and trait anxiety as treatments for the former may not be of long-term benefit for the latter. This distinction was originally proposed by Cattell and Scheier [69]. State anxiety refers to anxiety which is situation specific experienced at a particular moment, while trait anxiety refers to the tendency of an individual to experience anxiety which is chronic and pervasive across situations; it is considered an enduring characteristic of a person. In my search of the literature, it does not appear that benzodiazepines and 5-HT drugs are prescribed exclusively to either one. The discrepancies between results from the current anxiety tests and their lack of sensitivity to non-benzodiazepine drugs led animal scientists to believe that the introduction of this distinction between state and trait anxiety could resolve these issues, that some anxiolytics are effective in one case but not in another [27,29,47,148,155]. They also suggest that behavioral tests of state anxiety are distinct from behavioral tests of trait anxiety [14,62,73,75]. However, they do not indicate in which aspects these tests differ between themselves and contribute to state or trait anxiety. In fact, these suggestions are based on the belief that state anxiety equates to “normal anxiety” and trait anxiety equates to “pathological anxiety”, and as a consequence these two are not relieved by the same treatment [29,47]. This is a clear misunderstanding of the concepts state and trait anxieties [see 161,226,234]. Eysenck et al. [131] consider state anxiety as “the currently experienced level of anxiety”, and this “is determined interactively by trait

435 436 437 438 Q6 439 440 441 442 443 444

450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472

C

430 431

E

428 429

R

426 427

R

424 425

N C O

422 423

U

420 421

F

434 Q5

418 419

O

6.3. Sensitivity depending on anxiety types

417

R O

433

415 416

P

432

The lack of construct validity of the currents tests of anxiety account for the difficulty in determining consistently the strain of animals that demonstrates high or low levels of anxiety, and the strain of animals which is commonly affected by anxiolytic drugs [28,48,49,86,160,227, 235,260,310,334,335,396]. Baseline level of emotionality may have accounted for differences between strains [21,125,335,400] but it cannot account for the disparity of the results between separate experiments or between independent reports on the same strains of animals. A number of studies suggests that some strains of animals are best suited in detecting drugs that are anxiolytic while other strains of animals are best suited in detecting drugs that are anxiogenic, and that a particular strain of animals is suitable to detect the anxiolytic effects of a drug only in a particular anxiety test [65,75,260,389,396]. This implies that depending on the strains of animals, anxiety is either high or low, and a behavioral test needs to be selected according to its sensitivity to either one. This is an appealing proposition but, after 30 years of EPM, 20 years of LDB, and at least 70 years of OF scientists have yet to determine without equivoque a difference in anxiety level between strains of mice or rats that is stable and consistent across research experiments and between independent laboratories.

D

413 414

or test anxiety and by situational stress”. Therefore, the distinction between state and trait anxiety does not indicate two forms of anxiety as an emotional response, it only highlights the fact there are individuals that express anxiety very often when faced with a challenging or a stressful situation. State and trait anxiety inventory was developed by Spieleberg et al. [369] for this purpose. It is a psychometric test which contains separate self-report scales to measure two distinct anxiety concepts: state anxiety (A-State) and trait anxiety (A-Trait). Clinical studies, and a large number of cognitive neuroscience studies, rely on either or both scales from this inventory to determine differences between individuals in anxiety. Unlike animal behavioral tests of anxiety, these scales do not induce these differences. The current behavioral tests of anxiety in animals are inadequate to measure persistent anxiety in humans. They provide a stimulus, a condition or a situation that elicits a state of anxiety; whether this state of anxiety is acute or chronic depend on the conditions and characteristics of an individual. Consequently, the only way to measure trait anxiety would be to assess how often or how intensively an individual experiences anxious states [231]. Thus, in rodents, a high trait anxiety would correspond to an inner disposition to react anxiously in most anxiety-related test conditions. If two animals differ in this trait, this could be explained by differences in their genes and developmental environments [161,367]. In order to determine the enduring trait anxiety characteristic of some animals or strains of animals, one has to test each animal in a number of test situations that elicit variable levels of emotionality. High trait anxiety animals are expected to respond almost equally and frequently to all situations with a high level of anxiety whereas low trait anxiety animals will respond to a few number of situations with their level of anxiety likely to decrease on subsequent exposures.

E

6.2. Sensitivity to strain difference

T

412

5

473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501

7. Issue with pharmacological validation

502

In one of his early publication, Gray wrote that “(1) one can describe the psychological state which constitutes anxiety by studying the behavioural effects of drugs which reduce anxiety; and (2) that, by studying the physiological route by which the anti-anxiety drugs produce their behavioural effects, one can discover the physiological substrates of anxiety” [151]. Since then pharmacological validation became a pre-requisite for any behavioral test of anxiety. It is considered as a determinant of construct validity. Hence, anxiety tests are required only to predict anxiolytic properties of prescribed anxiolytic drugs as suggested by Belzung and Griebel [29] “Predictive validity implies that the animal model should be sensitive to clinically effective pharmacological agents”. This explains why, up to date, the current anxiety tests have yet to predict anything new. Their inability to predict anxiolytic compounds, which act through non-benzodiazepines mechanisms, led Rodgers et al. [331] to reiterate a statement made earlier by Lister [243] that “pharmacological validation alone does not make a test a model of anxiety”. However, it remains that most journal editors and reviewers do not look at a paper submission which does not include pharmacological validation. Is pharmacological validation of a behavioral test justified and necessary?

503 504

7.1. Animal models of human behavior

522

Pharmacological validation is not justified because if there were no anxiolytic drugs available we should still be able to assess anxiety in animals. Drugs have never been required to validate any of the behavioral tests that have been used to assess cognition, neither in humans nor in animals. Most if not all tests of anxiety in humans were designed and validated without the need for a drug [26,70,147,166,369]. Early animal studies on emotionality and anxiety did not need pharmacology too [13,25,58,130,277,281,365,378,385]. Behavioral tests are developed to measure a construct and the changes to defined parameters of a construct. Their use is not limited to the effect of drugs but includes strains and gender, lesion applications, genetic manipulations, neuronal and neurochemical changes and modifications. If a test is able to distinguish

523

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521

524 525 526 527 528 529 530 531 532 533 534

555

Pharmacological validity should be aimed at an animal model of an anxiety disorder as it implies that there is an identified biological target and mechanism that it would interfere with to modify this emotional maladaptive behavior [220,228,240,241,291,321]. Animals that display exaggerated or pathological anxiety could be found among strains of mice and rats (or a selected sub-strain) or produced by environmental, pharmacological, neurochemical or genetic manipulations. These animals would be considered to represent a model of “pathological anxiety”. They would demonstrate symptoms that a behavioral test should be able to detect and measure.

556

8. Issue with stress-induced potentiation of anxiety

557 558

582

Anxiety may be expressed in the EPM, the LDB and the OF but there is a difference between the assumption that a particular behavior response indicative of mental state or mental process is involved in a behavioral test and the actual measure(s) of that particular response in that test. My view is that the existing unconditioned tests of anxiety in animals involve fear-induced escape or avoidance rather than fearinduced anxiety. The presence of protective or dark space is likely to reduce this fear after prolonged or repeated exposures [81,169,389]. In order to insure that anxiety is consistently and reliably expressed by animals in the current tests, a number of scientists resorted to prior exposure of animals to acute stressors such as social defeat [327,407], electric shocks [22,376], forced swim [14,22,179], immobilization [10, 22,401], and exposure to a predator or a predator odor [7,17,206,327, 418,424,425]. It was thought that the results of the EPM are very robust when fear is potentiated by prior stressor [221,261,401], unfortunately this was not the case as inconsistencies soon emerged with acute and chronic exposure to these stressors [15,132,180,251,261,263,337,409]. The conflicting evidence from fear potentiation led to the suggestion that the defensive behavioral repertoire of animals during exposure to these stressors is more relevant to anxiety on its own than the anxiety tests themselves [11,33,39,206,306,357, but see, 251]. With the introduction of stress induced potentiation, the spatio-temporal parameters of the existing tests were complemented with ethological parameters [62,64,74,331,362,357,409], but now it seems that there is a complete switch from the existing unconditioned tests of anxiety to ethological assays [39,40,103,111,271,337,381].

583

9. Ethological parameters

584

Self-grooming, rearing, thigmotaxis and risk-assessment were introduced in the study of drug actions on behavior as complementary or replacement to spatio-temporal parameters in the assessment of anxiety in animals [64,330]. However, lack of sensitivity and consistency of spatio-temporal parameters does not seem to have been bettered by these ethological parameters as described below. Ethological parameters proved unreliable and subject to ambiguous interpretations. In addition, there is no concordance between results obtained with ethological parameters and those obtained with spatio-temporal parameters.

565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581

585 586 587 588 589 590 591 592 593

9.1.2. Rearing behavior It consists of animals standing on both hind paws in a vertical upright posture. It is considered an exploratory behavior [63,135], an orienting response [79], a means of sampling or scanning the environment [1,264], or a marker of environmental novelty [181,236]. Like other ethological parameters, rearing has been used as an indicator of

652

T

C

563 564

E

561 562

R

559 560

R

553 554

O

551 552

C

549 550

N

547 548

U

542 543

595 596

F

546

541

9.1.1. Self-grooming According to Bolles [42] and Spruijt et al. [372], self-grooming occupies up to 40% of the waking time of adult rats. It is considered as a body care behavior [203,372], a form of scent dissemination for olfactory communication and sexual attraction [97,133,134,417]. It is also suggested that it may be performed for body temperature regulation [384]. Another possible role of self-grooming which has not been fully addressed in the literature is self-stimulation and pleasure [257,422]. Some authors reported that self-grooming is displayed in situations involving stress and conflict [135,146,170,203,252,338] while others reported that self-grooming is observed after social conflict and fight, and argue that preoccupations with threats are of higher priority than self-grooming [20,372]. It is also reported that self-grooming is observed in subordinate animals in close proximity to a dominant animal [31,145,393] though some reported that dominant males and females are involved in more self-grooming than subordinates [106,273,274]. Excessive self-grooming has been proposed as a model of human obsessive compulsive disorder [360,387,421] and autism [262,363,364, 419,420]. Lister [243] warned against misinterpretation of this behavior which may have no clear obsessional dimension. Excessive selfgrooming could be stereotyped repetitive movements with no intentional purpose. Since the compulsive behavior is considered a ritual, which is performed to avoid intrusive thought, one would need to demonstrate at least that animals perform a behavioral ritual such as excessive grooming in anticipation of a punishment or exposure to an aversive stimulus. It is reported that self-grooming is observed only against walls and in the corners of an OF [74] and in the enclosed arms of the EPM [254,275]. It was reported to be reduced by both benzodiazepines and serotonergic drugs [16,24,74,118,351] but this was not confirmed in other studies [2,93,357,368]. Furthermore, it was found not to correlate with other indices of anxiety [137,203,229,269] and it seems suppressed or unaffected by exposure to a stressor [38,115,140,224,395]. To account for these discrepancies, some studies suggested that the magnitude of self-grooming responses is dependent on the type and intensity of the stressor [102,141,217] but these were also not confirmed in other studies with stressors of various intensity [18,36,170,202,395]. These conflicting reports are further complicated by a number of studies on anxiety in BTBR T + tf/J (BTBR) mice, which are considered a model of autism for their excessive self-grooming. In these studies, BTBR mice demonstrated either reduced or no anxiety compared to B6 mice in the EPM [30,71,286,364,420], the EZM [262,308], the light– dark test [420,286,364] and the OF [262,286,363,419]. However, they demonstrated increased anxiety in the EPM after being subjected to 90 second tail suspension [30]. This stress reactivity was not confirmed in another study [364]. One study, reported that BTBR mice show increased anxiety in the EPM but reduced anxiety in the EZM [308]. Excessive grooming has been observed in mutant mice model of OCD but its association with anxiety remains conflicting. In two studies, SAPAP3−/− mice and Slitrk5−/− were found to spent less time in the center of the OF [360,411] and in the open arms of the EPM [360,411], and less time in the lit chamber of the LDB [411] compared to their control. However, other mutant mice (shank 3) which display excessive grooming too demonstrated no difference with control in the OF, the light–dark box [303,406], and the EZM [406]. One study [303] reported reduced time in the open arms of the EPM while another study [406] reported no difference in the EZM compared to control.

O

7.2. Animal models of human pathology

539 540

594

R O

545

537 538

9.1. Self-grooming and rearing behavior

P

544

consistently and reliably between anxious and less anxious animals with any of these manipulations, then lack of sensitivity and reliability to the effect of a drug cannot invalidate this test but it rather provides clear evidence that such drug may be not anxiolytic or may have no specific effect on anxiety. A behavioral anxiety test should be able to detect and consistently predict any drug treatment that may have anxiolytic properties rather than trying to predict drugs retrieved from the apothecary shelves. If pharmacological validation is set to determine the validity of a behavioral test, then one has to rely on good luck or be content with me-too drugs in drug discovery.

D

535 536

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

E

6

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651

653 654 655 656 657

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722

9.1.3. Grooming vs. rearing Rearing is mostly used by animals to reach to something distal either physically (reach, touch and/or grasp) or sensorially (visual or smell) whereas self-grooming is a preoccupation with one's own body. When confronted with an anxiogenic stimulus or condition, self-grooming is likely to be inhibited [38,52,115,140,224,252] as it would distract attention from an impending threat and would interfere with defense mechanisms. However, this decrease in self-grooming would be expected to be accompanied by an increase in rearing [but see, 115,110] which is directed towards stimuli of the environment of immediate concern. Unfortunately, it does not appear to be the case in a large number of animal studies of anxiety. Grooming and rearing were found to be reduced in anxious mice in the EPM and the OF [65] in animals subjected to social defeat and exposed to the EPM [327]. They were also suppressed by chronic “moderate” environmental stress in the OF in mice [50,115,250] and by both acute and chronic exposure to cats in rats [38]. However, it is not the case between studies. Angrini et al. [16] observed increased rearing and grooming in rats exposed to the OF and these were attenuated with anxiolytic drugs. Other studies observed increased grooming and reduced rearing in anxious mice in the OF [129,227], the plus maze [129], and in rats exposed to cats [326] or to a predator odor [404]. It is also reported that exposure to moderate stress [bright light and white noise] increased grooming and had no effect on rearing in rats tested in an OF [229,338] and that exposure to cat odor reduced grooming and increased rearing in rats [264]. Another study reported that predator and non-predator (sheep) odors had no effect on rearing and grooming [208]. A number of studies reported that benzodiazepines decreased both rearing and grooming in the OF [16,56,57,264] and the EPM [96],

