Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, 62032 Camerino (MC), Italy
Received 18 January 2002; in revised form 24 February 2002; accepted 9 May 2002
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ABSTRACT |
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INTRODUCTION |
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Our previous studies aimed at investigating the neurochemical mechanisms underlying the effects of HPE showed that the antidepressant-like effect of HPE in the forced swimming test was inhibited by pretreatment with the neurotoxin 5-dihydroxytryptamine or by pretreatment with sigma-1 receptor antagonists, suggesting the involvement of serotonergic mechanisms, as well as of sigma receptors, in the antidepressant-like action of HPE (Perfumi et al., 2001). Neither 5-dihydroxytryptamine nor sigma-1 receptor antagonists influenced the effect of HPE on ethanol intake, suggesting that the antidepressant-like effect and the inhibitory effect on ethanol intake are not mediated by the same neurochemical mechanisms.
HPE contain a variety of biologically active compounds, including the naphthodianthrones hypericin and pseudohypericin, and the fluoroglucynol derivatives hyperforin, adhyperforin and several flavonoids (Nahrstedt and Butterweck, 1997; Barnes et al., 2001
; Jensen et al., 2001
). A large body of evidence suggests that hyperforin may represent the major component responsible for the antidepressant effect of HPE (Bhattacharya et al., 1998
; Chatterjee et al., 1998
; Laakmann et al., 1998
; Muller et al., 1998
; Kaehler et al., 1999
; Singer et al., 1999
; Vormfelde and Poser, 2000
). The antidepressant potency both in humans and in laboratory animals appears to be strictly related to the hyperforin content (Laakmann et al., 1998
; Di Carlo et al., 2001
). A recent paper from our group (Perfumi et al., 2001
) found a similar correlation also for the effect of HPE on alcohol intake, suggesting that hyperforin may account for this effect of HPE.
Hyperforin has been reported to inhibit the reuptake of a variety of neurotransmitters, such as -aminobutyric acid (GABA), 5-hydroxytryptamine (5-HT), dopamine (DA) and noradrenaline (NA), with IC50 of
50100 ng/ml, and l-glutamate, with IC50 of
500 ng/ml (Bennett et al., 1998
; Chatterjee et al., 1998
; Muller et al., 1998
; Kaehler et al., 1999
).
Recent reports have shown that in addition to influencing GABA reuptake, HPE and hyperforin bind to GABAA and GABAB receptors (Cott, 1997; Chatterjee et al., 1998
; Dimpfel et al., 1998
; Wonnemann et al., 2000
; Nathan, 2001
; Jensen et al., 2001
). A large body of evidence shows that GABA-ergic mechanisms modulate the motivational properties of ethanol and influence ethanol intake in rats and humans (Boyle et al., 1993
; Korpi, 1994
; Tomkins and Fletcher, 1996
; Koob et al., 1998
; McBride and Li, 1998
; Nowak et al., 1998
; Chester and Cunningham, 1999
; Addolorato et al., 2000
; Colombo et al., 2000
; Malatynska et al., 2001
).
On the basis of these findings, the present study was aimed at evaluating the possible involvement of GABA-ergic mechanisms in the effect of a CO2 Hypericum extract (HPCO2) on ethanol intake in alcohol-preferring rats, using selective GABAA and GABAB receptor antagonists.
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MATERIALS AND METHODS |
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At the age of 2 months, msP rats were selected for their preference for 10% (v/v) ethanol solution, offering them a free choice between water and 10% ethanol 24 h a day for 15 days. Water and 10% ethanol were offered in graduated drinking tubes equipped with metallic drinking spouts. The rats employed in the following experiments had a 24-h ethanol intake of 6 g/kg with a percentage ethanol preference [100 x ml of ethanol solution/ml of total fluids (water + 10% ethanol) ingested in 24 h] >80.
Animal testing was carried out according to the European Communities Council Directive of 24 November 1986 (86/609/EEC).
Drugs
The extract employed (HPCO2, provided by Indena, Milan, Italy) had a 24.33% content of hyperforin and 0.08% hypericin. The extract was emulsified in 0.1% Tween 80 and tap water, just before administration. It was given intragastrically (i.g.) at doses of 15 or 125 mg/kg; these doses were chosen on the basis of the results of our previous study (Perfumi et al., 2001).
