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BEHAVIOURAL EFFECTS OF PINOLINE IN THE RAT FORCED SWIMMING, OPEN FIELD AND ELEVATED PLUS-MAZE TESTS
Pharmacological Research, Vol. 34, No. 1/2, 1996
BEHAVIOURAL EFFECTS OF PINOLINE IN THE RAT FORCED SWIMMING, OPEN FIELD AND ELEVATED PLUS-MAZE TESTS ¨ HKLA, JAANUS HARRO and LEMBIT RA ¨ GO REIN PA Department of Pharmacology, University of Tartu, EE2400 Tartu, Estonia Accepted 10 May 1996 Pinoline (6-methoxy-1,2,3,4-tetrahydro-β-carboline) is a naturally occurring compound in the mammalian body which inhibits serotonin (5-hydroxytryptamine) uptake and monoamine oxidase-A activity. The present study was designed to assess potential antidepressant- or anxiolytic-like behavioural effects of pinoline in rat forced swimming, open field and elevated plus-maze tests. In the forced swimming test pinoline dose-dependently reduced the immobility time, starting from a dose of 8 mg kg −1. In the open field test pinoline reduced the total open field activity. This effect was significant at 20 mg kg−1, whereas the dose of 15 mg kg −1 only significantly reduced the number of rearings. In the plus-maze test pinoline decreased the total number of arm entries, the number of line crossings and time spent in the open part of the apparatus, but increased the number of approaches to the open part of the maze. The effects of pinoline were dose-dependent in all three behavioural tests used. Since antidepressant drugs reduce the immobility time in the forced swimming test but typically inhibit activity in open field and elevated plus-maze tests, the behavioural effects of pinoline resemble those of drugs with an antidepressant profile. 1996 The Italian Pharmacological Society KEY WORDS: 6-methoxytetrahydro- β-carboline, forced swimming, elevated plus-maze, open field, rat.
INTRODUCTION Beta-carbolines are a large group of pharmacologically active compounds which have been found in different plants. Some of those have been reported to occur as normal constituents of mammalian tissues including those of humans [1, 2]. One of the most interesting members of this group is 6-methoxy1,2,3,4-tetrahydro-β-carboline (6-MeO-THBC, 5methoxytryptoline), named pinoline from the words ‘pineal β -carboline’. It has been found in pineal and adrenal glands and in the brain in concentrations from 2 ng g −1 or below [3] up to a concentration of 21 µg g −1 [4, 5]. Pinoline inhibits the activity of monoamine oxidase-A (MAO-A) [6], serotonin (5-hydroxytryptamine, 5-HT) uptake [7] and binds to the imipramine recognition site [8]. Pinoline was initially proposed to function as an endogenous ligand of the imipramine recognition site [4, 9]. Now it is recognized that imipramine binds to the same domain in the serotonin transporter that binds 5-HT or at least to the site partly overlapping the 5-HT recognition site [10]. Acute administration of pinoline has been shown to increase the brain 5-HT levels [11]. This effect is Correspondence to: Dr Rein Pa¨hkla, Department of Pharmacology, ¨ likooli 18, EE2400 Tartu, Estonia. University of Tartu, U 1043–6618/96/070073–06/$25.00/0
common to most antidepressants. Pinoline has been reported to exert some pharmacological and behavioural effects characteristic of serotoninergic drugs [12, 13] but it has never been studied in behavioural tests suitable to assess the antidepressive potential of a compound. The purpose of this study was to compare the effect of pinoline with tricyclic antidepressants in the forced swimming test in rats. In order to measure whether any possible changes in immobility in the forced swimming test are related to changes in motor activity, the open field test was performed as previously described by Porsolt et al. [14]. In addition, we assessed the effect of pinoline on exploratory activity in the elevated plus-maze test to reveal possible anxiogenic- or anxiolytic-like properties of the compound.
