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Measuring anxiety- and locomotion-related behaviours in mice: a new way of using old tests

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Abstract

Rationale

Batteries of tests that are thought to measure different aspects of anxiety-related behaviour are used to characterise mice after genetic or pharmacological manipulation. However, because of the potentially confounding effects of repeated testing and natural intra-individual variations in behaviour over time, subjecting mice to a succession of tests is not ideal.

Objectives

The aim of this study was to investigate, in mice, the utility of an integrated apparatus that combines three classical tests of anxiety, the open field, elevated plus maze (EPM) and light/dark box.

Methods

Mice from four different strains (CD-1, BALB/cJ, DBA/2J, C57BL/6J) were used in a series of five experiments where their behaviour was observed for 15 min in the integrated apparatus. Responses to anxiety-modulating drugs and 2-day repeated testing were evaluated.

Results

CD-1 mice explored the apparatus thoroughly, providing measures from all areas throughout the entire testing session. Factor analysis showed that measures of locomotion and anxiety-related behaviour were dissociable. BALB/cJ, DBA/2J and C57BL/6J showed markedly different behavioural profiles, largely consistent with previous studies examining individual tests. Avoidance of aversive environments did not increase with repeated testing. In CD-1 mice, the anxiolytics diazepam and alprazolam (4 and 2 mg/kg, respectively) increased the approach towards the EPM open arms. Alprazolam also had sedative effects, whereas the anxiogenic pentylenetetrazole had no effects.

Conclusions

These findings suggest that the triple test is sensitive to genetic/pharmacological influences on anxiety and locomotion and that, by providing quasi-simultaneous measures from three different apparatuses, it may represent an alternative to the use of test batteries.

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References

  • Anisman H, Hayley S, Kelly O, Borowski T, Merali Z (2001) Psychogenic, neurogenic, and systemic stressor effects on plasma corticosterone and behavior: mouse strain-dependent outcomes. Behav Neurosci 115:443–454

    Article  PubMed  CAS  Google Scholar 

  • Bakshi VP, Kalin NH (2002) Animal models and endophenotypes of anxiety and stress disorders. In: Davis KL, Charney D, Coyle JT, Nemeroff C. (eds) Neuropsychopharmacology: the fifth generation of progress, Am Coll Neuropsychopharmacol, pp 883–900

  • Ballaz SJ, Akil H, Watson SJ (2007) Previous experience affects subsequent anxiety-like responses in rats bred for novelty seeking. Behav Neurosci 121:1113–1118

    Article  PubMed  Google Scholar 

  • Belzung C, Griebel G (2001) Measuring normal and pathological anxiety-like behaviour in mice: a review. Behav Brain Res 125:141–149

    Article  PubMed  CAS  Google Scholar 

  • Blanchard DC, Griebel G, Blanchard RJ (2001) Mouse defensive behaviors: pharmacological and behavioral assays for anxiety and panic. Neurosci Biobehav Rev 25:205–218

    Article  PubMed  CAS  Google Scholar 

  • Bourin M, Hascoet M, Mansouri B, Colombel M, Bradwejn J (1992) Comparison of behavioural effects after single and repeated administrations of four benzodiazepines in three mice behavioural models. J Psychiatry Neurosci 17:72–77

    PubMed  CAS  Google Scholar 

  • Brigman JL, Mathura P, Lu L, Williams RW, Holmes A (2009) Genetic relationship between anxiety related and fear-related behaviors in BXD recombinant inbred mice. Behav Pharmacol 20:204–209

    Article  PubMed  Google Scholar 

  • Carobrez AP, Bertoglio LJ (2005) Ethological and temporal analyses of anxiety-like behavior: the elevated plus-maze model 20 years on. Neurosci Biobehav Rev 29:1193–1205

    Article  PubMed  CAS  Google Scholar 

  • Carola V, D’Olimpio F, Brunamonti E, Mangia F, Renzi P (2002) Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res 134:49–57

    Article  PubMed  Google Scholar 

  • Choleris E, Thomas AW, Kavaliers M, Pratos FS (2001) 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 25:235–260

    Article  PubMed  CAS  Google Scholar 

  • Cole JC, Rodgers RJ (1995) Ethological comparison of the effects of diazepam and acute/chronic imipramine on the behaviour of mice in the elevated-plus maze. Pharmacol Biochem Behav 52:473–478

    Article  PubMed  CAS  Google Scholar 

  • Crabbe JC, Wahlsten D, Dudek BC (1999) Genetics of mouse behavior: interactions with laboratory environment. Science 284:1670–1672