(+)-Bicuculline, phaclofen and (RS)-baclofen (Tocris, Cookson Ltd, Bristol, UK) and CGP-36742 (a generous gift of Novartis Pharma AG, Basel, Switzerland) were used. Bicuculline was dissolved in distilled water immediately before intraperitoneal (i.p.) injection. CGP-36742 and baclofen were dissolved in 0.9% (w/v) NaCl immediately prior to i.p. injection; phaclofen, dissolved in saline, was administered by intracerebroventricular (i.c.v.) injection.
Intragastric administration
All animals used in the present study were implanted with i.g. catheters before experiments began. This procedure was adopted to avoid any possible disturbance to the animal during HPCO2 administration.
Rats were anaesthetized by i.p. injection of 100150 µl/100 g body weight of a solution containing ketamine (86.2 mg/ml) and acepromazine (1.3 mg/ml). A polyethylene catheter (PE-50; Clay Adams) was permanently implanted into the stomach, according to the method of Lukas and Moreton (1979). The PE tubing was run subcutaneously to reach the skin between the scapulae, where it was exteriorized. Prior to i.c.v. surgery or other treatments, rats were allowed a week to recover from i.g. surgery. Before the experiments, they were made familiar with the administration procedure.
Intracerebroventricular injection
Under ketamine/acepromazine anaesthesia, as described above, rats were implanted with a stainless-steel guide cannula for injection of phaclofen into the lateral ventricle. Coordinates, taken from the stereotaxic atlas of Paxinos and Watson (1986), were: 1 mm posterior and 2 mm lateral to bregma, 2 mm ventral from the surface of the skull. For drug injection, a stainless steel injector was inserted into the guide cannula (0.65 mm external diameter). The drug solution was injected at a volume of 2 µl by means of a 10 µl syringe; control rats received the same volume of vehicle.
Experimental procedure
In all experiments, a 10% (v/v) ethanol solution was offered for 2 h/day at the beginning of the dark phase (10:00) of the reverse light/dark cycle, while water and food were freely available during the entire day. Rats were made familiar with this schedule of access to ethanol for 3 weeks after surgery; this period was sufficient to have a rather stable 2-h ethanol intake.
The i.g. administration of HPCO2 was given 1 h before access to ethanol; our previous studies (Perfumi et al., 1999, 2001
) revealed that a shorter time interval between i.g. administration and access to ethanol results in lower effect of HPE on ethanol intake, at least in the first 30 min of access. Tap water and food were removed from the rats cage immediately before the i.g. treatment with either HPCO2 or vehicle, and they were offered again 1 h later together with 10% ethanol.
Ethanol, food and water intakes were recorded 15, 30, 60, 90 and 120 min after access to them.
Experiment 1. Effect of HPCO2 on ethanol intake following pretreatment with the selective GABAA receptor antagonist, bicuculline
First of all, in a preliminary experiment the effect of i.p. injections of bicuculline, 0.52 mg/kg, on ethanol intake was assessed in msP rats given ethanol 2 h/day. Rats were divided into four groups and each group received an i.p. dose of bicuculline or vehicle, 30 min before ethanol presentation. These doses of bicuculline have been reported to block effectively GABAA receptors (Nakagawa et al., 1995; Chester and Cunningham, 1999
; Zarrindast et al., 2001
).
The influence of bicuculline on the inhibitory effect of HPCO2 on ethanol intake was then investigated. Rats were divided into eight groups (that is four per dose of HPCO2), and treated as follows: two groups were i.p. injected with bicuculline vehicle, 30 min prior to i.g. vehicle injection (controls); two groups were i.p. injected with bicuculline vehicle, 30 min prior to i.g. injection of 15 or 125 mg/kg of HPCO2; two groups were i.p. injected with 2 mg/kg of bicuculline, prior to i.g. vehicle injection; two groups were i.p. injected with 2 mg/kg of bicuculline 30 min prior to i.g. injection of 15 or 125 mg/kg of HPCO2.
Experiment 2. Effect of HPCO2 on ethanol intake following pretreatment with the selective GABAB receptor antagonists CGP-36742 and phaclofen
This experiment evaluated the influence of the GABAB receptor antagonists, CGP-36742 (Olpe et al., 1993; Nakagawa et al., 1999
) and phaclofen (Humeniuk et al., 1993
) on the effect of HPCO2 on ethanol intake.
