- Email: [email protected]
Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate
European Journal of Pharmacology, 158 (1988) 207-212
207
Elsevier EJP 50580
Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate Antonio Armario *, Amadeu Gavaldh and Octavi Marti Departamento de Biologla Celular y Fisiologla, Facultad de Ciencias, Universidad A utbnoma de Barcelona, 08143 Bellaterra, Barcelona, Spain
Received 14 September 1988, revised MS received 30 September 1988, accepted 11 October 1988
The effect of desipramine administration and the duration of the daily exposure to forced swimming on some variables has been studied in adult male rats. Desipramine administration (15 mg/kg) significantly increased struggling behavior in the first and second 5-min periods of a single exposure to forced swimming. Swimming was reduced in the first 5 min and remained unchanged thereafter. Immobility was decreased in the second and the third 5-min periods. After a pre-exposure to forced swimming for 15 rain the day before, the drug was effective in increasing struggling behavior and reducing immobility during a subsequent 5-min test. Swimming was not modified. Daily exposure to forced swimming for 3 days caused a decline in struggling behavior and swimming, while increasing immobility and the defecation rate. The duration of daily exposure to forced swimming did not alter the changes in the variables measured. The present results indicate that a one-day test can be used to discriminate between saline- and desipramine-treated rats, and that struggling behavior could be a reliable measure of the positive action of antidepressants. The finding that behavioral changes over the 3 days were independent of the duration of exposure to swimming argues against the interpretation of the results which suggest that the responses are caused by the appearance of a behavioral despair state, and suggests that these behaviors might be trait-markers in the rat. In addition, the changes in struggling behavior and immobility over the 3 days cannot be attributed to a behavioral adaptation to the test because the defecation rate increased rather than decreased during successive forced swimming tests. Forced swimming test; Struggling behavior; Defecation; Immobility; Antidepressants
1. Introduction Considerable effort has been taken to establish animal models for the screening of antidepressant drugs. Of these models, the forced swimming test developed by Porsolt et al. (1977b; 1978) has gained considerable acceptance. When placed in a cylinder containing water (25°C; up to 15 cm deep) for 15 rain, rats initially display vigorous activity, apparently attempting to escape from the
* To whom all correspondence should be addressed.
tank. After a few minutes the animals become progressively immobile and float passively in the water, making only minor movements to maintain the head out of the water. The following day the rats are placed in the same tank for 5 min, and the time the rats remain immobile is measured. It has been reported that most antidepressant treatments (many drugs, R E M sleep deprivation, electroconvulsive shock) reduce immobility independently of changes in general motor activity, as usually assessed by open-field ambulation (Porsolt et al., 1977b; 1978; Porsolt, 1981). In addition, we have demonstrated recently that chronic exposure
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
208 to shock and a combination of various stressors applied in a random schedule increases immobility in Porsolt's test (Garcia-Mfirquez and Armario, 1987a), an effect that is prevented by the concomitant administration of clomipramine (GarciaMfirquez and Armario, 1987b). Since some forms of chronic stress are considered to be good animal models of depression (Willner, 1984), these data suggest that results from the forced swimming test could be related to the mood state of the animals. However, there are several theoretical and practical problems (Willner, 1984; Jesberger and Richardson, 1985). We wish to mention some of them here. First, the consideration of what is or is not immobility is subjective. Active rats usually show two apparently different behaviors (Weiss et al., 1981): (a) intense movements of the four limbs with the two front paws breaking the surface of the water or against the walls of the tank; (b) mild swimming, with movement of the four limbs or the hindlimbs. Whether or not the latter behavior should be categorized as activity is a matter of controversy and could, at least partially, explain the highly variable values, ranging from 50 to 250 s, for immobility reported in the literature (O'Neill and Valentino, 1982; Koyuncuoglu et al., 1982; Garcia-Mfirquez and Armario, 1987a). Second, it is not clear from the available data why only one swimming session is used for the screening of antidepressant drugs in mice (Porsolt et al., 1977a) and two sessions (see above) are used in rats. Third, Porsolt (1981) hypothesized that the increased immobility observed on the second exposure to water was because the rats knew, from previous experience, that escape was impossible. Therefore they used the term 'behavioural despair' to describe the cognitive state of the animals caused by this experimental situation. However, the possibility of escape from the tank on the first day did not alter the duration of immobility on the second day (O'Neill and Valentino, 1982). In addition, placing the rats in an empty tank on the first day increased immobility on the second day to the same extent as when the rats were placed in a tank containing water (Borsini et al., 1986). The present study was undertaken to thoroughly characterize the forced swimming test, and addressed some of the above-mentioned problems.
