An automated analysis of rat behavior in the forced swim test
Introduction
The forced swim test (FST), introduced by Porsolt in 1977 (Porsolt et al., 1977b) has been extensively used to investigate the effects of new drugs with potential antidepressant activity Borsini et al., 1991, Bourin, 1990, Healy et al., 1999, Wong et al., 2000. FST has also proven its usefulness for further characterizing in rats, neurochemical (Connor et al., 2000) or behavioral Detke et al., 1995, Redrobe et al., 1998 effects of drugs with known beneficial outcomes in the treatment of human depression (for a review, see Borsini and Meli, 1988). In addition, FST has been exploited for assessing animal models of depression induced by pharmacological manipulation (Kokkinidis et al., 1986) or breeding selection Overstreet et al., 1995, Tizabi et al., 2000 and as a model for the negative symptoms of schizophrenia Corbett et al., 1999, Noda et al., 2000.
FST consists of placing a rat into a tank filled with water. The procedure is generally divided into a preexposure session (pretest) lasting 15 min, followed 24 h later by a 5-min test session. Following an initial period of vigorous struggling, the animal adopts a typical posture, performing only those movements necessary to keep its head above water (i.e., floating). The total amount of time the animal demonstrates this behavior is then measured. The pretest session induces in naı̈ve rats a prolonged floating time (FT) during the test session. This prolonged FT during the second exposure to the tank has been interpreted by Porsolt as reflecting the animal's state of despair Porsolt et al., 1977b, Porsolt et al., 1978a, elicited by the inescapable nature of the tank, that was learned during pretest. Whilst this learned helplessness/despair interpretation has been questioned Borsini et al., 1986, De Pablo et al., 1989, Nishimura et al., 1988, increased FT during the test session has been repeatedly and consistently reduced by a large variety of drugs known for their efficacy in successfully treating human depression. This has led to the use of FT measurement for assessing the efficacy of antidepressant agents in the FST.
Because the measurement of FT is performed visually by human observers, it requires training and observer objectivity. Even when the observer is trained and objective, there still remains a problem of behavioral definition: FT is also referred to as immobility, but to remain afloat the animal must make slight movements and is therefore not strictly immobile. In addition, the measurement of FT is time consuming and is not readily compatible with experiments requiring large groups of animals and the testing of many drugs. In an attempt to overcome these limitations, two studies have reported automated measurement of mobility (i.e., the inverse of FT) in the FST. Shimazoe et al. (1987) used tremor sensors surrounding the tank to record water vibrations during rat swimming, and De Pablo et al. (1989) measured the variations in the frequency of the natural electromagnetic field of water induced by rat movements. These methods, although appropriate for the FST, require complex and dedicated equipment and one such set-up per animal. Here, we introduce a new automated method for measuring the rat's behavior in the FST. This is based on the determination of the rat's distance moved (DM) within the tank. We reasoned that if we could demonstrate that DM was closely correlated with FT, then DM could be used as the dependent measure in the FST. This would confer several advantages. The method is rapid and objective, and extends the use of the existing Noldus Ethovision Software to the FST. In addition to the gain in objectivity and rapidity, a further advantage of this method is that it uses a software that is already widely employed in laboratories. Indeed, the same software can be used in tasks such as the open field and therefore to identify compounds that induce false positive results in FST due to psychomotor effects Plaznik et al., 1985, Porsolt et al., 1977b, Tizabi et al., 1999, West et al., 1999. In order to test its validity, this new application of Noldus software was investigated and compared against FT in different experiments to measure (1) the hypothesized depressive-like state of rats during the withdrawal from repeated amphetamine treatment and (2) the known antidepressant-like activity of fluoxetine and desipramine.
Withdrawal of psychostimulants (cocaine or amphetamine) following their repeated intermittent administration has been shown to induce depressive-like symptoms in humans Jittiwutikan et al., 1997, Kosten et al., 1998, Schildkraut et al., 1971, Watson et al., 1972. In addition, several lines of evidence suggest that withdrawal from repeated amphetamine treatment in rodents induces an anhedonia- or more generally, a disphoria-like state as well as other symptoms resembling human depression. The former includes reduction of intracranial self-stimulation Borowski and Kokkinidis, 1992, Paterson et al., 2000, Wise and Munn, 1995; the latter include nocturnal hypoactivity Paulson et al., 1991, Paulson and Robinson, 1996, lower locomotor activity in a novel environment (Persico et al., 1995), and impaired sexual behavior (Barr et al., 1999). Furthermore, Kokkinidis et al. (1986) have reported increased duration of immobility in the FST in mice chronically treated with amphetamine. However, these behavioral effects were observed in animals receiving large doses of amphetamine, ranging from 1 to 12 mg/kg either via chronic repeated injections or via continuous delivery from subcutaneous osmotic minipumps (Paterson et al., 2000).
The first set of experiments of the present study aimed at investigating the effects of the withdrawal period from repeated injections of doses of amphetamine or cocaine that have been shown to produce behavioral sensitization (Hedou et al., 2001) on FT and DM in the FST. In order to assess the feasibility of administering sensitization-inducing treatments between pretest and test, we first had to validate the procedure of using a prolonged delay of 7 days between pretest and test as compared with the 24-h delay of the original description of the procedure (Experiment 1). Behavioral sensitization was induced by repeated, intermittent injections of 1.5 mg/kg amphetamine or 20 mg/kg cocaine for 5 days, and rat behavior in the FST was then measured following a 48-h withdrawal period (Experiment 2). In Experiment 3, DM and FT in the FST were measured following treatment with repeated injections of the classical antidepressant desipramine and the selective re-uptake inhibitor (SSRI) fluoxetine. These antidepressants are known for their efficacy in decreasing FT in FST Detke et al., 1995, Page et al., 1999 as well as in alleviating depressive symptoms in humans (Schatzberg, 2000).
Section snippets
Subjects
Male Wistar [Zur:WIST(HanIbm)] and Sprague–Dawley [Zur:SD(Crl:CD (SD)BR)] rats (ETH Research Unit, Schwerzenbach, Switzerland) weighing 250–300 g were group housed in a temperature- (21±1.0°C) and humidity- (55±5%) controlled room. They had free access to food (Nafag, 9431, Nafag Ecossan, Gossau, Switzerland) and water and were kept on a reversed 12-h light/dark cycle (lights on at 7:00 p.m.). Daily care was provided to the animals by in-house animal technicians. This included changing and
Experiment 1: Comparison of the effects of a 24-h vs. 7-day delay between pretest and test sessions on FT and DM in naı̈ve rats
In this experiment, either 24 h or 7 days separated pretest and test sessions. In both conditions, FT and DM were measured and compared (Fig. 1).
Discussion
The present study introduces a new automated method for the measurement of rat behavior in the FST. This measurement is based on the DM by the rat in the swim tank calculated by the Noldus Ethovision software instead of the usual visual assessment of rat FT. The usefulness of our method was demonstrated in three experiments. In all these experiments, DM was negatively correlated with the established measure of FT. The first experiment showed that the increase in FT during the test session was
Acknowledgements
This study was performed in partial fulfillment of the requirements for the PhD degree of Gaël Hédou from the ETH Zurich and was supported by the Swiss National Science foundation and the Swiss Federal Institute of Technology Zurich. The authors want to thank the Animal Facility team for their care of the animals.
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