European Journal of Pharmacology, 181 (1990) 35-41
35
Elsevier EJP 51324
Picrotoxin changes the effects of imipramine and desipramine in rats in the forced swimming test Alberto Fernfindez-Teruel, Rosa M. Escorihuela, Fernando Boix 1 and Adolf Tobefia Medical Psychology Unit, Department of Pharmacology and Psychiatry, School of Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain
Received 24 October 1989, revised MS received 14 February 1990, accepted 6 March 1990
The aim was to find whether the chloride channel blocker, picrotoxin, at subconvulsant doses could affect the activity of imipramine or desipramine in the 'forced swimming' test with rats. It was found that picrotoxin enhanced the anti-immobility effects of imipramine and desipramine whereas open field activity remained unaffected or was even decreased by the same treatments. The results seem consistent with recent reports showing direct interactions between several antidepressant drugs and the GABA A receptor/benzodiazepine receptor/chloride ionophore complex (GABAA/benzodiazepine/C1 complex). The results also conform with the hypothesis that a reduction in the functionality of this complex could be related to the clinical effects of antidepressant drugs. Forced swimming test; Picrotoxin; Imipramine; Desipramine; GABAA-benzodiazepine-C1 complex; Open field
1. Introduction
The possible involvement of GABAergic transmission in depression and in the mechanism of action of antidepressant treatments has been much studied (for reviews see Bartholini et al., 1985; 1986; Lloyd and Pichat, 1986). It has been shown that different chronic antidepressant treatments up-regulate G A B A B receptors (Lloyd et al., 1985) and down-regulate both benzodiazepine (SuranyiCadotte et al., 1985) and G A B A A recognition sites (Suzdak and Gianutsos, 1985), although some resuits are conflicting (Cross and Horton, 1988; Kimber et al., 1987; Pilc and Lloyd, 1984).
1 Present address: Institut far PhysiologischePsychologie, Psychologisches Institut, Universitat Diisseldorf, Universit~itstrasse 1, 4000 Diisseldorf, F.R.G. Correspondence to: A. Fern~mdez-Teruel, Medical Psychology Unit, Department of Pharmacology and Psychiatry, School of Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain.
Likewise, we have recently observed that chronic imipramine administration to rats decreased the GABA-stimulated 36C1- uptake in cerebral synaptoneurosomes (Fernfindez-Teruel et al., 1989b). Also, several antidepressant drugs and metabolites decrease the function of the G A B A A receptor-benzodiazepine receptor/chloride ionophore ( G A B A A / b e n z o d i a z e p i n e / C 1 ) complex in vitro by a direct interaction with the complex (Malatynska et al., 1988; Squires and Saeredup, 1985). Again in line with the hypothesis linking G A B A function with depression, there are several reports that some GABA-mimetic drugs have antidepressant-like properties in several animal models of depression, e.g. the 'olfactory bulbectomized rat' and the 'learned helplessness' test (Lloyd et al., 1983; 1987; Poncelet et al., 1987; Sanger et al., 1986), or the 'forced swimming' test with rats (Borsini et al., 1986a; Fernfindez-Teruel et al., 1988; Poncelet et al., 1987). Accordingly, the GABA-mimetics, progabide and fengabine, show
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
36 clinical antidepressant activity, their effectiveness being similar to that of imipramine (Musch, 1986; Weiss et al., 1986). Nevertheless, the functional meaning of the changes observed in the GABAA/benzodiazepine/C1 complex after antidepressant administration has not been clearly established, although some authors have suggested that the decrease in function could have a role in the therapeutic effects of antidepressant drugs (Squires and Saeredup, 1988). To further explore this possibility, we evaluated whether the chloride channel blocker, picrotoxin, at subconvulsant doses interacts with the effects of two tricyclic antidepressants (imipramine and desipramine) in the forced swimruing test with rats. This test is widely used for screening antidepressants.
