Department of Pharmacological Sciences and Experimental Medicine, University of Camerino, 62032 Camerino (MC), Italy
Received 16 October 1998; in revised form 12 January 1999; accepted 3 March 1999
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ABSTRACT |
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INTRODUCTION |
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Several reports indicate co-morbidity between depression and alcohol misuse (Deykin et al., 1987; Neighbors et al., 1992
; Grant and Harford, 1995
; Markou et al., 1998
; Merikangas et al., 1998
; Swendsen et al., 1998
). Depression may be secondary to ethanol misuse and can be exacerbated by ethanol withdrawal (Baving and Olbrich, 1996
). However, according to the self-medication hypothesis of addictive disorders (Khantzian, 1985
), ethanol may be misused to relieve a pre-existing depression (Deykin et al., 1987
; Weiss et al., 1992
). Co-morbidity, together with the observation that depression and alcoholism might imply similar changes in the regulation of some central neurotransmitters (Markou et al., 1998
), raise interest in the potential therapeutic effects of antidepressant drugs and of HPE in alcoholism.
Genetically selected alcohol-preferring rats represent an interesting animal model for studies concerning mechanisms involved in ethanol intake control. A line of genetically selected alcohol-preferring rats has been bred in the Department of Pharmacological Sciences and Experimental Medicine of the University of Camerino (Marche, Italy) from Sardinian alcohol-preferring (sP) rats (Agabio et al., 1996; Colombo, 1997
; Lobina et al., 1997
), the strain is referred to as Marchigian sP (msP) rats. In a recent study (Ciccocioppo et al., 1999a
), it was observed that ethanol-naive msP and sP rats display longer immobility in the FST in comparison to Sardinian alcohol-non-preferring rats. A positive association between high alcohol intake and a depression-like state has been suggested also in genetically selected alcohol-preferring AA rats (Kiianmaa et al., 1991
; Viglinskaya et al., 1995
) and in fawn-hooded rats (Overstreet et al., 1992
). Moreover, Ciccocioppo et al. (1999a) showed that voluntary ethanol drinking or intragastric (i.g.) ethanol administration to sP or msP rats markedly reduces their immobility score in the FST. These findings raise the question of whether the high ethanol preference and intake of sP and msP rats might somehow be related to the antidepressant-like action of ethanol, and whether antidepressant drugs in these rats are able to reduce ethanol consumption.
On the basis of these findings, the present study was aimed at evaluating the effect of HPE on ethanol intake in msP rats.
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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 67 g/kg with an ethanol preference [100 x ml of ethanol solution/ml of total fluids (water + 10% ethanol) ingested in 24 h] higher than 90%.
Drugs
The HPE employed in the present study was a generous gift from Indena S.p.A., Milan, Italy. It was a dry extract (code no: 4149040, prepared in October 1997) containing 0.3% hypericin. It was dissolved in distilled water, just before administration.
Administration i.g.
Rats were anaesthetized by i.p. injection of 100150 µl/100 g body wt of a solution containing ketamine (86.2 mg/ml) and acepromazine (1.3 mg/ml). A polyethylene catheter (PE-50, Clay Adams) was implanted permanently in the stomach, according to the method of Lukas and Moreton (1979). The PE tubing was run s.c. to reach the skin between the scapulae, where it was exteriorized. Rats were allowed a week to recover from surgery.
The i.g. PE catheter was adopted for extract administration, in order to avoid any possible disturbance to the animal at the time of the experiment. Before the experiments, rats were familiarized with the administration procedure.
Experimental procedure
Experiment 1: effect of acute i.g. administration of HPE on ethanol intake in msP rats, offered 10% ethanol for 2 h/day.
A 10% 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 familiarized with this schedule of access to ethanol for 3 weeks following surgery. This period was sufficient to produce a stable 2-h ethanol intake; during the 3 days before the start of the experiments, the mean ± SEM 2-h ethanol intake of the msP rats employed ranged from 1.33 ± 0.1 to 1.50 ± 0.07 g/kg.
