Animal Tests of Anxiety

Sandra E. File1, Arnold S. Lippa2, Bernard Beer2, Morgen T. Lippa2

1 King's College London, London, 2 DOV Pharmaceutical, Inc., Hackensack, New Jersey
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.3
DOI:  10.1002/0471142301.ns0803s26
Online Posting Date:  May, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Animal tests of anxiety are used to screen novel compounds for anxiolytic or anxiogenic activity, to investigate the neurobiology of anxiety, and to assess the impact of other occurrences such as exposure to predator odors or early rearing experiences. This unit presents protocols for the most commonly used animal tests of anxiety. The Geller‐Seifter conflict test, the social interaction test, light/dark exploration, the elevated plus‐maze, defensive burying, and the thirsty rat conflict. The protocols are described in terms of drug screening tests, but can be modified easily for other purposes.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Geller‐Seifter Conflict
  • Basic Protocol 2: Social Interaction
  • Basic Protocol 3: Light/Dark Exploration
  • Basic Protocol 4: Elevated Plus‐Maze Test
  • Basic Protocol 5: Defensive Burying
  • Basic Protocol 6: Thirsty Rat Conflict
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Geller‐Seifter Conflict

  Materials
  • Male or female rats, group housed
  • Rat food pellets
  • Drugs to be tested
  • Saline or other appropriate drug vehicle for control injections
  • Animal scale
  • Standard rat operant chamber (Skinner box) with floor of metal bars for delivery of footshock, and with house light, cue light triggered by the response, lever, and a pellet dispenser
  • Shock generator and scrambler (unit 8.4)

Basic Protocol 2: Social Interaction

  Materials
  • Adult male or adolescent male or female rats
  • Drugs to be tested
  • Saline or other vehicle for control injections
  • Single rat cages
  • Quiet test room away from disturbance
  • Video camera with automatic iris
  • Social interaction test arena: 60 × 60–cm wooden box with 35‐cm high walls, and infrared photocells mounted in the walls 4.5 and 12.5 cm from the floor, illuminated either brightly (300 radiometric lux) or dimly (30 radiometric lux)

Basic Protocol 3: Light/Dark Exploration

  Materials
  • Male mice of appropriate strain (see Critical Parameters), group housed
  • Drugs to be tested
  • Saline or vehicle for control injections
  • Quiet, darkened test room away from disturbance
  • Illuminated test chamber, screened from observers (Fig. )

Basic Protocol 4: Elevated Plus‐Maze Test

  Materials
  • Male or female rats or mice or male gerbils
  • Quiet test room away from disturbance
  • Video camera and monitor
  • Elevated plus‐maze apparatus (Fig. )
  • Automated test system or keyboard for scoring behaviors

Basic Protocol 5: Defensive Burying

  Materials
  • Male or female rats of desired strain and age
  • Drugs to be tested
  • Saline or other vehicle for control injections
  • Individual polycarbonate rat cages
  • Aspen chips
  • Absorbent bedding material (e.g., cat litter)
  • Quiet room away from disturbance
  • Video camera
  • Shock‐probe test apparatus (Fig. )
  • Multimeter