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats (Autonomous University of Barcelona) were used in all the experiments. The rats were housed in groups of 3-4 per cage, kept on a controlled light-dark schedule (light on between 0 8 : 0 0 ansd 20:00 h) and at constant room temperature (22 + 2° C) with ad lib access to food and water. 2.2. Drugs Imipramine and desipramine hydrochloride (Ciba-Geigy), and picrotoxin (SIGMA CHEMICAL) were dissolved in 0.9% NaC1 and injected i.p. in a volume of 2 ml/kg. Control treatments were injections of 0.9% NaC1 2 ml/kg. 2.3. Swim test Individual rats were forced to swim inside vertical plexiglass cylinders (height: 40 cm; diameter: 18 cm) containing 18 cm of water at 25 °C. The rats were removed after 15 min and allowed to dry for 15 min. The rats were replaced in the cylinders for 5 min 24 h later and the time of
immobility ws measured as described by Porsolt et al. (1978). The first drug injection was given at the end of the drying period. The second injection was given 5 h before the 5-min swim test. The last antidepressant injection was administered 1 h (antidepressants or corresponding vehicle), whereas the last picrotoxin (or the corresponding vehicle) injection was always administered 20 rain before the 5-min swim test (exp. 1-3). When only one injection of picrotoxin was given (exp. 4), it was also administered 20 rain before the 5-min swim session.
2.3.1. Experiments 1-3: effects of two or three concomitant injections of antidepressants and picrotoxin on immobility In experiments 1-3 rats weighing 386 + 19 g were used. Imipramine (exp. 1) was injected at 10, 20 and 30 m g / k g (IMI10, IMI20 and IMI30) and desipramine (exp. 2A) at 7.5 and 15 m g / k g (DMI7.5 and DMI15). In both experiments picrotoxin was either injected three times at 0.5 m g / k g (PIC0.5 treatment), or twice at 1 m g / k g plus a third injection of 0.6 m g / k g (PIC1 treatment). Experiment 2B was carried out in order to test the effects of higher doses of desipramine alone (20 and 25 m g / k g ) in the forced swimming test. In experiment 3 the rats were given two injections of desipramine (15 or 30 mg/kg) and two concomitant injections of picrotoxin (0.5 or 1 mg/kg) between the swim sessions. The first injection was given immediately after the drying period (15 min after the 15-min swim session), and the second injection was administered 60 (desipramine or vehicle) or 20 min (picrotoxin or vehicle) before the 5-rain swim session.
2.3.2. Experiment 4: effect of three antidepressant injections plus one picrotoxin injection on immobility Rats weighing 318 _+ 35 g were used. Desipramine (15 and 20 m g / k g ) and imipramine (20 mg/kg) were administered as in experiments 1-2. Only a single picrotoxin injection (0.5, 1 and 2 mg/kg) was given 20 min before the 5-min swim test.
37
2.4. Open field experiments In order to measure whether changes in immobility were related with changes in motor activity, the animals were treated with the most relevant drug treatments and were tested for activity in the open field test as previously described by Porsolt et al. (1978) and by Gomfi and Tobe?m (1978). The apparatus was composed of a circular arena (diameter: 83 cm) divided into 19 sectors of equal area marked on the floor. The arena was made of plywood and beige melamine plastic. The wall was white and 34 cm high. The animals had identical conditions of illumination and no noise as the rats used for the swim experiments. Drug treatment schedules were also identical to those for the swim experiments. After the last injection, each rat was placed in the center of the box, and scored as follows: ambulation, number of sectors entered with all four paws; rearing, standing on its hind legs. Each open field session lasted 5 min.
2.5. Statistical analysis Since differences between variances of groups were observed (Snedecor's F-test) all data were analyzed using the non-parametric Kruskal-Wallis analysis of variance, followed by Mann-Whitney's U-comparisons between pairs of groups.
300.
o
4(
-
250.
T r
c--
I ~ II1 0
201.
I
--
; 1
150.
