Protocol for Tail Suspension Test (TST)
Materials Required
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Principle
The Tail Suspension Test is a mouse behavioral assay in which an animal is suspended by the tail in an inescapable position, causing alternating escape-directed activity and immobility; the main readout is immobility time, and antidepressant-like treatments generally reduce immobility compared with vehicle controls[1][2][3][4].
The assay detects behavioral response to acute inescapable stress rather than a molecular event; immobility is interpreted as passive stress-coping behavior, while reduced immobility is used as a predictive screen for antidepressant-like activity, with important limitations related to strain, locomotor activity, and tail-climbing behavior[2][3][6][7][8][9].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Vehicle solution is used as the negative treatment control when drug or compound effects are tested[1][2][4].
• A known antidepressant drug may be used as a positive control because the assay was developed and validated as a screen for antidepressant-like drug effects[1][3][4].
• Mice are the standard experimental animals for TST; strain must be selected and reported because immobility and antidepressant response differ strongly among mouse strains[2][3][6][7][8][9].
• A known antidepressant drug may be used as a positive control because the assay was developed and validated as a screen for antidepressant-like drug effects[1][3][4].
• Mice are the standard experimental animals for TST; strain must be selected and reported because immobility and antidepressant response differ strongly among mouse strains[2][3][6][7][8][9].
Equipment and instruments
• Required equipment includes a suspension bar or hook, adhesive tape for tail attachment, an enclosure or testing box that prevents escape and visual interference, timer, video camera or automated activity recording system, and scoring software or blinded manual scoring method[2][4][5].Controls
• Use vehicle-treated controls and, when testing antidepressant-like effects, a known antidepressant positive control; include locomotor control testing when reduced immobility could reflect nonspecific motor stimulation[1][2][3][4].Experimental Procedure
Preparation Steps
• Acclimate mice to the testing room and keep lighting, noise, handling, testing order, and time of day consistent across groups; randomize animals and blind the scorer to treatment group[2][3][4].• Prepare the suspension setup so that each mouse is suspended by the tail with adhesive tape and cannot touch nearby surfaces or climb to the suspension bar[2][6].
• For strains prone to tail climbing, especially C57BL/6 mice, use an anti-climbing tail device or exclude animals that climb the tail, because tail climbing prevents valid immobility scoring[2][6].
Operation Steps
• Attach adhesive tape securely to the distal tail and suspend the mouse from the bar or hook so that the animal hangs freely without contacting walls or the floor[1][2][4].• Record the session immediately after suspension; the commonly reported TST duration is 6 min, although some studies score defined intervals within the session[1][2][4].
• Score immobility as the time during which the mouse hangs passively without active escape-directed movement; active behaviors may include struggling, swinging, curling, or other escape-directed movements[1][2][5][10].
• After the session, remove the mouse from the tape carefully and return it to the home cage or recovery cage according to approved animal-care procedures[2][4]• Data Acquisition and Analysis
The primary outcome is total immobility time during the defined scoring period; reduced immobility relative to vehicle control is commonly interpreted as antidepressant-like activity, but interpretation should consider locomotor effects, strain background, tail climbing, and other behavioral assays[1][2][3][4][6][7][8][9].
• Manual scoring should be performed from video by a blinded observer, or automated systems may be used when validated against manual scoring; the same immobility definition and scoring threshold must be applied to all groups[2][5].
• Report mouse strain, sex, age, body weight, treatment, timing of treatment, session duration, apparatus design, height or suspension configuration, use of anti-climb devices, exclusion criteria, scorer blinding, and statistical test[2][3][4][6].
Troubleshooting
Problem: Mouse climbs its tail.
Possible Cause: Some strains, especially C57BL/6 mice, show tail-climbing behavior that invalidates immobility measurement.Literature-supported Solution: Use an anti-climbing device or exclude animals that climb, and report this criterion[2][6].
Problem: Reduced immobility may not reflect antidepressant-like activity.
Possible Cause: Psychostimulant or motor-activating effects can reduce immobility without a true antidepressant-like mechanism.Literature-supported Solution: Include locomotor activity controls and interpret TST alongside complementary behavioral assays[1][2][3][4].
Problem: Results vary strongly between strains.
Possible Cause: Baseline immobility and drug responsiveness differ across mouse genetic backgrounds.Literature-supported Solution: Use a consistent strain, report strain explicitly, and avoid comparing results across strains without appropriate controls[6][7][8][9].
Problem: Immobility scoring is inconsistent.
Possible Cause: Manual scoring depends on how immobility and active movements are defined.Literature-supported Solution: Use blinded video scoring with predefined criteria or a validated automated recording system[2][5][10].
Verweise:
- [1]. Steru L, et al. The tail suspension test: a new method for screening antidepressants in mice. Psychopharmacology (Berl). 1985;85(3):367-370. [Content Brief]
- [2]. Can A, et al. The tail suspension test. J Vis Exp. 2012;(59):e3769. [Content Brief]
- [3]. Cryan JF, et al. The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. Neurosci Biobehav Rev. 2005;29(4-5):571-625. [Content Brief]
- [4]. Castagné V, et al. Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice. Curr Protoc Pharmacol. 2011;Chapter 5:Unit 5.8. [Content Brief]
- [5]. Steru L, et al. The automated tail suspension test: a computerized device which differentiates psychotropic drugs. Prog Neuropsychopharmacol Biol Psychiatry. 1987;11(6):659-671. [Content Brief]
- [6]. Mayorga AJ, et al. Limitations on the use of the C57BL/6 mouse in the tail suspension test. Psychopharmacology (Berl). 2001;155(1):110-112. [Content Brief]
- [7]. van der Heyden JA, et al. Strain differences in response to drugs in the tail suspension test for antidepressant activity. Psychopharmacology (Berl). 1987;92(1):127-130. [Content Brief]
- [8]. Trullas R, et al. Genetic differences in a tail suspension test for evaluating antidepressant activity. Psychopharmacology (Berl). 1989;99(2):287-288. [Content Brief]
- [9]. Vaugeois JM, et al. Individual differences in response to imipramine in the mouse tail suspension test. Psychopharmacology (Berl). 1997;134(4):387-391. [Content Brief]
- [10]. Berrocoso E, et al. Active behaviours produced by antidepressants and opioids in the mouse tail suspension test. Int J Neuropsychopharmacol. 2013;16(1):151-162. [Content Brief]
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