Basic Protocol 6: Thirsty Rat Conflict

  Materials
  • Male or female rats
  • 10% dextrose‐water solution for water bottle
  • Drug to be tested
  • Saline or other appropriate drug vehicle for control injections
  • Clear Plexiglas box (1/4‐in thick; 38 × 38 × 30 (h)–cm) with stainless steel grid floor and Plexiglas lid
  • Black Plexiglas compartment (1/4‐in thick; 10 × 10.5 × 10.5 (h)–cm)
  • Water bottle with metal drinking tube
  • Constant current shock generator with circuit for counting the number of shocks (e.g., Anxio‐Meter model 102; Columbus Instruments)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   Andrews, N. and File, S.E. 1993. Handling history of rats modifies behavioral effects of drugs in the elevated plus‐maze test of anxiety. Eur. J. Pharmacol. 235:109‐112.
   Becker, A. and Grecksch, G. 1996. Illumination has no effect on rats' behavior in the elevated plus‐maze. Physiol. Behav. 59:1175‐1177.
   Charrier, D., Dangoumau, L., Hamon, M., Puech, A.J., and Thiébot, M.‐H. 1994. Effects of 5‐HT1A receptor ligands on a safety signal withdrawal procedure of conflict in the rat. Pharmacol. Biochem. Behav. 48:281‐289.
   Crawley, J.N. and Davis, L.G. 1982. Baseline exploratory behavior predicts anxiolytic responsiveness to diazepam in five mouse strains. Brain Res. Bull. 8:609‐612.
   Fernandes, C. and File, S.E. 1996. The influence of open arm ledges and maze experience in the elevated plus‐maze. Pharmacol. Biochem. Behav. 54:31‐40.
   Fernandes, C., Gonzalez, M.I., Wilson, C.A. and File, S.E. 1999. Factor analysis reveals that female rat behaviour is characterised by activity, male rats are driven by sex and anxiety. Pharmacol. Biochem. Behav. 64:731‐738.
   File, S.E. 1980. The use of social interaction as a method for detecting anxiolytic activity of chlordiazepoxide‐like drugs. J. Neurosci. Methods 2:219‐238.
   File, S.E. 1990. Age and anxiety: Increased anxiety, decreased anxiolytic, but enhanced sedative, response to chlordiazepoxide in old rats. Hum. Psychopharmacol. 5:169‐173.
   File, S.E. 1991. Animal models of anxiety. In Biological Psychiatry (G. Racagni, N. Brunello,, and, T. Fukuda, eds.) pp. 596‐599. Elsevier, New York.
   File, S.E. 1993. The interplay of learning and anxiety in the elevated plus‐maze. Behav. Brain Res. 58:199‐202.
   File, S.E. 2000. NKP608, an NK1 receptor antagonist, has an anxiolytic action in the social interaction test in rats. Psychopharmacology 152:105‐109.
   File, S.E. 2001. Factors controlling measures of anxiety and responses to novelty in the mouse. Behav. Brain Res. 125:151‐157.
   File, S.E. and Andrews, N. 1991. Low but not high doses of buspirone reduce the anxiogenic effects of diazepam withdrawal. Psychopharmacology 105:578‐582.
   File, S.E. and Hyde, J.R.G. 1978. Can social interaction be used to measure anxiety? Br. J. Pharmacol. 62: 19‐24.
   File, S.E. and Seth, P. 2003. A review of 25 years of the social interaction test. Eur. J. Pharmacol. 463:35‐53.
   File, S.E. and Tucker, J.C. 1983. Prenatal treatment with clomipramine has an anxiolytic profile in the adolescent rat. Physiol. Behav. 31:57‐62.
   File, S.E. and Wardill, A.G. 1975. Validity of head‐dipping as a measure of exploration in a modified holeboard. Psychopharmacologia 44:53‐59.
   File, S.E. and 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 110:240‐244.
   File, S.E., Baldwin, H.A., and Aranko, K. 1987. Anxiogenic effects from benzodiazepine withdrawal are linked to the development of tolerance. Brain Res. Bull. 19:607‐610.
   File, S.E, Gonzalez, L.E., and Andrews, N. 1996. Comparative study of pre‐and post‐synaptic 5‐HT1A receptor modulation of anxiety in two ethological animal tests. J. Neurosci. 16:4810‐4815.
   File, S.E., Amarbirpal, M., Mangiarini, L., and Bates, G.P. 1998. Striking changes in anxiety in Huntington's disease transgenic mice. Brain Res. 805:234‐240.
   File, S.E., Cheeta, S., and Akanezi, C. 2001. Diazepam and nicotine increase social interaction in gerbils: A test for anxiolytic action. Brain Res. 888:311‐313.
   Hartley, D.E., Edwards, J.E., Spiller, C.E., Alom, N., Tucci, S., Seth, P., Forsling, M.L., and File, S.E. 2003. The soya isoflavone content of rat diet can increase anxiety and stress hormone release in the male rat. Psychopharmacology 167:46‐53.
   Higgins, G.A, Bradbury, A.J., Jones, B.J., and Oakley, N.R. 1988. Behavioural and biochemical consequences following activation of 5‐HT1‐like and GABA receptors in the dorsal raphé nucleus of the rat. Neuropharmacology 27:993‐1001.
   Hodges, H., Green, S., and Glenn, B. 1987. Evidence that the amygdala is involved in benzodiazepine and serotonergic effects on punished responding but not on discrimination. Psychopharmacology 92:491‐504.