CGP-36742. A preliminary experiment was run to evaluate whether the dose of 100 mg/kg of CGP-36742, employed in other studies, might effectively antagonize the effect on alcohol intake of the GABAB receptor agonist baclofen. Rats were divided into four groups: one group was i.p. injected with CGP-36742 vehicle, 10 min prior to i.p. vehicle injection (control). One group was i.p. treated with CGP-36742, 100 mg/kg, 10 min before i.p. injection of baclofen vehicle; one group was i.p. injected with GCP-36742 vehicle 10 min before i.p. injection of baclofen 5 mg/kg; one group was i.p. injected with 100 mg/kg of CGP-36742 10 min before i.p. injection of 5 mg/kg of baclofen.
To study the effect of CGP-36742, rats were divided into eight groups and treated as follows: two groups were i.p. injected with antagonist vehicle 10 min before i.g. injection with vehicle (controls); two groups were i.p. injected with 100 mg/kg of CGP-36742, 10 min prior to i.g. vehicle injection; two groups received i.p. 100 mg/kg of antagonist 10 min prior to i.g. injection of 15 or 125 mg/kg of HPCO2; two groups were i.p. injected with CGP-36742 vehicle, 10 min prior to i.g. injection of 15 or 125 mg/kg of HPCO2.
Phaclofen. In a preliminary experiment, the dose of 25 µg/rat of phaclofen was tested versus the effect of the GABAB receptor agonist, baclofen, on ethanol intake. In this experiment, rats were divided into four groups and treated as follows: one group received i.c.v. phaclofen vehicle, 10 min prior to i.p. vehicle injection (control); one group was i.c.v. pretreated with phaclofen, 25 µg/rat 10 min before i.p. injection of baclofen vehicle; one group, i.c.v. pretreated with phaclofen vehicle, received 10 min later an i.p. injection of 5 mg/kg of baclofen; one group was i.c.v. injected with 25 µg/rat of phaclofen and 10 min later was i.p. treated with 5 mg/kg of baclofen.
To test the influence of phaclofen pretreatment on the effect of HPCO2, rats implanted with an i.c.v. guide cannula were employed. They were divided into four groups that were treated as follows: two groups, i.c.v. pretreated with phaclofen vehicle, received i.g. injection of 125 mg/kg of HPCO2 or vehicle (controls); two groups were i.c.v. pretreated with phaclofen, 25 µg/rat 10 min before i.g. injection of 125 mg/kg of HPCO2 or vehicle.
Validation of the i.g. and i.c.v. cannulae
After completion of the experiments, rats were killed with an overdose of anaesthetic, and placement and perviousness of the i.g. and i.c.v. cannulae were evaluated.
Statistical analysis
Data are reported as means ± SEM. The results were analysed by multifactorial split-plot analysis of variance (ANOVA), with between-group comparisons for drug treatment and within-group comparisons for time. Post hoc comparisons were made by means of the NewmanKeuls test. Statistical significance was set at P < 0.05.
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RESULTS |
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The influence of bicuculline pretreatment on the effect of HPCO2 on ethanol intake is shown in Fig. 1. ANOVA revealed a highly significant treatment effect following the 15 mg/kg [F(3,26) = 6.8, P < 0.01] and the 125 mg/kg [F(3,26) = 31.9, P < 0.001] doses of HPCO2. Pairwise comparison revealed no significant effect for the antagonist pretreatment; HPCO2, 15 or 125 mg/kg, reduced 10% ethanol intake in rats pretreated with bicuculline vehicle to the same extent as in rats pretreated with bicuculline.
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Experiment 2. Effect of HPCO2 on ethanol intake following pretreatment with the selective GABAB receptor antagonists, CGP-36742 and phaclofen
CGP-36742. Pretreatment with CGP-36742 reversed the reduction of ethanol intake induced by i.p. treatment with 5 mg/kg of the GABAB selective agonist baclofen [F(3,24) = 6.1; P < 0.01] (Fig. 2). The dose of 5 mg/kg of baclofen was chosen since 10 mg/kg of baclofen evoked pronounced sedation and general suppression of the ingestive behaviour for the entire 2-h period of observation, while the dose of 2.5 mg/kg did not significantly reduce ethanol intake.
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Phaclofen
Intracerebroventricular injection of phaclofen, 25 µg/rat, significantly reduced the effect of baclofen on ethanol intake [F(3,24) = 17.8; P < 0.001] (Fig. 4).
