Defensive Responses to Predator Threat in the Rat and Mouse

D. Caroline Blanchard1, Robert J. Blanchard1, Guy Griebel2

1 University of Hawaii, Honolulu, Hawaii, 2 Sanofi‐Synthelabo, Paris, null
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.19
DOI:  10.1002/0471142301.ns0819s30
Online Posting Date:  February, 2005
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Abstract

Defensive responses include an array of specific behaviors, including flight, freezing, risk assessment, and defensive threat/attack, that are elicited by unconditioned threat stimuli such as predators or predator odors. Some individual defensive behaviors are selectively responsive to drugs effective against generalized anxiety disorder or panic, providing a rationale for their use in investigation of compounds that may be useful in treating these disorders. In addition, defensive behaviors toward predators and some predator odors show rapid conditioning to contextual stimuli, whereas other predator odors do not, although they too elicit defensiveness. This pattern suggests that the ability of a predator odor to predict danger may be a determinant of the degree to which that odor supports aversive conditioning. Predators and predator odors are also increasingly used in studies of brain systems potentially related to emotionality. These factors indicate the need for selective, reliable, and convenient tests of defensiveness to predators and predator odors using rat and mouse subjects.

Keywords: defensive behavior; cat exposure; cat odor; defensive conditioning; anxiety

     
 
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Table of Contents

  • Basic Protocol 1: Use of Mouse Defense Test Battery (MDTB) to Test Defensive Behaviors of Mice To an Anesthetized Rat
  • Basic Protocol 2: Use of Rat Exposure Test (RET) to Evaluate Mouse Defensive Responses to a Live Rat
  • Basic Protocol 3: Testing Rat Defensive Responses to Cat Odor and Conditioning to Associated Contextual Stimuli
  • Alternate Protocol 1: Use of Cat Odor to Elicit a Range of Defensive Behaviors when a Hiding Area is Available
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Use of Mouse Defense Test Battery (MDTB) to Test Defensive Behaviors of Mice To an Anesthetized Rat

  Materials
  • Adult male mice (e.g., Swiss strain)
  • Adult male rats
  • Drugs to be tested
  • CO 2
  • Laboratory detergent (mild)
  • Saline or other vehicle for control injections
  • Standard single mouse cages
  • Video camera (optional: television screen connected to video camera, located in an adjacent room)
  • Runway apparatus (Fig. ; custom‐made)
  • Quiet test room away from disturbance (run tests under red light)

Basic Protocol 2: Use of Rat Exposure Test (RET) to Evaluate Mouse Defensive Responses to a Live Rat

  Materials
  • Adult male mice
  • Adult rats (threat stimuli)
  • Control stimulus (plush toy animal about the same size as the rats to be used)
  • Drugs to be tested
  • Saline or other vehicle for control injections
  • 5% alcohol
  • Mild laboratory detergent
  • D‐amphetamine
  • Standard single mouse cages
  • Two identical RET apparatuses (custom‐made; Fig. )
  • Two quiet, darkened rooms, of similar dimensions and construction, free from disturbances
  • Video camera

Basic Protocol 3: Testing Rat Defensive Responses to Cat Odor and Conditioning to Associated Contextual Stimuli

  Materials
  • Adult male rats
  • Adult cat (odor donor)
  • Drugs to be tested
  • Saline or other vehicle for control injections
  • 9 × 9 × 2–cm Plexiglas blocks
  • Terry cloth
  • Single rat cages
  • Two quiet, darkened rooms of similar dimensions and construction, away from disturbances
  • Video camera
  • Two identical cat odor apparatuses (Fig. )

Alternate Protocol 1: Use of Cat Odor to Elicit a Range of Defensive Behaviors when a Hiding Area is Available

  Materials
  • Adult male rats
  • Access to an adult cat (odor donor, not brought into the lab)
  • Standard wool acrylic or synthetic nylon cat collars
  • Standard single rat cages
  • Two quiet, darkened rooms, of similar dimensions and construction, away from disturbances
  • Video camera
  • Two identical cat odor with hide box apparatuses (custom‐made; Fig. )
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Figures

