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Effects of diazepam, pentobarbital, scopolamine and the timing of saline injection on learned immobility in rats
Physiology & Behavior, Vol. 50. pp. 895-899. © Pergamon Press plc, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Effects of Diazepam, Pentobarbital, Scopolamine and the Timing of Saline Injection on Learned Immobility in Rats J U A N M. D E P A B L O , J A V I E R O R T I Z - C A R O , F E R N A N D O S A N C H E Z - S A N T E D
AND ANTONIO GUILLAMON 1
Departamento de Psicobiologfa, Universidad Nacional de Educaci6n a Distancia, Ciudad Universitaria P.O. Box 50487, 28040 Madrid, Spain R e c e i v e d 21 A u g u s t 1990 DE PABLO, J. M., J. ORTIZ-CARO, F. SANCHEZ-SANTED AND A. GUILLAMON. Effects of diazepam, pentobarbital, scopolamine and the timing of saline injection on learned immobility in rats. PHYSIOL BEHAV 50(5) 895-899, 1991.--The rat forced-swimming test (FST) is widely used for screening substances with a potential antidepressant effect. Rat immobility shown in the FST has been interpreted as "behavioral despair" and has been suggested as an animal model of human depression. In the following series of experiments, it is shown that pentobarbital and scopolamine administered immediately after the first phase, and diazepam administered 15 minutes before the first phase, behave as false positives in the FST. It is concluded that the learningmemory hypothesis seems to cope better with the behavior of rats during the FST than the "behavioral despair" hypothesis. It is also shown that the sensitivity of the FST is affected by the fact that the last saline injection, one hour before the second phase, increases the animals' mobility. Forced-swimming test Automatic recording Pentobarbital Behavioral despair Learned immobility Memory
Diazepam
Scopolamine
Saline injection
10, 22, 29). Finally, some substances that score as false positives in the FST also deteriorate memory processes (1,5): these include anticholinergics (7,12), convulsants (17,18), gabaergic substances (2,8) and anisomycin (6), an inhibitor of protein synthesis. In a previous paper (6), we showed that anisomycin acts as a false positive in the FST, thus supporting our hypothesis that the behavioral process underlying behavior in the FST is related to acquisition and consolidation mechanisms. The main purpose of the present study was to determine if diazepam, pentobarbital and scopolamine, three substances that are not antidepressants and that deteriorate memory (1), could behave as false positives in the FST. In Experiment 1, the diazepam and pentobarbital were administered at two different times: immediately after the first phase or one hour before the second phase. The two conditions were chosen because the effect of a substance on the process of acquisition and/or consolidation depends on the time of administration to the subject in relation to training. Martinez et al. (16) suggest the use of posttraining treatments for testing consolidation. Since the amnesic effects of scopolamine, an anticholinergic, are very well known (1), it could be predicted that its administration immediately after the first phase of the FST would affect the habitual decrease of mobility seen during the second phase of the FST, thus supporting the view that memory is the underlying behavioral process in the FST. Because diaz-
THE rat forced-swimming test (FST) was introduced by Porsolt et al. (21) as a new animal model sensitive to antidepressant treatments. Later, Porsolt (23) suggested that the FST could be considered a reliable animal model of human depression to be added to other current models in the field such as "learned helplessness" (24) and "separation" (27) models. Briefly, when rats or mice are forced to swim in a confined inescapable space (usually a cylinder), they tend to become immobile (float) after vigorous activity. This immobility was characterized by Porsolt as "behavioral despair." The FST is conducted in two consecutive phases for rats and sometimes for mice (17,18). The first phase usually lasts 15 minutes, while the second phase, carded out 24 hours later, only lasts 5 or 6 minutes. Potentially antidepressive substances are commonly administered to rats 23 hours 45 min, 5 hours and 1 hour before the second phase. The animals' behavior (duration of immobility or changes in mobility) is evaluated during the 5 min of the second phase of the test. There is data in the literature showing that the animal's experience with the FST plays an important role in swimming behavior: first, immobility increases (or swimming activity decreases) throughout the FST (6,22). Furthermore, swimming activity decreases in the second phase with respect to the first phase (6,30). Secondly, antidepressant treatments such as electroconvulsive shock (ECS) and REM sleep deprivation, which affect memory processes (25,26), also increase swimming activity in the FST (9, 1Requests for reprints should be addressed to A. Guillam6n.
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epam has been found to induce anterograde amnesia (3,28), it was administered 15 min before the first phase to one group of rats. Previous observations (6) and the behavior of control rats (injected only with saline) in Experiment 1 led us to believe that the timing of the saline injection in control groups could have different effects on rats' swimming behavior. This question was therefore addressed in Experiment 2. EXPERIMENT 1 METHOD
Animals Ninety-one male Wistar rats (ALIN, Madrid, Spain) weighing 170-230 g were used. Subjects were caged in groups of four or five. Food and water were continuously available in a room maintained at 21--2°C, with controlled light-dark cycle (light: 0700 to 1900 h).
Apparatus A Panlab Animal Activity System (PAAS, Panlab, Barcelona, Spain) and a Plexiglas cylinder (height: 50 cm; diameter: 19.5 cm) were used (6). The PAAS system consisted of a sensory unit, an electronic counter, a printer and a control unit. General activity (mobility) was evaluated as a function of the variations produced by the rats' swimming activity on the standard frequency (484 KHz) of the electromagnetic field of the sensory unit. Frequency variations were transformed into voltage changes, which, in turn, were converted into impulses that were collected by the counter when they reached a certain level.