759

9.2.1. Stretch-attend posture (SAP) It is performed by rats and mice when exploring a novel and/or anxiogenic object or environment [36]. It is described as standing still with forward-stretched elongation of the body and both head and forepaws extended outside a protected space [37]. It has been observed in the EPM or EZM [62,225,275,298], the LDB [34,62,225] and the OF [74,255,275,345] as well as in other ethological tests involving exposure to predators or their odors [17,206,418]. SAP is considered to reflect a risk assessment behavior [40,329]. It is associated with learning about the potential threat stimuli [307], reluctance to leave the confines/ security of the protected areas [37], and reflect approach/avoidance conflict [150,201,394,418]. High levels of anxiety are thought to be associated with an increased number of SAP [93,154,155,329,359]. However, in a number of studies the number of SAP in rats and mice is either very low (≤5) or not observed [65,245,255,298,312,409] or moderate (N5) to very high (N20) [17,96,135,185,275,334]. It is not clear if this is due to differences between the strains of animals, to the test conditions or to uncontrollable circumstances. In the plus maze, rats that were exposed to a cat demonstrated reduced arm entries and SAP [4,6] and rats that were exposed to cat odors demonstrated increased number and duration of SAP [4,206] whereas mice exposed to a cat demonstrated increased SAP which were higher than in mice exposed to cat odors. However, these predator stressors (i.e. cat or its odor) had no effect on open arm exploration [5]. This difference between rats and mice is complicated by other studies on strain differences. Anisman et al. [17] reported that in the rat

760 761

O

F

9.2. Stretch attend posture and head-dipping

R O

677 678

P

675 676

723 724

D

673 674

decreased grooming and had no effect on rearing in the OF [24,361], reduced rearing and no effect on grooming in the OF [74] or had no effect on both in the OF [56,57] and the LDB [187]. Other studies reported that in the EPM 5-HT anxiolytic drugs had no effect on rearing and grooming [93,362] while anxiogenic drugs reduced rearing with no effect on grooming [93]. When using ethological parameters, the nature of the stimulus may determine the direction of a response. It is possible that the first exposure to a novel environment, exposure to immediate (cat presence) and less immediate threat (cat odors) all involve anxiety but recruit different adaptive responses. In some cases, grooming would predominate over rearing and in other cases it is the opposite. However, one must provide some coherent explanation for the occurrence of these behaviors within defined and controlled conditions instead of the current messy state of affairs where confusion prevails due to lack of systematic and methodic investigation of these behaviors. Odors and in particular predator odors may increase grooming because odors diffuse and spread onto the animal space and may stick to animal fur. It is also possible that a predator releases some odors that are not detected by the experimenter; these odors are intended to promote obedience and submission from a receiver. Rearing may endow additional height to animals to reach and scan the surrounding environment, and to make a decision as to whether to proceed further away from safety or retract and withdraw to safety. Rearing is also demonstrated by some animals as a persuasive tool against attacks or intruders. In such situations, grooming would distract from rearing, and it is the immediacy of concern that would prioritize one type of behavior over the other [110] or would inhibit both in situation of aggression and fight [103]. In all our studies [3,120,122–124,272], animals exposed to an open space elevated platform do not display any rearing or self-grooming. Rearing is observed only in presence of an object, and both rearing and self-grooming are observed when a protected space is present. However, in an enclosed OF, animals do self-groom in the corners and do rear against the walls or in the presence of an object. This suggests that rearing and grooming are unrelated to animals' emotional responses.

E

671 672

T

669 670

C

667 668

E

665 666

R

664

R

662 663

N C O

660 661

anxiety in the EPM [129,235,330], the light–dark/box [84,89,235] and the OF [63,229]. Reductions in the number of rearing has been interpreted as heightened anxiety responses [29,329,331,359], and like grooming, it has been reported to occur mostly in the enclosed areas of the EPM [6,65,129,275,330,331] and against the walls and in the corners of the OF [63,65,74,229]. However, in a number of reports, there is no clear indication in which way rearing is indicative of or associated to anxiety or anxiolysis. In some studies, increased rearing seems to concord with increased anxiety [44,181,263,299,353] while in other studies decreased rearing seems to concord with increased anxiety [83,84,129,327,330,334,358,424]. In a number of these studies rearing was considered to relate to general locomotor or exploratory activity, and a correlation between the two was observed in the EPM [136], in the LDB [88] and in the OF [218,383]. However, this correlation analysis was not performed in most studies, and it proved neither consistent nor reliable in other studies [27,211,278,302, see Discussion section in 72]. A number of studies discriminate between measures of anxiety from measures of general activity. They relate activities in the open arms of the EPM, the lit area of the LDB and the central area of the OF to fear and anxiety, while activities in the enclosed arms, the dark area and the peripheral areas are related to general motor activity [29,93,229, 314]. Based on this discrimination ambulation, rearing, grooming which are performed in these areas, are not always considered to reflect anxiety [but see 93,327] though they are the main behavioral activities of animals that avoided the unprotected/lit spaces [6,63,65,74,129,229, 254,275,330,331]. Rodgers and Cole [327] make some exceptions as they wrote “total arm entries and rearing do not necessarily reflect a nonspecific treatment effect. Rather, in some circumstances, such changes may reflect additional signs of enhanced anxiety”. It seems that rearing is a specific indicator of anxiety as long as it fit well with the spatiotemporal parameter results: “As the drug produced a temporal redistribution of behaviour on the maze (reducing time centre/time closed, and increasing time open), the reduction in rearing can logically be attributed to a consequence of anxiolysis, rather than behavioural non-specificity” [330].

U

658 659

7

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758

762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786

810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851

O

F

868

R O

808 809

10. Alternatives to the current tests of anxiety The numerous methodological issues associated with the current behavioral tests for the assessment of anxiety in mice and rats cannot be resolved with some modifications to the layouts of the test apparatus or some changes to the test procedures. Any novel test of anxiety needs to expose animals to conditions which involve uninformative or ambiguous stimuli, and that the outcomes from the choice between these stimuli are uncertain. In an unfamiliar open space environment, the motivation to escape can be exploited to provide measures of anxiety. In this case, escape routes are made available, but the distant segments of these routes are left inaccessible to immediate or direct sensory perceptions. The experience of fear from the unfamiliar and open space is therefore complicated by the ambiguity of the choices and the uncertainty of the choice outcomes. The entry into any of the distal segment of the test environment is used to determine anxiety in animals. The above proposition is illustrated in the following descriptions of two open space anxiety tests. The first one is based on a modified version of the radial-maze in which animals need to cross an upward inclined bridge (15 cm length, ∠40°) to reach an arm (35 cm length) [Fig. 1]. In this test, mice explore several times the bridges and stop at the lines that separate the bridges from the arms [119]. This pattern of behavior is consistently observed in all mice. Some strains of mice take a long time to start crossing into the arms of the maze on a first or a second exposure to the test while other strains require more sessions. A recent study [126] shows that C57BL/6J ventured into a small number of arms in the first session, and made eight arm visits in the second session. CD-1 ventured into a small number of arms in the second session, and made eight arm visits in the third session. BALB/c ventured into a small number of arms in the third session, and made eight arm visits in the fifth session [see, Fig. 1]. The number of crossings into the arms was used as a measure of anxiety. It indicated that BALB/c mice expressed higher anxiety than the other two strains of mice. The second test is based on an elevated platform (80 cm × 80 cm) with downward inclined steep slopes (80 cm × 25 cm, ∠75°) attached to two opposite sides [Fig 2]. In this test, animals explore the platform but are hesitant to venture onto a slope [123,272]. Recent studies showed that C57/BL6J, CD-1 and BALB/c mice did move quickly away from the center, and explored the outer area of the platform; they spent most of the time in the areas adjacent to the slopes. However, C57/BL6J and CD-1 mice did cross onto the slopes while BALB/c mice remained the entire session on the platform [272]. The number of crossings onto the slopes was used as a measure of anxiety. BALB/c mice expressed higher anxiety than the other two strains of mice. In the open space of the 3D maze and the elevated platform with slopes, animals experience fear and try to escape. As there are no apparent refuges in proximity to escape to, animals would need to explore the distal parts of the test environment. In these tests, anxious

P

806 807

852 853

D

804 805

towards the unprotected arms looking for an escape route, hence demonstrating increased head dips [66,178,186]. It has been difficult to reconcile the view that escape or escape attempts from the unprotected arms reflect increased anxiety with the generally held view that entries into unprotected arms and head dipping indicate decreased anxiety [186,196,197,204,205,212,280]. As a consequence, two conflicting interpretations of the entries into the unprotected arms were proposed: “One is that their (rats) level of fear may have declined to the point where it is no longer sufficient to inhibit open-arm exploration; the other is that their (rats) level of fear may have increased to the point that it motivates a search for escape routes from the apparatus” [204,205]. These conflicting views of the same behavioral response appear to be overlooked due to the primacy attributed to pharmacological validity. They seem irrelevant if “clinically effective drugs” decrease animals' preference for the protected space of the EPM and the EZM.

T

802 803

C

800 801

E

798 799

R

796 797

9.2.2. Head-dipping Looking down over the edges of the EPM and the EZM is described as head-dipping [359]. This behavior was observed in protected and unprotected spaces. In the latter, animals are able to lean over the edge of the unprotected space but in the latter, they can lean over the edges of the open quadrant of the EZM while stretching from the enclosed quadrant, or head dip over the edges of the central area of the EPM from the enclosed arms. Hence, protected head-dips can be confounded with measure of stretch-attend postures. The number of head dips is considered an index of anxiety, and an increase of head-dips is associated with a decrease in anxiety [76,93,328]. However, treatments with chlordiazepoxide and diazepam were reported to increase open arm entries, and either increase [96,359, 409], decrease [304,398] or have no effect [53,350] on the frequency of head-dipping. Similar conflicting results were reported with buspirone, paroxetine and fluoxetine [117,216,325,332,359,362,398]. Head dipping is directed toward the outside of the maze; it can be viewed as an attempt by animals to find an escape route from a potentially dangerous environment [59,320,340]. Indeed, a number of studies reported that rats and mice peer over the edge of the unprotected space and jump to the floor [178,186,196,197,204,205,280,207,212,311,313]. When released on a platform, which was elevated by 50 cm from the ground, rats and mice displayed frequent head dips, and most of them jumped to the ground [127,272]. Comparable behavior was observed in rats exposed to an unstable EPM apparatus [212,213]. This suggests that head dipping contributes to risk assessment and escape response. Hence, animals that experience intense fear and anxiety may move

R

794 795

O

793

C

791 792

N

789 790

exposure test, in the EPM and in the OF, BALB/c mice displayed a high level of anxiety and elevated number of SAP compared to C57BL/6 mice. Makino et al. [255] reported also that BALB/c mice displayed increased SAP in the OF, however they found that C57BL/6 and DBA/2 mice never displayed such behavior whereas Podhorna and Brown [310] reported that C57BL/6 mice displayed high anxiety and made a high number of SAP than DBA/2 mice in the OF and in the EPM. This increase in SAP was also observed by Yang et al. [418] in C57BL/6 compared to BALB/c, and CD-1 mice in the rat exposure test. Other studies reported that SAP is unaffected in anxious strains of mice [176], that SAP did not discriminate between anxious and less anxious, stressed or non-stressed c57/BL6J mice [195] or proved inconsistent [6]. Benzodiazepine and non-benzodiazepine anxiolytics have been reported to reduce SAP in the plus maze and in the OF [93,74,155,156, 317,331,345,357,409]. Other studies reported that diazepam, chlordiazepoxide and fluoxetine did not affect SAP in the EPM [96,165,225,330, 362]. Similar mixed results were obtained with drugs that supposedly increased anxiety. They increased SAP in the EPM and EZM [142,359, 392], decreased SAP in the EPM [317] and in the LDB [225] or had no effect in the EPM [93,357,362]. Whether SAP is reduced or increased, it does not always concord with spatio-temporal parameters that are indicative of reduced or increased anxiety [211,357,362, see Section 5]. For instance, in one study on difference between strains of mice [211], the time spent in the lit chamber of the LDB was significantly high in FVB/N mice compared to BALB/c and C57 mice, the latter were no different from each other. However, the number of SAP was not different between FVB/N and C57, and it was low in these two strains compared to BALB/c. In another study on sex differences [245 female rats compared to male demonstrated low anxiety on spatio-temporal parameters in the EZM and white/dark box but high number of SAP in the EZM and not in the LDB. In a different study [357], 5-HT 1A agonist [ipsapirone] was reported to decrease SAP without effect on open-arm entries and time spent on open arms whereas 5-HT 2C agonist [TFMPP] and 5-HT 2A antagonist [SR 46350B] had anxiogenic effects with no major change on risk assessment. The above examples indicate clearly that there is no concordance between spatio-temporal and ethological parameters, and neither of these two provides a reliable measure of anxiety in animals.

U

787 788

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

E

8

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

854 855 856 857 858 859 860 861 862 863 864 865 866 867

869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915

9

O

F

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

P

D E T C E R

921

hideout consisted of a cylinder with small openings at the base; it occupied the central platform of the maze and the central area of the elevated platform. All three strains of mice demonstrated a preference for the enclosed space; this seems to reduce or eliminate the incentive to escape and to explore the distal segments of the test apparatus. The preference of safety seems to prevail over risk taking [12,272,290,414].

R

919 920

animals appeared unable to take risks, and showed increased amount of hesitations as measured by the number of crossings into the bridges [Fig. 1] and the amount of time spent in the areas adjacent to the slopes [Fig. 2]. When offered a hideout, both anxious (BALB/c) and less anxious (C57/BL6J and CD-1) strains of mice did not venture into the arms of the maze [121] and onto the slopes of the elevated platform [272]. The

N C O

917 918

U

916

R O

Fig. 1. Picture of the 3D maze in a raised arm configuration, and results showing the number of entries into the bridges (B) and arms (A) of the maze. Mice were left in the test apparatus until 8 arm visits were made or 10 min elapsed. All mice were unfamiliar with the test apparatus in the first session. They were not food or water deprived, and they were not offered any reward [see video http://www.ennassor.com/Anxiety/3DMaze/index.html].

Fig. 2. Picture of the elevated platform, and results showing the number of crossings onto the slopes. Mice were left in the test apparatus for 12 min [see videos http://www.ennassor.com/ Anxiety/Platform/index.html].

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

922 923 924 925 926 927

10

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx 30

300 Saline

Saline

Amphetamine 5mg/kg

250

Amphetamine 5mg/kg

25

Diazepam 1mg/kg

200

Number of crossings

Number of crossings

Diazepam 1mg/kg

150 100 50

20 15 10 5

0

0 s1

s2

s3

s1

s2 Sessions

s3

F

Sessions

945 946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975

11. Conclusion

R O

OF. However, it remains to be seen whether they can predict the anxio- 976 lytic effects of novel drug compounds. 977 978 979

12. Uncited reference

998 Q7

E

D

P

In the present report, I examined numerous issues that undermine the validity of the current tests of anxiety in rodents, and I highlighted various flaws in the aspects of the tests and the methodologies pursued. It is very apparent that these tests have no established construct validity. In my view, they do not provide unequivocal measures of anxiety; they may measure fear-induced escape/avoidance or just a spontaneous natural preference for enclosed or unlit spaces. This lack of construct validity accounts for the inconsistent and contradictory results in screening for novel anxiolytic drugs, and in mouse behavioral phenotyping. The current unconditioned tests of anxiety have been introduced, based on presumed face validity, to confirm the anxiolytic effect of benzodiazepines. The demonstrations that these tests were sensitive to these drugs, though without consistency, led scientists to make a big leap from face validity to predictive validity. It seems that predictive validity is limited to drugs that have been proposed by clinical studies, and it is apparently confused with sensitivity to the effects of these drugs. The present report concludes that the evidence in support of the validity of the EPM, LDB and the OF as anxiety tests is poor and methodologically questionable.