6
5
5
5
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5 i
6
0
0
0.5
I
10
10
10
20
0
0.5
I
0
1" IMI (r~/Ko) O PIC
,,
0
EXP,
1 6
6
20
20
30
30
30
0.5
1
0
0.5
I
1
Fig. 1. Effect of the combination of imipramine ([MI, three injections) plus picrotoxin (PIC, three injections) on the time of immobility in the 5-min swimming test. Means _+S.E. are shown, and the n u m b e r of animals used is given in each column. * P ~< 0.025 vs. control (IMI 0 plus PIC 0) group. * * P ~< 0.0025 vs. control group. • P ~< 0.025 vs. IMI 30 m g / k g plus PIC 0 m g / k g group (all comparisons were one-tailed).
ramine 30 m g / k g + picrotoxin 0.5 m g / k g (IMI30 + PICO.5) was injected, so that this group was significantly different from the control group 3O0,
2so
.T.
3. Results
In agreement with our previous results (Fernfindez-Teruel, 1989), picrotoxin enhanced the effects of imipramine when both drugs were administered simultaneous to rats in the forced swimming test. In experiment 1 (fig. 1), the significance shown in the Kruskal-Wallis analysis (H = 26.26, P = 0.0059) was corroborated by the significant Mann-Whitney's U-comparisons between control group (IMI0 + PIC0) and imipramine 20 m g / k g (IMI20 + PIC0), imipramine 20 m g / k g + picrotoxin 0.5 m g / k g (IMI20 + PIC0.5), imipramine 20 m g / k g + picrotoxin 1 m g / k g (IMI20 + PIC1) and imipramine 30 m g / k g + picrotoxin 1 m g / k g (IMI30 + PIC1) groups (P < 0.025; see fig. 1). The clearest effect was observed when imip-
DMI(r~/Kg)0
0
0
7.5 7.5 7.5
15
15
15
PIE
0.5
I
0
0.5
0
0,5
1
EXP.
2A
0
I
0
li 20
25
2B
Fig. 2. Effect of the combination of desipramine (DMI, three injections) plus picrotoxin (PIC, three injections) on the time of immobility in the 5-rain swimming test. M e a n s + S . E . are shown, and the n u m b e r of animals used is given in each column. Exp. 2A: * P ~< 0.05 vs. control (DMI 0 plus PIC 0) group. ** P ~< 0.02 vs. control, • P ~ 0.05 vs. DMI 15 m g / k g plus PIC 0 m g / k g group. Exp 2B: * P ~< 0.01 and * * P ~
35
Elsevier EJP 51324
Picrotoxin changes the effects of imipramine and desipramine in rats in the forced swimming test Alberto Fernfindez-Teruel, Rosa M. Escorihuela, Fernando Boix 1 and Adolf Tobefia Medical Psychology Unit, Department of Pharmacology and Psychiatry, School of Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain
Received 24 October 1989, revised MS received 14 February 1990, accepted 6 March 1990
The aim was to find whether the chloride channel blocker, picrotoxin, at subconvulsant doses could affect the activity of imipramine or desipramine in the 'forced swimming' test with rats. It was found that picrotoxin enhanced the anti-immobility effects of imipramine and desipramine whereas open field activity remained unaffected or was even decreased by the same treatments. The results seem consistent with recent reports showing direct interactions between several antidepressant drugs and the GABA A receptor/benzodiazepine receptor/chloride ionophore complex (GABAA/benzodiazepine/C1 complex). The results also conform with the hypothesis that a reduction in the functionality of this complex could be related to the clinical effects of antidepressant drugs. Forced swimming test; Picrotoxin; Imipramine; Desipramine; GABAA-benzodiazepine-C1 complex; Open field
1. Introduction
The possible involvement of GABAergic transmission in depression and in the mechanism of action of antidepressant treatments has been much studied (for reviews see Bartholini et al., 1985; 1986; Lloyd and Pichat, 1986). It has been shown that different chronic antidepressant treatments up-regulate G A B A B receptors (Lloyd et al., 1985) and down-regulate both benzodiazepine (SuranyiCadotte et al., 1985) and G A B A A recognition sites (Suzdak and Gianutsos, 1985), although some resuits are conflicting (Cross and Horton, 1988; Kimber et al., 1987; Pilc and Lloyd, 1984).
1 Present address: Institut far PhysiologischePsychologie, Psychologisches Institut, Universitat Diisseldorf, Universit~itstrasse 1, 4000 Diisseldorf, F.R.G. Correspondence to: A. Fern~mdez-Teruel, Medical Psychology Unit, Department of Pharmacology and Psychiatry, School of Medicine, Autonomous University of Barcelona, 08193 Bellaterra, Barcelona, Spain.