Procedure Thirty-three subjects were randomly assigned to six groups for the administration of diazepam or pentobarbital (kindly provided by Servicio de Restricci6n de Estupefacientes del Ministerio de Sanidad y Consumo) and saline immediately after the first phase: the control group ( N = 6 ) received saline (5 ml/kg) after training in the first phase; a second, third and fourth group ( N = 5 ) were injected a dose of diazepam (2.5, 7.5 mg/kg or 15 mg/kg respectively) dissolved in 5 ml/kg of saline after training in the first phase; and a fifth and sixth group (N = 6) were administered a dose of 7.5 or 15 mg/kg of pentobarbital 15 minutes after training in the first phase. Twenty-four raics were also randomly assigned to three different groups for the administration of scopolamine immediately after the first phase: the control group (N = 8) received 5 ml/kg of saline; two other groups ( N = 8 ) received 0.5 mg/kg and 1 mg/kg of scopolamine hydrochloride (Sigma) dissolved in 5 ml/kg of saline respectively. Twenty different subjects were randomly assigned to three groups for administration of diazepam and pentobarbital and saline one hour before the second phase: the control group (N = 6) received saline (5 ml/kg); a second and third group (N = 7) were injected a dose of diazepam or pentobarbital (7.5 mg/kg) respectively, dissolved in 5 ml/kg of saline. Finally, fourteen subjects were also randomly administered saline ( N = 6 ) or a single dose of diazepam (2.5 mg/kg) dissolved in 5 mt/kg ( N = 8 ) 15 minutes before the first phase. Training during the first phase lasted 15 minutes and testing in the second phase lasted 5 minutes. Individual rats were forced to swim inside an upright cylinder containing 15 cm of water at 25°C. Swimming activity (mobility) was automatically recorded during the two sessions. The data were statistically treated as
TABLE 1 EFFECT OF DIAZEPAM AND PENTOBARBITAL INJECTEI) IMMEDIATEI "~ AFTER THE FIRST PHASE ON RAT MOBILITY 1N TIlE FST
Treatment Control Diazepam Diazepam Diazepam Pentobarbital Pentobarbital
Doses
N
Saline 2.5 mg/kg 7.5 mg/kg 15 mg/kg 7.5 mg/kg t5 mg/kg
6 5 5 5 6 6
% of Impuise~ :L SEM 38.5 53.72 35.0 39.2 85,6 78.4
:. i i~43 - t2.3 :: 13.99 :: 14.99 _*__15.81" - 21.88
*p<0.05 with respect to the control group.
follows: mobility in the second phase was expressed as the percentage of change in relation to each subject's number of impulses during the first 5 minutes of the first phase, then submitted for Student's t-test (one tailed). RESULTS
Effect of the Administration of Diazepam and Pentobarbital Immediately After the First Phase of the FST As can be seen in Table 1, the rats treated with 7.5 mg/kg of pentobarbital showed significantly greater mobility than the control animals in the 5 min swim test, t(10)= 2.63, p<0.05. However, a 15 mg/kg dose of pentobarbital did not reach a statistically significant effect, t(10)= 1.77, p < 0 . 1 . Nevertheless, this group of rats did not differ from those injected with 7.5 mg/kg of pentobarbital in the level of mobility. None of the three doses of diazepam affected the rats' swimming activity.
Effect of the Administration of Scopolamine Immediately After the First Phase of the FST As can be seen in Table 2, the administration of 1 mg/kg of scopolamine immediately after the first phase increased the rats' mobility in the FST with respect to the control group, t(14)= 2.77, p<0.01; however, a lesser dose (0.5 mg/kg) did not affect mobility, t(14) = p < 0 . 1 0 .
Effect of the Administration of Diazepam or Pentobarbital One Hour Before the Second Phase of the FST As can be observed in Table 3, the administration of diazepam or pentobarbital one hour before the second phase did not affect the rats' swimming activity in the FST [diazepam vs.
TABLE 2 EFFECT OF SCOPOLAMINE INJECTED IMMEDIATELY AFTER THE FIRST PHASE ON RAT MOBILITY IN THE FST
Treatment Control Scopolamine Scopolamine
Doses
N
% of Impulses -+ SEM
Saline 0.5 mg/kg 1.0 mg/kg
8 8 8
45.4 _+ 8.88 60.98 ± 7.79 85.07 ± 12.5"
*p<0.01 with respect to the control group.
LEARNING AND STRESS IN THE FST
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TABLE 3 EFFECT OF D1AZEPAMAND PENTOBARBITALINJECTEDONE HOUR BEFORETHE SECONDPHASEON RAT MOBILITYIN THE FST Treatment Control Diazepam Pentobarbital
Doses
N
% of Impulses --- SEM
Saline 7.5 mg/kg 7.5 mg/kg
6 7 7
80.82 -+ 7.92 65.06 - 11.66 88.52 + 9.56
control: t(11)=1.17, 0.66, N.S.].
