Automated analysis of antidepressants’ effect in the forced swim test
Introduction
The forced swim test (FST) is a commonly used procedure of preclinical screening of drugs for the antidepressant activity (Borsini and Meli, 1988, Willner, 1990, Willner and Mitchell, 2002, Lucki, 1997, Tecott, 2003, Cryan and Mombereau, 2004, Yacoubi and Vaugeois, 2007). The test consists in placing rat or mouse in a vessel filled with water from which there is no escape. After a short period of escape-oriented behavior (swimming across the water surface, climbing at the vessel walls) the animal shows bouts of relative immobility and passive floating (Porsolt et al., 1977). The most of clinically effective antidepressants decrease the immobility time (Porsolt et al., 1977, Porsolt et al., 1978, Borsini and Meli, 1988, Willner, 1990, Willner and Mitchell, 2002). There are two protocols of the FST in mice. In the first and commonly used (long) protocol immobility is recorded for 240 s after a 120 s adaptation period (Crowley et al., 2004). In the second (short) protocol, immobility time is recorded for 180 s after a 40 s adaptation period (Nikulina et al., 1991).
In spite of apparent simplicity, the FST bears the main unresolved problem concerning the determination of immobility. Various observers define immobility as a state of passive floating when animal's movements are reduced down to required to keep the head above the water (Porsolt et al., 1977). However this definition is arbitrary and cannot be interpreted unambiguous by different observers. So, creation of an objective and automatic procedure for immobility time evaluation is an important problem of preclinical psychopharmacology.
The first attempt to automate the immobility registration was made by Nomura et al. (1982) who put a small water-wheel in the water tank. A mouse trying to escape runs the wheel and the number of runs serves as a negative measure of immobility. A good negative correlation between immobility time and the number of the wheel runs was found (Nikulina et al., 1991). However some animals rapidly learned to escape from water without running the wheel.
Digital video is more progressive and universal technique of behavior registration (Noldus et al., 2001). Crowley et al. (2004) applied the Smart video tracking system to automation of immobility time measurement in the FST. They used a threshold algorithm when movements with horizontal velocity lower than 2 cm/s were qualified as immobility. However, a low horizontal velocity is not an adequate measure of immobility, since it does not include climbing behavior. Other authors used the VideoTrack Quantization software and animal activity was classified according to a threshold algorithm by the following states: immobility (<450), swimming (450–750) and struggling (>750) (Mason et al., 2009). However, the authors did not present any biological or statistical grounds for the threshold choice.
The aim of the study was further development of threshold algorithm approach including hardware- and software-based ameliorations of the immobility registration and development of objective procedure of threshold choice. Here we compared three methods of the immobility registration: visual by a rater and automated by the alteration of animal's horizontal velocity and silhouette. The silhouette alteration includes both horizontal swimming and climbing behavior. The main task of the study was a statistical analysis of the structure of the forced swim behavior and development of an objective automatic procedure of threshold choice. The created procedure of immobility registration was then validated using tricyclic antidepressant imipramine and comparing with visual registration. We also intended to compare two protocols (long and short) of the FST. At last we developed new hardware and software ameliorating the registration of immobility time in the FST.
Section snippets
Animals
Experiments were carried out on adult males of two highly inbred mouse strains: C57BL/6J (n = 22) and BALB/cJ (n = 21) which are differed in the fur color (black and white, respectively) and frequently used in preclinical study of antidepressant. The strains are maintained for more than 50 generations with close inbreeding. All animals were 8–12 weeks of aged and weighing 25 ± 2 g. After weaning males were kept in groups of 5–8 in cage of 40 cm × 30 cm × 20 cm. Two to three days prior to the experiment
Results
The time intervals of immobility bouts calculated with the ImmobilityTest software were in a good agreement with the dynamics of mouse silhouette and velocity alterations (Fig. 1).
Discussion
Being introduced by Porsolt et al. (1977) the FST is the most commonly used test for antidepressant activity (Borsini and Meli, 1988, Willner, 1990, Willner and Mitchell, 2002, Lucki, 1997, Tecott, 2003, Cryan and Mombereau, 2004, Yacoubi and Vaugeois, 2007). Although the FST has low face and construct validities its predictive validity for a large group of clinically effective antidepressant drugs is high (Willner, 1990, Willner and Mitchell, 2002). From time to time the main idea of the FST
Conclusion
Four new modification of the FST, namely the inverted lighting, silhouette alteration method for the evaluation of animal movement, automated choice of the threshold and the permutation test for drug's effect were invented. The inverted lighting provides the maximal contrast between animal and background and completely eliminates the noise produced with glares of light reflecting from water surface. Silhouette contains more information on animal's movement and its alteration is more sensitive
Acknowledgements
This work was supported by the interdisciplinary integration project of Siberian Branch of Russian Academy of Sciences (grant no. 18), programs “Fundamental Science to Medicine” (grant no. 21.1) and “Molecular and Cellular Biology” of the Presidium of Russian Academy of Sciences (grant no. 22.9).
References (31)
- et al.
Increased water temperature renders single-housed C57BL/6J mice susceptible to antidepressant treatment in the forced swim test
Behav Brain Res
(2008) Role of the serotonergic system in the forced swimming test
Neurosci Biobehav Rev
(1995)- et al.
Induction of hyperlocomotion in mice exposed to a novel environment by inhibition of serotonin reuptake. A pharmacological characterization of diverse classes of antidepressant agents
Pharmacol Biochem Behav
(2002) - et al.
Automated tests for measuring the effects of antidepressants in mice
Pharmacol Biochem Behav
(2004) - et al.
Lipopolysaccharide induces delayed FosB/DeltaFosB immunostaining within the mouse extended amygdala, hippocampus and hypothalamus, that parallel the expression of depressive-like behavior
Psychoneuroendocrinology
(2007) - et al.
Automated measurement of special preference in the open field test with transmitted lighting
J Neurosci Methods
(2008) - et al.
Genetic differences in the tail-suspension test and its relationship to imipramine response in 11 inbred strains of mice
Biol Psychiatry
(2001) - et al.
Differential sensitivity to SSRI and tricyclic antidepressants in juvenile and adult mice of three strains
Eur J Pharmacol
(2009) - et al.
A new behavioral test for antidepressant drugs
Eur J Pharmacol
(1982) - et al.
Use of a platform in an automated open-field to enhance assessment of anxiety-like behaviors in mice
J Neurosci Methods
(2007)
Behavioral dispair in rats: a new model sensitive to antidepressant treatments
Eur J Pharmacol
Animal models of depression: an overview
Pharmacol Ther
Molecular physiology of norepinephrine and serotonin transporters
J Exp Biol
Is the forced swimming test a suitable model for revealing antidepressant activity?
Psychopharmacology
Strain-dependent antidepressant-like effects of citalopram on the mouse tail suspension test
Psychopharmacology (Berlin)
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