Psychopharmacology (1999) 144 : 61–66
© Springer-Verlag 1999
ORIGINAL INVESTIGATION
V.S.N. Rao · F.A. Santos · W.G. Paula · R.M. Silva A.R. Campos
Effects of acute and repeated dose administration of caffeine and pentoxifylline on diazepam-induced mouse behavior in the hole-board test Received: 18 August 1998 / Final version: 16 December 1998
Abstract Rationale: The behavioral effects of methyl xanthines and their interactions with benzodiazepines have not been clearly established in animal models of anxiety. Objective : The present study extended the previous studies to determine the effects of acute and repeated administration of caffeine, a non-specific phosphodiesterase (PDE) inhibitor and pentoxyfylline, a specific type-4 phosphodiesterase (PDE4) inhibitor on (1) baseline anxiety-like behavior and (2) the response to an acute challenge with diazepam on anxiety-like behavior in the hole-board test. Methods: Mice were observed for the number of head-dips they made into the holes of the hole-board apparatus during a 5-min period, starting 30 min after acute (20 mg/kg) and repeated oral dose (20 mg / kg, twice a day for 4 days) administration of caffeine and pentoxifylline. In separate experiments, the response to an acute challenge with graded doses of diazepam (0.375–3 mg/kg, SC) was observed in naive mice or mice on acute and repeated dose regimen with methyl xanthines. Results: Mice on acute but not after repeated dose regimen demonstrated a significantly increased number of hole-dips, indicating an anxiolytic-like effect of methylxanthines. Diazepam at the lower doses (0.375 and 0.75 mg/kg) but not at the highest doses (1.5 and 3 mg/kg) examined produced a significant anxiolyticlike effect. After an acute dose exposure of mice to caffeine and pentoxifylline, a rightward shift in the dose-response curve of diazepam was observed and particularly at 1.5 mg /kg dose, the net effect of diazepam was significantly enhanced which was, however, impaired upon repeated administration, more so
V.S.N. Rao (*) · F.A. Santos · W.G. Paula · R.M. Silva · A.R. Campos Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Cel Nunes de melo, 1127, Porangabussu, POB 3157, 60430–270 Fortaleza, Ceará, Brazil e-mail :
[email protected], Fax: +55-85-243-9333
with caffeine than with pentoxifylline. Conclusions: It is concluded that the xanthine drugs exert anxiolyticlike activity similar to diazepam in the hole-board test. In addition, they seem to modulate the anxiolytic effects of diazepam after both acute and repeated administration, probably as a result of an endogenous adenosinergic mechanism which may have therapeutic significance. Key words Xanthine drug · Caffeine · Pentoxifylline · Diazepam · Hole-board · Head dip
Introduction Caffeine (1,3,7-trimethylxanthine), an alkaloid found in coffee, tea, chocolate and other foods is one of the most widely used stimulants in the human population (Graham 1978). Studies have implied that the ability of caffeine to inhibit phospodiesterases, the enzymes responsible for the hydrolytic inactivation of cAMP and cGMP (Daly 1977), might be involved in its stimulant effects, but it is unlikely that this property of caffeine is of relevance, since relatively higher doses of caffeine are required to produce phosphodiesterase inhibition (Marangos et al. 1984). Evidence also points towards its antagonism of adenosine receptors in the brain and to an increase in the the rate of catecholamine synthesis which accounts for the cortical arousal (Berkowitz et al. 1970; Waldeck 1971; Lin et al. 1980; Daly et al. 1981; Nehling et al. 1992). Its adenosine receptor antagonism may greatly contribute to stimulant properties of coffee consumption, since adenosine released during normal cortical metabolic activity acts to depress synaptic activity and inhibit neurotransmitter release. Studies have also shown that moderate intake of caffeine exerts beneficial effects on mood/anxiety, whereas an excessive intake of caffeine can cause increases in anxiety (Liebermann et al. 1987; Zahn and