MATERIALS AND METHODS
Animals Adult male Wistar rats weighing 270–350 g were used in the forced swimming test and adult female Wistar rats weighing 210–270 g in the open field and elevated plus-maze tests. Animals obtained from Grindex (Riga, Latvia) were housed at the Laboratory Animal Department of the Faculty of Medicine of 1996 The Italian Pharmacological Society
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University of Tartu in polypropylene cages in groups of five for at least 4 weeks prior to testing. The animal room had a 12:12 h light–dark cycle with lights on at 0700 Food and drinking water were available ad libitum. All experiments were performed between 1200 and 1900.
Drug treatment Drugs used in the present experiments were 6-MeOTHBC, imipramine hydrochloride and amitriptyline hydrochloride (all from Sigma, St Louis, MO, USA). Pinoline was dispersed in an aqueous suspension of Tween 85 (1% vol.), imipramine and amitriptyline were dissolved in saline. Drugs were injected intraperitoneally in a volume of 1 ml kg −1 . Control groups received injections of Tween 85 watery solution of the same concentration, or saline, respectively.
Forced swimming test The technique first characterized by Porsolt and colleagues [14] was used. Briefly, rats were forced to swim in a glass box (floor 19×29 cm, height 39 cm) containing 25 cm of fresh water maintained at 25°C. On the first day of an experiment, rats were placed in water for 15 min. After the swimming session, each rat was removed from the water, partially dried with a paper towel, given an injection of the drug and returned to their home cage. On the following day, rats were given the drug treatment 30 min before forced swimming. The total duration of immobility during the first 5 minutes of the swimming session was recorded. The rat was judged to be immobile when it made only the necessary movements to keep its head above water level.
Open field test In the open field test rats were placed at the center of a rectangular arena 100×100 cm with 40-cm-high side walls. The arena was divided into 16 equal squares. Parameters registered 30 min after drug treatment during 4 min were the number of squares visited (with all four feet on one square) and the number of rearings. Both these measures were summed to yield total open field activity.
Elevated plus-maze test This test was conducted as first described by Handley and Mithani [15] with some modifications [16]. The plus-maze was made of wood, with two opposite open arms, 50×10 cm without any walls and two opposite closed arms of the same size with 40cm-high side walls and an end wall. The arms were connected by a central platform 10×10 cm. Both open arms were divided into three parts of equal size by lines which also separated the central platform from all arms. The central platform and open arms formed the ‘open part’ of the apparatus. The maze was elevated to a height of 50 cm from the floor. An entry into open arms was counted when the rat crossed the line
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between the central arena and an open arm with all four paws. The rat was considered to explore the open part of the apparatus when it had clearly crossed the line between a closed arm and the central arena with both its forepaws. The rat was placed on the central platform of the maze facing an open arm 30 min after injection. Behavioural measures taken during 4 min included: (a) the number of line crossings; (b) time spent in the open part of the apparatus; (c) the number of open arm entries; (d) the total number of arm entries; (e) the number of approaches to the open part. The latter measure reflects risk assessment activity of animals and was recorded when a rat approached the central platform but did not cross the line between the closed arm and the platform.
Statistical analysis One-way analysis of variance (ANOVA) was used to compare the data from all experiments. Post-hoc Fisher’s PLSD test was applied to compare group means with the control.
RESULTS
Forced swimming test Results of the forced swimming test are presented in Fig. 1. ANOVA indicated a significant difference among experimental groups in rats treated with imipramine [F(2,18)=4.61; P<0.05], amitriptyline [F(2,20)=5.57; P<0.05] and in both experiments with pinoline [F(2,21)=10.97; P<0.001 and F(4,35)=3.62; P<0.05]. Fisher’s PLSD test revealed that imipramine significantly reduced the duration of immobility at both doses tested and amitriptyline at the dose level of 30 mg kg−1. Pinoline reduced the immobility at the dose level of 20 mg kg −1 in the first pilot experiment (immobility time 151.3±8.8 s for control group and 93.5±11.5 s for 20 mg kg −1 group; P<0.01). In another independent experiment with multiple dose levels, the effect of pinoline was dose-dependent and already significant at the dose of 8 mg kg−1.