Fifteen rats were used for this experiment. According to a within-subject design, each rat received 125, 250 or 500 mg/kg HPE i.g. at intervals of 34 days. The i.g. administration of HPE was given 1 h before access to ethanol; a preliminary study revealed that a shorter time interval between i.g. administration and access to ethanol results in a smaller effect of HPE on ethanol intake, at least in the first 30 min of access.
Experiment 2: effect of acute i.g. administration of HPE on food intake in food-deprived msP rats. In Experiment 1, immediately after 10% ethanol presentation, msP rats were strongly motivated for ethanol, but showed low food intake and negligible water intake. To assess the selectivity of HPE on ingestive behaviour, the present experiment evaluated the effect of HPE on food intake of msP rats following a period of food deprivation.
Rats were food-deprived for 22 h, from 12:00 to 10:00; they had water, but no ethanol was available, during the deprivation period. HPE or vehicle were given i.g. at 09:00, i.e. 1 h before access to food. Food intake was determined by weighing the pellets remaining in the food cup and taking into account spillage; food-associated drinking was determined by reading the volume of water in the graduated burette. Both food and food-associated drinking were measured at 30, 60, 90, and 120 min after access to food. The 10% ethanol solution was offered at the end of the experiment.
Nine rats were employed for this experiment. According to a within-subject design, each rat received both vehicle and HPE, 125 or 250 mg/kg, at intervals of 7 days. The dose of 500 mg/kg was not included in the experiment, since, in Experiment 1, it proved to evoke immobility and in preliminary tests it produced a general inhibition of ingestive behaviour.
Experiment 3: time course of ethanol intake following acute i.g. administration of HPE in msP rats, offered 10% ethanol for 12 h/day. In Experiment 1, rats treated with HPE showed a lower ethanol intake, in comparison to controls, during the entire 2-h period of observation. In the present experiment, 10% ethanol solution was offered for 12 h/day at the beginning of the dark phase (10:00) to evaluate the time course of ethanol intake following HPE administration over a longer period. Food and water were freely available during the entire day. Rats were made familiar with this schedule of access to ethanol for 3 weeks after i.g. surgery.
Two groups of msP rats were employed: one group received a single i.g. vehicle dose, whereas the other group received 250 mg/kg HPE i.g. The i.g. administration took place 1 h before access to ethanol.
Experiment 4: effect of i.g. administration of HPE on the immobility time of msP rats in the FST. The swimming sessions were conducted by placing the rat in individual glass cylinders 30 cm in diameter, containing water at a temperature of 2325°C. The water was 30-cm deep rather than 18-cm deep, as reported in the original method of Porsolt et al. (1977). This change was adopted according to a recent study by Detke and Lucki (1995), showing that a deeper water level eliminates the false negative of selective serotonin reuptake inhibitors. At this water depth, rats could touch the bottom of the jar with their tail, but they could not support themselves with their hindlimbs. The first 15-min swimming session (pretest) was conducted between 10:00 and 12:00; 24 h later, rats were again placed in water for the 5-min test. Following each swimming session, rats were removed from the cylinder, dried with paper towels, placed in a heated chamber for 20 min and then returned to their home cages. Test sessions were video-taped (Canon VC-20 colour videocamera) and analysed by means of a Panasonic (NV-HD650EG) video-cassette recorder. The time spent immobile was measured by an experienced observer who was blind to the treatment conditions.
The rats employed in this experiment did not have access to ethanol for 2 weeks before the FST. HPE was given i.g. during the period between the two swimming sessions. In the first part of the experiment, it was given in a single i.g. dose of 250 mg/kg, 1 h before the FST. In the second part of the experiment, the HPE was administered at doses of 125 or 250 mg/kg, given three times (24, 12, and 1 h) before the test. A multiple dosing regime was employed, since a single drug administration is usually not sufficient to reveal the anti-immobility effect of antidepressant drugs.
