Automated analysis of antidepressants’ effect in the forced swim test

doi:10.1016/j.jneumeth.2010.06.002

Journal of Neuroscience Methods

Volume 191, Issue 1, 15 August 2010, Pages 26-31

https://doi.org/10.1016/j.jneumeth.2010.06.002 Get rights and content

Abstract

The forced swim test (FST) is a commonly used procedure of preclinical screening of drugs for the antidepressant activity. It has high predictive validity for a large group of antidepressant drugs blocking serotonin and noradrenaline reuptakes and improvement of immobility time evaluation in the FST is an important problem of preclinical psychopharmacology. Here a new automated version of the FST was developed. This version includes 4 inventions: (1) transmitted lighting instead of reflected lighting, (2) mouse silhouette tracking, (3) automated choice of immobility threshold and (4) the permutation test of drug's effect. Experiment was carried out on adult males of C57BL/6J and BALB/cJ mouse strains. The mice were treated with tricyclic antidepressant imipramine (15 and 30 mg/kg, i.p.). Mouse was placed in water tank, its movements were recorded by a rater and the silhouette alterations were automatically tracked. The sequence of silhouette alterations was scanned for immobility bouts with a threshold algorithm. Threshold was gradually altered and the value which maximized the difference between control and treated groups was chosen. The immobility values obtained with the procedure were compared with the permutation test. The data obtained with this procedure did not differ from those obtained by the rater. Imipramine dose dependently attenuated immobility time in C57BL/6 mice without any effect on BALB/c mice. The new procedure has been implemented in the EthoStudio software. It provides an objective automated evaluation of immobility time in the FST.

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).

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Automated analysis of antidepressants’ effect in the forced swim test

Abstract

Introduction

Section snippets

Animals

Results

Discussion

Conclusion

Acknowledgements

Behav Brain Res

Neurosci Biobehav Rev

Pharmacol Biochem Behav

Pharmacol Biochem Behav

Psychoneuroendocrinology

J Neurosci Methods

Biol Psychiatry

Eur J Pharmacol

Eur J Pharmacol

J Neurosci Methods

Eur J Pharmacol

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)