Open field test The effect of pinoline on the activity of rats in the open field test is shown on Figure 2. ANOVA indicated an overall effect of treatment for the total open field activity [F(4.35)=2.66; P<0.05] and for the number of rearings [F(4,35)=3.62; P<0.05] but the effect missed significance for the number of squares visited. Fisher’s PLSD test revealed that pinoline significantly reduced the total open field activity of rats at the dose of 20 mg kg −1 (P<0.05). The effect of the dose 15 mg kg−1 was just below the conventional significance level (P=0.06), but at this dose, pinoline significantly reduced (P<0.01) the number of rearings.
Elevated plus-maze The effect of treatment with different doses of pino-
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75 40 Number of squares visited
200 Immobility time (s)
A 150 *
**
100
50
30 20 10 0
0
Control
Control
15 mg/kg
10 mg/kg
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
15
200
B Number of rearings
B Immobility time (s)
A
150
100
**
50
10
5 **
0 0
Control
Control
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
30 mg/kg
10 mg/kg
Immobility time (s)
C
150 * * 100
***
50
Total open field activity
50 200
C 40 30
10 0
0
Control
2 mg/kg
8 15 mg/kg mg/kg
20 mg/kg
Fig. 1. Effects of treatment with imipramine (A), amitriptyline (B) and pinoline (C) on immobility time of rats in the forced swimming test. Data are presented as mean±SEM (n=8). Doses of different treatments are presented in abscissas of figures. *P<0.05, **P<0.01 and ***P<0.005 vs control; Fisher’s PLSD test after significant ANOVA.
line is presented in Table I. Pinoline significantly decreased the number of line crossings on open parts
*
20
Control
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
Fig. 2. The effect of pinoline (2–20 mg/kg) on (A) the number of squares visited in the open field, (B) the number of rearings and (C) the total open field activity (mean±SEM, n=8). Data were analysed using the one-way ANOVA and Fisher’s PLSD test for the difference from the control group. *P<0.05, **P<0.01.
[F(4,35)=8.19; P<0.0001], time spent in the open part [F(4,35)=5.77; P<0.01] and the total number of arm entries [F(4,35)=6.17; P<0.001]. The effects of pinoline on these three measures were already statistically
Table I The effect of pinoline (2–20 mg/kg) on rat behaviour in the elevated plus-maze test Dose Control 2 mg/kg 8 mg/kg 15 mg/kg 20 mg/kg
Crossed squares on open parts
Time spent on open parts (s)
Number of approaches
Number of open arm entries
Total number of arm entries
16.3±2.6 8.6±1.5† 8.3±1.6† 5.9±1.7‡ 2.9±1.0§
57.5±10.0 29.4±4.0† 36.3±4.2* 30.0±7.8† 14.4±4.3§
1.6±0.6 3.3±0.7* 2.8±0.4 1.0±0.4 2.4±0.5
0.75±0.4 0 0 0.5±0.3 0
6.8±0.9 4.4±0.5* 4.8±0.6* 4.1±0.7† 2.3±0.5§
Data are expressed as mean values±SEM (n=8 in each group). *P<0.05, †P<0.01, ‡P<0.001 and §P<0.0001 vs vehicle control; Fisher’s PLSD test after significant ANOVA.
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significant from dose 2 mg kg −1 and were highly significant (P<0.0001) at the dose level 20 mg kg−1. Pinoline increased the number of approaches to the open part [F(4,35)=2.92; P<0.05]; this effect was significant only at the dose 2 mg kg−1.