T

943 944

C

941 942

E

939 940

R

937 938

R

935 936

O

933 934

C

931 932

Rats and mice demonstrate a spontaneous natural preference for unlit and protected spaces. This species-specific behavior may have evolved to facilitate hiding, which is essential for animal survival. It is an adaptive response to avoid attack and predation. A number of species retreat to a refuge when confronted with threatening stimuli, and they may wait for hours until they feel that the danger has passed. It remains to be demonstrated whether they experience anxiety or not during the waiting time. However, this cannot be as elevated as when they are forced into an open space, and the options for escape are ambiguous or uninformative. The 3D maze proved insensitive to the anxiolytic effect of diazepam and chlordiazepoxide [121]. In fact, these drugs appear to increase anxiety in C57/BL6J and CD-1 mice; they reduced the number of entries into the arms. They had no effect on BALB/c mice. However, in the elevated platform, diazepam reduced anxiety in BALB/c mice; it increased the number of crossings onto the slopes [123,124]. The effects of benzodiazepines on cognition account for the conflicting evidence from these two tests. In the elevated platform, there are only two slopes. The choice between these two is less cognitively challenging than the choice between the eight arms of the maze. Hence, one would predict that an anxiolytic drug devoid of impairing effects on cognition would facilitate crossings into the arms of the 3D-maze. Indeed, a recent experiment with fluoxetine seems to confirm this prediction. BALB/c mice pre-treated for two weeks with fluoxetine crossed into the arms of the maze in their first exposure to the test, which suggests reduced fear and anxiety in this strain of mice. In the 3D maze and the elevated platform, the crossing into the arms and the slopes is a consistent and reliable indicator of anxiety in animals. Unlike the current tests, the 3D maze and the elevated platform discriminate systematically between high and low anxiety strains of mice. In the 3D maze, both anxious (BALB/c) and less anxious (C57BL/ 6J and CD-1) strains of mice venture frequently into the far end of the bridges; the anxious one do so for the whole 12 min session while the less anxious strain ventures into the arms after repeated hesitations [Fig. 1]. In the elevated platform, both anxious and less anxious strains of mice explore the outer area of the maze, and spend a significant amount of time in the areas adjacent to the slopes. The less anxious strains of mice do venture onto the slopes after repeated hesitations while the most anxious remain most of the 12 min test session in the areas adjacent to the slopes [Fig. 2]. BALB/c mice crossed onto the slopes of the elevated platform when treated with diazepam [Fig. 3]. Hyperactivity observed with amphetamine did not lead BALB/c mice to cross onto the slopes [Fig. 3]. The sensitivity of the 3D maze and the elevated platform remains to be challenged in independent laboratories. These tests were developed to overcome the methodological shortcomings of the current test of anxiety. Therefore, one cannot expect the 3D maze and the elevated platform to replicate the findings of the EPM, the light–dark box or the

N

929 930

U

928

O

Fig. 3. The number of crossings on the surface of the platform (left) was significantly increased in diazepam and amphetamine treated mice, and it was significantly high the latter compared to the former. However, only BALB/c mice treated with diazepam crossed onto the slopes (right). None of the saline and amphetamine treated mice did cross (right). The effects of amphetamine and diazepam were observed across three consecutive 12 min sessions, one session a day [see 123,124].

980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997

[68]

999

References

1000

[1] Abeelen JHF. Genetics of rearing behavior in mice. Behav Genet 1970;1:71–6. [2] Abrams JK, Johnson PL, Hay-Schmidt A, Mikkelsen JD, Shekhar A, Lowry CA. Serotonergic systems associated with arousal and vigilance behaviors following administration of anxiogenic drugs. Neuroscience 2005;133:983–97. [3] Abuhamdah RM, van Rensburg R, Lethbridge NL, Ennaceur A, Chazot PL. Effects of methimepip and JNJ-5207852 in Wistar rats exposed to an open-field with and without object and in Balb/c mice exposed to a radial-arm maze. Front Syst Neurosci 2012;6:54. [4] Adamec R, Kent P, Anisman H, Shallow T, Merali Z. Neural plasticity, neuropeptides and anxiety in animals—implications for understanding and treating affective disorder following traumatic stress in humans. Neurosci Biobehav Rev 1998;23:301–18. [5] Adamec R, Walling S, Burton P. Long-lasting, selective, anxiogenic effects of feline predator stress in mice. Physiol Behav 2004;83:401–10. [6] Adamec R, Bartoszyk GD, Burton P. Effects of systemic injections of Vilazodone, a selective serotonin reuptake inhibitor and serotonin 1A receptor agonist, on anxiety induced by predator stress in rats. Eur J Pharmacol 2004;504:65–77. [7] Adamec R, Burton P, Blundell J, Murphy DL, Holmes A. Vulnerability to mild predator stress in serotonin transporter knockout mice. Behav Brain Res 2006;170:126–40. [8] Ader R, Friedman SB, Grota LJ. Emotionality' and adrenal cortical function: effects of strain, tests, and the 24-hour corticosterone rhythm. Anim Behav 1967;15:37–44.

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1001 1002 1003 1004 1005 1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

[48] [49]

[50]

[51] [52] [53]

[54] [55] [56] [57]

D

[58]

C

E

R

R

N C O

F

[47]

O

[46]

R O

[45]

disorder: advances in research and practice. New York: Guilford Press; 2004. p. 77–108. Borta A, Schwarting RK. Inhibitory avoidance, pain reactivity, and plus-maze behavior in Wistar rats with high versus low rearing activity. Physiol Behav 2005;84:387–96. Bourin M. Animal models of anxiety: are they suitable for predicting drug action in humans? Pol J Pharmacol 1997;49:79–84. Bourin M, Hascoët M. The mouse light/dark box test. Eur J Pharmacol 2003;463:55–65. Bourin M, Petit-Demoulière B, Dhonnchadha BN, Hascöet M. Animal models of anxiety in mice. Fundam Clin Pharmacol 2007;21:567–74. Bouwknecht JA, Paylor R. Behavioral and physiological mouse assays for anxiety: a survey in nine mouse strains. Behav Brain Res 2002;136:489–501. Bouwknecht JA, van der Gugten J, Groenink L, Olivier B, Paylor RE. Effects of repeated testing in two inbred strains on flesinoxan dose–response curves in three mouse models for anxiety. Eur J Pharmacol 2004;494:35–44. Bouwknecht JA, Spiga F, Staub DR, Hale MW, Shekhar A, Lowry CA. Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors; relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus. Brain Res Bull 2007;72:32–43. Bouwknecht JA, Paylor R. Pitfalls in the interpretation of genetic and pharmacological effects on anxiety-like behaviour in rodents. Behav Pharmacol 2008;19:385–402. Bramley GN, Waas JR. Laboratory and field evaluation of predator odors as repellents for kiore (Rattus exulans) and ship rats (R. rattus). J Chem Ecol 2001;27:1029–47. Braun AA, Skelton MR, Vorhees CV, Williams MT. Comparison of the elevated plus and elevated zero mazes in treated and untreated male Sprague–Dawley rats: effects of anxiolytic and anxiogenic agents. Pharmacol Biochem Behav 2011;97:406–15. Brimblecombe RW. Effects of psychotropic drugs on open-field behaviour in rats. Psychopharmacologia 1963;4:139–47. Brioni JD, O'Neill AB, Kim DJ, Decker MW. Nicotinic receptor agonists exhibit anxiolytic-like effects on the elevated plus-maze test. Eur J Pharmacol 1993;238:1–8. Britton DR, Britton KT, Dalton D, Vale W. Effects of naloxone on anti-conflict and hyperphagic actions of diazepam. Life Sci 1981;29:1297–302. Britton DR, Britton KT. A sensitive open field measure of anxiolytic drug activity. Pharmacol Biochem Behav 1981;15:577–82. Broadhurst PL. Emotionality and the Yerkes–Dodson law. J Exp Psychol 1957;54:345–52. Brown GR, Nemes C. The exploratory behaviour of rats in the hole-board apparatus: is head-dipping a valid measure of neophilia? Behav Processes 2008;78:442–8. Buchanan RW, Keefe RS, Lieberman JA, Barch DM, Csernansky JG, Goff DC, Marder SR. A randomized clinical trial of MK-0777 for the treatment of cognitive impairments in people with schizophrenia. Biol Psychiatry 2011;69:442–9. Cai WH, Blundell J, Han J, Greene RW, Powell CM. Postreactivation glucocorticoids impair recall of established fear memory. J Neurosci 2006;26:9560–6. Calatayud F, Belzung C, Aubert A. Ethological validation and the assessment of anxiety-like behaviours: methodological comparison of classical analyses and structural approaches. Behav Process 2004;67:195–206. Carli M, Prontera C, Samanin R. Effect of 5-HT1A agonists on stress-induced deficit in open field locomotor activity of rats: evidence that this model identifies anxiolytic-like activity. Neuropharmacology 1989;28:471–6. Carobrez AP, Bertoglio LJ. Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 2005;29:1193–205. Carola V, D'Olimpio F, Brunamonti E, Mangia F, Renzi P. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res 2002;134:49–57. Casarrubea M, Roy V, Sorbera F, Magnusson MS, Santangelo A, Arabo A, et al. Temporal structure of the rat's behavior in elevated plus maze test. Behav Brain Res 2013;237:290–9. Castner SA, Arriza JL, Roberts JC, Mrzljak L, Christian EP, Williams GV. Reversal of ketamine-induced working memory impairments by the GABAAalpha2/3 agonist TPA023. Biol Psychiatry 2010;67:998–1001. Catherall DR. How fear differs from anxiety. Traumatology 2003;9:76–92. Cattell RB, Scheier IM. The meaning and measurement of neuroticism and anxiety. New York: Ronald Press; 1961. Cattell RB, Shrader RR, Barton K. The definition and measurement of anxiety as a trait and a state in the 12- to 17-year range. Br J Soc Clin Psychol 1974;13:173–82. Chadman KK. Fluoxetine but not risperidone increases sociability in the BTBR mouse model of autism. Pharmacol Biochem Behav 2011;97:586–94. Chaouloff F, Durand M, Mormède P. Anxiety- and activity-related effects of diazepam and chlordiazepoxide in the rat light/dark and dark/light tests. Behav Brain Res 1997;85:27–35. Chapillon P, Manneché C, Belzung C, Caston J. Rearing environmental enrichment in two inbred strains of mice: 1. Effects on emotional reactivity. Behav Genet 1999;29:41–6. Choleris E, Thomas AW, Kavaliers M, Prato FS. A detailed ethological analysis of the mouse open field test: effects of diazepam, chlordiazepoxide and an extremely low frequency pulsed magnetic field. Neurosci Biobehav Rev 2001;25:235–60. Clément Y, Le Guisquet AM, Venault P, Chapouthier G, Belzung C. Pharmacological alterations of anxious behaviour in mice depending on both strain and the behavioural situation. PLoS One 2009;4:e7745. Cole JC, Rodgers RJ. Ethological comparison of the effects of diazepam and acute/ chronic imipramine on the behaviour of mice in the elevated plus-maze. Pharmacol Biochem Behav 1995;52:473–8. Collinson N, Dawson GR. On the elevated plus-maze the anxiolytic-like effects of the 5-HT(1A) agonist, 8-OH-DPAT, but not the anxiogenic-like effects of the

P

[44]

E

[59]

T

[9] Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor in anxiety disorders. Biol Psychiatry 2009;66:627–35. [10] Albonetti ME, Farabollini F. Behavioural responses to a single and repeated restraint in male and female rats. Behav Process 1992;28:97–110. [11] Albrechet-Souza L, de Carvalho MC, Franci CR, Brandão ML. Increases in plasma corticosterone and stretched-attend postures in rats naive and previously exposed to the elevated plus-maze are sensitive to the anxiolytic-like effects of midazolam. Horm Behav 2007;52:267–73. [12] Alstott J, Timberlake W. Effects of rat sex differences and lighting on locomotor exploration of a circular open field with free-standing central corners and without peripheral walls. Behav Brain Res 2009;96:214–9. [13] Amsel A, Cole KF. Generalization of fear-motivated interference with water intake. J Exp Psychol 1953;46:243–7. [14] Andreatini R, Bacellar LFS. Animal models: trait or state measure? The test–retest reliability of the elevated plus-maze and behavioral despair. Prog NeuroPsychopharmacol Biol Psychiatry 2000;24:549–60. [15] Andreatini R, Bacellar LFS. The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze. Braz J Med Biol Res 1999;32:1121–6. [16] Angrini M, Leslie JC, Shephard RA. Effects of propranolol, buspirone, pCPA, reserpine, and chlordiazepoxide on open-field behavior. Pharmacol Biochem Behav 1998;59:387–97. [17] Anisman H, Hayley S, Kelly O, Borowski T, Merali Z. Psychogenic, neurogenic, and systemic stressor effects on plasma corticosterone and behavior: mouse strain-dependent outcomes. Behav Neurosci 2001;115:443–54. [18] Audet MC, Goulet S, Doré FY. Repeated subchronic exposure to phencyclidine elicits excessive atypical grooming in rats. Behav Brain Res 2006;167:103–10. [19] Aulich D. Escape versus exploratory activity: an interpretation of rats' behaviour in the open field and a light–dark preference test. Behav Processes 1976;1:153–64. [20] Aureli F, van Schaik CP. Post-conflict behaviour in longtailed macaques (Macaca fascicularis). Ethology 1991;89:101–14. [21] Avgustinovich DF, Lipina TV, Bondar NP, Alekseyenko OV, Kudryavtseva NN. Features of the genetically defined anxiety in mice. Behav Genet 2000;30:101–9. [22] Baas JM, Grillon C, Böcker KB, Brack AA, Morgan III CA, Kenemans JL, et al. Benzodiazepines have no effect on fear-potentiated startle in humans. Psychopharmacology (Berl) 2002;161:233–47. [23] Barlow DH. Anxiety and its disorders: the nature and treatment of anxiety and panic. New York: Guilford Press; 2002. [24] Barros HMT, Tannhauser SL, Tannhauser MAL, Tannhauser M. The effects of GABAergic drugs on grooming behaviour in the open field. Pharmacol Toxicol 1994;74:339–44. [25] Beach FA, Fowler H. Effects of “situational anxiety” on sexual behavior in male rats. J Comp Physiol Psychol 1959;52:245–8. [26] Beck AT, Epstein N, Brown G, Steer RA. An inventory for measuring clinical anxiety: psychometric properties. J Consult Clin Psychol 1988;56:893–7. [27] Belzung C, Le Pape G. Comparison of different behavioral test situations used in psychopharmacology for measurement of anxiety. Physiol Behav 1994;56:623–8. [28] Belzung C, Le Guisquet AM, Crestani F. Flumazenil induces benzodiazepine partial agonist-like effects in BALB/c but not C57BL/6 mice. Psychopharmacology (Berl) 2000;148:24–32. [29] Belzung C, Griebel G. Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav Brain Res 2001;125:141–9. [30] Benno R, Smirnova Y, Vera S, Liggett A, Schanz N. Exaggerated responses to stress in the BTBR T + tf/J mouse: an unusual behavioral phenotype. Behav Brain Res 2009;197:462–5. [31] Benton D, Brain PF. Behavioural comparisons of isolated, dominant and subordinate mice. Behav Process 1979;4:211–9. [32] Berry RB, Werner DF, Wang X, Jablonski MM, Homanics GE, Mittleman G, et al. Mice with targeted genetic reduction of GABAA receptor a1 subunits display performance differences in Morris water maze tasks. Neurobiol Learn Mem 2008;90:580–3. [33] Berton F, Vogel E, Belzung C. Modulation of mice anxiety in response to cat odor as a consequence of predators diet. Physiol Behav 1998;65:247–54. [34] Bisong SA, Brown R, Osim EE. Comparative effects of Rauwolfia vomitoria and chlorpromazine on locomotor behaviour and anxiety in mice. J Ethnopharmacol 2010;132:334–9. [35] Blagden JC, Craske MG. Effects of active and passive rumination and distraction: a pilot replication with anxious mood. J Anxiety Disord 1996;10:243–52. [36] Blanchard RJ, Blanchard DC. Antipredator defensive behaviors in a visible burrow system. J Comp Psychol 1989;103:70–82. [37] Blanchard RJ, Blanchard DC, Rodgers J, Weiss SM. The characterization and modelling of antipredator defensive behavior. Neurosci Biobehav Rev 1990;14:463–72. [38] Blanchard RJ, Nikulina JN, Sakai RR, McKittrick C, McEwen B, Blanchard DC. Behavioral and endocrine change following chronic predatory stress. Physiol Behav 1998;63:561–9. [39] Blanchard DC, Griebel G, Blanchard RJ. The mouse defense test battery: pharmacological and behavioral assays for anxiety and panic. Eur J Pharmacol 2003;463:97–116. [40] Blanchard DC, Griebel G, Pobbe R, Blanchard RJ. Risk assessment as an evolved threat detection and analysis process. Neurosci Biobehav Rev 2011;35:991–8. [41] Blednov YA, Jung S, Alva H, Wallace D, Rosahl T, Whiting PJ, et al. Deletion of the alpha1 or beta2 subunit of GABAA receptors reduces actions of alcohol and other drugs. J Pharmacol Exp Ther 2003;304:30–6. [42] Bolles RC. Grooming behaviour in the rat. J Comp Physiol Psychol 1960;53:306–10. [43] Borkovec TD, Alcaine O, Behar E. Avoidance theory of worry and generalized anxiety disorder. In: Heimberg RG, Turk CL, Mennin DS, editors. Generalized anxiety