   Howard, J.L. and Pollard, G.T. 1991. Effects of drugs on punished behavior: Preclinical test for anxiolytics. In Psychopharmacology of Anxiolytics and Antidepressants (S.E. File, ed.) pp. 131‐153. Pergamon Press, Elmsford, N.Y.
   Johnston, A.L. and File, S.E. 1991. Sex differences in animal tests of anxiety. Physiol. Behav. 49:245‐250.
   Jones, N., Duxon, M.S., and King, S.M. 2002. Ethopharmacological analysis of the unstable elevated exposed plus‐maze, a novel model of extreme anxiety: Predictive validity and sensitivity to anxiogenic agents. Psychopharmacology 16:314‐323.
   Lippa, A.S., Nash, P.A., and Greenblatt, E.N. 1977. Pre‐clinical neuropsychopharmacological testing procedures for anxiolytic drugs. In The Anxiolytics, Vol. 3 (S. Fielding, and, H. Lal, eds.) pp. 41‐81. Futura, New York.
   Lippa, A.S., Coupet, J., Greenblatt, E.N., Klepner, C., and Beer, B. 1979. A synthetic non‐benzodiazepine ligand for benzodiazepine receptors: A probe for investigating neuronal substrates of anxiety. Pharmacol. Biochem. Behav. 11:99‐106.
   Lister, R.G. 1987. The use of a plus‐maze to measure anxiety in the mouse. Psychopharmacology 92:180‐185.
   Mathis, C., Paul, S.M., and Crawley, J.N. 1994. Characterization of benzodiazepine‐sensitive behaviors in the A/J and C57BL/6J inbred strains of mice. Behav. Genet. 24:171‐180.
   Pellow, S. and File, S.E. 1986. Anxiolytic and anxiogenic drug effects in exploratory activity in an elevated plus‐maze: A novel test of anxiety in the rat. Pharmacol. Biochem. Behav. 24:525‐529.
   Rodgers, R.J., Cole, J.C., Aboualfa, K., and Stephenson, L.H. 1995. Ethopharmacological analysis of the effects of putative “anxiogenic” agents in the mouse elevated plus‐maze. Pharmacol. Biochem. Behav. 52:805‐813.
   Thiébot, M.‐H., Dangoumau, L., Richard, G., and Puech, A.J. 1991. Safety signal withdrawal: A behavioural paradigm sensitive to both “anxiolytic” and “anxiogenic” drugs under identical experimental conditions. Psychopharmacology 103:415‐424.
   Treit, D., Pinel, J.P.J., and Fibiger, H.C. 1981. Conditioned defensive burying: A new paradigm for the study of anxiolytic agents. Pharmacol. Biochem. Behav. 15:619‐626.
   Treit, D., Menard, J., and Royan, C. 1993. Anxiogenic stimuli in the elevated plus‐maze. Pharmacol. Biochem. Behav. 44:463‐469.
   Trullas, R. and Skolnick, P. 1993. Differences in fear motivated behaviors among inbred mouse strains. Psychopharmacology 111:323‐331.
   Varty, G.B., Morgan, C.A., Cohen‐Williams, M.E., Coffin, V.L., and Carey, G.J. 2002. The gerbil elevated plus‐maze. I. Behavioral characterization and pharmacological validation. Neuropsychopharmacology 27:357‐370.
   Zangrossi, H. and File, S.E. 1992. Behavioral consequences in animal tests of anxiety and exploration of exposure to cat odor. Brain Res. Bull. 29:381‐388.
Key References
   Blumstein, L.K. and Crawley, J.N. 1983. Further characterization of a simple automated exploratory model for the anxiolytic effects of benzodiazepines. Pharmacol. Biochem. Behav. 18:37‐40.
   Costall, B., Jones, B.J., Kelly, M.E., Naylor, R.J., and Tomkins, D.M. 1989. Exploration of mice in a black and white test box: Validation as a model of anxiety. Pharmacol. Biochem. Behav. 32:777‐785.
   Crawley, J.N. 1981. Neuropharmacological specificity of a simple animal model for the behavioral actions of benzodiazepines. Pharmacol. Biochem. Behav. 15:695‐699.
  File, 1980. See above.
   File, S.E. 1993. The social interaction test of anxiety. Neurosci. Protocols 93‐010‐01‐01‐07.
  File and Hyde, 1978. See above.
  File and Seth, 2003. See above.
   Howard, J.L. and Pollard, G.T. 1991. Effects of drugs on punished behavior: Preclinical test for anxiolytics. In Psychopharmacology of Anxiolytics and Antidepressants (S.E. File, ed.) pp. 131‐153. Pergamon Press, New York.
  Lister, 1987. See above.
   Pellow, S., Chopin, P., File, S.E., and Briley, M. 1985. Validation of open:closed arm entries in an elevated plus‐maze as a measure of anxiety in the rat. J. Neurosci. Methods 14:149‐167.
   Treit, D. 1985. Animal models for the study of anti‐anxiety agents: A review. Neurosci. Biobehav. Rev. 9:203‐222.
   Treit, D. 1991. Defensive burying: A pharmacological animal model for specific fears? In Animal Models of Psychiatric Disorders, Vol. 3 (P. Soubrie, P. Simon, and D. Widlocher, eds.) pp. 1‐19. S. Karger, Basel.
   Vogel, J.R., Beer, B., and Clody, D.E. 1971. A simple and reliable conflict procedure for testing anti‐anxiety agents. Psychopharmacologia (Berl.) 21:1‐7.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library