DISCUSSION The results of the experiments presented demonstrate that pinoline significantly and reliably reduced the immobility time in the forced swimming test. The effect of pinoline was similar to the effect of imipramine and amitriptyline. In the open field experiment, pinoline reduced the activity of rats at higher doses. Therefore, the reduction of immobility in the forced swimming test was apparently not related to increased motor activity of rats. Observed increase of activity of rats in the forced swimming test and reduction in the open field test was similar to the effect of tricyclic antidepressants as described already in the classic paper by Porsolt et al. [14]. The elevated plus-maze test has been validated for testing the anxiolytic and anxiogenic effects of the drugs [17]. Routinely, the open/total arm entries ratio is used to assess the effect of a drug on anxiety level whereas the number of total arm entries is taken to reflect a general locomotor activity [18]. In the present experiment it was not possible to use the conventional open/total arm entries ratio because all animals had a very low exploratory activity in the plus-maze. However, several authors have shown that a positive correlation exists between the total number of arm entries and validated anxiety measures [19–21]. Therefore, according to the decrease in the number of total arm entries and more specifically, time spent in the open part of the apparatus, pinoline in our experiment showed an anxiogenic-like effect. This interpretation of the test is supported by an increased number of approaches to the open part. This ethological measure also termed ‘closed arm returns’, is consistently reduced by anxiolytic drugs and enhanced by anxiogenic treatments [19, 22–24]. Several β -carbolines are anxiogenic, acting as inverse agonists at benzodiazepine receptors [1, 25]. However, all tetrahydro-β-carbolines including 6MeO-THBC have been shown to lack significant affinity for these binding sites [26, 27]. Therefore, the anxiogenic-like effect of pinoline in the plus-maze test is probably not mediated by benzodiazepine receptors. Since pinoline has been demonstrated to increase brain serotonin levels, the anxiogenic-like inhibitory effect of pinoline on rat exploratory behaviour is possibly a result of the serotoninergic action of the drug. Serotonin receptor agonists have an anxiogeniclike profile in the elevated plus-maze test [28, 29]. Most of the studies with selective serotonin reuptake inhibitors and tricyclic antidepressants reveal that
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they are inactive [18, 30] or anxiogenic-like [29–31] in the elevated plus-maze test after acute administration. In general, a reduction in 5-HT neurotransmission tends to reduce anxiety whereas stimulation of the 5-HT system usually results in an anxiogenic effect in different animal models of anxiety [30, 32]. In our laboratory conditions tricyclic antidepressants imipramine and desipramine and the selective serotonin reuptake inhibitor citalopram tended to reduce both the number of total arm entries and the activity of rats in open arms of the plus-maze [33]. Therefore, the anxiogenic-like effect of pinoline in the plus-maze is well explained by increased serotonin levels in the brain. However, it has never been convincingly shown that the anti-exploratory effect of antidepressant drugs is a true anxiogenic effect. The serotonergic nature of pinoline action may at least partly explain its effect in the forced swimming test. Tricyclic antidepressants which non-selectively inhibit the reuptake of serotonin and monoamines in brain synaptosomes reduce the immobility time in the forced swimming test [14, 34]. However, selective 5HT uptake inhibitors femoxetine, zimelidine, fluoxetine and sertraline reduce immobility time at high doses, whereas paroxetine and citalopram are ineffective [35, 36]. The reason for this discrepancy is not known. Pinoline has been reported to inhibit 5-HT uptake in rat brain synaptosomes with an IC50 of about 1 µM; the IC 50 for dopamine and noradrenaline uptake was 40–50 times higher [7]. Pinoline also inhibits specifically and reversibly the monoamine oxidase A with IC50 determined in different studies: 1.6 µM [37], 5 µM [38] and 8 µM [6]. Although MAO inhibition appears at higher concentrations, this may contribute to the antidepressant-like effect of pinoline in the present experiments. In addition, it is not excluded that pinoline may act partly through unique and specific binding sites. An autoradiographic study by Airaksinen et al. [39] revealed that a part of bound tritiated 6-MeO-THBC remains distinct from both MAO-A and the 5-HT transporter. Pinoline appears rather non-toxic in acute toxicity studies. LD50 in mice for 6-MeO-THBC following intraperitoneal administration was 235 mg kg−1 [12]. In comparison, LD50 for intraperitoneally administered amitriptyline was 65 mg kg−1 and for imipramine 90 mg kg−1 [40]. In conclusion, pinoline exerted an antidepressantlike effect in Porsolt’s test without an increase in general locomotor activity. The reduction of activity of rats in the open field and plus-maze tests seen after pinoline is in line with the behavioural profile of various antidepressants which also reduce exploration after acute administration of low doses. The dosedependent antidepressant-like effect of pinoline in behavioural tests in combination with a wide safety margin suggest that this compound should be further studied as a potential antidepressant.