U

1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108

11

[60]

[61] [62]

[63]

[64]

[65]

[66]

[67]

[68] [69] [70] [71] [72]

[73]

[74]

[75]

[76]

[77]

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125 1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185 1186 1187 1188 1189 1190 1191 1192 1193 1194

[89]

[90] [91]

[92]

[93] [94]

[95]

[96]

[97] [98]

[99]

[100]

[101] [102]

[103]

[104]

[105] [106] [107]

[108]

F

O

R O

[88]

P

[87]

D

[86]

[109] Diaz MR, Chappell AM, Christian DT, Anderson NJ, McCool BA. Dopamine D3-like receptors modulate anxiety-like behavior and regulate GABAergic transmission in the rat lateral/basolateral amygdala. Neuropsychopharmacology 2011;36:1090–103. [110] Dielenberg RA, Carrive P, McGregor IS. The cardiovascular and behavioral response to cat odor in rats: unconditioned and conditioned effects. Brain Res 2001;897:228–37. [111] Dielenberg RA, McGregor IS. Defensive behavior in rats towards predatory odors: a review. Neurosci Biobehav Rev 2001;25:597–609. [112] Dinsmoor JA. Punishment: I. The avoidance hypothesis. Psychol Rev 1954;61:34–46. [113] Dixon CI, Morris HV, Breen G, Desrivieres S, Jugurnauth S, Steiner RC, et al. Cocaine effects on mouse incentive-learning and human addiction are linked to alpha2 subunit-containing GABAA receptors. Proc Natl Acad Sci U S A 2010;107:2289–94. [114] Drapier D, Bentué-Ferrer D, Laviolle B, Millet B, Allain H, Bourin M, et al. Effects of acute fluoxetine, paroxetine and desipramine on rats tested on the elevated plus-maze. Behav Brain Res 2007;176:202–9. [115] Ducottet C, Aubert A, Belzung C. Susceptibility to subchronic unpredictable stress is related to individual reactivity to threat stimuli in mice. Behav Brain Res 2004;155:291–9. [116] Dulawa SC, Holick KA, Gundersen B, Hen R. Effects of chronic fluoxetine in animal models of anxiety and depression. Neuropsychopharmacology 2004;29:1321–30. [117] Dygalo NN, Shishkina GT, Kalinina TS, Yudina AM, Ovchinnikova ES. Effect of repeated treatment with fluoxetine on tryptophan hydroxylase-2 gene expression in the rat brainstem. Pharmacol Biochem Behav 2006;85:220–7. [118] Enginar N, Hatipoglu I, Firtina M. Evaluation of the acute effects of amitriptyline and fluoxetine on anxiety using grooming analysis algorithm in rats. Pharmacol Biochem Behav 2008;89:450–5. [119] Ennaceur A, Michalikova S, van Rensburg R, Chazot PL. Models of anxiety: responses of mice to novelty and open spaces in a 3D maze. Behav Brain Res 2006;174:9–38. [120] Ennaceur A, Michalikova S, Chazot PL. Models of anxiety: rats' responses to novelty in an open space and an enclosed space. Behav Brain Res 2006;171:26–49. [121] Ennaceur A, Michalikova S, van Rensburg R, Chazot PL. Are benzodiazepines really anxiolytic? Evidence from a 3D maze spatial navigation task. Behav Brain Res 2008;188:136–53. [122] Ennaceur A, Michalikova S, Chazot PL. Do rats really express neophobia towards novel objects? Experimental evidence from exposure to novelty and to an object recognition task in an open space and an enclosed space. Behav Brain Res 2009;197:417–34. [123] Ennaceur A, Michalikova S, van Rensburg R, Chazot PL. Distinguishing anxiolysis and hyperactivity in an open space behavioral test. Behav Brain Res 2010;207:84–98. [124] Ennaceur A, Michalikova S, van Rensburg R, Chazot PL. Tolerance, sensitization and dependence to diazepam in Balb/c mice exposed to a novel open space anxiety test. Behav Brain Res 2010;209:154–64. [125] Ennaceur A, Michalikova S, van Rensburg R, Chazot PL. MK-801 increases the baseline level of anxiety in mice introduced to a spatial memory task without prior habituation. Neuropharmacology 2011;61:981–91. [126] Ennaceur A. Omission of the habituation procedure in the acquisition of a working memory task — evidence from Balb/c, C57/BL6J, and CD-1 mice. Behav Brain Res 2011;223:203–10. [127] Ennaceur A. Open space anxiety test in rodents: the elevated platform with steep slopes. Methods Mol Biol 2012;829:177–91. [128] Epstein S. The nature of anxiety with emphasis upon its relationship to expectancy. In: Spielberger CD, editor. Anxiety: Current trends in theory and research, vol. 2. New York: Academic Press; 1972. p. 291–337. [129] Escorihuela RM, Fernández-Teruel A, Gil L, Aguilar R, Tobeña A, Driscoll P. Inbred Roman high- and low-avoidance rats: differences in anxiety, novelty-seeking, and shuttlebox behaviors. Physiol Behav 1999;67:19–26. [130] Estes WK, Skinner BF. Some quantitative properties of anxiety. J Exp Psychol 1941;29:390–400. [131] Eysenck MW, Derakshan N, Santos R, Calvo MG. Anxiety and cognitive performance: attentional control theory. Emotion 2007;7:336–53. [132] Fendt M, Endres T. 2,3,5-Trimethyl-3-thiazoline (TMT), a component of fox odor — just repugnant or really fear-inducing? Neurosci Biobehav Rev 2008;32:1259–66. [133] Ferkin MH, Leonard ST, Heath LA, Paz-y-Miño C. Self-grooming as a tactic used by prairie voles, Microtus ochrogaster, to enhance sexual communication. Ethology 2001;107:939–49. [134] Ferkin MH, Li HZ. A battery of olfactory-based screens for phenotyping the social and sexual behaviors of mice. Physiol Behav 2005;85:489–99. [135] Fernández Espejo E. Structure of the mouse behaviour on the elevated plus-maze test of anxiety. Behav Brain Res 1997;86:105–12. [136] File SE. Chlordiazepoxide-induced ataxia, muscle relaxation and sedation in the rat: effects of muscimol, picrotoxin and naloxone. Pharmacol Biochem Behav 1982;17:1165–70. [137] File SE, Mabbutt PS, Walker JH. Comparison of adaptive responses in familiar and novel environments: modulatory factors. Ann N Y Acad Sci 1988;525:69–79. [138] File SE. Animal models of different anxiety states. Adv Biochem Psychopharmacol 1995;48:93–113. [139] Fischer BD, Atack JR, Platt DM, Reynolds DS, Dawson GR, Rowlett JK. Contribution of GABA(A) receptors containing a3 subunits to the therapeutic-related and side effects of benzodiazepine-type drugs in monkeys. Psychopharmacology (Berl) 2011;215:311–9. [140] Flügge G, Kramer M, Fuchs E. Chronic subordination stress in male tree shrews: replacement of testosterone affects behaviour and central alpha2-adrenoceptors. Physiol Behav 2001;73:293–300. [141] Fokkema DS, Smit K, van der Gugten J, Koolhaas J. A coherent pattern among social behavior, blood pressure, corticosterone and catecholamine measures in individual male rats. Physiol Behav 1988;42:485–9.

T

[85]

C

[84]

E

[83]

R

[82]

R

[81]

O

[80]

C

[79]

N

[78]

5-HT(1A) partial agonist, buspirone, are blocked by the 5-HT1A antagonist, WAY 100635. Psychopharmacology (Berl) 1997;132:35–43. Collinson N, Kuenzi FM, Jarolimek W, Maubach KA, Cothliff R, Sur C, et al. Enhanced learning and memory and altered GABAergic synaptic transmission in mice lacking the alpha 5 subunit of the GABAA receptor. J Neurosci 2002;22:5572–80. Colorado RA, Shumake J, Conejo NM, Gonzalez-Pardo H, Gonzalez-Lima F. Effects of maternal separation, early handling, and standard facility rearing on orienting and impulsive behavior of adolescent rats. Behav Processes 2006;71:51–8. Cone JD. Hierarchical views of anxiety: what do they profit us? Behav Ther 1998;29:325–32. Cook MN, Crounse M, Flaherty L. Anxiety in the elevated zero-maze is augmented in mice after repeated daily exposure. Behav Genet 2002;32:113–8. Copland AM, Balfour DJK. Spontaneous activity and brain 5-hydroxyindole levels measured in rats tested in two designs of elevated X-maze. Life Sci 1987;41:57–64. Costall B, Hendric CA, Kelly ME, Nayior RJ. Actions of sulpiride and tiapride in a simple model of anxiety in mice. Neuropharmacology 1987;26:295–300. Costall B, Jones BJ, Kelly ME, Naylor RJ, Tomkins DM. Exploration of mice in a black and white test box: validation as a model of anxiety. Pharmacol Biochem Behav 1989;32:777–85. Cottone P, Sabino V, Steardo L, Zorrilla EP. FG 7142 specifically reduces meal size and the rate and regularity of sustained feeding in female rats: evidence that benzodiazepine inverse agonists reduce food palatability. Neuropharmacology 2007;32:1069–81. Crabbe JC, Wahlsten D, Dudek BC. Genetics of mouse behavior: interactions with laboratory environment. Science 1999;284:1670–2. Crawley JN, Goodwin FK. Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiazepines. Pharmacol Biochem Behav 1980;13:167–70. Crawley JN. Neuropharmacologic specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol Biochem Behav 1981;15:695–9. Crawley JN, Skolnick P, Paul SM. Absence of intrinsic antagonist actions of benzodiazepine antagonist on an exploratory model of anxiety in the mouse. Neuropharmacology 1984;5:531–7. Crawley JN. Exploratory behavior models of anxiety in mice. Neurosci Biobehav Rev 1985;9:37–44. Crestani F, Lorez M, Baer K, Essrich C, Benke D, Laurent JP, et al. Decreased GABAAreceptor clustering results in enhanced anxiety and a bias for threat cues. Nat Neurosci 1999;2:833–9. Crestani F, Keist R, Fritschy JM, Benke D, Vogt K, Prut L, et al. Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors. Proc Natl Acad Sci U S A 2002;99:8980–5. Cruz AP, Frei F, Graeff FG. Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 1994;49:171–6. Cryan JF, Dev KK. The glutamatergic system as a potential therapeutic target for the treatment of anxiety disorders. In: Blanchard RJ, Blanchard DC, Griebel G, Nutt D, editors. Handbook of Anxiety and Fear, vol. 17. Elsevier; 2008. p. 269–301. Cunha C, Monfils M-H, LeDoux JE. GABAC receptors in the lateral amygdala: a possible novel target for the treatment of fear and anxiety disorders? Front Behav Neurosci 2010;4:6. Dalvi A, Rodgers RJ. Behavioral effects of diazepam in the murine plus-maze: flumazenil antagonism of enhanced head dipping but not the disinhibition of open-arm avoidance. Pharmacol Biochem Behav 1999;62:727–34. Daly JA, Hogg E, Sacks D, Smith M, Zimring L. Sex and relationship affect social selfgrooming. J Nonverbal Behav 1983;7:83–189. Davis M, Shi C. The extended amygdala: are the central nucleus of the amygdala and the bed nucleus of the stria terminalis differentially involved in fear versus anxiety? Ann N Y Acad Sci 1999;877:281–91. Davis M, Walker DL, Miles L, Grillon C. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology 2010;35:105–35. Dawson GR, Crawford SP, Collinson N, Iversen SD, Tricklebank MD. Evidence that the anxiolytic-like effects of chlordiazepoxide on the elevated plus maze are confounded by increases in locomotor activity. Psychopharmacology (Berl) 1995;118:316–23. Dawson GR, Tricklebank MD. Use of the elevated plus maze in the search for novel anxiolytic agents. Trends Pharmacol Sci 1995;16:33–6. de Boer SF, Slangen JL, van der Gugten J. Effects of chlordiazepoxide and buspirone on plasma catecholamine and corticosterone levels in rats under basal and stress conditions. Endocrinol Exp 1990;24:229–39. de Boer SF, Koolhaas JM. 5-HT1A and 5-HT1B receptor agonists and aggression: a pharmacological challenge of the serotonin deficiency hypothesis. Eur J Pharmacol 2005;526:125–39. de la Mora MP, Gallegos-Cari A, Arizmendi-García Y, Marcellino D, Fuxe K. Role of dopamine receptor mechanisms in the amygdaloid modulation of fear and anxiety: structural and functional analysis. Prog Neurobiol 2010;90:198–216. Degroot A, Nomikos GG. Genetic deletion of muscarinic M4 receptors is anxiolytic in the shock-probe burying model. Eur J Pharmacol 2006;531:183–6. Di Bitetti MS. Evidence for an important social role of allogrooming in a platyrrhine primate. Anim Behav 1997;54:199–211. Di Lio A, Benke D, Besson M, Desmeules J, Daali Y, Wang ZJ, et al. HZ166, a novel GABAA receptor subtype-selective benzodiazepine site ligand, is antihyperalgesic in mouse models of inflammatory and neuropathic pain. Neuropharmacology 2011;60:626–32. Dias R, Sheppard WF, Fradley RL, Garrett EM, Stanley JL, Tye SJ, et al. Evidence for a significant role of α3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines. J Neurosci 2005;25:10682–8.