Likewise, we have recently observed that chronic imipramine administration to rats decreased the GABA-stimulated 36C1- uptake in cerebral synaptoneurosomes (Fernfindez-Teruel et al., 1989b). Also, several antidepressant drugs and metabolites decrease the function of the G A B A A receptor-benzodiazepine receptor/chloride ionophore ( G A B A A / b e n z o d i a z e p i n e / C 1 ) complex in vitro by a direct interaction with the complex (Malatynska et al., 1988; Squires and Saeredup, 1985). Again in line with the hypothesis linking G A B A function with depression, there are several reports that some GABA-mimetic drugs have antidepressant-like properties in several animal models of depression, e.g. the 'olfactory bulbectomized rat' and the 'learned helplessness' test (Lloyd et al., 1983; 1987; Poncelet et al., 1987; Sanger et al., 1986), or the 'forced swimming' test with rats (Borsini et al., 1986a; Fernfindez-Teruel et al., 1988; Poncelet et al., 1987). Accordingly, the GABA-mimetics, progabide and fengabine, show
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
36 clinical antidepressant activity, their effectiveness being similar to that of imipramine (Musch, 1986; Weiss et al., 1986). Nevertheless, the functional meaning of the changes observed in the GABAA/benzodiazepine/C1 complex after antidepressant administration has not been clearly established, although some authors have suggested that the decrease in function could have a role in the therapeutic effects of antidepressant drugs (Squires and Saeredup, 1988). To further explore this possibility, we evaluated whether the chloride channel blocker, picrotoxin, at subconvulsant doses interacts with the effects of two tricyclic antidepressants (imipramine and desipramine) in the forced swimruing test with rats. This test is widely used for screening antidepressants.
2. Materials and methods
2.1. Animals Male Sprague-Dawley rats (Autonomous University of Barcelona) were used in all the experiments. The rats were housed in groups of 3-4 per cage, kept on a controlled light-dark schedule (light on between 0 8 : 0 0 ansd 20:00 h) and at constant room temperature (22 + 2° C) with ad lib access to food and water. 2.2. Drugs Imipramine and desipramine hydrochloride (Ciba-Geigy), and picrotoxin (SIGMA CHEMICAL) were dissolved in 0.9% NaC1 and injected i.p. in a volume of 2 ml/kg. Control treatments were injections of 0.9% NaC1 2 ml/kg. 2.3. Swim test Individual rats were forced to swim inside vertical plexiglass cylinders (height: 40 cm; diameter: 18 cm) containing 18 cm of water at 25 °C. The rats were removed after 15 min and allowed to dry for 15 min. The rats were replaced in the cylinders for 5 min 24 h later and the time of
immobility ws measured as described by Porsolt et al. (1978). The first drug injection was given at the end of the drying period. The second injection was given 5 h before the 5-min swim test. The last antidepressant injection was administered 1 h (antidepressants or corresponding vehicle), whereas the last picrotoxin (or the corresponding vehicle) injection was always administered 20 rain before the 5-min swim test (exp. 1-3). When only one injection of picrotoxin was given (exp. 4), it was also administered 20 rain before the 5-min swim session.
2.3.1. Experiments 1-3: effects of two or three concomitant injections of antidepressants and picrotoxin on immobility In experiments 1-3 rats weighing 386 + 19 g were used. Imipramine (exp. 1) was injected at 10, 20 and 30 m g / k g (IMI10, IMI20 and IMI30) and desipramine (exp. 2A) at 7.5 and 15 m g / k g (DMI7.5 and DMI15). In both experiments picrotoxin was either injected three times at 0.5 m g / k g (PIC0.5 treatment), or twice at 1 m g / k g plus a third injection of 0.6 m g / k g (PIC1 treatment). Experiment 2B was carried out in order to test the effects of higher doses of desipramine alone (20 and 25 m g / k g ) in the forced swimming test. In experiment 3 the rats were given two injections of desipramine (15 or 30 mg/kg) and two concomitant injections of picrotoxin (0.5 or 1 mg/kg) between the swim sessions. The first injection was given immediately after the drying period (15 min after the 15-min swim session), and the second injection was administered 60 (desipramine or vehicle) or 20 min (picrotoxin or vehicle) before the 5-rain swim session.