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Rapoport 1987; Quinlan et al. 1997). Further, chronic caffeine ingestion results in behavioral depression of activity, in marked contrast to the behavioral stimulation caused by acute administration of caffeine, probably as a result of adenosine receptor up-regulation (Holtzman et al. 1991). More recent studies imply that the overall behavioral effect of xanthines (stimulation/depression) can depend critically on their relative activities as adenosine antagonists and phosphodiesterase (PDE) inhibitors (Howell et al. 1997). Although chronic effects of non-specific PDE inhibitors like caffeine and theophylline have been extensively studied, the behavioral effects of type-4 specific phosphodiesterase (PDE4) inhibitors such as pentoxifylline and rolipram have not been clearly established. Benzodiazepines produce their sedative, hypnotic, anxiolytic and anticonvulsant effects by interactions with GABAA receptors (Goodchild 1993; Shader and Greenblatt 1993). As the dose of benzodiazepine is increased, anxiolytic effects are produced first, followed by anticonvulsant effects and then a reduction in muscle tone, followed by sedation and hypnosis. Despite the development of anxiolytic drugs acting on the serotonin system, benzodiazepines still dominate the management of patients with anxiety disorders. However, some disadvantages, including sedation, ataxia, memory impairment, and abuse liability, are associated with benzodiazepine medication (Costa and Guidotti 1996). It is possible that adenosine participates in some of these effects, as it is known to be a potent endogenous depressant (Fredholm 1982). Benzodiazepine-induced sedation seems to be reversed by smaller doses of xanthines, which have adenosine blocking properties (Niemand et al. 1984). Previous studies that looked at xanthine and benzodiazepine interactions yielded confounding results, an enhancement or antagonism of the diazepam effect (Beer et al. 1972; Polc et al. 1981; Coffin and Spealman 1985). To evaluate more precisely the interaction effects, in the present study, we first explored the behavioral effects of caffeine and pentoxifylline on anxiety-like behavior in the hole-board apparatus following their acute and repeated dose administration. We then investigated the influence of acute and repeated treatment with these xanthines on the anxiolytic effect of diazepam.
Materials and methods Animals Six-week-old male Swiss mice obtained from the Central Animal House, Federal University of Ceará, Fortaleza, weighing 23–25 g were used. Prior to use, the mice were housed in polypropylene cages in groups of eight, under controlled conditions of temperature (23 ± 2°C), humidity (55–60%) and a 12-h light-dark cycle with lights on at 0700 hours. The animals were allowed free access to pelletted diet (Purina chow) and tap water except during the experiments. In the hole-board test, each mouse was tested only once
and six to eight animals were used in each group. The experimental protocol was approved by the Animal Care and Use Committee of this University.
Drugs Diazepam, caffeine hydrochloride and pentoxifylline were obtained from Sigma Chemical Co. (St Louis, Mo., USA). Diazepam was suspended in physiological saline using Tween 80 (2 drops/10 ml). The other drugs used were dissolved in physiological saline. Corresponding vehicles were used for control injections in each instance.
Hole-board test The method for hole-board test in mice was similar to the one described earlier (Bradley et al. 1968; Dorr et al. 1971). In the first series of experiments, the effects of xanthine drugs, caffeine (20 mg/ kg PO) and pentoxifylline (20 mg/kg PO) or vehicle (10 ml /kg PO) on the baseline anxiety-like behavior in the holeboard were observed 30 min after an acute or a repeated administration. In repeated dose regimen, the xanthines were administered twice a day (0800 and 1600 hours) for a consecutive period of 4 days, the last dose 30 min prior to the test. In preliminary experiments, caffeine at doses of 10 and 20 mg/kg and pentoxifylline at 20 and 40 mg/ kg enhanced the number of head dips of mice in the hole-board test. Since the test dose of 20 mg/ kg of either xanthine was able to produce a more consistent anxiolytic-like effect, this dose was selected for the current study. The number of head-dips into the holes of the hole-board apparatus (Ugo Basile, Italy) was counted for each animal during a 5-min period. To avoid disturbing environmental factors, the experimental procedure was carried out in a silent room under dim lights. In the second series of experiments, groups of mice that did not have any prior exposure to diazepam were treated with vehicle (10 ml /kg), or diazepam (0.375, 0.75, 1.5 and 3 mg/ kg, SC) 30 min before the test. In the third series, the pretreatment effects of xanthine drugs were noted on the head-dipping response of diazepam after both acute and repeated administration of caffeine or pentoxifylline.