U

1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

E

12

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 1358 1359 1360 1361 1362 1363 1364 1365 1366

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

N C O

R

R

E

C

D

P

R O

O

F

pharmacology and preclinical translational models. Pharmacol Biochem Behav 2012;100:775–800. [175] Hascoët M, Bourin M. A new approach to the light/dark procedure in mice. Pharmacol Biochem Behav 1998;60:645–53. [176] Heisler LK, Chu HM, Brennan TJ, Danao JA, Bajwa P, Parsons LH, et al. Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Acad Sci U S A 1998;95:15049–54. Hendrie CA, Eilam D, Weiss SM. Effects of diazepam and buspirone on the behaviour of wild voles (Microtus socialis) in two models of anxiety. Pharmacol Biochem Behav 1998;58:573–6. [177] Hendrie CA, Eilam D, Weiss SM. Effects of diazepam and buspirone on the behaviour of wild voles (Microtus socialis) in two models of anxiety. Pharmacol Biochem Behav 1997;58:573–6. [178] Hendrie CA, Van Driel KS, Talling JC, Inglis IR. PBI creams: a spontaneously mutated mouse strain showing wild animal-type reactivity. Physiol Behav 2001;74:621–8. [179] Hilakivi LA, Lister RG. Correlations between behavior of mice in Porsolt's swim test and in tests of anxiety, locomotion, and exploration. Behav Neural Biol 1990;53:153–9. [180] Hill MN, Gorzalka BB. Enhancement of anxiety-like responsiveness to the cannabinoid CB1 receptor agonist HU-210 following chronic stress. Eur J Pharmacol 2004;499:291–5. [181] Ho Y-J, Eichendorff J, Schwarting RKW. Individual response profiles of male Wistar rats in animal models for anxiety and depression. Behav Brain Res 2002;136:1–12. [182] Hofmann M, Kordás KS, Gravius A, Bölcskei K, Parsons CG, Dekundy A, et al. Assessment of the effects of NS11394 and L-838417, a2/3 subunit-selective GABA(A) [corrected] receptor-positive allosteric modulators, in tests for pain, anxiety, memory and motor function. Behav Pharmacol 2012;23:790–801. [183] Hogg S. A review of the validity and variability of the elevated plus-maze as an animal model of anxiety. Pharmacol Biochem Behav 1996;54:21–30. [184] Holmes A, Rodgers RJ. Responses of Swiss–Webster mice to repeated plus-maze experience: further evidence for a qualitative shift in emotional state? Pharmacol Biochem Behav 1998;60:473–88. [185] Holmes A, Rodgers RJ. Influence of spatial and temporal manipulations on the anxiolytic efficacy of chlordiazepoxide in mice previously exposed to the elevated plus-maze. Neurosci Biobehav Rev 1999;23:971–80. [186] Holmes A, Parmigiani S, Ferrari PF, Palanza P, Rodgers RJ. Behavioral profile of wild mice in the elevated plus-maze test for anxiety. Physiol Behav 2000;71:509–16. [187] Holmes A, Iles JP, Mayell SJ, Rodgers RJ. Prior test experience compromises the anxiolytic efficacy of chlordiazepoxide in the mouse light/dark exploration test. Behav Brain Res 2001;122:159–67. [188] Holmes A, Heilig M, Rupniak NM, Steckler T, Griebel G. Neuropeptide systems as novel therapeutic targets for depression and anxiety disorders. Trends Pharmacol Sci 2003;24:580–8. [189] Holmes A, Kinney JW, Wrenn CC, Li Q, Yang RJ, Ma L, et al. Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze. Neuropsychopharmacology 2003;28:1031–44. [190] Holmes A, Cryan JF. Modeling human anxiety and depression in mutant mice. In: Fisch GS, Flint J, editors. Contemporary clinical neuroscience: transgenic and knockout models of neuropsychiatric disorders. Totowa, NJ: Humana Press Inc.; 2006. p. 237–63. [191] Horváth J, Szögi T, Müller G, Szegedi V. The anxiolytic buspirone shifts coping strategy in novel environmental context of mice with different anxious phenotype. Behav Brain Res 2013;250:32–8. [192] Ide S, Sora I, Ikeda K, Minami M, Uhl GR, Ishihara K. Reduced emotional and corticosterone responses to stress in mu-opioid receptor knockout mice. Neuropharmacology 2010;58:241–7. [193] Itoi K, Sugimoto N. The brainstem noradrenergic systems in stress, anxiety and depression. J Neuroendocrinol 2010;22:355–61. [194] Jacobson LH, Cryan JF. Evaluation of the anxiolytic-like profile of the GABAB receptor positive modulator CGP7930 in rodents. Neuropharmacology 2008;54:854–62. [195] Jakovcevski M, Schachner M, Morellini F. Individual variability in the stress response of C57BL/6J male mice correlates with trait anxiety. Genes Brain Behav 2008;7:235–43. [196] Jones N, Duxon MS, King SM. Ethopharmacological analysis of the unstable elevated exposed plus maze, a novel model of extreme anxiety: predictive validity and sensitivity to anxiogenic agents. Psychopharmacology (Berl) 2002;161:314–23. [197] Jones N, King SM, Duxon MS. Further evidence for the predictive validity of the unstable elevated exposed plus-maze, a model of extreme anxiety in rats: differential effects of fluoxetine and chlordiazepoxide. Behav Pharmacol 2002;13:525–35. [198] June HL, Harvey SC, Foster KL, McKay PF, Cummings R, Garcia M, et al. GABA(A) receptors containing (alpha)5 subunits in the CA1 and CA3 hippocampal fields regulate ethanol-motivated behaviors: an extended ethanol reward circuitry. J Neurosci 2001;21:2166–77. [199] June HL, Foster KL, McKay PF, Seyoum R, Woods JE, Harvey SC, et al. The reinforcing properties of alcohol are mediated by GABA(A1) receptors in the ventral pallidum. Neuropsychopharmacology 2003;28:2124–37. [200] Kabbaj M, Devine DP, Savage VR, Akil H. Neurobiological correlates of individual differences in novelty-seeking behavior in the rat: differential expression of stress-related molecules. J Neurosci 2000;20:6983–8. [201] Kaesermann HP. Stretched attend posture, a non-social form of ambivalence, is sensitive to a conflict-reducing drug action. Psychopharmacology (Berl) 1986;89:31–7. [202] Kalueff AV, Tuohimaa P. Grooming analysis algorithm for neurobehavioural stress research. Brain Res Protocol 2004;13:151–8. [203] Kalueff AV, Tuohimaa P. The grooming analysis algorithm discriminates between different levels of anxiety in rats: potential utility for neurobehavioural stress research. J Neurosci Methods 2005;143:169–77.

E

T

[142] Fone KCF, Shalders K, Fox ZD, Arthur R, Marsden CA. Increased 5-HT2C receptor responsiveness occurs on rearing rats in social isolation. Psychopharmacology (Berl) 1996;123:346–52. [143] Frankowska M, Filip M, Przegalinski E. Effects of GABAB receptor ligands in animal tests of depression and anxiety. Pharmacol Rep 2007;59:645–55. [144] Gentsch C, Lichtsteiner M, Feer H. Locomotor activity, defecation score and corticosterone levels during an open field exposure: a comparison among individually and group-housed rats, and genetically selected rat lines. Physiol Behav 1981;27:183–6. [145] Gioiosa L, Chiarotti F, Alleva E, Laviola G. A trouble shared is a trouble halved: social context and status affect pain in mouse dyads. PLoS One 2009;4:e4143. [146] Gispen WH, Isaacson RL. ACTH-induced excessive grooming in the rat. Pharmacol Ther 1981;12:209–46. [147] Gleser G, Ulett G. The Saslow Screening Test as a measure of anxiety-proneness. J Clin Psychol 1952;8:279–83. [148] Goes TC, Antunes FD, Teixeira-Silva F. Trait and state anxiety in animal models: Is there correlation? Neurosci Lett 2009;450:266–9. [149] Gomes KS, Nunes-De-Souza RL. Implication of the 5-HT2A and 5-HT2C (but not 5HT1A) receptors located within the periaqueductal gray in the elevated plusmaze test–retest paradigm in mice. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:1261–9. [150] Grant EC, MacKintosh JH. A comparison of the social postures of some common laboratory rodents. Behaviour 1963;21:246–59. [151] Gray JA. The neuropsychology of anxiety. Br J Psychol 1978;69:417–34. [152] Gray JA, McNaughton N. The neuropsychology of anxiety: an enquiry into the functions of the septo-hippocampal system. Oxford University Press; 2000. [153] Gregus A, Wintink AJ, Davis AC, Kalynchuk LE. Effect of repeated corticosterone injections and restraint stress on anxiety and depression-like behavior in male rats. Behav Brain Res 2005;156:105–14. [154] Grewal SS, Shepherd JK, Bill DJ, Fletcher A, Dourish CT. Behavioural and pharmacological characterisation of the canopy stretched attend posture test as a model of anxiety in mice and rats. Psychopharmacology (Berl) 1997;133:29–38. [155] Griebel G, Sanger DJ, Perrault G. Further evidence for differences between nonselective and BZ-1 (ω1) selective, benzodiazepine receptor ligands in murine models of “state” and “trait” anxiety. Neuropharmacology 1996;35:1081–91. [156] Griebel G, Rodgers RJ, Perrault G, Sanger DJ. Risk assessment behavior: evaluation of utility in the study of 5-HT-related drugs in the rat elevated plus-maze test. Pharmacol Biochem Behav 1997;57:817–27. [157] Griebel G, Perrault G, Sanger DJ. Characterization of the behavioral profile of the non-peptide CRF receptor antagonist CP-154,526 in anxiety models in rodents. Comparison with diazepam and buspirone. Psychopharmacology (Berl) 1998;138:55–66. [158] Griebel G, Cohen C, Perrault G, Sanger DJ. Behavioral effects of acute and chronic fluoxetine in Wistar–Kyoto rats. Physiol Behav 1999;67:315–20. [159] Griebel G. Is there a future for neuropeptide receptor ligands in the treatment of anxiety disorders? Pharmacol Ther 1999;82:1–61. [160] Griebel G, Belzung C, Perrault G, Sanger DJ. Differences in anxiety-related behaviours and in sensitivity to diazepam in inbred and outbred strains of mice. Psychopharmacology (Berl) 2000;148:164–70. [161] Gross C, Hen R. The developmental origins of anxiety. Nat Rev Neurosci 2004;5:545–52. [162] Hajizadeh Moghaddam A, Roohbakhsh A, Rostami P, Heidary-Davishani A, Zarrindast MR. GABA and histamine interaction in the basolateral amygdala of rats in the plus-maze test of anxiety-like behaviors. Pharmacology 2008;82:59–66. [163] Hall C, Ballachey EL. A study of the rat's behavior in a field. A contribution to method in comparative psychology. Univ Calif Publ Psychol 1932;6:1–12. [164] Hall FS, Huang S, Fong GW, Sundstrom JM, Pert A. Differential basis of strain and rearing effects on open-field behavior in Fawn Hooded and Wistar rats. Physiol Behav 2000;71:525–32. [165] Haller J, Barna I, Barsvari B, Gyimesi Pelczer K, Yasar S, Panlilio LV, et al. Interactions between environmental aversiveness and the anxiolytic effects of enhanced cannabinoid signaling by FAAH inhibition in rats. Psychopharmacology (Berl) 2009;204:607–16. [166] Hamilton M. The assessment of anxiety states by rating. Br J Med Psychol 1959;32:50–5. [167] Handa RJ, Cross MK, George M, Gordon BH, Burgess LH, Cabrera TM, et al. Neuroendocrine and neurochemical responses to novelty stress in young and old male F344 rats: effects of d-fenfluramine treatment. Pharmacol Biochem Behav 1993;46:101–9. [168] Handley SL, Mithani S. Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ‘fear’-motivated behaviour. Naunyn Schmiedebergs Arch Pharmacol 1984;327:1–5. [169] Handley SL, McBlane JW. An assessment of the elevated X-maze for studying anxiety and anxiety-modulating drugs. J Pharmacol Toxicol Methods 1993;29:129–38. [170] Hannigan Jr JH, Isaacson RL. Conditioned excessive grooming in the rat after footshock: effect of naloxone and situational cues. Behav Neural Biol 1981;33:280–92. [171] Harada K, Aota M, Inoue T, Matsuda R, Mihara T, Yamaji T, et al. Anxiolytic activity of a novel potent serotonin 5-HT2C receptor antagonist FR260010: a comparison with diazepam and buspirone. Eur J Pharmacol 2006;553:171–84. [172] Harro J, Oreland L, Vasar E, Bradwejn J. Impaired exploratory behaviour after DSP-4 treatment in rats: implications for the increased anxiety after noradrenergic denervation. Eur Neuropsychopharmacol 1995;5:447–55. [173] Harvey SC, Foster KL, McKay PF, Carroll MR, Seyoum R, Woods II JE, et al. The GABA(A) receptor alpha1 subtype in the ventral pallidum regulates alcoholseeking behaviors. J Neurosci 2002;22:3765–75. [174] Harvey BH, Shahid M. Metabotropic and ionotropic glutamate receptors as neurobiological targets in anxiety and stress-related disorders: focus on

U

1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452

13

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 1476 1477 1478 1479 1480 1481 1482 1483 1484 1485 1486 1487 1488 1489 1490 1491 1492 1493 1494 1495 1496 1497 1498 1499 1500 1501 1502 1503 1504 1505 1506 1507 1508 1509 1510 1511 1512 1513 1514 1515 1516 1517 1518 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534 1535 1536 1537 1538