    Article  PubMed  CAS  Google Scholar 

  • Crawley JN (1981) Neuropharmacologic specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol Biochem Behav 15:695–699

    Article  PubMed  CAS  Google Scholar 

  • Crawley JN, Paylor R (1997) A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Behav 31:197–211

    Article  PubMed  CAS  Google Scholar 

  • Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC, Miner LL, Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R (1997) Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology 132:107–124

    Article  PubMed  CAS  Google Scholar 

  • Cruz APM, Frei F, Graeff FG (1994) Ethopharmacological analysis of rat behavior on the elevated plus-maze. Pharmacol Biochem Behav 49:171–176

    Article  PubMed  CAS  Google Scholar 

  • Cryan JF, Holmes A (2005) The ascent of mouse: advances in modeling human depression and anxiety. Nat Rev Drug Discov 4:775–790

    Article  PubMed  CAS  Google Scholar 

  • Dalvi A, Rodgers RJ (1996) GABAergic influences on plus-maze behaviour in mice. Psychopharmacology 128:380–397

    Article  PubMed  CAS  Google Scholar 

  • Dalvi A, Rodgers RJ (1999) Behavioural 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 62:727–734

    Article  PubMed  CAS  Google Scholar 

  • Evans AK, Lowry CA (2007) Pharmacology of the β-Carboline FG-7142, a partial inverse agonist at the benzodiazepine allosteric site of the GABAA receptor: neurochemical, neurophysiological, and behavioral effects. CNS Drug Rev 13:475–501

    Article  PubMed  CAS  Google Scholar 

  • File SE (1990) One-trial tolerance to the anxiolytic effects of chlordiazepoxide in the plus-maze. Psychopharmacology 100:281–282

    Article  PubMed  CAS  Google Scholar 

  • File SE (1991) The biological basis of anxiety. In: Meltzer HY, Nerozzi D (eds) Current practices and future developments in the pharmacotherapy of mental disorders. Elsevier, Amsterdam, pp 159–165

    Google Scholar 

  • File SE, Zangrossi H Jr (1993) “One-trial tolerance” to the anxiolytic actions of benzodiazepines in the elevated plus-maze, or the development of a phobic state? Psychopharmacology 110:240–444

    Article  PubMed  CAS  Google Scholar 

  • Finn DA, Rutledge-Gorman MT, Crabbe JC (2003) Genetic animal models of anxiety. Neurogenetics 4:109–135

    PubMed  Google Scholar 

  • Hall CS (1934) Emotional behavior in the rat. I. Defecation and urination as measures of individual differences in emotionality. J Comp Psychol 18:385–403

    Article  Google Scholar 

  • Handley SL, McBlane JW (1993) 5HT drugs in animal models of anxiety. Psychopharmacology 112:13–20

    Article  PubMed  CAS  Google Scholar 

  • Handley SL, Mithani S (1984) Effects of alpha-adrenoceptor agonists and antagonists in a maze-exploration model of ‘fear’-motivated behaviour. Naunyn-Schmiedeberg’s Arch Pharmacol 327:1–5

    Article  CAS  Google Scholar 

  • Holmes A, Rodgers RJ (1998) Responses of Swiss-Webster mice to repeated plus-maze experience: further evidence for a qualitative shift in emotional state? Pharmacol Biochem Behav 60:473–488

    Article  PubMed  CAS  Google Scholar 

  • Holmes A, Iles JP, Mayell SJ, Rodgers RJ (2001) Prior test experience compromises the anxiolytic efficacy of chlordiazepoxide in the mouse light/dark exploration test. Behav Brain Res 122:159–167

    Article  PubMed  CAS  Google Scholar 

  • Holmes A, Kinney JW, Wrenn CC, Li Q, Yang RJ, Ma L, Vishwanath J, Saavedra MC, Innerfield CE, Jacoby AS, Shine J, Iismaa TP, Crawley JN (2003) Galanin GAL-R1 receptor null mutant mice display increased anxiety-like behavior specific to the elevated plus-maze. Neuropsychopharmacology 28:1031–1044

    PubMed  CAS  Google Scholar 

  • Izidio GS, Lopes DM, Spricigo L Jr, Ramos A (2005) Common variations in the pretest environment influence genotypic comparisons in models of anxiety. Genes Brain Behav 4:412–419

    Article  PubMed  CAS  Google Scholar 

  • Johnston AL, File SE (1989) Sodium phenobarbitone reverses the anxiogenic effects of compounds acting at three different central sites. Neuropharmacology 28:83–88