2. Materials and methods
Male Sprague-Dawley rats were used. They were maintained under standard conditions (lights on from 07:00 to 19:00 h, temperature 22°C) in groups of three to four per cage for at least 1 week before the start of the experiments. Food and water were provided ad libitum. The rats were placed (in the morning) in a cylindric tank containing up to 15 cm of water (25 ° C). The time spent making the two following behaviors was measured with a stop-watch: (a) 'struggling', which occurred when the rats were moving all four limbs with the two front limbs breaking the surface of the water or touching the walls; (b) 'immobility', which occurred when the animals remained floating with all limbs motionless. The rats spent the remainder of the time swimming and this behavior was categorized as 'swimming'. The defecation rate was measured as the number of boluses eliminated by each animal during the first 5 rain of exposure to the tank. Rats approximately 45-day-old were used in the first experiment. They were assigned to saline or desipramine ( D M I ) groups and were injected i.p. with saline or D M I (15 m g / k g ) 24 and 1 h before the test. D M I hydrochloride, which was kindly donated by Ciba Geigy, was used. The dose of D M I is expressed in terms of the salt. The tests were carried out following either the protocol described by Porsolt et al. (1977b), which consists of a 15-rain swimming session before drug administration and a second, 5-min swimming session during which the behavior was recorded, or one in which there was only one 15-min swimming session, the behavior was recorded every 5 min. Approximately 50-day-old rats were used in the second experiment. The rats were assigned to two groups. One in which the rats were placed in the water tank for 5 min on 3 consecutive days. In the other group the rats were placed in the water tank for 15 min on 3 consecutive days. In the latter case the behavior was recorded every 5 rain. This experiment was repeated with approximately 60day-old rats (experiment 3). The statistical significance of the results was analyzed with Student's t-test or Mann-Whitney's U-test when the samples did not meet the require-
209
!
TABLE 1
200
Effect of the duration of the forced swimming test on the defecation rate during the first 5 min of each daily test. Means and S.E.M. (n = 7) are presented. The A N O V A revealed that the number of forced swimming tests had a significant effect (P < 0.05) but that of the duration (5 vs. 15 rain) of the daily tests did not.
.lit.
150 10C
5~n 15min
n,, S
SW
Fig. 1. Effect of D M I administration (15 m g / k g ) on the duration (s) of struggling behavior (S), swimming (SW) and immobility (I) in a 5-min exposure to the forced swimming test. The animals were exposed to a pre-test session of forced swimming for 15 min. Means and S.E.M. (n = 7) are presented. Open bars indicate saline-treated rats and closed bars DMItreated rats. ** P < 0.01 vs. saline-treated rats.
ments for Student's t-test (unequal variances, non-normal distribution). In other cases, the twoway ANOVA with repeated measures for one factor (within and between days measures) was used.
3. Results The administration of D M I to rats, tested for 5 min after a previous 15-min test the day before,
150I
/
SWIM
]
4.9±0.9 4.4±0.5
IMMOBILITY
225
'°°1" ~hI**
5
5 0 ~ 7 5 10
Thirdday
3.4±0.6 3.3±0.5
300
**
1o
5
Secondday
2.6±0.8 3.1±0.8
significantly increased struggling behavior (P < 0.01) and reduced immobility (P < 0.01). Swimming was not modified by the drug (fig. 1). Figure 2 depicts the duration of the struggling behavior, swimming and immobility of rats treated with saline or DMI during the first 15 min of forced swimming. The struggling behavior was significantly increased by DMI during the first and the second 5-min periods in the water tank (in both cases P < 0 . 0 1 vs. saline-treated group). Swimming was significantly reduced (P < 0.01) by the drug during the first 5 min of the test only. The duration of immobility was not significantly reduced by DMI during the first 5 rain, but was during the second and third periods (P < 0.01 vs. saline-treated rats). Figure 3 shows the influence of the duration of the swimming test on struggling behavior, swimming and immobility. The ANOVA showed that
I
]'el*STRUGGLING
Firstday
15
5
150
10
15
n,/111 5
10
15
Fig. 2. Effect of DMI treatment (15 m g / k g ) on the duration (s) of struggling behavior, swimming and immobility in a 15-min exposure to forced swimming. The rats did not have a pre-test swimming session. Means and S.E.M. (n = 14-15) are presented. Open bars indicate saline-treated rats and closed bars DMI-treated rats. N u m b e r s under bars indicate the successive 5-min periods of the test. ** P < 0.01 vs. saline-treated rats.
210
STRUGGLING
100 75
SWIM
100
*
75
50
30G •
225
5O
IMMOBILITY
//Y
25,
T !