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ACKNOWLEDGEMENTS This study was supported by grants from the Estonian Science Foundation, No. 10 to JH, and No. 1363 to LR.
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BEHAVIOURAL EFFECTS OF PINOLINE IN THE RAT FORCED SWIMMING, OPEN FIELD AND ELEVATED PLUS-MAZE TESTS ¨ HKLA, JAANUS HARRO and LEMBIT RA ¨ GO REIN PA Department of Pharmacology, University of Tartu, EE2400 Tartu, Estonia Accepted 10 May 1996 Pinoline (6-methoxy-1,2,3,4-tetrahydro-β-carboline) is a naturally occurring compound in the mammalian body which inhibits serotonin (5-hydroxytryptamine) uptake and monoamine oxidase-A activity. The present study was designed to assess potential antidepressant- or anxiolytic-like behavioural effects of pinoline in rat forced swimming, open field and elevated plus-maze tests. In the forced swimming test pinoline dose-dependently reduced the immobility time, starting from a dose of 8 mg kg −1. In the open field test pinoline reduced the total open field activity. This effect was significant at 20 mg kg−1, whereas the dose of 15 mg kg −1 only significantly reduced the number of rearings. In the plus-maze test pinoline decreased the total number of arm entries, the number of line crossings and time spent in the open part of the apparatus, but increased the number of approaches to the open part of the maze. The effects of pinoline were dose-dependent in all three behavioural tests used. Since antidepressant drugs reduce the immobility time in the forced swimming test but typically inhibit activity in open field and elevated plus-maze tests, the behavioural effects of pinoline resemble those of drugs with an antidepressant profile. 1996 The Italian Pharmacological Society KEY WORDS: 6-methoxytetrahydro- β-carboline, forced swimming, elevated plus-maze, open field, rat.
INTRODUCTION Beta-carbolines are a large group of pharmacologically active compounds which have been found in different plants. Some of those have been reported to occur as normal constituents of mammalian tissues including those of humans [1, 2]. One of the most interesting members of this group is 6-methoxy1,2,3,4-tetrahydro-β-carboline (6-MeO-THBC, 5methoxytryptoline), named pinoline from the words ‘pineal β -carboline’. It has been found in pineal and adrenal glands and in the brain in concentrations from 2 ng g −1 or below [3] up to a concentration of 21 µg g −1 [4, 5]. Pinoline inhibits the activity of monoamine oxidase-A (MAO-A) [6], serotonin (5-hydroxytryptamine, 5-HT) uptake [7] and binds to the imipramine recognition site [8]. Pinoline was initially proposed to function as an endogenous ligand of the imipramine recognition site [4, 9]. Now it is recognized that imipramine binds to the same domain in the serotonin transporter that binds 5-HT or at least to the site partly overlapping the 5-HT recognition site [10]. Acute administration of pinoline has been shown to increase the brain 5-HT levels [11]. This effect is Correspondence to: Dr Rein Pa¨hkla, Department of Pharmacology, ¨ likooli 18, EE2400 Tartu, Estonia. University of Tartu, U 1043–6618/96/070073–06/$25.00/0
common to most antidepressants. Pinoline has been reported to exert some pharmacological and behavioural effects characteristic of serotoninergic drugs [12, 13] but it has never been studied in behavioural tests suitable to assess the antidepressive potential of a compound. The purpose of this study was to compare the effect of pinoline with tricyclic antidepressants in the forced swimming test in rats. In order to measure whether any possible changes in immobility in the forced swimming test are related to changes in motor activity, the open field test was performed as previously described by Porsolt et al. [14]. In addition, we assessed the effect of pinoline on exploratory activity in the elevated plus-maze test to reveal possible anxiogenic- or anxiolytic-like properties of the compound.
MATERIALS AND METHODS
Animals Adult male Wistar rats weighing 270–350 g were used in the forced swimming test and adult female Wistar rats weighing 210–270 g in the open field and elevated plus-maze tests. Animals obtained from Grindex (Riga, Latvia) were housed at the Laboratory Animal Department of the Faculty of Medicine of 1996 The Italian Pharmacological Society
74
University of Tartu in polypropylene cages in groups of five for at least 4 weeks prior to testing. The animal room had a 12:12 h light–dark cycle with lights on at 0700 Food and drinking water were available ad libitum. All experiments were performed between 1200 and 1900.