2.3.2. Experiment 4: effect of three antidepressant injections plus one picrotoxin injection on immobility Rats weighing 318 _+ 35 g were used. Desipramine (15 and 20 m g / k g ) and imipramine (20 mg/kg) were administered as in experiments 1-2. Only a single picrotoxin injection (0.5, 1 and 2 mg/kg) was given 20 min before the 5-min swim test.
37
2.4. Open field experiments In order to measure whether changes in immobility were related with changes in motor activity, the animals were treated with the most relevant drug treatments and were tested for activity in the open field test as previously described by Porsolt et al. (1978) and by Gomfi and Tobe?m (1978). The apparatus was composed of a circular arena (diameter: 83 cm) divided into 19 sectors of equal area marked on the floor. The arena was made of plywood and beige melamine plastic. The wall was white and 34 cm high. The animals had identical conditions of illumination and no noise as the rats used for the swim experiments. Drug treatment schedules were also identical to those for the swim experiments. After the last injection, each rat was placed in the center of the box, and scored as follows: ambulation, number of sectors entered with all four paws; rearing, standing on its hind legs. Each open field session lasted 5 min.
2.5. Statistical analysis Since differences between variances of groups were observed (Snedecor's F-test) all data were analyzed using the non-parametric Kruskal-Wallis analysis of variance, followed by Mann-Whitney's U-comparisons between pairs of groups.
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--
; 1
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6
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6
0
0
0.5
I
10
10
10
20
0
0.5
I
0
1" IMI (r~/Ko) O PIC
,,
0
EXP,
1 6
6
20
20
30
30
30
0.5
1
0
0.5
I
1
Fig. 1. Effect of the combination of imipramine ([MI, three injections) plus picrotoxin (PIC, three injections) on the time of immobility in the 5-min swimming test. Means _+S.E. are shown, and the n u m b e r of animals used is given in each column. * P ~< 0.025 vs. control (IMI 0 plus PIC 0) group. * * P ~< 0.0025 vs. control group. • P ~< 0.025 vs. IMI 30 m g / k g plus PIC 0 m g / k g group (all comparisons were one-tailed).
ramine 30 m g / k g + picrotoxin 0.5 m g / k g (IMI30 + PICO.5) was injected, so that this group was significantly different from the control group 3O0,
2so
.T.
3. Results
In agreement with our previous results (Fernfindez-Teruel, 1989), picrotoxin enhanced the effects of imipramine when both drugs were administered simultaneous to rats in the forced swimming test. In experiment 1 (fig. 1), the significance shown in the Kruskal-Wallis analysis (H = 26.26, P = 0.0059) was corroborated by the significant Mann-Whitney's U-comparisons between control group (IMI0 + PIC0) and imipramine 20 m g / k g (IMI20 + PIC0), imipramine 20 m g / k g + picrotoxin 0.5 m g / k g (IMI20 + PIC0.5), imipramine 20 m g / k g + picrotoxin 1 m g / k g (IMI20 + PIC1) and imipramine 30 m g / k g + picrotoxin 1 m g / k g (IMI30 + PIC1) groups (P < 0.025; see fig. 1). The clearest effect was observed when imip-
DMI(r~/Kg)0
0
0
7.5 7.5 7.5
15
15
15
PIE
0.5
I
0
0.5
0
0,5
1
EXP.
2A
0
I
0
li 20
25
2B
Fig. 2. Effect of the combination of desipramine (DMI, three injections) plus picrotoxin (PIC, three injections) on the time of immobility in the 5-rain swimming test. M e a n s + S . E . are shown, and the n u m b e r of animals used is given in each column. Exp. 2A: * P ~< 0.05 vs. control (DMI 0 plus PIC 0) group. ** P ~< 0.02 vs. control, • P ~ 0.05 vs. DMI 15 m g / k g plus PIC 0 m g / k g group. Exp 2B: * P ~< 0.01 and * * P ~