N.S.; pentobarbital vs. control: t ( l l ) =
Effect of Administration of Diazepam 15 Minutes Before the First Phase of the FST Table 4 shows that a single injection of 2.5 mg/kg of diazepam administered 15 min before the first phase increases the rats' mobility in the second phase of the FST, t(12)=2.75, p<0.01. Finally, if we compare the performance of the control groups of the four assays (see Tables 1, 2, 3 and 4), it can be seen that rats injected with saline one hour before the second phase present more mobility than the other control groups, t(10)= 3.27, p<0.01; t(12)= 3.21, p<0.01; t(10)=5.65, p<0.001. No treatment affected the behavior of rats during the first phase. EXPERIMENT 2 Unexpectedly, in Experiment 1 it was observed that the control group injected with saline one hour before the second phase presented greater mobility than those injected 15 min before or immediately after the first phase. This might have methodological and theoretical implications for the interpretation of the FST. If the pure saline injection behaves as a false positive, depending upon its timing, it poses serious constraints for the use of the FST as a technique for screening antidepressant substances, at least when subjects are injected one hour before the second phase of the test. Differences in mobility shown by the control group in Experiment 1 might be dependent upon our specific procedure, i.e., we injected the subjects only one time during the entire test. However, since introduction of the test by Porsolt, the common procedure has been to inject animals 23 hours 45 min, 5 hours and 1 hour before the second phase. Thus in the present experiment we have compared the performance in the FST of rats injected with saline, taking into account the number of injections and their timing. METHOD
Animals Forty-three male rats of the Wistar strain (ALIN, Madrid, Spain) were used. They possessed similar characteristics and
Treatment
N
% of Impulses _ SEM
Uninjected Control 1 hour before the second phase Immediately after the first phase 15 min before the first phase 23.45, 5 and 1 h before second phase Three doses (2 h interval)
9 6 7 6 8
56.12 80.5 45.42 38.27 82.66
7
42.82 _
-+ -+ + -+ ---
10.6 8* 12.5t:~ ll.6t:~ 4.79* 6.4"t~:
*p<0.05 with respect to uninjected control group. 1"p<0.01 with respect to 1 hour before second phase group. :~p<0.01 with respect to 23.45, 5 and 1 h before second phase group.
housing to those reported in Experiment 1.
Procedure The subjects were randomly assigned to the following conditions with respect to the number and time of injections: group not injected (Uninjected Control group; N = 9 ) ; group injected one hour before the second phase (N = 6); group injected immediately after the first phase ( N = 7 ) ; group injected 15 minutes before the first phase ( N = 6 ) ; group injected three times (23 hours 45 minutes, 5 hours and 1 hour before the second phase, usual control group for screening antidepressant substances with the FST; N = 8), and group injected three times with an interval of two hours beginning 15 minutes before the first phase (N = 7). Subjects swam 15 minutes in the first phase and 24 hours later they swam 5 minutes in the second phase, as in Experiment 1. The data was also recorded and analysed as in Experiment 1. RESULTS As shown in Table 5, the group commonly used according to the literature for screening antidepressant substances and the group injected one hour before the second phase presented greater mobility than the uninjected group [t(15)= 2.28, p < 0 . 0 5 and t(13)= 1.79, p<0.05, respectively]. No statistically significant differences were seen between the uninjected control group and the group injected three times with an interval of two hours beginning 15 minutes before the first phase, t(14)= 1.07, N.S. Nor was the difference between the uninjected control group and the group injected immediately after the first phase found to be significant, t(14)=0.71, N.S. Also, no differences were seen between the uninjected control group and the group injected 15 minutes before first phase, t(13) = 1.16, N.S. For other comparisons, see Table 5. GENERAL DISCUSSION
TABLE 4 EFFECT OF DIAZEPAM INJECTED 15 MINUTESBEFORE THE FIRSTPHASEON RAT MOBILITYIN THE FST Treatment
Doses
N
% of Impulses _ SEM
Control Diazepam
Saline 2.5 mg/kg
6 8
26.24 _ 7.07 79.44 _ 17"
*p<0.01 with respect to the control group.
TABLE 5 EFFECT OF NUMBERAND TIME OF SALINEINJECTION ON RAT MOBILITYIN THE FST
Pentobarbital, scopolamine and diazepam behave as false positives in the FST. A single dose of 7.5 mg/kg of pentobarbital administered immediately after the first phase, but not one hour before the second phase of the FST, increased mobility in animals during the 5-minute test of the second phase. A similar treatment with diazepam did not, nevertheless, affect rat mobility in the second phase of the FST. However, when diazepam was administered 15 minutes before the first phase it increased rat mobility 24 hours later during the second phase. A single
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DE PABiA) t,,l \ i
dose of 1 mg/kg of scopolamine administered immediately after the first phase also increased mobility. Although a decrease in mobility is currently being accepted in the literature as an indication of "behavioral despair" (23), we have proposed that the behavioral process involved in the FST is learning to be immobile (6). Our results with pentobarbital (a barbiturate), diazepam (an anxiolytic) and scopolamine (an anticholinergic), three substances that are not antidepressants, support our previous hypothesis. Reports in the literature show that pentobarbital deteriorates memory in humans and other species (1). Interestingly enough, diazepam, which is reputed to induce anterograde amnesia (3, 19, 28), only increases mobility in the FST when injected before the first phase. The fact that an anticholinergic substance such as scopolamine behaves as a false positive in the FST with a procedure that directly addresses swimming activity supports once again our hypothesis that learning and memory ore the behavioral processes involved in the behavior of rats in the FST. It should be remembered that cholinergic mechanisms in the CNS are directly involved in mediating diverse learning and memory phenomena (1). In Experiment 1, we observed statistically significant differences among the control (saline) groups, while the only procedural difference among these four groups was the time at which the saline injection was administered. When this problem was addressed in Experiment 2, it was observed that indeed the injection of saline one hour before the second phase acted as a false positive. Furthermore, rats injected with saline three times, 23 hours 45 min, 5 hours and 1 hour before the second phase (the usual control group in the FST for testing antidepressant substances), presented greater mobility than the uninjected rats. This increment in mobility does not depend on the number of injections, since three injections of saline scheduled before and after the first phase did not increase mobility during the second phase. Thus the sensitivity of the FST is affected by the fact that the last saline injection, one hour before the second phase, increases animals' mobility. There are reports in the literature showing that chronic stress decreases the duration of immobility in mice (20). However, to our knowledge, the possible stressing effect of saline injection in the FST has not been studied. Porsolt's data (22) comparing saline-injected (24, 5 and 1 hour before the second phase) and uninjected rats shows no differences between these groups. The discrepancies between our results in Experiment 2 and those reported by Porsolt might be explained by differences in procedures: Porsolt estimated by observation the duration of immobility, whereas we automatically measured swimming activity. Thus it could be possible that mobility and