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DISCUSSION |
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HPE, as well as the active constituent hyperforin, have been shown to act as potent inhibitors of GABA reuptake (Dimpfel et al., 1998; Wonnemann et al., 2000
; Nathan, 2001
). In addition, a recent study has shown that hyperforin facilitates GABA release from synaptosomes (Chatterjee et al., 2001
). Binding studies have also demonstrated that HPE exhibit affinity for the GABA receptors (Cott, 1997
; Gobbi et al., 1999
). At a functional level, the GABA-mimetic properties of HPE are indicated by their anxiolytic effect, which is blocked by pretreatment with the benzodiazepine antagonist flumazenil (Kumar et al., 2000
; Vandenbogaerde et al., 2000
).
A large body of evidence suggests that the GABA-ergic system is involved in the regulation of ethanol consumption in rodents (Smith et al., 1999). For example, it has been demonstrated that the GABAB receptor agonist baclofen elicits a dose-dependent reduction of voluntary alcohol intake in alcohol-preferring rats (Colombo et al., 2000
), and similar findings were obtained in the present study. Moreover, other studies have observed that this drug reduces the intensity of alcohol withdrawal in ethanol-dependent rats, shows anticraving properties, and reduces alcohol intake in humans (Addolorato et al., 2000
; Colombo et al., 2000
).
Also the GABAA receptor subtype is involved in the regulation of ethanol consumption; however, its role is less clear. In effect, it has been reported that ethanol intake is reduced by both selective GABAA receptor agonists and antagonists. For example, it has been shown that administration of the GABAA receptor agonist muscimol in the prefrontal cortex, in the pedunculopontine nucleus or in nucleus accumbens decreases ethanol self-administration in rats (Hodge et al., 1995; Samson and Chappell, 2001a
,b
). Conversely, other studies have reported that ethanol intake is increased following peripheral administration of the GABAA receptor agonist THIP or central intra-raphe injection of THIP or muscimol, and their effect is prevented by administration of the GABAA receptor antagonists picrotoxin or bicuculline (Boyle et al., 1993
; Tomkins and Fletcher, 1996
). Moreover, injection of the GABAA receptor antagonists picrotoxin or bicuculline into the ventral tegmental area decreased ethanol, but not saccharin, consumption (Nowak et al., 1998
).
Based on these findings, the present study investigated the effect of the GABAA receptor antagonist, bicuculline, and of the GABAB receptor antagonists, CGP-36742 and phaclofen, on the effect of HPCO2. The antagonists were used at doses capable of antagonizing the effect of GABA receptor agonists, but not enough to modify ethanol drinking per se. Moreover, to avoid the occurrence of ceiling or floor effects, and to evaluate both the reduction or the increase of the effects of HPE under the influence of the antagonists, the extract was given at two different doses. The results revealed that the GABAA and GABAB receptor antagonists used did not modify the inhibitory effect on alcohol intake induced by HPCO2.
The dose of bicuculline used, which is able to antagonize the effect of GABAA receptor agonists (Chester and Cunningham, 1999; Zarrindast et al., 2001
), did not modify the effect of the two HPCO2 doses tested. Bicuculline neither reduced the effect of the higher dose of HPCO2 (125 mg/kg), nor increased the effect of the lower dose tested (15 mg/kg). Doses of the GABAB receptor antagonists, which significantly inhibited the effect of the GABAB receptor agonist, baclofen, did not modify the inhibitory effect of HPCO2 on ethanol intake. Taken together, these findings suggest that the effects of HPE on ethanol intake are not mediated by GABA agonist actions.
Thus, the mechanisms accounting for ethanol intake inhibition in alcohol-preferring rats by HPE remain unknown. On the basis of recent studies suggesting that HPE and hyperforin act as non-selective reuptake inhibitors for many neurotransmitters, with affinity for various neurotransmitter receptors, transporters and enzymes (Chatterjee et al., 1999; Singer et al., 1999
; Wonnemann et al., 2000
; Jensen et al., 2001
; Muller et al., 2001
; Nathan, 2001
), it is tempting to speculate that the effect of HPE on ethanol intake might be the result of interference with multiple biological systems. It is interesting to note that combination therapy has been proposed as a strategy to improve the efficacy of pharmacological treatments to reduce ethanol intake (Rezvani et al., 2000
). In this regard, it is possible to hypothesize that the involvement of different neurochemical systems is responsible for the high efficacy, potency and reliability of the inhibitory effect of HPE on ethanol intake.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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