!
5 10 DAY 1
I
15
T T 5
10 2
15
5
10 3
15
I
I
5 10 DAY 1
I
15
I
5
I
10 2
I
15
I
5
I
10 3
I
15
VT /
5 10 DAY I
T 15
5
10 2
15
5
I0 3
15
Fig. 3. Effect of the duration of the forced swimming test on the duration (s) of struggling behavior, swimming and immobility in the test. Means (n = 7) are presented. Triangles indicate the rats that were exposed daily, for 3 days (1, 2, 3), to a 5-min period of forced swimming and the circles indicate the rats that were exposed for 15 min. In the latter case, the three behaviors were recorded every 5 min (5, 10, 15). The arrows indicate the first 5 min of each day. A significant within-session and between-session effect on struggling behavior, swimming and immobility was found. In contrast, the effect of the duration of the daily exposure to the test was not significant.
there was a significant 'within session' reduction of struggling behavior (P < 0.001) and swimming (P < 0.001) every day, with a parallel increase in immobility (P < 0.001). W h e n the results f r o m rats that were tested daily for 5 min were c o m p a r e d with those from rats that were tested for 15 min, it was found that there were no significant overall differences in struggling, swimming or immobility. The A N O V A revealed a daily decline in the duration of struggling (P < 0.001) and swimming (P < 0.005), and a progressive increase in the duration of immobility (P < 0.001). In the latter case the interaction group b y days approached significance (P = 0.07). Table 1 shows the defecation rate of rats that were tested for 5 or 15 min for 3 days. We did not find that the duration of the daily swimming test had a significant effect on the defecation rate but that the n u m b e r of previous exposures (P < 0.05) did. The latter experiment was repeated (experiment 3) and the results were very similar to those obtained in experiment 2, without there being any evidence of an interaction between the two main factors with regard to immobility ( P = 0.386). These data are therefore not shown.
4. Discussion As expected, D M I significantly increased struggling behavior and decreased immobility of rats that had previously undergone a 15-min forced swimming test; swimming was not modified. D M I administration significantly increased struggling behavior of rats that did not have previous experience of the forced swimming test during the first and second 5-min periods of the test. Swimming was, on the contrary, significantly reduced during the first 5 min of the test and remained unaltered thereafter. Immobility was not reduced in the first 5 rain but was in the two following periods. These data suggest that a single exposure to the swimming test can distinguish between saline- and antidepressant ( D M I ) - t r e a t e d rats. This is compatible with the fact that only one swimming session is used for the screening of antidepressants in mice (Porsolt et al., 1977b). In addition, our results indicate that D M I treatment significantly increases struggling behavior whereas it does not influence or tends to decrease swimming. It therefore appears that struggling behavior is more reliably modified by
211
DMI than swimming is. This might be explained by assuming that struggling behavior is more directly related to attempts to escape than is swimming. In addition, swimming might be more sensitive than struggling behavior to drugs that alter locomotor activity. Thus, Kitada et al. (1981) observed that both caffeine and methamphetamine decrease immobility in the test without increasing escape-directed behavior. From our results it can be concluded that struggling behavior is a less subjective and more reliable measure of antidepressant action than is immobility in the forced swimming test. If the immobility of the rats in the water tank represents a state of behavioral despair (Porsolt, 1981), it would be expected that a more prolonged experience with the situation would increase the amount of time that the rats would remain immobile. The results from two different experiments (2 and 3) indicate that this was not the case. Likewise, the decline in struggling behavior with time was not influenced by the duration of the daily swimming test. These data are consistent with those reported by Borsini et al. (1986) who demonstrated that the presence of water in the tank was not necessary for the increase in immobility observed on the second day, and with those of O'Neill and Valentino (1982) who found that escapability did not prevent the increase in immobility on the second vs. the first day of test. It has been suggested that the change in activity from the first to the second day could be related to adaptation to an aversive situation (Hawkins et al., 1978). However, this appears unlikely in view of the fact that we have found an increase in the defecation rate after three daily exposures to swimming, and defecation is considered to be a good measure of emotional reactivity in aversive situations (Gray, 1971). The apparent lack of a relationship between emotional reactivity and immobility in the forced swimming test is supported by our finding that chronically shocked rats showed greater immobility than non-chronically stressed rats on the second day of exposure to forced swimming (Garcia-Mfirquez and Armario, 1987a), whereas the adrenocorticotropin response was similar in the two experimental groups (unpublished data). The pituitary-adrenal response
seems to be a very good index of stress experienced by the animals in aversive situations (Hennessy and Levine, 1978; Kant et al., 1983; Armario et al., 1986). It appears that the tendency to adopt passive behavior in a situation like the forced swimming test, in which there is a considerable demand for energy, is strong and requires only a brief period of previous exposure to a situation entirely or partly similar to that presently encountered. The rats could rapidly adopt passive behavior in aversive situations unless the opposite (active) behavior would actually be effective. This interpretation fits well with the 'searching-waiting' theory elaborated by Thierry et al. (1984). These authors suggest that animals are continuously choosing between active or passive strategies when faced with environmental changes, their ultimate choice depending on both external and internal cues. Based on all the aforementioned considerations, the present results could be explained alternatively by assuming that the behavior of rats in the forced swimming test is a trait rather than a state-dependent marker, the influence of antidepressants and other treatments (chronic stress) on the test being the consequence of neurochemical changes related to the maintenance of mood rather than of cognitive alterations.
