Application of Experimental Stressors in Laboratory Rodents

Stephen C. Heinrichs1, George F. Koob2

1 Boston College, Chestnut Hill, Massachusetts, 2 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 8.4
DOI:  10.1002/0471142301.ns0804s34
Online Posting Date:  February, 2006
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This unit presents eight separate stressor protocols for laboratory rodents. Stress induction is a critical element in the study of neural and neuroendocrine mechanisms involved in establishing and maintaining a state of stress. The first four procedures, immobilization, footshock, swimming, and noise, involve acute exposure to noxious stimuli. The next three procedures, social isolation, resident/intruder aggression, and maternal deprivation, induce social disruption by withdrawal from a group housing condition, attack within the unfamiliar territory of a dominant male, or segregation of a preweanling pup from its mother, respectively. The final procedure, sleep deprivation, involves passive denial of the opportunity to sleep. Support protocols are provided to address the need for environmental acclimation and calming procedures prior to any stressā€related studies (including, for rats, handling of the animals as a calming measure) and to detail a simple method of quantifying the response to a given stressor by direct measurement of levels of the stress hormones adrenocorticotropic hormone (ACTH) and corticosterone.

Keywords: stress; rat; mouse; ACTH; corticosterone; anxiety

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Restraint Stressor
  • Basic Protocol 2: Electric Footshock Stressor
  • Basic Protocol 3: Swim Stressor
  • Basic Protocol 4: Noise Stressor
  • Basic Protocol 5: Social Isolation Stressor
  • Basic Protocol 6: Resident/Intruder Stressor
  • Basic Protocol 7: Maternal Separation Stressor
  • Basic Protocol 8: Sleep Deprivation Stressor
  • Support Protocol 1: Colony Maintenance
  • Support Protocol 2: Rat Handling
  • Support Protocol 3: ACTH/Corticosterone Determinations
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Restraint Stressor

  • Naive experimental animal (rat or mouse)
  • Hemicylindrical acrylic restraint tube (Braintree Scientific), 4.5‐cm diameter, 12 cm long for a 150‐ to 300‐g rat, or 3‐cm diameter, 8 cm long for a 10‐ to 40‐g mouse
  • Absorbent pad
  • Timer

Basic Protocol 2: Electric Footshock Stressor

  • Experimental animal (rat or mouse)
  • 70% ethanol
  • Multimeter set to read milliamperes (mA)
  • Shock generator (Coulbourn Instruments)
  • Covered enclosure with electrified grid floor (Coulbourn Instruments)
  • Holding cage

Basic Protocol 3: Swim Stressor

  • Experimental animal (rat or mouse)
  • Chlorinated tap water containing 2 mg/liter granular chlorinating concentrate (available from local pool and spa store), maintained at 22° ± 1°C
  • Cylindrical water pool (Nalgene): plastic container 45 cm tall × 30 cm in diameter for a rat or 25 cm tall × 15 cm in diameter for a mouse
  • Thermometer
  • Stopwatch
  • Bath‐sized terry cloth towels

Basic Protocol 4: Noise Stressor

  • Experimental animal (rat or mouse)
  • Noise delivery and startle reactivity measurement system (e.g., SR‐LAB; San Diego Instruments) or
  • Sound generation and amplification equipment (e.g., pure tone/white noise generators and loudspeakers from a music store; personal computer with a sound card and powered speakers) and a decibel meter
  • Acoustically isolated chamber or room
  • Ear protection for human investigator

Basic Protocol 5: Social Isolation Stressor

  • Experimental animals: same‐sex rats or mice from 21 days (weaning) to 3 months of age
  • Opaque polyethylene group rodent cages (Nalgene), 40 × 30 × 10 cm for mice or 50 × 40 × 20 cm for rats
  • Opaque polyethylene single rodent cages (Nalgene), 30 × 15 × 10 cm for mice or 40 × 20 × 20 cm for rats
NOTE: An in‐house breeding colony is the preferred source for juveniles in order to avoid potential maternal stress effects on normal development of offspring provided by a commercial breeder.

Basic Protocol 6: Resident/Intruder Stressor

  • Experimental animals: several mixed‐gender pairs of male and female Long‐Evans rats; or Swiss‐Webster male mice
  • Resident cage, 72 × 52 × 35 cm for rats or 30 × 20 × 15 cm for mice
  • Wooden hutch (for rats only): two 30 × 15‐cm pieces of plywood joined at right angles
  • Intruder protective enclosure made of Plexiglas/wire mesh, 19 × 20 × 17.5 cm for rats or 10 × 15 × 10 cm for mice

Basic Protocol 7: Maternal Separation Stressor

  • Experimental animals: pregnant in‐house dams of the desired strain of rat or mouse
  • Rodent group caging with bedding, preferably wood shavings
  • Electric heating pad

Basic Protocol 8: Sleep Deprivation Stressor

  • Tap water containing 2 mg/liter granular chlorinating concentrate (available from local pool/spa store), room temperature
  • Experimental animal (mouse or rat)
  • Water pool, with pool floor extending >12.5 cm in all directions from the central point (e.g., a standard polycarbonate rodent housing cage)
  • Immersible cylindrical pedestals (e.g., inverted glass beakers), small (2 cm‐diameter for mice or 6.5 cm‐diameter for rats) and large (6‐cm diameter for mice or 16‐cm diameter for rats

Support Protocol 1: Colony Maintenance

  • Rats, adult
  • Laboratory coat
  • Indelible marking pen
  • Portable scale
  • Flat surface