D

P

R O

O

F

[239] Liebsch G, Landgraf R, Engelmann M, Lörscher P, Holsboer F. Differential behavioural effects of chronic infusion of CRH 1 and CRH 2 receptor antisense oligonucleotides into the rat brain. J Psychiatr Res 1999;33:153–63. [240] Linden A-M, Schoepp DD. Metabotropic glutamate receptor targets for neuropsychiatric disorders. Drug Discov Today Ther Strateg 2006;3:507–17. [241] Lipska BK, Weinberger DR. To model a psychiatric disorder in animals: schizophrenia as a reality test. Neuropsychopharmacology 2000;23:223–39. [242] Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology (Berl) 1987;92:180–5. [243] Lister RG. Ethologically-based animal models of anxiety disorders. Pharmacol Ther 1990;46:321–40. [244] Lohr JM, Olatunji BO, Sawchuk CN. A functional analysis of danger and safety signals in anxiety disorders. Clin Psychol Rev 2007;27:114–26. [245] Lopez-Aumatell R, Guitart-Masip M, Vicens-Costa E, Gimenez-Llort L, Valdar W, Johannesson M, et al. Fearfulness in a large N/Nih genetically heterogeneous rat stock: differential profiles of timidity and defensive flight in males and females. Behav Brain Res 2008;188:41–55. [246] Lovibond PF, Saunders JC, Weidemann G, Mitchell CJ. Evidence for expectancy as a mediator of avoidance and anxiety in a laboratory model of human avoidance learning. Q J Exp Psychol 2008;61:1199–216. [247] Löw K, Crestani F, Keist R, Benke D, Brünig I, Benson JA, et al. Molecular and neuronal substrate for the selective attenuation of anxiety. Science 2000;290:131–4. [248] Lowry CA, Johnson PL, Hay-Schmidt A, Mikkelsen J, Shekhar A. Modulation of anxiety circuits by serotonergic systems. Stress 2005;8:233–46. [249] Lund TD, Rovis T, Chung WC, Handa RJ. Novel actions of estrogen receptor-beta on anxiety-related behaviors. Endocrinology 2005;146:797–807. [250] Luo DD, An SC, Zhang X. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress. Brain Res Bull 2008;77:8–12. [251] MacLean RR, Datta S. The relationship between anxiety and sleep-wake behavior after stressor exposure in the rat. Brain Res 2007;1164:72–80. [252] Maestripieri D, Martel FL, Nevison CM, Simpson MJA, Keverne EB. Anxiety in rhesus monkey infants in relation to interactions with their mother and other social companions. Dev Psychobiol 1992;24:571–81. [253] Maier SF. Learned helplessness: relationships with fear and anxiety. In: Standford C, Salmon P, editors. Stress: an integrated approach. London: Academic Press; 1993. p. 207–43. [254] Mairesse J, Viltart O, Salomé N, Giuliani A, Catalani A, Casolini P, et al. Prenatal stress alters the negative correlation between neuronal activation in limbic regions and behavioral responses in rats exposed to high and low anxiogenic environments. Psychoneuroendocrinology 2007;32:765–76. [255] Makino J, Kato K, Maes FW. Temporal structure of open-field behavior in inbred strains of mice. Jpn Psychol Res 1991;33:145–52. [256] Marks IM. Fears, phobias, and rituals. Panic, anxiety, and their disorders. New York: Oxford University Press; 1987. [257] Marler P. Communications in monkeys and apes. In: DeVore I, editor. Primate behavior. New York: Holt, Rinehart & Winston Inc.; 1965. p. 544–84. [258] Márquez C, Nadal R, Armario A. Influence of reactivity to novelty and anxiety on hypothalamic–pituitary–adrenal and prolactin responses to two different novel environments in adult male rats. Behav Brain Res 2006;168:13–22. [259] Martin JR, Moreau JL, Jenck F, Cumin R. Acute and chronic administration of buspirone fails to yield anxiolytic-like effects in a mouse operant punishment paradigm. Pharmacol Biochem Behav 1993;46:905–10. [260] Mathiasen LS, Mirza NR, Rodgers RJ. Strain- and model-dependent effects of chlordiazepoxide, L-838,417 and zolpidem on anxiety-like behaviours in laboratory mice. Pharmacol Biochem Behav 2008;90:19–36. [261] Matuszewich L, Karney JJ, Carter SR, Janasik SP, O'Brien JL, Friedman RD. The delayed effects of chronic unpredictable stress on anxiety measures. Physiol Behav 2007;90:674–81. [262] McFarlane HG, Kusek GK, Yang M, Phoenix JL, Bolivar VJ, Crawley JN. Autism-like behavioral phenotypes in BTBR T + tf/J mice. Genes Brain Behav 2008;7:152–63. [263] McGregor IS, Schrama L, Ambermoon P, Dielenberg RA. Not all ‘predator odours’ are equal: cat odour but not 2,4,5 trimethylthiazoline (TMT; fox odour) elicits specific defensive behaviours in rats. Behav Brain Res 2002;129:1–16. [264] McGregor IS, Hargreaves GA, Apfelbach R, Hunt GE. Neural correlates of cat odorinduced anxiety in rats: region-specific effects of the benzodiazepine midazolam. J Neurosci 2004;24:4134–44. [265] McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, et al. Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype. Nat Neurosci 2000;3:587–92. [266] McNaughton N, Gray JA. Anxiolytic action on the behavioural inhibition system implies multiple types of arousal contribute to anxiety. J Affect Disord 2000;61:161–76. [267] McNaughton N, Corr PJ. A two-dimensional neuropsychology of defense: fear/ anxiety and defensive distance. Neurosci Biobehav Rev 2004;28:285–305. [268] McNaughton N, Corr PJ. The neuropsychology of fear and anxiety: a foundation for reinforcement sensitivity theory. In: Corr PJ, editor. The reinforcement sensitivity theory of personality. Cambridge University Press; 2008. p. 44–94. [269] Meerlo P, Sgoifo A. Long-lasting consequences of a social conflict in rats: behavior during the interaction predicts subsequent changes in daily rhythms of heart rate, temperature, and activity. Behav Neurosci 1999;113:1283–90. [270] Mendes-Gomes J, Miguel TT, Amaral VC, Nunes-de-Souza RL. Corticosterone does not change open elevated plus maze-induced antinociception in mice. Horm Behav 2011;60:408–13. [271] Merali Z, Levac C, Anisman H. Validation of a simple, ethologically relevant paradigm for assessing anxiety in mice. Biol Psychiatry 2003;54:552–65.

N

C

O

R

R

E

C

T

[204] Kalynchuk LE, Pinel JP, Treit D, Kippin TE. Changes in emotional behavior produced by long-term amygdala kindling in rats. Biol Psychiatry 1997;41:438–51. [205] Kalynchuk LE, Pinel JP, Treit D. Long-term kindling and interictal emotionality in rats: effect of stimulation site. Brain Res 1998;779:149–57. [206] Kalynchuk LE, Gregus A, Boudreau D, Perrot-Sinal TS. Corticosterone increases depression-like behavior, with some effects on predator odor-induced defensive behavior, in male and female rats. Behav Neurosci 2004;118:1365–77. [207] Kataoka M, Yamamori S, Suzuki E, Watanabe S, Sato T, Miyaoka H, et al. A single amino acid mutation in SNAP-25 induces anxiety-related behavior in mouse. PLoS One 2011;6:e25158. [208] Kemble ED, Bolwahnn BL. Immediate and long-term effects of novel odors on risk assessment in mice. Physiol Behav 1997;61:543–9. [209] Kent JM, Mathew SJ, Gorman JM. Molecular targets in the treatment of anxiety. Biol Psychiatry 2002;52:1008–30. [210] Kim H, Shimojo S, O'Doherty JP. Is avoiding an aversive outcome rewarding? Neural substrates of avoidance learning in the human brain. PLoS Biol 2006;4:e233. [211] Kim S, Lee S, Ryu S, Suk J, Park C. Comparative analysis of the anxiety-related behaviors in four inbred mice. Behav Process 2002;60:181–90. [212] King SM. Escape-related behaviors in an unstable, elevated and exposed environment: I. A new behavioral model of extreme anxiety. Behav Brain Res 1999;98:113–26. [213] King SM. Escape-related behaviors in an unstable, elevated and exposed environment: II. Long-term sensitization after repetitive electrical stimulation of the rodent midbrain defense system. Behav Brain Res 1999;98:127–42. [214] Knabl J, Witschi R, Hösl K, Reinold H, Zeilhofer UB, Ahmadi S, et al. Reversal of pathological pain through specific spinal GABAA receptor subtypes. Nature 2008;451:330–4. [215] Knabl J, Zeilhofer UB, Crestani F, Rudolph U, Zeilhofer HU. Genuine antihyperalgesia by systemic diazepam revealed by experiments in GABAA receptor point-mutated mice. Pain 2009;141:233–8. [216] Kõks S, Beljajev S, Koovit I, Abramov U, Bourin M, Vasar E. 8-OH-DPAT, but not deramciclane, antagonizes the anxiogenic-like action of paroxetine in an elevated plus-maze. Psychopharmacology (Berl) 2001;153:365–72. [217] Komorowska J, Pisula W. Does changing levels of stress affect the characteristics of grooming behavior in rats? Int J Comp Psychol 2003;16:237–46. [218] Kondratova AA, Dubrovsky YV, Antoch MP, Kondratov RV. Circadian clock proteins control adaptation to novel environment and memory formation. Aging 2010;2:285–97. [219] Kopp C, Rudolph U, Low K, Tobler I. Modulation of rhythmic brain activity by diazepam: GABA(A) receptor subtype and state specificity. Proc Natl Acad Sci U S A 2004;101:3674–9. [220] Korpi ER, Sinkkonen ST. GABA(A) receptor subtypes as targets for neuropsychiatric drug development. Pharmacol Ther 2006;109:12–32. [221] Korte SM, De Boer SF. A robust animal model of state anxiety: fear-potentiated behaviour in the elevated plus-maze. Eur J Pharmacol 2003;463:163–75. [222] Kralic JE, O'Buckley TK, Khisti RT, Hodge CW, Homanics GE, Morrow AL. GABA(A) receptor alpha-1 subunit deletion alters receptor subtype assembly, pharmacological and behavioral responses to benzodiazepines and zolpidem. Neuropharmacology 2002;43:685–94. [223] Kralic JE, Wheeler M, Renzi K, Ferguson C, O'Buckley TK, Grobin AC, et al. Deletion of GABAA receptor alpha 1 subunit-containing receptors alters responses to ethanol and other anesthetics. J Pharmacol Exp Ther 2003;305:600–7. [224] Kramer M, Hiemke C, Fuchs E. Chronic psychosocial stress and antidepressant treatment in tree shrews: time-dependant behavioural and endocrine effects. Neurosci Biobehav Rev 1999;23:937–47. [225] Lacosta S, Merali Z, Anisman H. Behavioral and neurochemical consequences of lipopolysaccharide in mice: anxiogenic-like effects. Brain Res 1999;818:291–303. [226] Lader M. Anxiety: its nature and treatment. S Afr Med J 1975;49:939–43. [227] Lalonde R, Strazielle C. Relations between open-field, elevated plus-maze, and emergence tests as displayed by C57/BL6J and BALB/c mice. J Neurosci Methods 2008;171:48–52. [228] Lampis V, Maziade M, Battaglia M. Animal models of human anxiety disorders: reappraisal from a developmental psychopathology vantage point. Pediatr Res 2011;69:77R–84R. [229] Lamprea MR, Cardenas FP, Setem J, Morato S. Thigmotactic responses in an openfield. Braz J Med Biol Res 2008;41:135–40. [230] Landgraf R, Wigger A, Holsboer F, Neumann ID. Hyper-reactive hypothalamo– pituitary–adrenocortical axis in rats bred for high anxiety-related behaviour. J Neuroendocrinol 1999;11:405–7. [231] Lazarus RS. Emotion and adaptation. Oxford University Press; 1991. [232] LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci 2000;23:155–84. [233] Leggio GM, Micale V, Le Foll B, Mazzola C, Nobrega JN, Drago F. Dopamine D3 receptor knock-out mice exhibit increased behavioral sensitivity to the anxiolytic drug diazepam. Eur Neuropsychopharmacol 2011;21:325–32. [234] Leonardo ED, Hen R. Anxiety as a developmental disorder. Neuropsychopharmacology 2008;33:134–40. [235] Lepicard EM, Joubert C, Hagneau I, Perez-Diaz F, Chapouthier G. Differences in anxiety-related behavior and response to diazepam in BALB/cByJ and C57BL/6J strains of mice. Pharmacol Biochem Behav 2000;67:739–48. [236] Lever C, Burton S, O'Keefe J. Rearing on hind legs, environmental novelty, and the hippocampal formation. Rev Neurosci 2006;17:111–33. [237] Levin ED, Bencan Z, Cerutti DT. Anxiolytic effects of nicotine in zebrafish. Physiol Behav 2007;90:54–8. [238] Lewis DA, Cho RY, Carter CS, Eklund K, Forster S, Kelly MA, et al. Subunit-selective modulation of GABA type A receptor neurotransmission and cognition in schizophrenia. Am J Psychiatry 2008;165:1585–93.

U

1539 1540 1541 1542 1543 1544 1545 1546 1547 1548 1549 1550 1551 1552 1553 1554 1555 1556 1557 1558 1559 1560 1561 1562 1563 1564 1565 1566 1567 1568 1569 1570 1571 1572 1573 1574 1575 1576 1577 1578 1579 1580 1581 1582 1583 1584 1585 1586 1587 1588 1589 1590 1591 1592 1593 1594 1595 1596 1597 1598 1599 1600 1601 1602 1603 1604 1605 1606 1607 1608 1609 1610 1611 1612 1613 1614 1615 1616 1617 1618 1619 1620 1621 1622 1623 1624

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

E

14

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1625 1626 1627 1628 1629 1630 1631 1632 1633 1634 1635 1636 1637 1638 1639 1640 1641 1642 1643 1644 1645 1646 1647 1648 1649 1650 1651 1652 1653 1654 1655 1656 1657 1658 1659 1660 1661 1662 1663 1664 1665 1666 1667 1668 1669 1670 1671 1672 1673 1674 1675 1676 1677 1678 1679 1680 1681 1682 1683 1684 1685 1686 1687 1688 1689 1690 1691 1692 1693 1694 1695 1696 1697 1698 1699 1700 1701 1702 1703 1704 1705 1706 1707 1708 1709 1710