Acknowledgement This work was supported by a grant from the FIS (86/967).
References Armario, A., J.L. Montero and J. Balasch, 1986, Sensitivity of corticosterone and some metabolic variables to graded levels of low intensity stresses in adult male rats, Physiol. Behav. 37, 559. Borsini, F., G. Volterra and A. Meli, 1986, Does the behavioral 'despair' test measure 'despair'?, Physiol. Behav. 38, 385. Garcia-M~rquez, C. and A. Armario, 1987a, Chronic stress depresses exploratory activity and behavioral performance in the forced swimming test without altering ACTH response to a novel acute stressor, Physiol. Behav. 40, 33. Garcla-Mhrquez, C. and A. Armario, 1987b, Interaction between chronic stress and clomipramine treatment in rats.
212 Effects on exploratory activity, behavioral despair, and pituitary-adrenal function, Psychopharmacology 93, 77. Gray, J.A., 1971, The Psychology of Fear and Stress (Weidenfeld and Nicolson, London). Hawkins, J., R.A. Hicks, N. Phillips and J.D. Moore, 1978, Swimming rats and human depression, Nature 274, 512. Hennessy, M.B. and S. Levine, 1978, Sensitive pituitary-adrenal responsiveness to varying intensities of psychological stimulation, Physiol. Behav. 21,295. Jesberger, J.A. and J.S. Richardson, 1985, Animal models of depression: parallels and correlates to severe depression in humans, Biol. Psychiat. 20, 764. Kant, G.J., E.H. Mougey, L.L. Pennington and J.L. Meyerhoff, 1983, Graded footshock stress elevates pituitary cyclic AMP and plasma B-endorphin, B-LPH, corticosterone and prolactin, Life Sci. 33, 2657. Kitada, Y., T. Miyauchi, A. Satoh and S. Satoh, 1981, Effects of antidepressants in the rat forced swimming test, European J. Pharmacol. 72, 145. Koyuncuoglu, H., L. Eroglu and T. Altug, 1982; Effects of L-aspartic acid, L-asparagine and/or L-asparaginase on forced swimming-induced immobility, analgesia, and decrease in rectal temperature in rats, Experientia 38, 117. O'Neill, K.A. and D. Valentino, 1982, Escapability and generalization: effect on 'behavioral despair', European J. Pharmacol. 78, 379.
Porsolt, R.D., 1981, Behavioral despair~ in: Antidepressants: Neurochemical and Behavioral Perspectives, eds. S.J. Enna. J.B. Malick and E. Richelson (Raven Press, New York) p. 121. Porsolt, R.D., G. Anton, N. Blavet and M. Jalfre, 1978~ Behavioral despair in rats: a new model sensitive to antidepressant treatments, Euroepan J. Pharmacol. 47, 379. Porsolt, R.D., A. Bertin and M. Jalfre, 1977a, Behavioral despair in mice: a primary screening test for antidepressants, Arch. Int. Pharmacodyn. 229, 327. Porsolt, R.D., M. Le Pichon and M. Jalfre, 1977b, Depression: a new model sensitive to antidepressant treatment, Nature 266, 730. Thierry, B., L. Steru, R. Chermat and P. Simon, 1984, Searching-waiting strategy: a candidate for an evolutionary model of depression, Behav. Neural Biol. 41, 180. Weiss, J.M., P.A. Goodman, B.G. Losito, S. Corrigan, J.M. Charry and W.H. Bailey, 1981, Behavioral depression produced by an uncontrollable stressor: relationship to norepinephrine, dopamine, and serotonin levels in various region of rat brain, Brain Res. Rev. 3, 167. Willner, P., 1984, The validity of animal models of depression, Psychopharmacology 83, 1.