not duration of immobility is affected by the stres~ reduced t~'~ the saline injection administered one hour befi)re the second phase. Interestingly, as can be seen in Table 3, aeither pento barbital nor diazepam were able to block this eltect when in-jected one hour before the second phase. Furthernlore, we have shown that the automatic procedure presents greater sensitivi!!~ than the direct observation of the duration of immobility (61. Taking into account the results of this and previous studies (6), it appears that the learning-memory hypothesis copes with the behavior of rats during the FST better than the "behavioral despair" hypothesis previously suggested in the literature (23). As we have pointed out (6), there is data in the literature showing that antidepressant substances impair memoD' both in humans (4, 13, 14) and rats (11,15). Judd et al. (11) suggest that the effect of amitriptyline and imipramine on human memory is similar to that of scopolamine. In rats it was reported that imipramine, amitriptyline, desipramine and mianserine impair avoidance responses (11,15). As we previously have pointed out. some antidepressant substances selected by means of the FST might be selected because of their effect on m e m o u . Although at this moment it is not possible to discard other actions of antidepressants affecting mobility in the FST (viz., on general activity or reactivity), in our opinion, the learning-memory hypothesis seems to cope better with the behavior of rats during the FST than the "behavioral despair" hypothesis for several reasons: first, it can explain the effect of anticholinergics, anxiolytics, convulsants and anisomycin, and it is also able to explain the effect of antidepressant substances, REM sleep deprivation and electroconvulsive shock in the FST. Second, it can cope with the fact that a single injection of imipramine (6), pentobarbital, diazepam or scopolamine is effective in the FST; it is known that antidepressant treatments are therapeutically effective after chronic administration only. And third, the learning-memory hypothesis springs from the experimental analysis of rats' behavior, whereas the "behavioral despair" hypothesis is an anthropomorphic interpretation of immobility in the FST. This interpretation has led to the following circular reasoning: since antidepressants change rats' immobility in the FST, such a behavior is a symptom of animal depression, and, as a result, it is assumed that substances that reduce rats' immobility as antidepressants do are antidepressants. ACKNOWLEDGEMENTS This work was supported by grants PB87-0621 and PB88/158 from the DGICYT. The authors wish to express their appreciation to A. A. Caminero, Mary Frances Litzler, M. de Amaral, L. Carrillo, L. Troca and G. Moreno for their technical assistance.
REFERENCES 1. Bartus, R. T.; Dean, R. L.; Flicker, Ch. Cholinergic psychopharmacology: An integration of human and animal research on memory. In: Meltzer, H. Y., ed. Psychopharmacology: The third generation of progress. New York: Raven; 1987:219-232. 2. Borsini, F.; Evangelista, S.; Meli, A. Effect of gabaergic drugs in the behavioral "despair" test in rats. Eur. J. Pharmacol. 121:265268; 1986. 3. Cole, S. O. Effects of benzodiazepines on acquisition and performance: A critical assessment. Neurosci. Biobehav. Rev. 10:265272; 1986. 4. Curran, H. V.; Shine, Ph.; Lader, M. Effects of repeated doses of fluvoxamine, mianserine and placebo on memory and measures of sedation. Psychopharmacology (Berlin) 89:360-363; 1984. 5. Davis, H. P.; Squire, L. R. Protein synthesis and memory: A review. Psychol. Bull. 96:518-559; 1984. 6. De Pablo, J. M.; Parra, A.; Segovia, S.; Guillarnon, A. Learned immobility explains the behavior of rats in the forced swimming
test. Physiol. Behav. 46:229-237; 1989. 7. Duncan, G. E.; Breese, G. R.; Criswell, H.; Stumpf, W. E.; Mueller, R. A.; Cobey, J. B. Effects of antidepressant drugs injected into amygdala on behavioral responses of rats in the forced swim test. J. Pharmacol. Exp. Ther. 238:758-762; 1986. 8. Fernandez Teruel, A.; Boix, F.; Escorihuela, R. M.; Yafiez, P.; Tobefia, A. Sodium valproate reduces immobility in the behavioral "despair" test in rats. Eur. J. Pharmacol. 152:1-7; 1988. 9. Hawkins, J.; Phillips, N.; Moore, J. D.; Gilliland, M. A.; Dunbar, S.; Hicks, R. A. Emotionality and REMD: A rat swimming model. Physiol. Behav. 25:167-171; 1980. 10. Hodgson, J. A. Swimming immobility of REM sleep deprived rats reared in different environments. Physiol. Behav. 32:17-22; 1984. 11. Judd, L. L.; Squire, L. R.; Butters, N.; Salmo, D. M.; Paller, K. A. Effects of psychotropic drugs on cognition and memory in normal humans and animals. In: Meltzer, H. Y., ed. Psychopharmacology: The third generation of progress. New York: Raven; 1987:
LEARNING AND STRESS IN THE FST
1467-1475. 12. Kitada, Y.; Miyauchi, T.; Satoh, A.; Satoh, S. Effects of antidepressants in the rat forced swimming test. Eur. J. Pharmacol. 72: 145-152; 1981. 13. Kupfer, D. J.; Detre, T. P. Tricyclic and monoamine-oxidase inhibitor antidepressants: Clinical use. In: Iversen, L. L.; Iversen, S. D.; Snyder, S. H., eds. Handbook of psychopharmacology, vol. 14. New York: Plenum; 1978:199-232. 14. Lamping, D. L.; Spring, B.; Gelenberg, A. J. Effects of two antidepressants on memory performance in depressed outpatients: A double-blind study. Psychopharmacology (Berlin) 84:254-261; 1984. 15. Lucki, I.; Nobler, M. S. The acute effects of antidepressant drugs on the performance of conditioned avoidance behavior in rats. Pharmacol. Biochem. Behav. 22:261-264; 1985. 16. Martinez, J. L., Jr.; Jensen, R. A.; McGaugh,.J.L. Facilitation of memory consolidation. In: Deutsch, J. A., ed. The physiological basis of memory. New York: Academic; 1983:49-70. 17. Nagatani, T.; Sugihara, T.; Kodaira, R. The effect of diazepam and of agents which change gabaergic functions in immobility in mice. Eur. J. Pharmacol. 97:271-275; 1984. 18. Nagatani, T.; Yamamoto, T.; Sugihara, T.; Ueki, S. The effect of agonists at the GABA-benzodiazepine receptor complex on the duration of immobility of mice in the forced swimming test. Eur. J. Pharmacol. 142:17-22; 1987. 19. Petersen, R. C.; Ghoneim, M. M. Diazepam and human memory: Influence on acquisition, retrieval and state-dependent learning. Prog. Neuropsychopharmacol. 4:81-89; 1980. 20. Platt, J. E.; Stone, E. A. Chronic restraint stress elicits a positive antidepressant response on the forced swim test. Eur. J. Pharmacol. 82:179-181; 1982.