Drug treatment Drugs used in the present experiments were 6-MeOTHBC, imipramine hydrochloride and amitriptyline hydrochloride (all from Sigma, St Louis, MO, USA). Pinoline was dispersed in an aqueous suspension of Tween 85 (1% vol.), imipramine and amitriptyline were dissolved in saline. Drugs were injected intraperitoneally in a volume of 1 ml kg −1 . Control groups received injections of Tween 85 watery solution of the same concentration, or saline, respectively.
Forced swimming test The technique first characterized by Porsolt and colleagues [14] was used. Briefly, rats were forced to swim in a glass box (floor 19×29 cm, height 39 cm) containing 25 cm of fresh water maintained at 25°C. On the first day of an experiment, rats were placed in water for 15 min. After the swimming session, each rat was removed from the water, partially dried with a paper towel, given an injection of the drug and returned to their home cage. On the following day, rats were given the drug treatment 30 min before forced swimming. The total duration of immobility during the first 5 minutes of the swimming session was recorded. The rat was judged to be immobile when it made only the necessary movements to keep its head above water level.
Open field test In the open field test rats were placed at the center of a rectangular arena 100×100 cm with 40-cm-high side walls. The arena was divided into 16 equal squares. Parameters registered 30 min after drug treatment during 4 min were the number of squares visited (with all four feet on one square) and the number of rearings. Both these measures were summed to yield total open field activity.
Elevated plus-maze test This test was conducted as first described by Handley and Mithani [15] with some modifications [16]. The plus-maze was made of wood, with two opposite open arms, 50×10 cm without any walls and two opposite closed arms of the same size with 40cm-high side walls and an end wall. The arms were connected by a central platform 10×10 cm. Both open arms were divided into three parts of equal size by lines which also separated the central platform from all arms. The central platform and open arms formed the ‘open part’ of the apparatus. The maze was elevated to a height of 50 cm from the floor. An entry into open arms was counted when the rat crossed the line
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between the central arena and an open arm with all four paws. The rat was considered to explore the open part of the apparatus when it had clearly crossed the line between a closed arm and the central arena with both its forepaws. The rat was placed on the central platform of the maze facing an open arm 30 min after injection. Behavioural measures taken during 4 min included: (a) the number of line crossings; (b) time spent in the open part of the apparatus; (c) the number of open arm entries; (d) the total number of arm entries; (e) the number of approaches to the open part. The latter measure reflects risk assessment activity of animals and was recorded when a rat approached the central platform but did not cross the line between the closed arm and the platform.
Statistical analysis One-way analysis of variance (ANOVA) was used to compare the data from all experiments. Post-hoc Fisher’s PLSD test was applied to compare group means with the control.
RESULTS
Forced swimming test Results of the forced swimming test are presented in Fig. 1. ANOVA indicated a significant difference among experimental groups in rats treated with imipramine [F(2,18)=4.61; P<0.05], amitriptyline [F(2,20)=5.57; P<0.05] and in both experiments with pinoline [F(2,21)=10.97; P<0.001 and F(4,35)=3.62; P<0.05]. Fisher’s PLSD test revealed that imipramine significantly reduced the duration of immobility at both doses tested and amitriptyline at the dose level of 30 mg kg−1. Pinoline reduced the immobility at the dose level of 20 mg kg −1 in the first pilot experiment (immobility time 151.3±8.8 s for control group and 93.5±11.5 s for 20 mg kg −1 group; P<0.01). In another independent experiment with multiple dose levels, the effect of pinoline was dose-dependent and already significant at the dose of 8 mg kg−1.
Open field test The effect of pinoline on the activity of rats in the open field test is shown on Figure 2. ANOVA indicated an overall effect of treatment for the total open field activity [F(4.35)=2.66; P<0.05] and for the number of rearings [F(4,35)=3.62; P<0.05] but the effect missed significance for the number of squares visited. Fisher’s PLSD test revealed that pinoline significantly reduced the total open field activity of rats at the dose of 20 mg kg −1 (P<0.05). The effect of the dose 15 mg kg−1 was just below the conventional significance level (P=0.06), but at this dose, pinoline significantly reduced (P<0.01) the number of rearings.