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

N C O

R

R

E

C

D

P

R O

O

F

[305] Perkins AM, Kemp SE, Corr PJ. Fear and anxiety as separable emotions: an investigation of the revised reinforcement sensitivity theory of personality. Emotion 2007;7:252–61. [306] Perrot-Sinal TS, Gregus A, Boudreau D, Kalynchuk LE. Sex and repeated restraint stress interact to affect cat odor-induced defensive behavior in adult rats. Brain Res 2004;1027:161–72. [307] Pinel JPJ, Mana MJ. Adaptive interactions of rats with dangerous inanimate objects: support for a cognitive theory of defensive behavior. In: Blanchard RJ, Brain PF, Blanchard DC, Parmigiani S, editors. Ethoexperimental approaches to the study of behavior. Dordrecht: Kluwer Academic Publishers; 1989. p. 137–50. [308] Pobbe RL, Defensor EB, Pearson BL, Bolivar VJ, Blanchard DC, Blanchard RJ. General and social anxiety in the BTBR T + tf/J mouse strain. Behav Brain Res 2011;216:446–51. [309] Podhorna J, Franklin KB. Long-lasting increase in anxiety after electrolytic lesions of the pedunculopontine tegmental nucleus. Behav Neurosci 1999;113:550–7. [310] Podhorna J, Brown RE. Strain differences in activity and emotionality do not account for differences in learning and memory performance between C57BL/6 and DBA/2 mice. Genes Brain Behav 2002;1:96–110. [311] Pohl J, Olmstead MC, Wynne-Edwards KE, Harkness K, Menard JL. Repeated exposure to stress across the childhood–adolescent period alters rats' anxiety- and depression-like behaviors in adulthood: the importance of stressor type and gender. Behav Neurosci 2007;121:462–74. [312] Pohorecky LA. Psychosocial stress and chronic ethanol ingestion in male rats: effects on elevated plus maze behavior and ultrasonic vocalizations. Physiol Behav 2008;94:432–47. [313] Price EO. Some behavioral differences between wild and domestic Norway rats: gnawing and platform jumping. Anim Learn Behav 1973;1:312–6. [314] Prut L, Belzung C. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 2003;463:3–33. [315] Prut L, Prenosil G, Willadt S, Vogt K, Fritschy JM, Crestani F. A reduction in hippocampal GABAA receptor alpha5 subunits disrupts the memory for location of objects in mice. Genes Brain Behav 2010;9:478–88. [316] Rachman S, Craske M, Tallman K. Does escape behavior strengthen agoraphobic avoidance? A replication. Behav Ther 1986;17:366–84. [317] Ramos A, Berton O, Mormede P, Chaouloff F. A multiple-test study of anxietyrelated behaviours in six inbred rat strains. Behav Brain Res 1997;85:57–69. [318] Ramos A, Mormede P. Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobehav Rev 1998;22:33–57. [319] Reiss S. Trait anxiety: it's not what you think it is. J Anxiety Disord 1997;11:201–14. [320] Renner MJ. Neglected aspects of exploratory and investigatory behavior. Psychobiology 1990;18:16–22. [321] Ressler KJ, Mayberg HS. Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic. Nat Neurosci 2007;10:1116–24. [322] Reynolds LM, Engin E, Tantillo G, Lau HM, Muschamp JW, Carlezon WA, et al. Differential roles of GABAA receptor subtypes in benzodiazepine-induced enhancement of brain-stimulation reward. Neuropsychopharmacology 2012;37:2531–40. [323] Rhudy JL, Meagher MW. Fear and anxiety: divergent effects on human pain thresholds. Ann NY Acad Sci 1997;821:305–31. [324] Rizk A, Curley J, Robertson J, Raber J. Anxiety and cognition in histamine H3 receptor−/− mice. Eur J Neurosci 2004;19:1992–6. [325] Robert G, Drapier D, Bentué-Ferrer D, Renault A, Reymann JM. Acute and chronic anxiogenic-like response to fluoxetine in rats in the elevated plus-maze: modulation by stressful handling. Behav Brain Res 2011;220:344–8. [326] Rodgers RJ, Blanchard DC, Wong LK, Blanchard RJ. Effects of scopolamine on antipredator defense reactions in wild and laboratory rats. Pharmacol Biochem Behav 1990;36:575–83. [327] Rodgers RJ, Cole JC. Anxiety enhancement in the murine elevated plus maze by immediate prior exposure to social stressors. Physiol Behav 1993;53:383–8. [328] Rodgers RJ, Johnson NJ. Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety. Pharmacol Biochem Behav 1995;52:297–303. [329] Rodgers RJ. Animal models of ‘anxiety’: where next? Behav Pharmacol 1997;8:477–96. [330] Rodgers RJ, Dalvi A. Anxiety, defence and the elevated plus-maze. Neurosci Biobehav Rev 1997;21:801–10. [331] Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 1997;30:289–304. [332] Rodgers RJ, Cutler MG, Jackson JE. Behavioural effects in mice of subchronic buspirone, ondansetron and tianeptine. II. The elevated plus-maze. Pharmacol Biochem Behav 1997;56:295–303. [333] Rodgers RJ, Haller J, Holmes A, Halasz J, Walton TJ, Brain PF. Corticosterone response to the plus-maze: high correlation with risk assessment in rats and mice. Physiol Behav 1999;68:47–53. [334] Rodgers RJ, Boullier E, Chatzimichalaki P, Cooper GD, Shorten A. Contrasting phenotypes of C57BL/6JOlaHsd, 129S2/SvHsd and 129/SvEv mice in two exploration-based tests of anxiety-related behaviour. Physiol Behav 2002;77:301–10. [335] Rodgers RJ, Davies B, Shore R. Absence of anxiolytic response to chlordiazepoxide in two common background strains exposed to the elevated plus-maze: importance and implications of behavioural baseline. Genes Brain Behav 2002;1:242–51. [336] Roelofs J, Huibers M, Peeters F, Arntz A, van Os J. Rumination and worrying as possible mediators in the relation between neuroticism and symptoms of depression and anxiety in clinically depressed individuals. Behav Res Ther 2008;46:1283–9. [337] Rosen JB, Pagani JH, Rolla KL, Davis C. Analysis of behavioral constraints and the neuroanatomy of fear to the predator odor trimethylthiazoline: a model for animal phobias. Neurosci Biobehav Rev 2008;32:1267–76.

E

T

[272] Michalikova S, van Rensburg R, Chazot PL, Ennaceur A. Anxiety responses in Balb/c, c57 and CD-1 mice exposed to a novel open space test. Behav Brain Res 2010;207:402–17. [273] Miczek KA. Intraspecies aggression in rats: effects of d-amphetamine and chlordiazepoxide. Psychopharmacologia 1974;39:275–301. [274] Miczek KA, Barry H. Δ9-Tetrahydrocannabinol and aggressive behavior in rats. Behav Biol 1974;11:261–7. [275] Mikics E, Barsy B, Barsvári B, Haller J. Behavioral specificity of non-genomic glucocorticoid effects in rats: effects on risk assessment in the elevated plusmaze and the open-field. Horm Behav 2005;48:152–62. [276] Milic M, Divljakovic J, Rallapalli S, van Linn ML, Timic T, Cook JM, et al. The role of a1 and a5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats. Behav Pharmacol 2012;23:191–7. [277] Miller NE. Studies of fear as an acquirable drive: I. Fear as motivation and fearreduction as reinforcement in the learning of new responses. J Exp Psychol 1948;38:89–101. [278] Milner LC, Crabbe JC. Three murine anxiety models: results from multiple inbred strain comparisons. Genes Brain Behav 2008;7:496–505. [279] Misslin R, Cigrang M. Does neophobia necessarily imply fear or anxiety? Behav Process 1986;12:45–50. [280] Mombereau C, Kaupmann K, Froestl W, Sansig G, van der Putten H, Cryan JF. Genetic and pharmacological evidence of a role for GABA(B) receptors in the modulation of anxiety- and antidepressant-like behavior. Neuropsychopharmacology 2004;29:1050–62. [281] Montgomery KC. The relation between fear induced by novel stimulation and exploratory behavior. J Comp Physiol Psychol 1955;48:254–60. [282] Moreira FA, Wotjak CT. Cannabinoids and anxiety. Curr Top Behav Neurosci 2010;2:429–50. [283] Morris HV, Dawson GR, Reynolds DS, Atack JR, Stephens DN. Both alpha2 and alpha3 GABAA receptor subtypes mediate the anxiolytic properties of benzodiazepine site ligands in the conditioned emotional response paradigm. Eur J Neurosci 2006;23:2495–504. [284] Morris HV, Dawson GR, Reynolds DS, Atack JR, Rosahl TW, Stephens DN. Alpha2containing GABAA receptors are involved in mediating stimulant effects of cocaine. Pharmacol Biochem Behav 2008;90:9–18. [285] Morris HV, Nilsson S, Dixon CI, Stephens DN, Clifton PG. a1- and a2-containing GABAA receptor modulation is not necessary for benzodiazepine-induced hyperphagia. Appetite 2009;52:675–83. [286] Moy SS, Nadler JJ, Young NB, Perez A, Holloway LP, Barbaro RP, et al. Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains. Behav Brain Res 2007;176:4–20. [287] Munro G, Erichsen HK, Rae MG, Mirza NR. A question of balance—positive versus negative allosteric modulation of GABA(A) receptor subtypes as a driver of analgesic efficacy in rat models of inflammatory and neuropathic pain. Neuropharmacology 2011;61:121–32. [288] Muris P. Normal and abnormal fear and anxiety in children and adolescents. Elsevier Science; 2010. [289] Murray F, Smith DW, Hutson PH. Chronic low dose corticosterone exposure decreased hippocampal cell proliferation, volume and induced anxiety and depression like behaviours in mice. Eur J Pharmacol 2008;583:115–27. [290] Nemati F, Whishaw IQ. The point of entry contributes to the organization of exploratory behavior of rats on an open field: an example of spontaneous episodic memory. Behav Brain Res 2007;182:119–28. [291] Nestler EJ, Hyman SE. Animal models of neuropsychiatric disorders. Nat Neurosci 2010;13:1161–9. [292] Nic Dhonnchadha BA, Bourin M, Hascoët M. Anxiolytic-like effects of 5-HT2 ligands on three mouse models of anxiety. Behav Brain Res 2003;140:203–14. [293] Oh JE, Zupan B, Gross S, Toth M. Paradoxical anxiogenic response of juvenile mice to fluoxetine. Neuropsychopharmacology 2009;34:2197–207. [294] Ohl F. Testing for anxiety. Clin Neurosci Res 2003;3:233–8. [295] Öhman A, Mineka S. Fears, phobias, and preparedness: toward an evolved module of fear and fear learning. Psychol Rev 2001;108:483–522. [296] Öhman A. Fear and anxiety. In: Lewis M, Haviland-Jones JM, Barrett LF, editors. Handbook of emotions. The Guilford Press; 2008. p. 709–29 [ch 44]. [297] Oliveira-Dos-Santos AJ, Matsumoto G, Snow BE, Bai D, Houston FP, Whishaw IQ, et al. Regulation of T cell activation, anxiety, and male aggression by RGS2. Proc Natl Acad Sci U S A 2000;97:12272–7. [298] Pähkla R, Kask A, Rägo L. Differential effects of beta-carbolines and antidepressants on rat exploratory activity in the elevated zero-maze. Pharmacol Biochem Behav 2000;65:737–42. [299] Panickar KS. Dose–response analysis of the effects of buspirone on rearing in rats. J Psychopharmacol 1991;5:72–6. [300] Pape HC, Jüngling K, Seidenbecher T, Lesting J, Reinscheid RK. Neuropeptide S: a transmitter system in the brain regulating fear and anxiety. Neuropharmacology 2010;58:29–34. [301] Patel S, Hillard CJ. Pharmacological evaluation of cannabinoid receptor ligands in a mouse model of anxiety: further evidence for an anxiolytic role for endogenous cannabinoid signaling. JPET 2006;318:304–11. [302] Pawlak CR, Schwarting RK. Object preference and nicotine consumption in rats with high versus low rearing activity in a novel open field. Pharmacol Biochem Behav 2002;73:679–87. [303] Peça J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, et al. Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 2011;472:437–42. [304] Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 1985;14:149–67.

U

1711 1712 1713 1714 1715 1716 1717 1718 1719 1720 1721 1722 1723 1724 1725 1726 1727 1728 1729 1730 1731 1732 1733 1734 1735 1736 1737 1738 1739 1740 1741 1742 1743 1744 1745 1746 1747 1748 1749 1750 1751 1752 1753 1754 1755 1756 1757 1758 1759 1760 1761 1762 1763 1764 1765 1766 1767 1768 1769 1770 1771 1772 1773 1774 1775 1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 1791 1792 1793 1794 1795 1796

15

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1797 1798 1799 1800 1801 1802 1803 1804 1805 1806 1807 1808 1809 1810 1811 1812 1813 1814 1815 1816 1817 1818 1819 1820 1821 1822 1823 1824 1825 1826 1827 1828 1829 1830 1831 1832 1833 1834 1835 1836 1837 1838 1839 1840 1841 1842 1843 1844 1845 1846 1847 1848 1849 1850 1851 1852 1853 1854 1855 1856 1857 1858 1859 1860 1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882

D

P

R O

O

F

[370] Spielberger CD. Conceptual and methodological issues in anxiety research. Anxiety Curr Trends Theory Res 1972;2:481–93. [371] Spooren W, Lesage A, Lavreysen H, Gasparini F, Steckler T. Metabotropic glutamate receptors: their therapeutic potential in anxiety. Curr Top Behav Neurosci 2010;2:391–413. [372] Spruijt BM, van Hooff JA, Gispen WH. Ethology and neurobiology of grooming behavior. Physiol Rev 1992;72:825–52. [373] Stachowicz K, Klodzinska A, Palucha-Poniewiera A, Schann S, Neuville P, Pilc A. The group III mGlu receptor agonist ACPT-I exerts anxiolytic-like but not antidepressant-like effects, mediated by the serotonergic and GABA-ergic systems. Neuropharmacology 2009;57:227–34. [374] Stapinski LA, Abbott MJ, Rapee RM. Evaluating the cognitive avoidance model of generalised anxiety disorder: impact of worry on threat appraisal, perceived control and anxious arousal. Behav Res Ther 2010;48:1032–40. [375] Steckler T. Developing small molecule nonpeptidergic drugs for the treatment of anxiety disorders: is the challenge still ahead? Curr Top Behav Neurosci 2010;2:415–28. [376] Steenbergen HL, Farabollini F, Heinsbroek RPW, Van de Poll NE. Sex-dependent effects of aversive stimulation on holeboard and elevated plus-maze behavior. Physiol Behav 1990;48:571–6. [377] Steimer T. The biology of fear- and anxiety-related behaviors. Dialogues Clin Neurosci 2002;4:231–49. [378] Strongman KT. The effect of anxiety on food intake in the rat. Q J Exp Psychol 1965:255–60. [379] Sziray N, Kuki Z, Nagy KM, Markó B, Kompagne H, Lévay G. Effects of single and simultaneous lesions of serotonergic and noradrenergic pathways on open-space and bright-space anxiety-like behavior in two animal models. Behav Brain Res 2010;209:93–8. [380] Takahashi LK. Role of CRF1 and CRF2 receptors in fear and anxiety. Neurosci Biobehav Rev 2001;25:627–36. [381] Takahashi LK, Nakashima BR, Hong H, Watanabe K. The smell of danger: a behavioral and neural analysis of predator odor-induced fear. Neurosci Biobehav Rev 2005;29:1157–67. [382] Täuber M, Calame-Droz E, Prut L, Rudolph U, Crestani F. Alpha2-gammaaminobutyric acid (GABA)A receptors are the molecular substrates mediating precipitation of narcosis but not of sedation by the combined use of diazepam and alcohol in vivo. Eur J Neurosci 2003;18:2599–604. [383] Thiel CM, Muller CP, Huston JP, Schwarting RK. High versus low reactivity to a novel environment: behavioural, pharmacological and neurochemical assessments. Neuroscience 1999;93:243–51. [384] Thiessen DD, Graham M, Perkins J, Marcks S. Temperature regulation and social grooming in the Mongolian gerbil (Meriones unguiculatus). Behav Biol 1977;19:279–88. [385] Thompson WR. Influence of prenatal maternal anxiety on emotionality in young rats. Science 1957;125:698–9. [386] Thorsell A. Brain neuropeptide Y and corticotropin-releasing hormone in mediating stress and anxiety. Exp Biol Med (Maywood) 2010;235:1163–7. [387] Ting JT, Feng G. Neurobiology of obsessive–compulsive disorder: insights into neural circuitry dysfunction through mouse genetics. Curr Opin Neurobiol 2011;21:842–8. [388] Torres AA. Anxiety versus escape conditioning and tranquilizing action. J Comp Physiol Psychol 1961;54:349–53. [389] Treit D, Menard J, Royan C. Anxiogenic stimuli in the elevated plus-maze. Pharmacol Biochem Behav 1993;44:463–9. [390] Treit D, Engin E, McEown K. Animal models of anxiety and anxiolytic drug action. Curr Top Behav Neurosci 2010;2:121–60. [391] Trent NL, Menard JL. The ventral hippocampus and the lateral septum work in tandem to regulate rats' open-arm exploration in the elevated plus-maze. Physiol Behav 2010;101:141–52. [392] Troelsen KB, Nielsen EØ, Mirza NR. Chronic treatment with duloxetine is necessary for an anxiolytic-like response in the mouse zero maze: the role of the serotonin transporter. Psychopharmacology (Berl) 2005;181:741–50. [393] Troisi A, Schino G. Environmental and social influences on autogrooming behaviour in a captive group of Java monkeys. Behaviour 1987;100:292–302. [394] Van der Poel AM. A note on “stretched attention”, a behavioural element indicative of an approach-avoidance conflict in rats. Anim Behav 1979;27:446–50. [395] van Erp AM, Kruk MR, Meelis W, Willekens-Bramer DC. Effect of environmental stressors on time course, variability and form of self-grooming in the rat: handling, social contact, defeat, novelty, restraint and fur moistening. Behav Brain Res 1994;65:47–55. [396] van Gaalen MM, Steckler T. Behavioural analysis of four mouse strains in an anxiety test battery. Behav Brain Res 2000;115:95–106. [397] van Rijnsoever C, Täuber M, Choulli MK, Keist R, Rudolph U, Mohler H, et al. Requirement of alpha5-GABAA receptors for the development of tolerance to the sedative action of diazepam in mice. J Neurosci 2004;24:6785–90. [398] Varty GB, Morgan CA, Cohen-Williams ME, Coffin VL, Carey GJ. The gerbil elevated plus-maze I: behavioral characterization and pharmacological validation. Neuropsychopharmacology 2002;27:357–70. [399] Vinkers CH, van Oorschot R, Korte SM, Olivier B, Groenink L. 5-HT1A receptor blockade reverses GABA(A) receptor alpha3 subunit-mediated anxiolytic effects on stress-induced hyperthermia. Psychopharmacology (Berl) 2010;211:123–30. [400] Võikar V, Kõks S, Vasar E, Rauvala H. Strain and gender differences in the behavior of mouse lines commonly used in transgenic studies. Physiol Behav 2001;72:271–81. [401] Vyas A, Chattarji S. Modulation of different states of anxiety-like behavior by chronic stress. Behav Neurosci 2004;118:1450–4.