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21. Porsolt, R. D.; Le Pichon, M.; Jalfre, M. Depression: A new animal model sensitive to antidepressant treatments. Nature 266:730732; 1977. 22. Porsolt, R. D.; Anton, G.; Blavet, N.; Jalfre, M. Behavioural despair in rats: A new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47:379-391; 1978. 23. Porsolt, R. D. Behavioral despair. In: Enna, S. J.; Malick, J. B.; Richelson, E., eds. Antidepressants: Neurochemical, behavioral and clinical perspectives. New York: Raven; 1981:121-139. 24. Seligman, M. E. P. Helplessness: On depression, development and death. San Francisco: Freeman; 1975. 25. Smith, C. Sleep states and learning: A review of the animal literature. Neurosci. Biobehav. Rev. 9:157-168; 1985. 26. Sneider, A. M.; Plough, M. Electroconvulsive shock and memory. In: Deutsch, J. A., ed. The physiological basis of memory. New York: Academic; 1983:352-365. 27. Suomi, S. J.; Harlow, H. F. Production and alleviation of depressive behaviors in monkeys. In: Maser, J. D.; Seligman, M. E. P., eds. Psychopathology: Experimental models. San Francisco: Freeman; 1977:131-173. 28. Taylor, J. L.; Tinldenberg, J. R. Cognitive impairment and benzodiazepines. In: Meltzer, H. Y., ed. Psychopharmacology: The third generation of progress. New York: Raven; 1987:1449-1454. 29. Van Luijtelaar, E. L. J. M.; Coenen, A. M. L. Paradoxical sleep deprivation and the immobility response in the rat: Effects of desipramine and phentolamine. Sleep 8:49-55; 1985. 30. Veldhuis, H. D.; De Korte, C. C. M. M.; De Kloet, E. R. Glucocorticoids facilitate the retention of acquired immobility during forced swimming. Eur. J. Pharmacol. 115:211-217; 1985.
0031-9384/91 $3.00 + .00
Effects of Diazepam, Pentobarbital, Scopolamine and the Timing of Saline Injection on Learned Immobility in Rats J U A N M. D E P A B L O , J A V I E R O R T I Z - C A R O , F E R N A N D O S A N C H E Z - S A N T E D
AND ANTONIO GUILLAMON 1
Departamento de Psicobiologfa, Universidad Nacional de Educaci6n a Distancia, Ciudad Universitaria P.O. Box 50487, 28040 Madrid, Spain R e c e i v e d 21 A u g u s t 1990 DE PABLO, J. M., J. ORTIZ-CARO, F. SANCHEZ-SANTED AND A. GUILLAMON. Effects of diazepam, pentobarbital, scopolamine and the timing of saline injection on learned immobility in rats. PHYSIOL BEHAV 50(5) 895-899, 1991.--The rat forced-swimming test (FST) is widely used for screening substances with a potential antidepressant effect. Rat immobility shown in the FST has been interpreted as "behavioral despair" and has been suggested as an animal model of human depression. In the following series of experiments, it is shown that pentobarbital and scopolamine administered immediately after the first phase, and diazepam administered 15 minutes before the first phase, behave as false positives in the FST. It is concluded that the learningmemory hypothesis seems to cope better with the behavior of rats during the FST than the "behavioral despair" hypothesis. It is also shown that the sensitivity of the FST is affected by the fact that the last saline injection, one hour before the second phase, increases the animals' mobility. Forced-swimming test Automatic recording Pentobarbital Behavioral despair Learned immobility Memory
Diazepam
Scopolamine
Saline injection
10, 22, 29). Finally, some substances that score as false positives in the FST also deteriorate memory processes (1,5): these include anticholinergics (7,12), convulsants (17,18), gabaergic substances (2,8) and anisomycin (6), an inhibitor of protein synthesis. In a previous paper (6), we showed that anisomycin acts as a false positive in the FST, thus supporting our hypothesis that the behavioral process underlying behavior in the FST is related to acquisition and consolidation mechanisms. The main purpose of the present study was to determine if diazepam, pentobarbital and scopolamine, three substances that are not antidepressants and that deteriorate memory (1), could behave as false positives in the FST. In Experiment 1, the diazepam and pentobarbital were administered at two different times: immediately after the first phase or one hour before the second phase. The two conditions were chosen because the effect of a substance on the process of acquisition and/or consolidation depends on the time of administration to the subject in relation to training. Martinez et al. (16) suggest the use of posttraining treatments for testing consolidation. Since the amnesic effects of scopolamine, an anticholinergic, are very well known (1), it could be predicted that its administration immediately after the first phase of the FST would affect the habitual decrease of mobility seen during the second phase of the FST, thus supporting the view that memory is the underlying behavioral process in the FST. Because diaz-