Experiment 5: effect of i.g. administration of HPE on the locomotor activity of msP rats. The open field, used to measure locomotor activity, consisted of a wooden chamber (45 cm high) with a circular base (75 cm diameter). The floor was divided into 12 sections of similar area by two concentric circles and radial segments. The apparatus was placed in a sound-proof room, illuminated by a white 80-W lamp placed 200 cm over the centre of the arena.
Two groups of seven and eight msP rats were used; 1 day before the test, rats were confined in the open arena for 20 min to allow them to habituate to the testing apparatus. Afterwards, animals received three i.g. administrations of HPE, 250 mg/ kg, or vehicle, 24, 12, and 1 h before the test. Rats were placed in the open field for 10 min; the rat's behaviour in the test session was video-taped, analysed, and scored. The following parameters were measured: number of line crossings (number of sections entered with the four limbs); time spent in locomotor activity; and number of rearing reactions.
Experiment 6: effect of acute i.g. injection of HPE on blood-alcohol levels (BAL) following i.g. ethanol administration in msP rats.
Twelve msP rats were employed; food was removed from their cages at 07:00, i.e. 2 h before the beginning of the experiment. At 09:00, six rats received an i.g. dose of 250 mg/kg HPE, while the other six received an i.g. intubation of vehicle. One hour later, all the rats employed received i.g. administration of a 0.7 g/kg dose of ethanol, as a 10% solution; this is the amount voluntarily ingested by msP rats shortly (25 min) after access to 10% ethanol, when this solution is offered for 2 h/day (Ciccocioppo et al., 1999b). Blood samples (50100 µl) were taken from the tail vein 15, 30, 60, and 120 min after i.g. ethanol administra- tion. BAL were measured by gas chromatography according to the method of Cingolani et al. (1991).
Validation of the i.g. cannula
After completion of the experiments, rats were killed with an overdose of anaesthetic and the placement of the i.g. cannula was evaluated.
Statistical analysis
Data from the first two experiments were analysed by ANOVA, with repeated measures. Data from Experiments 3 and 6 were analysed by split-plot multifactorial analysis of variance, with between-group comparisons for drug treatment and within-group comparisons for time after treatment. Post-hoc comparisons were made by means of Dunnett's test. The results of Experiment 4 were analysed by one-way ANOVA, followed by the NewmanKeuls test. The results of Experiment 5 were analysed by Student's t-test. Statistical significance was set at P < 0.05.
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RESULTS |
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In response to 500 mg/kg HPE, but not at the lower doses tested, rats appeared somewhat sedated and immobile during the first 2 h after administration.
Experiment 2: effect of acute i.g. administration of HPE on food intake in food-deprived msP rats
The i.g. dose of 250 mg/kg HPE produced only a modest reduction in food intake (Fig. 2A) and in food-associated drinking (Fig. 2B
) in food-deprived rats. ANOVA revealed no statistically significant effect either on food intake [F(1,8) = 1.7; P > 0.05] or on food-associated drinking [F(1,8) = 3.9; P > 0.05].
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Experiment 4: effect of i.g. administration of HPE on the immobility time of msP rats in the FST
Following acute i.g. administration of 250 mg/kg HPE, given 1 h before the FST, the immobility time of treated rats was not significantly different from that of controls (Fig. 4A).
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Experiment 5: effect of i.g. administration of HPE on the locomotor activity of msP rats
Statistical analysis revealed no significant difference between controls and treated rats as far as number of line crossing (mean + SEM) (35.8 ± 3.8 in controls vs 31.6 ± 5.7 in treated rats), rearing reactions (14.1 ± 3.3 in controls vs 15.0 ± 0.7 in treated rats), and time spent in locomotor activity (85.8 ± 3.8 s in controls vs 78.6 ± 4.1 s in treated rats) were concerned.