207
Elsevier EJP 50580
Forced swimming test in rats: effect of desipramine administration and the period of exposure to the test on struggling behavior, swimming, immobility and defecation rate Antonio Armario *, Amadeu Gavaldh and Octavi Marti Departamento de Biologla Celular y Fisiologla, Facultad de Ciencias, Universidad A utbnoma de Barcelona, 08143 Bellaterra, Barcelona, Spain
Received 14 September 1988, revised MS received 30 September 1988, accepted 11 October 1988
The effect of desipramine administration and the duration of the daily exposure to forced swimming on some variables has been studied in adult male rats. Desipramine administration (15 mg/kg) significantly increased struggling behavior in the first and second 5-min periods of a single exposure to forced swimming. Swimming was reduced in the first 5 min and remained unchanged thereafter. Immobility was decreased in the second and the third 5-min periods. After a pre-exposure to forced swimming for 15 rain the day before, the drug was effective in increasing struggling behavior and reducing immobility during a subsequent 5-min test. Swimming was not modified. Daily exposure to forced swimming for 3 days caused a decline in struggling behavior and swimming, while increasing immobility and the defecation rate. The duration of daily exposure to forced swimming did not alter the changes in the variables measured. The present results indicate that a one-day test can be used to discriminate between saline- and desipramine-treated rats, and that struggling behavior could be a reliable measure of the positive action of antidepressants. The finding that behavioral changes over the 3 days were independent of the duration of exposure to swimming argues against the interpretation of the results which suggest that the responses are caused by the appearance of a behavioral despair state, and suggests that these behaviors might be trait-markers in the rat. In addition, the changes in struggling behavior and immobility over the 3 days cannot be attributed to a behavioral adaptation to the test because the defecation rate increased rather than decreased during successive forced swimming tests. Forced swimming test; Struggling behavior; Defecation; Immobility; Antidepressants
1. Introduction Considerable effort has been taken to establish animal models for the screening of antidepressant drugs. Of these models, the forced swimming test developed by Porsolt et al. (1977b; 1978) has gained considerable acceptance. When placed in a cylinder containing water (25°C; up to 15 cm deep) for 15 rain, rats initially display vigorous activity, apparently attempting to escape from the
* To whom all correspondence should be addressed.
tank. After a few minutes the animals become progressively immobile and float passively in the water, making only minor movements to maintain the head out of the water. The following day the rats are placed in the same tank for 5 min, and the time the rats remain immobile is measured. It has been reported that most antidepressant treatments (many drugs, R E M sleep deprivation, electroconvulsive shock) reduce immobility independently of changes in general motor activity, as usually assessed by open-field ambulation (Porsolt et al., 1977b; 1978; Porsolt, 1981). In addition, we have demonstrated recently that chronic exposure
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
208 to shock and a combination of various stressors applied in a random schedule increases immobility in Porsolt's test (Garcia-Mfirquez and Armario, 1987a), an effect that is prevented by the concomitant administration of clomipramine (GarciaMfirquez and Armario, 1987b). Since some forms of chronic stress are considered to be good animal models of depression (Willner, 1984), these data suggest that results from the forced swimming test could be related to the mood state of the animals. However, there are several theoretical and practical problems (Willner, 1984; Jesberger and Richardson, 1985). We wish to mention some of them here. First, the consideration of what is or is not immobility is subjective. Active rats usually show two apparently different behaviors (Weiss et al., 1981): (a) intense movements of the four limbs with the two front paws breaking the surface of the water or against the walls of the tank; (b) mild swimming, with movement of the four limbs or the hindlimbs. Whether or not the latter behavior should be categorized as activity is a matter of controversy and could, at least partially, explain the highly variable values, ranging from 50 to 250 s, for immobility reported in the literature (O'Neill and Valentino, 1982; Koyuncuoglu et al., 1982; Garcia-Mfirquez and Armario, 1987a). Second, it is not clear from the available data why only one swimming session is used for the screening of antidepressant drugs in mice (Porsolt et al., 1977a) and two sessions (see above) are used in rats. Third, Porsolt (1981) hypothesized that the increased immobility observed on the second exposure to water was because the rats knew, from previous experience, that escape was impossible. Therefore they used the term 'behavioural despair' to describe the cognitive state of the animals caused by this experimental situation. However, the possibility of escape from the tank on the first day did not alter the duration of immobility on the second day (O'Neill and Valentino, 1982). In addition, placing the rats in an empty tank on the first day increased immobility on the second day to the same extent as when the rats were placed in a tank containing water (Borsini et al., 1986). The present study was undertaken to thoroughly characterize the forced swimming test, and addressed some of the above-mentioned problems.