THE rat forced-swimming test (FST) was introduced by Porsolt et al. (21) as a new animal model sensitive to antidepressant treatments. Later, Porsolt (23) suggested that the FST could be considered a reliable animal model of human depression to be added to other current models in the field such as "learned helplessness" (24) and "separation" (27) models. Briefly, when rats or mice are forced to swim in a confined inescapable space (usually a cylinder), they tend to become immobile (float) after vigorous activity. This immobility was characterized by Porsolt as "behavioral despair." The FST is conducted in two consecutive phases for rats and sometimes for mice (17,18). The first phase usually lasts 15 minutes, while the second phase, carded out 24 hours later, only lasts 5 or 6 minutes. Potentially antidepressive substances are commonly administered to rats 23 hours 45 min, 5 hours and 1 hour before the second phase. The animals' behavior (duration of immobility or changes in mobility) is evaluated during the 5 min of the second phase of the test. There is data in the literature showing that the animal's experience with the FST plays an important role in swimming behavior: first, immobility increases (or swimming activity decreases) throughout the FST (6,22). Furthermore, swimming activity decreases in the second phase with respect to the first phase (6,30). Secondly, antidepressant treatments such as electroconvulsive shock (ECS) and REM sleep deprivation, which affect memory processes (25,26), also increase swimming activity in the FST (9, 1Requests for reprints should be addressed to A. Guillam6n.
895
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896
epam has been found to induce anterograde amnesia (3,28), it was administered 15 min before the first phase to one group of rats. Previous observations (6) and the behavior of control rats (injected only with saline) in Experiment 1 led us to believe that the timing of the saline injection in control groups could have different effects on rats' swimming behavior. This question was therefore addressed in Experiment 2. EXPERIMENT 1 METHOD
Animals Ninety-one male Wistar rats (ALIN, Madrid, Spain) weighing 170-230 g were used. Subjects were caged in groups of four or five. Food and water were continuously available in a room maintained at 21--2°C, with controlled light-dark cycle (light: 0700 to 1900 h).
Apparatus A Panlab Animal Activity System (PAAS, Panlab, Barcelona, Spain) and a Plexiglas cylinder (height: 50 cm; diameter: 19.5 cm) were used (6). The PAAS system consisted of a sensory unit, an electronic counter, a printer and a control unit. General activity (mobility) was evaluated as a function of the variations produced by the rats' swimming activity on the standard frequency (484 KHz) of the electromagnetic field of the sensory unit. Frequency variations were transformed into voltage changes, which, in turn, were converted into impulses that were collected by the counter when they reached a certain level.
Procedure Thirty-three subjects were randomly assigned to six groups for the administration of diazepam or pentobarbital (kindly provided by Servicio de Restricci6n de Estupefacientes del Ministerio de Sanidad y Consumo) and saline immediately after the first phase: the control group ( N = 6 ) received saline (5 ml/kg) after training in the first phase; a second, third and fourth group ( N = 5 ) were injected a dose of diazepam (2.5, 7.5 mg/kg or 15 mg/kg respectively) dissolved in 5 ml/kg of saline after training in the first phase; and a fifth and sixth group (N = 6) were administered a dose of 7.5 or 15 mg/kg of pentobarbital 15 minutes after training in the first phase. Twenty-four raics were also randomly assigned to three different groups for the administration of scopolamine immediately after the first phase: the control group (N = 8) received 5 ml/kg of saline; two other groups ( N = 8 ) received 0.5 mg/kg and 1 mg/kg of scopolamine hydrochloride (Sigma) dissolved in 5 ml/kg of saline respectively. Twenty different subjects were randomly assigned to three groups for administration of diazepam and pentobarbital and saline one hour before the second phase: the control group (N = 6) received saline (5 ml/kg); a second and third group (N = 7) were injected a dose of diazepam or pentobarbital (7.5 mg/kg) respectively, dissolved in 5 ml/kg of saline. Finally, fourteen subjects were also randomly administered saline ( N = 6 ) or a single dose of diazepam (2.5 mg/kg) dissolved in 5 mt/kg ( N = 8 ) 15 minutes before the first phase. Training during the first phase lasted 15 minutes and testing in the second phase lasted 5 minutes. Individual rats were forced to swim inside an upright cylinder containing 15 cm of water at 25°C. Swimming activity (mobility) was automatically recorded during the two sessions. The data were statistically treated as
TABLE 1 EFFECT OF DIAZEPAM AND PENTOBARBITAL INJECTEI) IMMEDIATEI "~ AFTER THE FIRST PHASE ON RAT MOBILITY 1N TIlE FST
Treatment Control Diazepam Diazepam Diazepam Pentobarbital Pentobarbital
Doses
N
Saline 2.5 mg/kg 7.5 mg/kg 15 mg/kg 7.5 mg/kg t5 mg/kg
6 5 5 5 6 6
% of Impuise~ :L SEM 38.5 53.72 35.0 39.2 85,6 78.4
:. i i~43 - t2.3 :: 13.99 :: 14.99 _*__15.81" - 21.88
*p<0.05 with respect to the control group.
follows: mobility in the second phase was expressed as the percentage of change in relation to each subject's number of impulses during the first 5 minutes of the first phase, then submitted for Student's t-test (one tailed). RESULTS
Effect of the Administration of Diazepam and Pentobarbital Immediately After the First Phase of the FST As can be seen in Table 1, the rats treated with 7.5 mg/kg of pentobarbital showed significantly greater mobility than the control animals in the 5 min swim test, t(10)= 2.63, p<0.05. However, a 15 mg/kg dose of pentobarbital did not reach a statistically significant effect, t(10)= 1.77, p < 0 . 1 . Nevertheless, this group of rats did not differ from those injected with 7.5 mg/kg of pentobarbital in the level of mobility. None of the three doses of diazepam affected the rats' swimming activity.