Experiment 6: effect of acute i.g. injection of HPE on BAL levels following i.g. ethanol administration in msP rats
The mean BAL in controls and in HPE-treated rats, following i.g. administration of a 0.7 g/kg dose of ethanol, are reported in Table 1. ANOVA revealed neither a statistically significant effect of HPE treatment [F(1,10) = 1.1; P < 0.05], nor a significant treatmenttime interaction [F(3,30) = 0.7; P > 0.05], but a significant time effect [F(3,30) = 25.4; P < 0.001].
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DISCUSSION |
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The finding that HPE does not significantly affect food intake, at doses that reduce ethanol intake, may represent an interesting advantage in comparison to selective serotonin reuptake inhibitors. The latter reduce ethanol intake in both humans and rats, but their effect is usually associated with a pronounced inhibitory effect on food intake (Ciccocioppo et al., 1997), which is an undesirable effect in the treatment of alcoholic patients.
Another interesting finding of the present study is that the effect on ethanol intake is not strictly related to the antidepressant-like effect of HPE in msP rats. As a matter of fact, the two effects can be dissociated: the first can be observed after a single administration of 125 mg/kg, while the latter requires repeated HPE treatment at doses higher than 125 mg/kg.
As far as the mechanism of action is concerned, the only information provided by the present study is that the effect of HPE on ethanol intake is not related to changes in the pharmacokinetics of ethanol.
The present study employed whole HPE, which is known to contain at least seven classes of compounds (phenylpropanes, flavonol glycosides, biflavones, proanthocyanidins, xanthones, naphthodianthrones, phloroglucinols) (Nahrstedt and Butterweck, 1997). The fractions containing flavonoids and the naphthodianthrone, hypericin, were found to be the most active on immobility time in the FST by Nahrstedt and Butterweck (1997); moreover, recent studies by Chatterjee et al. (1998a,b) suggested that the phloroglucinol derivative hyperforin may represent an interesting antidepressant component of HPE. However, at present we do not know which compounds are responsible for the effect of HPE on ethanol intake.
A variety of neurochemical and biochemical effects have been reported for HPE in the literature. It can reduce serotonin reuptake (Perovic and Muller, 1995; Muller et al., 1997
); the flavonoids of the HPE can cause monoamine oxidase inhibition (Cott, 1997
; Nahrstedt and Butterweck, 1997
). It is also noteworthy that components of HPE show remarkable affinity for 5-HT1A receptors (Cott, 1997
). All these effects can influence serotonin neurotransmission. HPE has been shown to reduce not only serotonin reuptake, but also noradrenaline, dopamine and l-glutamate reuptake (Muller et al., 1997
; Chatterjee et al., 1998a
,b
). Butterweck et al. (1997) showed that the effects of HPE in the FST may be mediated, at least in part, by activation of dopaminergic and opioid mechanisms. Finally, Cott (1997) reported affinity of crude HPE for GABAA, GABAB, adenosine, and benzodiazepine receptors; the same author also reported that hypericin shows affinity for the N-methyl-d-aspartate receptor in the micromolar range.
Further studies are under way to establish whether the effects of HPE on ethanol intake are due to a particular active principle with a selective mechanism of action. The identification of the active principle might allow the development of new pharmacological and pharmaceutical research; it might also allow the administration of an active principle without the simultaneous administration of other compounds of HPE that may be responsible for adverse side-effects, such as photosensitivity (Brockmoller et al., 1997).
However, it could also be hypothesized that the effects of HPE on ethanol intake may be the result of the effect of a cocktail of compounds present in the extract and that the overall effects of HPE may be due to interaction with many (if not all) of the neurochemical systems mentioned above. Several studies have documented that pharmacological manipulation of the central serotonergic, dopaminergic, opioid, GABAergic, and glutamatergic mechanisms can influence ethanol consumption (for review, see Weiss and Koob, 1991).
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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