Elevated plus-maze The effect of treatment with different doses of pino-
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75 40 Number of squares visited
200 Immobility time (s)
A 150 *
**
100
50
30 20 10 0
0
Control
Control
15 mg/kg
10 mg/kg
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
15
200
B Number of rearings
B Immobility time (s)
A
150
100
**
50
10
5 **
0 0
Control
Control
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
30 mg/kg
10 mg/kg
Immobility time (s)
C
150 * * 100
***
50
Total open field activity
50 200
C 40 30
10 0
0
Control
2 mg/kg
8 15 mg/kg mg/kg
20 mg/kg
Fig. 1. Effects of treatment with imipramine (A), amitriptyline (B) and pinoline (C) on immobility time of rats in the forced swimming test. Data are presented as mean±SEM (n=8). Doses of different treatments are presented in abscissas of figures. *P<0.05, **P<0.01 and ***P<0.005 vs control; Fisher’s PLSD test after significant ANOVA.
line is presented in Table I. Pinoline significantly decreased the number of line crossings on open parts
*
20
Control
2 mg/kg
8 mg/kg 15 mg/kg 20 mg/kg
Fig. 2. The effect of pinoline (2–20 mg/kg) on (A) the number of squares visited in the open field, (B) the number of rearings and (C) the total open field activity (mean±SEM, n=8). Data were analysed using the one-way ANOVA and Fisher’s PLSD test for the difference from the control group. *P<0.05, **P<0.01.
[F(4,35)=8.19; P<0.0001], time spent in the open part [F(4,35)=5.77; P<0.01] and the total number of arm entries [F(4,35)=6.17; P<0.001]. The effects of pinoline on these three measures were already statistically
Table I The effect of pinoline (2–20 mg/kg) on rat behaviour in the elevated plus-maze test Dose Control 2 mg/kg 8 mg/kg 15 mg/kg 20 mg/kg
Crossed squares on open parts
Time spent on open parts (s)
Number of approaches
Number of open arm entries
Total number of arm entries
16.3±2.6 8.6±1.5† 8.3±1.6† 5.9±1.7‡ 2.9±1.0§
57.5±10.0 29.4±4.0† 36.3±4.2* 30.0±7.8† 14.4±4.3§
1.6±0.6 3.3±0.7* 2.8±0.4 1.0±0.4 2.4±0.5
0.75±0.4 0 0 0.5±0.3 0
6.8±0.9 4.4±0.5* 4.8±0.6* 4.1±0.7† 2.3±0.5§
Data are expressed as mean values±SEM (n=8 in each group). *P<0.05, †P<0.01, ‡P<0.001 and §P<0.0001 vs vehicle control; Fisher’s PLSD test after significant ANOVA.
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significant from dose 2 mg kg −1 and were highly significant (P<0.0001) at the dose level 20 mg kg−1. Pinoline increased the number of approaches to the open part [F(4,35)=2.92; P<0.05]; this effect was significant only at the dose 2 mg kg−1.