  •   FigureFigure 8.19.1 The runway apparatus is an oval runway made of black Plexiglas, 0.40 m wide, 0.30 m high, and 4.4 m in total length, consisting of two 2‐m straight segments joined by two 0.4‐m curved segments and separated by a median wall (2.0 × 0.30 × 0.06–m). The apparatus is elevated to a height of 0.80 m from the floor to enable the experimenter to easily hold the stimulus rat while minimizing visual contact with the mouse. The floor is marked every 20 cm to facilitate distance and activity measurement, and doors, 60 cm apart, (not shown on this sketch) are located near one end of the apparatus.
  •   FigureFigure 8.19.2 This photograph shows flight, which is locomotion directed away from the oncoming threat source.
  •   FigureFigure 8.19.3 This photograph shows risk assessment behavior. During the chase, subject stops, then orients its head towards the hand‐held rat.
  •   FigureFigure 8.19.4 This photograph shows defensive upright posture and biting. Upon forced contact with the hand‐held rat, subject displays a typical terminal defense response, consisting of sonic vocalization, upright posture, and defensive attack behavior.
  •   FigureFigure 8.19.5 This photograph shows a typical escape attempt. Following the removal of the rat from the runway cage, subject attempts to escape from the place where it has been confronted with the threatening stimulus.
  •   FigureFigure 8.19.6 The RET apparatus apparatus is a 46.0 × 24.0 × 21.0–cm clear polycarbonate exposure cage covered with a metal lid. The exposure cage is divided into two equal sized compartments by a wall‐to‐wall 0.25‐ to 0.5‐in. wire mesh screen. The home chamber is a 7 × 7 × 12–cm box constructed of black Plexiglas on three sides and clear Plexiglas on one side to facilitate videotaping. The home chamber is connected to the larger exposure cage by a clear Plexiglas tube tunnel 4.4 cm in diameter, 13 cm in length, and elevated 1.5 cm from the floor of the two chambers.
  •   FigureFigure 8.19.7 Each cat odor apparatus is a 100 × 15 × 50–cm white Plexiglas box, with the front wall of clear Plexiglas to allow videotaping. The compartment is divided by lines on the floor into three segments, each 33.3 cm in length.
  •   FigureFigure 8.19.8 The apparatus for the cat odor test with hide box is a rectangular enclosure with Perspex or Plexiglas walls (60‐cm length × 26‐cm width × 36‐cm height, with a metal grid or wire mesh floor, mounted above a tray containing absorbent material. Across one end of the chamber is a hide box or barrier delineating a 21‐cm deep space, with a 6 × 6–cm square hole permitting the subject to enter the hiding space. On the opposite end of the chamber from the hide box, an alligator clip is positioned 4 cm above the floor. During testing this clip holds a portion of a cloth cat collar. Photobeam detectors are mounted to detect activity in the hiding area and approaches to the cat collar. An additional set of photobeams (not shown in figure) placed just in front of the hide box can be used to detect “head outs.” Placement of the collar in front of, or behind a wire mesh barrier can be manipulated, as shown. A videocamera is mounted on the roof of the apparatus. As an alternative, photobeams and photobeam detectors may be omitted, and a side‐mounted camera used to record behaviors.
  •   FigureFigure 8.19.9 (A) Effect of buspirone (5.0, 10.0, and 20.0 mg/kg) on location within the RET: chamber, tunnel, or surface area. Enhanced time near the threat stimulus reflects reduced avoidance. (B) Effect of diazepam (0.5, 1.0, and 3.0 mg/kg) on the proportion of time spent in stretch approach while the animal is in each of three locations within the RET; chamber, tunnel, or surface area. An asterisk (*) indicates significant differences from controls (p < .05). Asterisks (**) represent p < .01 or less.
  •   FigureFigure 8.19.10 Cue and context conditioning to cat odor in the rat. (A) Contact with a block cue. (B) Duration of stretch behaviors (risk assessment). Each of these behaviors is shown during initial exposure to a cat odor block, or to a similar block without odor as control, and during subsequent tests to the context alone, or to the cue alone, presented in a different context. An asterisk (*) indicates significant cat‐odor and control group differences ( p < 0.05 or less).
  •   FigureFigure 8.19.11 Effect of midazolam (0.375 mg/kg) on (A) approach time (time spent in close proximity to collar) and (B) hide time (time spent with most or all of the body inside the hide box). Data are from the habituation session (no collar present), cat odor session (worn or unworn collar present in chamber), and context conditioning test (rats were returned to the test box on the day after cat odor). Groups received either unworn cat collar in cat odor session and no drugs (control), saline in both cat odor exposure session and test for context conditioning (SAL/SAL), midazolam in cat odor session followed by saline in context test day (MDZ/SAL), or saline in cat collar session followed by midazolam in context test (SAL/MDZ). An asterisk (*) indicates significant differences from no‐odor controls (p < 0.05 or less); # indicates significant differences from group SAL/SAL (p < 0.05).