2. Materials and methods
Male Sprague-Dawley rats were used. They were maintained under standard conditions (lights on from 07:00 to 19:00 h, temperature 22°C) in groups of three to four per cage for at least 1 week before the start of the experiments. Food and water were provided ad libitum. The rats were placed (in the morning) in a cylindric tank containing up to 15 cm of water (25 ° C). The time spent making the two following behaviors was measured with a stop-watch: (a) 'struggling', which occurred when the rats were moving all four limbs with the two front limbs breaking the surface of the water or touching the walls; (b) 'immobility', which occurred when the animals remained floating with all limbs motionless. The rats spent the remainder of the time swimming and this behavior was categorized as 'swimming'. The defecation rate was measured as the number of boluses eliminated by each animal during the first 5 rain of exposure to the tank. Rats approximately 45-day-old were used in the first experiment. They were assigned to saline or desipramine ( D M I ) groups and were injected i.p. with saline or D M I (15 m g / k g ) 24 and 1 h before the test. D M I hydrochloride, which was kindly donated by Ciba Geigy, was used. The dose of D M I is expressed in terms of the salt. The tests were carried out following either the protocol described by Porsolt et al. (1977b), which consists of a 15-rain swimming session before drug administration and a second, 5-min swimming session during which the behavior was recorded, or one in which there was only one 15-min swimming session, the behavior was recorded every 5 min. Approximately 50-day-old rats were used in the second experiment. The rats were assigned to two groups. One in which the rats were placed in the water tank for 5 min on 3 consecutive days. In the other group the rats were placed in the water tank for 15 min on 3 consecutive days. In the latter case the behavior was recorded every 5 rain. This experiment was repeated with approximately 60day-old rats (experiment 3). The statistical significance of the results was analyzed with Student's t-test or Mann-Whitney's U-test when the samples did not meet the require-
209
!
TABLE 1
200
Effect of the duration of the forced swimming test on the defecation rate during the first 5 min of each daily test. Means and S.E.M. (n = 7) are presented. The A N O V A revealed that the number of forced swimming tests had a significant effect (P < 0.05) but that of the duration (5 vs. 15 rain) of the daily tests did not.
.lit.
150 10C
5~n 15min
n,, S
SW
Fig. 1. Effect of D M I administration (15 m g / k g ) on the duration (s) of struggling behavior (S), swimming (SW) and immobility (I) in a 5-min exposure to the forced swimming test. The animals were exposed to a pre-test session of forced swimming for 15 min. Means and S.E.M. (n = 7) are presented. Open bars indicate saline-treated rats and closed bars DMItreated rats. ** P < 0.01 vs. saline-treated rats.
ments for Student's t-test (unequal variances, non-normal distribution). In other cases, the twoway ANOVA with repeated measures for one factor (within and between days measures) was used.
3. Results The administration of D M I to rats, tested for 5 min after a previous 15-min test the day before,
150I
/
SWIM
]
4.9±0.9 4.4±0.5
IMMOBILITY
225
'°°1" ~hI**
5
5 0 ~ 7 5 10
Thirdday
3.4±0.6 3.3±0.5
300
**
1o
5
Secondday
2.6±0.8 3.1±0.8
significantly increased struggling behavior (P < 0.01) and reduced immobility (P < 0.01). Swimming was not modified by the drug (fig. 1). Figure 2 depicts the duration of the struggling behavior, swimming and immobility of rats treated with saline or DMI during the first 15 min of forced swimming. The struggling behavior was significantly increased by DMI during the first and the second 5-min periods in the water tank (in both cases P < 0 . 0 1 vs. saline-treated group). Swimming was significantly reduced (P < 0.01) by the drug during the first 5 min of the test only. The duration of immobility was not significantly reduced by DMI during the first 5 rain, but was during the second and third periods (P < 0.01 vs. saline-treated rats). Figure 3 shows the influence of the duration of the swimming test on struggling behavior, swimming and immobility. The ANOVA showed that
I
]'el*STRUGGLING
Firstday
15
5
150
10
15
n,/111 5
10
15
Fig. 2. Effect of DMI treatment (15 m g / k g ) on the duration (s) of struggling behavior, swimming and immobility in a 15-min exposure to forced swimming. The rats did not have a pre-test swimming session. Means and S.E.M. (n = 14-15) are presented. Open bars indicate saline-treated rats and closed bars DMI-treated rats. N u m b e r s under bars indicate the successive 5-min periods of the test. ** P < 0.01 vs. saline-treated rats.