N

C

O

R

R

E

C

T

[338] Roth KA, Katz RJ. Stress, behavioral arousal, and open field activity a reexamination of emotionality in the rat. Neurosci Biobehav Rev 1979;3:247–63. [339] Rowlett JK, Lelas S. Comparison of zolpidem and midazolam self-administration under progressive-ratio schedules: consumer demand and labor supply analyses. Exp Clin Psychopharmacol 2007;15:328–37. [340] Roy V, Chapillon P, Jeljeli M, Caston J, Belzung C. Free versus forced exposure to an elevated plus-maze: evidence for new behavioral interpretations during test and retest. Psychopharmacology (Berl) 2009;2(03):131–41. [341] Rudolph U, Möhler H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr Opin Pharmacol 2006;6:18–23. [342] Russell JA. Stress, anxiety and the amygdala. Stress 2005;8:207–8. [343] Salkovskis PM. Obsessional–compulsive problems: a cognitive–behavioural analysis. Behav Res Ther 1985;23:571–83. [344] Salomé N, Salchner P, Viltart O, Sequeira H, Wigger A, Landgraf R, et al. Neurobiological correlates of high (HAB) versus low anxiety-related behavior (LAB): differential Fos expression in HAB and LAB rats. Biol Psychiatry 2004;55:715–23. [345] Salomons AR, Pinzon NE, Boleij H, Kirchhoff S, Arndt SS, Nordquist RE, et al. Differential effects of diazepam and MPEP on habituation and neuro-behavioural processes in inbred mice. Behav Brain Funct 2012;8:30. [346] Salum C, Roque-da-Silva AC, Morato S. Conflict as a determinant of rat behavior in three types of elevated plus-maze. Behav Processes 2003;63:87–93. [347] Savic MM, Huang S, Furtmüller R, Clayton T, Huck S, Obradovic DI, et al. Are GABAA receptors containing alpha5 subunits contributing to the sedative properties of benzodiazepine site agonists? Neuropsychopharmacology 2008;33:332–9. [348] Savic MM, Milinkovic MM, Rallapalli S, Clayton Sr T, Joksimovic S, Van Linn M, et al. The differential role of alpha1- and alpha5-containing GABA(A) receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats. Int J Neuropsychopharmacol 2009;12:1179–93. [349] Savic MM, Majumder S, Huang S, Edwankar RV, Furtmüller R, Joksimovic S, et al. Novel positive allosteric modulators of GABAA receptors: do subtle differences in activity at alpha1 plus alpha5 versus alpha2 plus alpha3 subunits account for dissimilarities in behavioral effects in rats? Prog Neuropsychopharmacol Biol Psychiatry 2010;34:376–86. [350] Sayin Ü, Purali N, Özkan T, Altug T, Büyükdevrim S. Vigabatrin has an anxiolytic effect in the elevated plus-maze test of anxiety. Pharmacol Biochem Behav 1992;43:529–35. [351] Schino G, Perretta G, Taglioni AM, Monaco V, Troisi A. Primate displacement activities as an ethopharmacological model of anxiety. Anxiety 1996;2:186–91. [352] Schoenfeld WN. An experimental approach to anxiety, escape, and avoidance behavior. In: Hoch PH, Zubin J, editors. Anxiety. New York: Grune & Stratton; 1950. p. 70–99. [353] Schwarting RKW, Thiel CM, Müller CP, Huston JP. Relationship between anxiety and serotonin in the ventral striatum. Neuroreport 1998;9:1025–9. [354] Schwartz GE, Weinberger DA. Patterns of emotional responses to affective situations: relations among happiness, sadness, anger, fear, depression, and anxiety. Motiv Emot 1980;4:175–91. [355] Selcher JC, Nekrasova T, Paylor R, Landreth GE, Sweatt JD. Mice lacking the ERK1 isoform of MAP kinase are unimpaired in emotional learning. Learn Mem 2001;8:11–9. [356] Selleri S, Bruni F, Costagli C, Costanzo A, Guerrini G, Ciciani G, et al. A novel selective GABA(A) alpha1 receptor agonist displaying sedative and anxiolytic-like properties in rodents. J Med Chem 2005;48:6756–60. [357] Setem J, Pinheiro AP, Motta VA, Morato S, Cruz AP. Ethopharmacological analysis of 5-HT ligands on the rat elevated plus-maze. Pharmacol Biochem Behav 1999;62:515–21. [358] Shepard JD, Myers DA. Strain differences in anxiety-like behavior: association with corticotropin-releasing factor. Behav Brain Res 2008;186:239–45. [359] Shepherd JK, Grewal SS, Fletcher A, Bill DJ, Dourish CT. Behavioural and pharmacological characterisation of the elevated ‘zero-maze’ as an animal model of anxiety. Psychopharmacology (Berl) 1994;116:56–64. [360] Shmelkov SV, Hormigo A, Jing D, Proenca CC, Bath KG, Milde T, et al. Slitrk5 deficiency impairs corticostriatal circuitry and leads to obsessive–compulsive-like behaviors in mice. Nat Med 2010;16:598–602. [361] Silva MT, Alves CR, Santarem EM. Anxiogenic-like effect of acute and chronic fluoxetine on rats tested on the elevated plus-maze. Braz J Med Biol Res 1999;32:333–9. [362] Silva RCB, Brandão ML. Acute and chronic effects of gepirone and fluoxetine in rats tested in the elevated plus-maze: an ethological analysis. Pharmacol Biochem Behav 2000;65:209–16. [363] Silverman JL, Tolu SS, Barkan CL, Crawley JN. Repetitive self-grooming behavior in the BTBR mouse model of autism is blocked by the mGluR5 antagonist MPEP. Neuropsychopharmacology 2010;35:976–89. [364] Silverman JL, Yang M, Turner SM, Katz AM, Bell DB, Koenig JI, et al. Low stress reactivity and neuroendocrine factors in the BTBR T + tf/J mouse model of autism. Neuroscience 2010;171:1197–208. [365] Singh SD. Conditioned emotional response in the rat: I. Constitution and situational determinants. J Comp Physiol Psychol 1959;52:574–8. [366] Smith KS, Engin E, Meloni EG, Rudolph U. Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice. Neuropharmacology 2012;63:250–8. [367] Sousa N, Almeida OF, Wotjak CT. A hitchhiker's guide to behavioral analysis in laboratory rodents. Genes Brain Behav 2006;5:5–24. [368] Spasojević N, Gavrilović L, Varagić VV, Dronjak S. Effects of chronic diazepam treatments on behavior on individually housed rats. Arch Biol Sci Belgrade 2007;59:113–7. [369] Spielberger CD, Gorsuch RL, Lushene RE. The State-Trait Anxiety Inventory: test manual. Palo Alto, CA: Consulting Psychologist Press; 1970.

U

1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922 1923 1924 1925 1926 1927 1928 1929 1930 1931 1932 1933 1934 1935 1936 1937 1938 1939 1940 1941 1942 1943 1944 1945 1946 1947 1948 1949 1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

E

16

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 2024 2025 2026 2027 2028 2029 2030 2031 2032 2033 2034 2035 2036 2037 2038 2039 2040 2041 2042 2043 2044 2045 2046 2047 2048 2049 2050 2051 2052 2053 2054

A. Ennaceur / Physiology & Behavior xxx (2014) xxx–xxx

O

R O

P

U

N C O

R

R

E

C

T

E

2129

[416] Wiltgen BJ, Godsil BP, Peng Z, Saab F, June HL, Linn ML, et al. The alpha1 subunit of the GABA(A) receptor modulates fear learning and plasticity in the lateral amygdala. Front Behav Neurosci 2009;3:37. [417] Wolff JO, Watson MW, Thomas SA. Is self-grooming by male prairie voles a predictor of mate choice? Ethology 2002;108:169–79. [418] Yang M, Augustsson H, Markham CM, Hubbard DT, Webster D, Wall PM, et al. The rat exposure test: a model of mouse defensive behaviors. Physiol Behav 2004;81:465–73. [419] Yang M, Zhodzishsky V, Crawley JN. Social deficits in BTBR T + tf/J mice are unchanged by cross-fostering with C57BL/6J mothers. Int J Dev Neurosci 2007;25:515–21. [420] Yang M, Clarke AM, Crawley JN. Postnatal lesion evidence against a primary role for the corpus callosum in mouse sociability. Eur J Neurosci 2009;29:1663–77. [421] Yang XW, Lu XH. Molecular and cellular basis of obsessive–compulsive disorder-like behaviors: emerging view from mouse models. Curr Opin Neurol 2011;24:114–8. [422] Yerkes RM. Genetic aspects of grooming, a socially important primate behaviour pattern. J Soc Psychol 1933;4:3–25. [423] Yokoyama F, Yamauchi M, Oyama M, Okuma K, Onozawa K, Nagayama T, et al. Anxiolytic-like profiles of histamine H3 receptor agonists in animal models of anxiety: a comparative study with antidepressants and benzodiazepine anxiolytic. Psychopharmacology (Berl) 2009;205:177–87. [424] Zangrossi Jr H, File SE. Chlordiazepoxide reduces the generalised anxiety, but not the direct responses, of rats exposed to cat odor. Pharmacol Biochem Behav 1992;43:1195–200. [425] Zangrossi Jr H, File SE. Behavioral consequences in animal tests of anxiety and exploration of exposure to cat odor. Brain Res Bull 1992;29:381–8. [426] Zarrindast MR, Moghadam AH, Rostami P, Roohbakhsh A. The effects of histaminergic agents in the central amygdala of rats in the elevated plus-maze test of anxiety. Behav Pharmacol 2005;16:643–9. [427] Zinbarg RE. Concordance and synchrony in measures of anxiety and panic reconsidered: A hierarchical model of anxiety and panic. Behav Ther 1998;29:301–23. [428] Zuckerman M. Psychobiology of personality. Cambridge, UK: Cambridge University Press; 1991. [429] Zvolensky MJ, Lejuez CW, Eifert GH. Prediction and control: operational definitions for the experimental analysis of anxiety. Behav Res Ther 2000;38:653–63.

F

[402] Wahlsten D, Metten P, Phillips TJ, Boehm II SL, Burkhart-Kasch S, Dorow J, et al. Different data from different labs: lessons from studies of gene–environment interaction. J Neurobiol 2003;54:283–311. [403] Wahlsten D, Rustay NR, Metten P, Crabbe JC. In search of a better mouse test. Trends Neurosci 2003;26:132–6. [404] Wallace KJ, Rosen JB. Predator odor as an unconditioned fear stimulus in rats: elicitation of freezing by trimethylthiazoline, a component of fox feces. Behav Neurosci 2000;114:912–22. [405] Walsh RN, Cummins RA. The open-field test: a critical review. Psychol Bull 1976;83:482–504. [406] Wang X, McCoy PA, Rodriguiz RM, Pan Y, Je HS, Roberts AC, et al. Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum Mol Genet 2011;20:3093–108. [407] Watt MJ, Burke AR, Renner KJ, Forster GL. Adolescent male rats exposed to social defeat exhibit altered anxiety behavior and limbic monoamines as adults. Behav Neurosci 2009;123:564–76. [408] Wei Q, Lu XY, Liu L, Schafer G, Shieh KR, Burke S, et al. Glucocorticoid receptor overexpression in forebrain: a mouse model of increased emotional lability. Proc Natl Acad Sci U S A 2004;101:11851–6. [409] Weiss SM, Wadsworth G, Fletcher A, Dourish CT. Utility of ethological analysis to overcome locomotor confounds in elevated maze models of anxiety. Neurosci Biobehav Rev 1998;23:265–71. [410] Weisstaub NV, Zhou M, Lira A, Lambe E, González-Maeso J, Hornung JP, et al. Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science 2006;313:536–40. [411] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature 2007;448:894–900. [412] Welker WI. “Free” versus “forced” exploration of a novel situation by rats. Psychol Rep 1957;3:95–108. [413] Welker WI. Escape, exploratory, and food-seeking responses of rats in novel situation. J Comp Physiol Psychol 1959;52:106–11. [414] Whishaw IQ, Gharbawie OA, Benjamin JC, Lehmann H. The exploratory behavior of rats in an open environment optimizes security. Behav Brain Res 2006;171:230–9. [415] Wieronska JM, Stachowicz K, Nowak G, Pilc A. The loss of glutamate-GABA harmony in anxiety disorders. In: Kalinin V, editor. Anxiety disorders; 2011. p. 135–58 [chapter 8].

D

2055 2056 2057 2058 2059 2060 2061 2062 2063 2064 2065 2066 2067 2068 2069 2070 2071 2072 2073 2074 2075 2076 2077 2078 2079 2080 2081 2082 2083 2084 2085 2086 2087 2088 2089 2090 2091

17

Please cite this article as: Ennaceur A, Unconditioned tests of anxiety — Pitfalls and disappointments, Physiol Behav (2014), http://dx.doi.org/ 10.1016/j.physbeh.2014.05.032

2092 2093 2094 2095 2096 2097 2098 2099 2100 2101 2102 2103 2104 2105 2106 2107 2108 2109 2110 2111 2112 2113 2114 2115 2116 2117 2118 2119 2120 2121 2122 2123 2124 2125 2126 2127 2128