DISCUSSION The results of the experiments presented demonstrate that pinoline significantly and reliably reduced the immobility time in the forced swimming test. The effect of pinoline was similar to the effect of imipramine and amitriptyline. In the open field experiment, pinoline reduced the activity of rats at higher doses. Therefore, the reduction of immobility in the forced swimming test was apparently not related to increased motor activity of rats. Observed increase of activity of rats in the forced swimming test and reduction in the open field test was similar to the effect of tricyclic antidepressants as described already in the classic paper by Porsolt et al. [14]. The elevated plus-maze test has been validated for testing the anxiolytic and anxiogenic effects of the drugs [17]. Routinely, the open/total arm entries ratio is used to assess the effect of a drug on anxiety level whereas the number of total arm entries is taken to reflect a general locomotor activity [18]. In the present experiment it was not possible to use the conventional open/total arm entries ratio because all animals had a very low exploratory activity in the plus-maze. However, several authors have shown that a positive correlation exists between the total number of arm entries and validated anxiety measures [19–21]. Therefore, according to the decrease in the number of total arm entries and more specifically, time spent in the open part of the apparatus, pinoline in our experiment showed an anxiogenic-like effect. This interpretation of the test is supported by an increased number of approaches to the open part. This ethological measure also termed ‘closed arm returns’, is consistently reduced by anxiolytic drugs and enhanced by anxiogenic treatments [19, 22–24]. Several β -carbolines are anxiogenic, acting as inverse agonists at benzodiazepine receptors [1, 25]. However, all tetrahydro-β-carbolines including 6MeO-THBC have been shown to lack significant affinity for these binding sites [26, 27]. Therefore, the anxiogenic-like effect of pinoline in the plus-maze test is probably not mediated by benzodiazepine receptors. Since pinoline has been demonstrated to increase brain serotonin levels, the anxiogenic-like inhibitory effect of pinoline on rat exploratory behaviour is possibly a result of the serotoninergic action of the drug. Serotonin receptor agonists have an anxiogeniclike profile in the elevated plus-maze test [28, 29]. Most of the studies with selective serotonin reuptake inhibitors and tricyclic antidepressants reveal that
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they are inactive [18, 30] or anxiogenic-like [29–31] in the elevated plus-maze test after acute administration. In general, a reduction in 5-HT neurotransmission tends to reduce anxiety whereas stimulation of the 5-HT system usually results in an anxiogenic effect in different animal models of anxiety [30, 32]. In our laboratory conditions tricyclic antidepressants imipramine and desipramine and the selective serotonin reuptake inhibitor citalopram tended to reduce both the number of total arm entries and the activity of rats in open arms of the plus-maze [33]. Therefore, the anxiogenic-like effect of pinoline in the plus-maze is well explained by increased serotonin levels in the brain. However, it has never been convincingly shown that the anti-exploratory effect of antidepressant drugs is a true anxiogenic effect. The serotonergic nature of pinoline action may at least partly explain its effect in the forced swimming test. Tricyclic antidepressants which non-selectively inhibit the reuptake of serotonin and monoamines in brain synaptosomes reduce the immobility time in the forced swimming test [14, 34]. However, selective 5HT uptake inhibitors femoxetine, zimelidine, fluoxetine and sertraline reduce immobility time at high doses, whereas paroxetine and citalopram are ineffective [35, 36]. The reason for this discrepancy is not known. Pinoline has been reported to inhibit 5-HT uptake in rat brain synaptosomes with an IC50 of about 1 µM; the IC 50 for dopamine and noradrenaline uptake was 40–50 times higher [7]. Pinoline also inhibits specifically and reversibly the monoamine oxidase A with IC50 determined in different studies: 1.6 µM [37], 5 µM [38] and 8 µM [6]. Although MAO inhibition appears at higher concentrations, this may contribute to the antidepressant-like effect of pinoline in the present experiments. In addition, it is not excluded that pinoline may act partly through unique and specific binding sites. An autoradiographic study by Airaksinen et al. [39] revealed that a part of bound tritiated 6-MeO-THBC remains distinct from both MAO-A and the 5-HT transporter. Pinoline appears rather non-toxic in acute toxicity studies. LD50 in mice for 6-MeO-THBC following intraperitoneal administration was 235 mg kg−1 [12]. In comparison, LD50 for intraperitoneally administered amitriptyline was 65 mg kg−1 and for imipramine 90 mg kg−1 [40]. In conclusion, pinoline exerted an antidepressantlike effect in Porsolt’s test without an increase in general locomotor activity. The reduction of activity of rats in the open field and plus-maze tests seen after pinoline is in line with the behavioural profile of various antidepressants which also reduce exploration after acute administration of low doses. The dosedependent antidepressant-like effect of pinoline in behavioural tests in combination with a wide safety margin suggest that this compound should be further studied as a potential antidepressant.
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ACKNOWLEDGEMENTS This study was supported by grants from the Estonian Science Foundation, No. 10 to JH, and No. 1363 to LR.
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