210
STRUGGLING
100 75
SWIM
100
*
75
50
30G •
225
5O
IMMOBILITY
//Y
25,
T !
!
5 10 DAY 1
I
15
T T 5
10 2
15
5
10 3
15
I
I
5 10 DAY 1
I
15
I
5
I
10 2
I
15
I
5
I
10 3
I
15
VT /
5 10 DAY I
T 15
5
10 2
15
5
I0 3
15
Fig. 3. Effect of the duration of the forced swimming test on the duration (s) of struggling behavior, swimming and immobility in the test. Means (n = 7) are presented. Triangles indicate the rats that were exposed daily, for 3 days (1, 2, 3), to a 5-min period of forced swimming and the circles indicate the rats that were exposed for 15 min. In the latter case, the three behaviors were recorded every 5 min (5, 10, 15). The arrows indicate the first 5 min of each day. A significant within-session and between-session effect on struggling behavior, swimming and immobility was found. In contrast, the effect of the duration of the daily exposure to the test was not significant.
there was a significant 'within session' reduction of struggling behavior (P < 0.001) and swimming (P < 0.001) every day, with a parallel increase in immobility (P < 0.001). W h e n the results f r o m rats that were tested daily for 5 min were c o m p a r e d with those from rats that were tested for 15 min, it was found that there were no significant overall differences in struggling, swimming or immobility. The A N O V A revealed a daily decline in the duration of struggling (P < 0.001) and swimming (P < 0.005), and a progressive increase in the duration of immobility (P < 0.001). In the latter case the interaction group b y days approached significance (P = 0.07). Table 1 shows the defecation rate of rats that were tested for 5 or 15 min for 3 days. We did not find that the duration of the daily swimming test had a significant effect on the defecation rate but that the n u m b e r of previous exposures (P < 0.05) did. The latter experiment was repeated (experiment 3) and the results were very similar to those obtained in experiment 2, without there being any evidence of an interaction between the two main factors with regard to immobility ( P = 0.386). These data are therefore not shown.
4. Discussion As expected, D M I significantly increased struggling behavior and decreased immobility of rats that had previously undergone a 15-min forced swimming test; swimming was not modified. D M I administration significantly increased struggling behavior of rats that did not have previous experience of the forced swimming test during the first and second 5-min periods of the test. Swimming was, on the contrary, significantly reduced during the first 5 min of the test and remained unaltered thereafter. Immobility was not reduced in the first 5 rain but was in the two following periods. These data suggest that a single exposure to the swimming test can distinguish between saline- and antidepressant ( D M I ) - t r e a t e d rats. This is compatible with the fact that only one swimming session is used for the screening of antidepressants in mice (Porsolt et al., 1977b). In addition, our results indicate that D M I treatment significantly increases struggling behavior whereas it does not influence or tends to decrease swimming. It therefore appears that struggling behavior is more reliably modified by
211
DMI than swimming is. This might be explained by assuming that struggling behavior is more directly related to attempts to escape than is swimming. In addition, swimming might be more sensitive than struggling behavior to drugs that alter locomotor activity. Thus, Kitada et al. (1981) observed that both caffeine and methamphetamine decrease immobility in the test without increasing escape-directed behavior. From our results it can be concluded that struggling behavior is a less subjective and more reliable measure of antidepressant action than is immobility in the forced swimming test. If the immobility of the rats in the water tank represents a state of behavioral despair (Porsolt, 1981), it would be expected that a more prolonged experience with the situation would increase the amount of time that the rats would remain immobile. The results from two different experiments (2 and 3) indicate that this was not the case. Likewise, the decline in struggling behavior with time was not influenced by the duration of the daily swimming test. These data are consistent with those reported by Borsini et al. (1986) who demonstrated that the presence of water in the tank was not necessary for the increase in immobility observed on the second day, and with those of O'Neill and Valentino (1982) who found that escapability did not prevent the increase in immobility on the second vs. the first day of test. It has been suggested that the change in activity from the first to the second day could be related to adaptation to an aversive situation (Hawkins et al., 1978). However, this appears unlikely in view of the fact that we have found an increase in the defecation rate after three daily exposures to swimming, and defecation is considered to be a good measure of emotional reactivity in aversive situations (Gray, 1971). The apparent lack of a relationship between emotional reactivity and immobility in the forced swimming test is supported by our finding that chronically shocked rats showed greater immobility than non-chronically stressed rats on the second day of exposure to forced swimming (Garcia-Mfirquez and Armario, 1987a), whereas the adrenocorticotropin response was similar in the two experimental groups (unpublished data). The pituitary-adrenal response
seems to be a very good index of stress experienced by the animals in aversive situations (Hennessy and Levine, 1978; Kant et al., 1983; Armario et al., 1986). It appears that the tendency to adopt passive behavior in a situation like the forced swimming test, in which there is a considerable demand for energy, is strong and requires only a brief period of previous exposure to a situation entirely or partly similar to that presently encountered. The rats could rapidly adopt passive behavior in aversive situations unless the opposite (active) behavior would actually be effective. This interpretation fits well with the 'searching-waiting' theory elaborated by Thierry et al. (1984). These authors suggest that animals are continuously choosing between active or passive strategies when faced with environmental changes, their ultimate choice depending on both external and internal cues. Based on all the aforementioned considerations, the present results could be explained alternatively by assuming that the behavior of rats in the forced swimming test is a trait rather than a state-dependent marker, the influence of antidepressants and other treatments (chronic stress) on the test being the consequence of neurochemical changes related to the maintenance of mood rather than of cognitive alterations.