Effect of the Administration of Scopolamine Immediately After the First Phase of the FST As can be seen in Table 2, the administration of 1 mg/kg of scopolamine immediately after the first phase increased the rats' mobility in the FST with respect to the control group, t(14)= 2.77, p<0.01; however, a lesser dose (0.5 mg/kg) did not affect mobility, t(14) = p < 0 . 1 0 .
Effect of the Administration of Diazepam or Pentobarbital One Hour Before the Second Phase of the FST As can be observed in Table 3, the administration of diazepam or pentobarbital one hour before the second phase did not affect the rats' swimming activity in the FST [diazepam vs.
TABLE 2 EFFECT OF SCOPOLAMINE INJECTED IMMEDIATELY AFTER THE FIRST PHASE ON RAT MOBILITY IN THE FST
Treatment Control Scopolamine Scopolamine
Doses
N
% of Impulses -+ SEM
Saline 0.5 mg/kg 1.0 mg/kg
8 8 8
45.4 _+ 8.88 60.98 ± 7.79 85.07 ± 12.5"
*p<0.01 with respect to the control group.
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TABLE 3 EFFECT OF D1AZEPAMAND PENTOBARBITALINJECTEDONE HOUR BEFORETHE SECONDPHASEON RAT MOBILITYIN THE FST Treatment Control Diazepam Pentobarbital
Doses
N
% of Impulses --- SEM
Saline 7.5 mg/kg 7.5 mg/kg
6 7 7
80.82 -+ 7.92 65.06 - 11.66 88.52 + 9.56
control: t(11)=1.17, 0.66, N.S.].
N.S.; pentobarbital vs. control: t ( l l ) =
Effect of Administration of Diazepam 15 Minutes Before the First Phase of the FST Table 4 shows that a single injection of 2.5 mg/kg of diazepam administered 15 min before the first phase increases the rats' mobility in the second phase of the FST, t(12)=2.75, p<0.01. Finally, if we compare the performance of the control groups of the four assays (see Tables 1, 2, 3 and 4), it can be seen that rats injected with saline one hour before the second phase present more mobility than the other control groups, t(10)= 3.27, p<0.01; t(12)= 3.21, p<0.01; t(10)=5.65, p<0.001. No treatment affected the behavior of rats during the first phase. EXPERIMENT 2 Unexpectedly, in Experiment 1 it was observed that the control group injected with saline one hour before the second phase presented greater mobility than those injected 15 min before or immediately after the first phase. This might have methodological and theoretical implications for the interpretation of the FST. If the pure saline injection behaves as a false positive, depending upon its timing, it poses serious constraints for the use of the FST as a technique for screening antidepressant substances, at least when subjects are injected one hour before the second phase of the test. Differences in mobility shown by the control group in Experiment 1 might be dependent upon our specific procedure, i.e., we injected the subjects only one time during the entire test. However, since introduction of the test by Porsolt, the common procedure has been to inject animals 23 hours 45 min, 5 hours and 1 hour before the second phase. Thus in the present experiment we have compared the performance in the FST of rats injected with saline, taking into account the number of injections and their timing. METHOD
Animals Forty-three male rats of the Wistar strain (ALIN, Madrid, Spain) were used. They possessed similar characteristics and
Treatment
N
% of Impulses _ SEM
Uninjected Control 1 hour before the second phase Immediately after the first phase 15 min before the first phase 23.45, 5 and 1 h before second phase Three doses (2 h interval)
9 6 7 6 8
56.12 80.5 45.42 38.27 82.66
7
42.82 _
-+ -+ + -+ ---
10.6 8* 12.5t:~ ll.6t:~ 4.79* 6.4"t~:
*p<0.05 with respect to uninjected control group. 1"p<0.01 with respect to 1 hour before second phase group. :~p<0.01 with respect to 23.45, 5 and 1 h before second phase group.
housing to those reported in Experiment 1.
Procedure The subjects were randomly assigned to the following conditions with respect to the number and time of injections: group not injected (Uninjected Control group; N = 9 ) ; group injected one hour before the second phase (N = 6); group injected immediately after the first phase ( N = 7 ) ; group injected 15 minutes before the first phase ( N = 6 ) ; group injected three times (23 hours 45 minutes, 5 hours and 1 hour before the second phase, usual control group for screening antidepressant substances with the FST; N = 8), and group injected three times with an interval of two hours beginning 15 minutes before the first phase (N = 7). Subjects swam 15 minutes in the first phase and 24 hours later they swam 5 minutes in the second phase, as in Experiment 1. The data was also recorded and analysed as in Experiment 1. RESULTS As shown in Table 5, the group commonly used according to the literature for screening antidepressant substances and the group injected one hour before the second phase presented greater mobility than the uninjected group [t(15)= 2.28, p < 0 . 0 5 and t(13)= 1.79, p<0.05, respectively]. No statistically significant differences were seen between the uninjected control group and the group injected three times with an interval of two hours beginning 15 minutes before the first phase, t(14)= 1.07, N.S. Nor was the difference between the uninjected control group and the group injected immediately after the first phase found to be significant, t(14)=0.71, N.S. Also, no differences were seen between the uninjected control group and the group injected 15 minutes before first phase, t(13) = 1.16, N.S. For other comparisons, see Table 5. GENERAL DISCUSSION
TABLE 4 EFFECT OF DIAZEPAM INJECTED 15 MINUTESBEFORE THE FIRSTPHASEON RAT MOBILITYIN THE FST Treatment
Doses
N
% of Impulses _ SEM
Control Diazepam
Saline 2.5 mg/kg
6 8
26.24 _ 7.07 79.44 _ 17"
*p<0.01 with respect to the control group.