Support Protocol 2: Rat Handling

  • Experimental animals (mice or rats)
  • 50 mg/ml EDTA (ethylenediaminetetraacetic acid, tetrasodium salt)
  • 5000 kallikrein inhibitor units (KIU)/ml aprotinin
  • ACTH immunometric assay kit (Nichols Institute Diagnostics;
  • Corticosterone radioimmunoassay kit (MP Biomedicals;
  • Indwelling vascular catheter apparatus (see step annotation)
  • 12 × 75‐mm centrifuge tubes, ice cold
  • Tabletop centrifuge
  • Scintillation counter
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Literature Cited

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   Balcombe, J.P., Barnard, N.D., and Sandusky, C. 2004. Laboratory routines cause animal stress. Contemp. Top. Lab. Anim. Sci. 43:42‐51.
   Bale, T.L., Picetti, R., Contarino, A., Koob, G.F., Vale, W.W., and Lee, K.F. 2002. Mice deficient for both corticotropin‐releasing factor receptor 1 (CRFR1) and CRFR2 have an impaired stress response and display sexually dichotomous anxiety‐like behavior. J. Neurosci. 22:193‐199.
   Berglund, B., Lindvall, T. and Schwela, D.H. (eds.) 1999. Guidelines for Community Noise. World Health Organization, Geneva.
   Britton, K.T., McLeod, S., Koob, G.F., and Hauger, R. 1992. Pregnane steroid alphaxalone attenuates anxiogenic behavioral effects of corticotropin releasing factor and stress. Pharmacol. Biochem. Behav. 41:399‐403.
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   Heinrichs, S.C. and Koob, G.F. 2004. Corticotropin‐releasing factor in brain: A role in activation, arousal and affect regulation. J. Pharm. Exp. Ther. 311:427‐440.
   Heinrichs, S.C. and Seyfried, T.N. 2005. Behavioral seizure correlates in animal models of epilepsy: A roadmap for assay selection, data interpretation and the search for causal mechanisms. Epilepsy Behav. In press.
   Heinrichs, S.C., Menzaghi, F., Pich, E.M., Badwin, H.A., Russwick, S., Britton, K.T., and Koob, G.F. 1994. Anti‐stress action of a corticotropin‐releasing factor antagonist on behavioral reactivity to stressors of varying type and intensity. Neuropsychopharmacology 11:179‐186.
   Institute for Laboratory Animal Research. 2003. Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. The National Academies Press, Washington, DC.
   Levine, S. and Wiener, S.G. 1988. Psychoendocrine aspects of mother‐infant relationships in nonhuman primates. Psychoneuroendocrinology 13:143‐154.
   Mason, J.W. 1971. A re‐evaluation of the concept of “non‐specificity” in stress theory. J. Psychiatr. Res. 8:323‐333.
   Michaud, D.S., McLean, J., Keith, S.E., Ferrarotto, C., Hayley, S., Khan, S.A., Anisman, H., and Merali, Z. 2003. Differential impact of audiogenic stressors on Lewis and Fischer rats: Behavioral, neurochemical and endocrine variations. Neuropsychopharmacology 28:1068‐1081.
   Miczek, K.A. 1979. A new test for aggression in rats without aversive stimulation: Differential effects of D‐amphetamine and cocaine. Psychopharmacology 60:253‐259.
   Morrison, A.R., Evans, H.L., Ator, N.A., and Nakamura, R.K. (eds.) 2002. Methods and Welfare Considerations in Behavioral Research with Animals: Report of a National Institutes of Health Workshop. NIH publication no. 02‐5083. U.S. Government Printing Office, Washington, DC.
   Natelson, B.H., Tapp, W.M., Adamus, J.E., Mittler, J.C., and Levin, B.E. 1981. Humoral indices of stress in rats. Physiol. Behav. 26:1049‐1054.
   Pitman, D.L., Ottenweller, J.E., and Natelson, B.H. 1988. Plasma corticosterone levels during repeated presentation of two intensities of restraint stress: Chronic stress and habituation. Physiol. Behav. 43:47‐55.
   Pryce, C.R., Ruedi‐Bettschen, D., Dettling, A.C., Feldon, J. 2005. Early‐life environmental manipulations in rodents and primates: Potential animal models in depression research. In Handbook of Stress and the Brain, Part 2. (T. Steckler, N.H. Kalin, and J.M.H.N. Reul, eds.) pp. 23‐50. Elsevier, Amsterdam.
   Rechtschaffen, A., Bergmann, B.M., Everson, C.A., Kushida, C.A., and Gilliland, M.A. 1989. Sleep deprivation in the rat: X. Integration and discussion of the findings. Sleep 12:68‐87.
   Selye, H. 1976. The Stress of Life. McGraw‐Hill, New York.
   Vetter, D.E., Li, C., Zhao, L., Contarino, A., Liberman, M.C., Smith, G.W., Marchuk, Y., Koob, G.F., Heinemann, S.F., Vale, W., and Lee, K.F. 2002. Urocortin‐deficient mice show hearing impairment and increased anxiety‐like behavior. Nat. Genet. 31:363‐369.
   Valzelli, L. 1973. The “isolation syndrome” in mice. Psychopharmacologia 31:305‐320.
Key References
   Balcombe et al., 2004. See above.
  This meta‐analysis of available literature supports the thesis that significant increases in valid indices of stress are predictable consequences of routine laboratory procedures.
   Selye, 1976. See above.
  The seminal text written by the father of stress biology; part autobiography, part scientific discourse, and thoroughly entertaining and informative.
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