Acknowledgement This work was supported by a grant from the FIS (86/967).
References Armario, A., J.L. Montero and J. Balasch, 1986, Sensitivity of corticosterone and some metabolic variables to graded levels of low intensity stresses in adult male rats, Physiol. Behav. 37, 559. Borsini, F., G. Volterra and A. Meli, 1986, Does the behavioral 'despair' test measure 'despair'?, Physiol. Behav. 38, 385. Garcia-M~rquez, C. and A. Armario, 1987a, Chronic stress depresses exploratory activity and behavioral performance in the forced swimming test without altering ACTH response to a novel acute stressor, Physiol. Behav. 40, 33. Garcla-Mhrquez, C. and A. Armario, 1987b, Interaction between chronic stress and clomipramine treatment in rats.
212 Effects on exploratory activity, behavioral despair, and pituitary-adrenal function, Psychopharmacology 93, 77. Gray, J.A., 1971, The Psychology of Fear and Stress (Weidenfeld and Nicolson, London). Hawkins, J., R.A. Hicks, N. Phillips and J.D. Moore, 1978, Swimming rats and human depression, Nature 274, 512. Hennessy, M.B. and S. Levine, 1978, Sensitive pituitary-adrenal responsiveness to varying intensities of psychological stimulation, Physiol. Behav. 21,295. Jesberger, J.A. and J.S. Richardson, 1985, Animal models of depression: parallels and correlates to severe depression in humans, Biol. Psychiat. 20, 764. Kant, G.J., E.H. Mougey, L.L. Pennington and J.L. Meyerhoff, 1983, Graded footshock stress elevates pituitary cyclic AMP and plasma B-endorphin, B-LPH, corticosterone and prolactin, Life Sci. 33, 2657. Kitada, Y., T. Miyauchi, A. Satoh and S. Satoh, 1981, Effects of antidepressants in the rat forced swimming test, European J. Pharmacol. 72, 145. Koyuncuoglu, H., L. Eroglu and T. Altug, 1982; Effects of L-aspartic acid, L-asparagine and/or L-asparaginase on forced swimming-induced immobility, analgesia, and decrease in rectal temperature in rats, Experientia 38, 117. O'Neill, K.A. and D. Valentino, 1982, Escapability and generalization: effect on 'behavioral despair', European J. Pharmacol. 78, 379.
Porsolt, R.D., 1981, Behavioral despair~ in: Antidepressants: Neurochemical and Behavioral Perspectives, eds. S.J. Enna. J.B. Malick and E. Richelson (Raven Press, New York) p. 121. Porsolt, R.D., G. Anton, N. Blavet and M. Jalfre, 1978~ Behavioral despair in rats: a new model sensitive to antidepressant treatments, Euroepan J. Pharmacol. 47, 379. Porsolt, R.D., A. Bertin and M. Jalfre, 1977a, Behavioral despair in mice: a primary screening test for antidepressants, Arch. Int. Pharmacodyn. 229, 327. Porsolt, R.D., M. Le Pichon and M. Jalfre, 1977b, Depression: a new model sensitive to antidepressant treatment, Nature 266, 730. Thierry, B., L. Steru, R. Chermat and P. Simon, 1984, Searching-waiting strategy: a candidate for an evolutionary model of depression, Behav. Neural Biol. 41, 180. Weiss, J.M., P.A. Goodman, B.G. Losito, S. Corrigan, J.M. Charry and W.H. Bailey, 1981, Behavioral depression produced by an uncontrollable stressor: relationship to norepinephrine, dopamine, and serotonin levels in various region of rat brain, Brain Res. Rev. 3, 167. Willner, P., 1984, The validity of animal models of depression, Psychopharmacology 83, 1.