TABLE 5 EFFECT OF NUMBERAND TIME OF SALINEINJECTION ON RAT MOBILITYIN THE FST
Pentobarbital, scopolamine and diazepam behave as false positives in the FST. A single dose of 7.5 mg/kg of pentobarbital administered immediately after the first phase, but not one hour before the second phase of the FST, increased mobility in animals during the 5-minute test of the second phase. A similar treatment with diazepam did not, nevertheless, affect rat mobility in the second phase of the FST. However, when diazepam was administered 15 minutes before the first phase it increased rat mobility 24 hours later during the second phase. A single
~gx
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dose of 1 mg/kg of scopolamine administered immediately after the first phase also increased mobility. Although a decrease in mobility is currently being accepted in the literature as an indication of "behavioral despair" (23), we have proposed that the behavioral process involved in the FST is learning to be immobile (6). Our results with pentobarbital (a barbiturate), diazepam (an anxiolytic) and scopolamine (an anticholinergic), three substances that are not antidepressants, support our previous hypothesis. Reports in the literature show that pentobarbital deteriorates memory in humans and other species (1). Interestingly enough, diazepam, which is reputed to induce anterograde amnesia (3, 19, 28), only increases mobility in the FST when injected before the first phase. The fact that an anticholinergic substance such as scopolamine behaves as a false positive in the FST with a procedure that directly addresses swimming activity supports once again our hypothesis that learning and memory ore the behavioral processes involved in the behavior of rats in the FST. It should be remembered that cholinergic mechanisms in the CNS are directly involved in mediating diverse learning and memory phenomena (1). In Experiment 1, we observed statistically significant differences among the control (saline) groups, while the only procedural difference among these four groups was the time at which the saline injection was administered. When this problem was addressed in Experiment 2, it was observed that indeed the injection of saline one hour before the second phase acted as a false positive. Furthermore, rats injected with saline three times, 23 hours 45 min, 5 hours and 1 hour before the second phase (the usual control group in the FST for testing antidepressant substances), presented greater mobility than the uninjected rats. This increment in mobility does not depend on the number of injections, since three injections of saline scheduled before and after the first phase did not increase mobility during the second phase. Thus the sensitivity of the FST is affected by the fact that the last saline injection, one hour before the second phase, increases animals' mobility. There are reports in the literature showing that chronic stress decreases the duration of immobility in mice (20). However, to our knowledge, the possible stressing effect of saline injection in the FST has not been studied. Porsolt's data (22) comparing saline-injected (24, 5 and 1 hour before the second phase) and uninjected rats shows no differences between these groups. The discrepancies between our results in Experiment 2 and those reported by Porsolt might be explained by differences in procedures: Porsolt estimated by observation the duration of immobility, whereas we automatically measured swimming activity. Thus it could be possible that mobility and
not duration of immobility is affected by the stres~ reduced t~'~ the saline injection administered one hour befi)re the second phase. Interestingly, as can be seen in Table 3, aeither pento barbital nor diazepam were able to block this eltect when in-jected one hour before the second phase. Furthernlore, we have shown that the automatic procedure presents greater sensitivi!!~ than the direct observation of the duration of immobility (61. Taking into account the results of this and previous studies (6), it appears that the learning-memory hypothesis copes with the behavior of rats during the FST better than the "behavioral despair" hypothesis previously suggested in the literature (23). As we have pointed out (6), there is data in the literature showing that antidepressant substances impair memoD' both in humans (4, 13, 14) and rats (11,15). Judd et al. (11) suggest that the effect of amitriptyline and imipramine on human memory is similar to that of scopolamine. In rats it was reported that imipramine, amitriptyline, desipramine and mianserine impair avoidance responses (11,15). As we previously have pointed out. some antidepressant substances selected by means of the FST might be selected because of their effect on m e m o u . Although at this moment it is not possible to discard other actions of antidepressants affecting mobility in the FST (viz., on general activity or reactivity), in our opinion, the learning-memory hypothesis seems to cope better with the behavior of rats during the FST than the "behavioral despair" hypothesis for several reasons: first, it can explain the effect of anticholinergics, anxiolytics, convulsants and anisomycin, and it is also able to explain the effect of antidepressant substances, REM sleep deprivation and electroconvulsive shock in the FST. Second, it can cope with the fact that a single injection of imipramine (6), pentobarbital, diazepam or scopolamine is effective in the FST; it is known that antidepressant treatments are therapeutically effective after chronic administration only. And third, the learning-memory hypothesis springs from the experimental analysis of rats' behavior, whereas the "behavioral despair" hypothesis is an anthropomorphic interpretation of immobility in the FST. This interpretation has led to the following circular reasoning: since antidepressants change rats' immobility in the FST, such a behavior is a symptom of animal depression, and, as a result, it is assumed that substances that reduce rats' immobility as antidepressants do are antidepressants. ACKNOWLEDGEMENTS This work was supported by grants PB87-0621 and PB88/158 from the DGICYT. The authors wish to express their appreciation to A. A. Caminero, Mary Frances Litzler, M. de Amaral, L. Carrillo, L. Troca and G. Moreno for their technical assistance.
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