Videos

Literature Cited

   Blanchard, R.J. and Blanchard, D.C. 1989. Anti‐predator defensive behaviors in a visible burrow system. J. Comp. Psychol. 103:70‐82.
   Blanchard, D.C., Blanchard, R.J., Tom, P., and Rodgers, R.J. 1990. Diazepam alters risk assessment in an anxiety/defense test battery. Psychopharmacology 101:511‐518.
   Blanchard, D.C., Griebel, G., and Blanchard, R.J. 2003a. The mouse defense test battery: Pharmacological and behavioral assays for anxiety and panic. Eur. J. Pharmacol. 463:97‐116.
   Blanchard, D.C., Li, C.‐I., Hubbard, D., Markham, C., Yang, M., Takahashi, L.K., and Blanchard, R.J. 2003b. Dorsal premammillary nucleus differentially modulates defensive behaviors induced by different threat stimuli. Neurosci. Lett. 345:145‐148.
   Blanchard, D.C., Markham, C., Yang, M., Hubbard, D., Madarang, E., and Blanchard, R.J. 2003c. Failure to produce conditioning with low‐dose trimethylthiazoline or cat feces as unconditioned stimuli. Behav. Neurosci. 1172:360‐368.
   Blanchard, R.J., Yang, M., Li, C.‐I., Garvacio, A., and Blanchard, D.C. 2001. Cue and context conditioning of defensive behaviors to cat odor stimuli. Neurosci. Biobehav. Rev. 26:587‐595.
   Dielenberg, R.A. and McGregor, I.S. 2001. Defensive behavior in rats towards predatory odors: A review. Neurosci. Biobehav. Rev. 25:597‐609.
   Dielenberg, R.A. and McGregor, I.S. 1999. Habituation of the hiding response to cat odor in rats Rattus norvegicus. J. Comp. Psychol. 113:376‐387.
   Dielenberg, R.A., Arnold, J.C., and McGregor, I.S. 1999. Low‐dose midazolam attenuates predatory odor avoidance in rats. Pharmacol. Biochem. Behav. 62:197‐201.
   Dielenberg, R.A., Carrive, P., and McGregor, I.S. 2001. The cardiovascular and behavioral response to cat odor in rats: Unconditioned and conditioned effects. Brain Res. 897:228‐237.
   Griebel, G., Blanchard, D.C., and Blanchard, R.J. 1996. Evidence that the behaviors in the mouse defense test battery relate to different emotional states: A factor analytic study. Physiol. Behav. 60:1255‐1260.
   Griebel, G., Blanchard, D.C., Jung, A., and Blanchard, R.J. 1995. A model of ‘antipredator’ defense in Swiss‐Webster mice: Effects of benzodiazepine receptor ligands with different intrinsic activities. Behav. Pharmacol. 6:732‐745.
   Griebel, G., Sanger, D.J., and Perrault, G. 1997. Genetic differences in the mouse defense test battery. Aggress. Behav. 23:19‐31.
   McGregor, I.S. and Dielenberg, R.A. 1999. Differential anxiolytic efficacy of a benzodiazepine on first versus second exposure to a predatory odor in rats. Psychopharmacology Berl. 147:174‐181.
   McGregor, I.S., Schrama, L., Ambermoon, P., and Dielenberg, R.A. 2002. Not all ‘predator odours’ are equal: Cat odour but not 2,4,5 trimethylthiazoline TMT; fox odour elicits specific defensive behaviors in rats. Behav. Brain Res. 129:1‐16.
   Yang, M., Augustsson, H., Markham, C.M., Hubbard, D.T., Webster, D., Wall, P.M., Blanchard, R.J., and Blanchard, D.C. 2004. The rat exposure test: A model of mouse defensive behaviors. Physiol. Behav. 8:465‐473.
Key References
   Blanchard et al., 2003a. See above.
  Provides a general review of the development of the MDTB and reviews the effects of 70+ drugs in this procedure.
   Dielenberg and McGregor, 2001. See above
  Provides a general review of cat odor tests and findings, with particular attention to the development and use of the cat odor test with hide box.
   Yang et al., 2004. See above
  Describes procedures and measures of the RET and compares effects of two inbred and two outbred mouse strains in this test.
   Blanchard et al., 2003b. See above
  Describes procedures and measures for the cat odor test and compares conditioning for cat fur/skin odor, cat feces, and TMT in this test.
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