    Article  PubMed  CAS  Google Scholar 

  • Jones GH, Schneider C, Schneider HH, Seidler J, Cole BJ, Stephens DN (1994) Comparison of several benzodiazepine receptor ligands in two models of anxiolytic activity in the mouse: an analysis based on fractional receptor occupancies. Psychopharmacology 114:191–199

    Article  PubMed  CAS  Google Scholar 

  • Jung ME, Lal H, Gatch MB (2002) The discriminative stimulus effects of pentylenetetrazol as a model of anxiety: recent developments. Neurosci Biobehav Rev 26:429–439

    Article  PubMed  CAS  Google Scholar 

  • Lister RG (1990) Ethologically-based animal models of anxiety disorders. Pharmacol Ther 46:321–340

    Article  PubMed  CAS  Google Scholar 

  • Miller LG, Woolverton S, Greenblatt DJ, Lopez F, Roy RB, Shader RI (1989) Chronic benzodiazepine administration. IV. Rapid development of tolerance and receptor downregulation associated with alprazolam administration. Biochem Pharmacol 38:3773–3777

    Article  PubMed  CAS  Google Scholar 

  • Millstein RA, Holmes A (2007) Effects of repeated maternal separation on anxiety- and depression-related phenotypes in different mouse strains. Neurosci Biobehav Rev 31:3–17

    Article  PubMed  Google Scholar 

  • Milner LC, Crabbe JC (2008) Three murine anxiety models: results from multiple inbred strain comparisons. Genes Brain Behav 7:496–505

    Article  PubMed  CAS  Google Scholar 

  • Norcross M, Poonam M, Enoch AJ, Karlsson RM, Brigman JL, Cameron HA, Harvey-White J, Holmes A (2008) Effects of adolescent fluoxetine treatment on fear-, anxiety- or stress-related behaviors in C57BL/6J or BALB/cJ mice. Psychopharmacology 200:413–424

    Article  PubMed  CAS  Google Scholar 

  • Nutt DJ, Lister RG (1988) Strain differences in response to a benzodiazepine receptor inverse agonist (FG 7142) in mice. Psychopharmacology 94:435–436

    Article  Google Scholar 

  • Paylor R, Spencer CM, Yuva-Paylor LA, Pieke-Dahl S (2006) The use of behavioral test batteries, II: effect of test interval. Physiol Behav 87:95–102

    Article  PubMed  CAS  Google Scholar 

  • Pellow S, Chopin P, File SE, Briley M (1985) Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Meth 14:149–167

    Article  CAS  Google Scholar 

  • Prut L, Belzung C (2003) The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463:3–33

    Article  PubMed  CAS  Google Scholar 

  • Ramos A (2008) Animal models of anxiety: do I need multiple tests? Trends Pharmacol Sci 29:493–498

    Article  PubMed  CAS  Google Scholar 

  • Ramos A, Mormede P (1998) Stress and emotionality: a multidimensional and genetic approach. Neurosci Biobehav Rev 22:33–57

    Article  PubMed  CAS  Google Scholar 

  • Ramos A, Mellerin Y, Mormede P, Chaouloff F (1998) A genetic and multifactorial analysis of anxiety-related behaviours in Lewis and SHR intercrosses. Behav Brain Res 96:195–205

    Article  PubMed  CAS  Google Scholar 

  • Ramos A, Pereira E, Martins GC, Wehrmeister TD, Izídio GS (2008) Integrating the open field, elevated plus maze and light/dark box to assess different types of emotional behaviors in one single trial. Behav Brain Res 193:277–288

    Article  PubMed  Google Scholar 

  • Rodgers RJ, Cole JC (1994) The elevated plus-maze: pharmacology, methodology and ethology. In: Cooper SJ, Hendrie CA (eds) Ethology and psychopharmacology. Wiley, Chichester, pp 9–44

    Google Scholar 

  • Rodgers RJ, Johnson NJT (1995) Factor analysis of spatiotemporal and ethological measures in the murine plus-maze test of anxiety. Pharmacol Biochem Behav 52:297–303

    Article  PubMed  CAS  Google Scholar 

  • Rodgers RJ, Shepherd JK (1993) Influence of prior maze experience on behaviour and response to diazepam in the elevated plus-maze and light-dark tests of anxiety in mice. Psychopharmacology 113:237–242

    Article  PubMed  CAS  Google Scholar 

  • Rodgers RJ, Cole JC, Aboualfa K, Stephenson H (1995) Ethopharmacological analysis of the effects of putative ‘anxiogenic’ agents in the mouse elevated plus-maze. Pharmacol Biochem Behav 52:805–813

    Article  PubMed  CAS  Google Scholar 

  • Rodgers RJ, Davies B, Shore R (2002) 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 1:242–251

    Article  PubMed  CAS  Google Scholar 

  • Rogers DC, Jones DNC, Nelson PR, Jones CM, Quilter CA, Robinson TL, Hagan JJ (1999) Use of SHIRPA and discriminant analysis to characterise marked differences in the behavioural phenotype of six inbred mouse strains. Behav Brain Res 105:207–217

    Article  PubMed  CAS  Google Scholar 

  • Shekhar A, McCann UD, Meaney MJ, Blanchard DC, Davis M, Frey KA, Liberzon I, Overall KL, Shear MK, Tecott LH, Winsky L (2001) Summary of a National Institute of Mental Health workshop: developing animal models of anxiety disorders. Psychopharmacology 157:327–339

    Article  PubMed  CAS  Google Scholar 

  • Shimada T, Matsumoto K, Osanai M, Matsuda H, Tarasawa K, Watanabe H (1995) The modified light/dark transition test in mice: evaluation of classic and putative anxiolytic and anxiogenic drugs. Gen Pharmacol 26:205–210

    PubMed  CAS  Google Scholar 

  • Sulcova A, Krsiak M (1989) Differences among nine 1, 4-bezodiazepines: an ethopharmacological evaluation in mice. Psychopharmacology 97:157–159

    Article  PubMed  CAS  Google Scholar 

  • Treit D (1985) Animal models for the study of anti-anxiety agents: a review. Neurosci Biobehav Rev 9:203–222

    Article  PubMed  CAS  Google Scholar 

  • Trullas R, Skolnick P (1993) Differences in fear motivated behaviors among inbred mouse strains. Psychopharmacology 111:323–331

    Article  PubMed  CAS  Google Scholar 

  • Van der Staay FJ (2006) Animal models of behavioral dysfunctions: basic concepts and classifications, and an evaluation strategy. Brain Res Rev 52:131–159

    Article  PubMed  Google Scholar 

  • Voikar V, Vasar E, Rauvala H (2004) Behavioral alterations induced by repeated testing in C57BL/6J and 129S2/SV mice: implications for phenotyping screens. Genes Brain Behav 3:27–38

    Article  PubMed  CAS  Google Scholar 

  • Wada T, Fukuda N (1991) Effects of DN-2327, a new anxiolytic, diazepam and buspirone on exploratory activity of the rat in an elevated plus-maze. Psychopharmacology 104:444–450

    Article  PubMed  CAS  Google Scholar 

  • Wahlsten D, DA BachmanovA F, Crabbe JC (2006) Stability of inbred mouse strain differences in behavior and brain size between laboratories and across decades. Proc Natl Acad Sci USA 103:16364–16369

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Dr H. Robertson for kindly providing DZP and APZ, R. Gunn for providing technical assistance and B. Eisner (all from Dalhousie University) for building the triple apparatus. L. Fraser was funded by an NSERC PGS-D and A. Ramos had a fellowship from CAPES/Brazil.

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Authors and Affiliations

  1. Psychology Department and Neuroscience Institute, Dalhousie University, Halifax, Nova Scotia, Canada, B3H 4J1

    Leanne M. Fraser, Richard E. Brown, Ahmed Hussin, Mara Fontana, Ashley Whittaker & Timothy P. O’Leary

  2. Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcoholism and Alcohol Abuse, NIH, 5625 Fishers Lane Room 2N09, Rockville, MD, 20852-9411, USA

    Lauren Lederle & Andrew Holmes

  3. Laboratório de Genética do Comportamento, Departamento de Biologia Celular, Embriologia e Genética, Universidade Federal de Santa Catarina, 88.040-900, Florianópolis, Santa Catarina, Brazil

    André Ramos

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  1. Leanne M. Fraser
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  3. Ahmed Hussin
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Correspondence to André Ramos.

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L. M. Fraser was funded by NSERC, Canada and A. Ramos had a fellowship from CAPES, Brazil.

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Fraser, L.M., Brown, R.E., Hussin, A. et al. Measuring anxiety- and locomotion-related behaviours in mice: a new way of using old tests. Psychopharmacology 211, 99–112 (2010). https://doi.org/10.1007/s00213-010-1873-0

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