Statistical analysis Results are expressed as mean ± SEM. Data were analysed by oneway analysis of variance (ANOVA) followed by Student-Newman Keuls test for multiple comparisons. In order to evaluate the impact of adenosine receptor blockade on the treatment effects, multifactorial ANOVA followed by the Scheffé F test were used. P values of 0.05 or less were considered statistically significant.
Results Caffeine and pentoxifylline In the hole-board, caffeine and pentoxifylline given acutely at a dose of 20 mg/ kg each caused a significant increase in head dips, this effect being attenuated after repeated administration (Fig. 1). The potency of single dose caffeine (upper panel) to increase the number of head dips disappeared after repeated treatment of mice with this drug [F(2,19) = 13.79, P < 0.002]. On
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Fig. 1 Effects of acute and repeated administration of caffeine (20 mg/ kg) and pentoxifylline (20 mg / kg) on baseline anxiety-like behavior of mice in the hole-board test. Data are presented as mean number of head dips ± SEM. VEH Vehicle. Different from VEH group; P < 0.05 (upper panel); different from VEH group; P < 0.05 (lower panel ); 1 different from acute caffeine; P < 0.05 (upper panel ); 1 different from acute pentoxifylline P < 0.05 (lower panel )
the other hand, pentoxifylline (lower panel) partially retained its effect after repeated administration [F(2,19) = 5.88, P < 0.015], although this effect failed to reach an acceptable level of statistical significance. Combining all the data into a factorial ANOVA indicated a significant effect on the number of head dips in the hole-board [F(4,25) = 8.2, P < 0.002]. However, the follow-up analysis did not indicate any significant differences in the treatment effects between caffeine and pentoxifylline following their acute or repeated administration.
Fig. 2 Effects of acute and repeated administration of caffeine (20 mg/ kg) on diazepam-induced anxiety-like behavior of mice in the hole-board test. Data are presented as mean values ± SEM. VEH Vehicle, clear bars; CON control, vehicle-treated animals that received acute single dose caffeine but no diazepam, shaded bars (middle panel); or repeated dose caffeine but no diazepam, striped bars (lower panel ); dark bars, different doses of diazepam alone (0.375, 0.75, 1.5 and 3 mg /kg, upper panel) or in association with acute caffeine (middle panel) or repeated caffeine (lower panel). The data for VEH and CON (acute or repeated caffeine) are the same as in Fig. 1. *Different from VEH group, P < 0.05; # different from 1.5 mg/kg diazepam alone (middle panel), P < 0.05; @ different from acute caffeine + 1.5 mg/ kg diazepam (lower panel ), P < 0.05
Diazepam
dose of caffeine (20 mg/ kg), the total head dips to 1.5 mg / kg diazepam were significantly increased [F(5,34) = 7.19, P < 0.0001] while for other doses, ANOVA failed to reveal any significant change (Fig. 2, middle panel). In contrast, the total head dips to different doses of diazepam in mice that received repeated caffeine were not significantly different from those of vehicle treated subjects (Fig. 2, lower panel). Repeated caffeine treatment not only reversed its acute influence on 1.5 mg / kg diazepam [F(5,34) = 2.06,
The effects of diazepam, administered 30 min before testing on the hole-board, are shown in the upper panels of Figs. 2 and 3. Compared to vehicle treated control, diazepam treated animals manifested significantly increased levels of head dips at doses of 0.375 and 0.75 mg/kg [F(4,30) = 17.03, P < 0.0001]. Diazepam treatment failed significantly to alter the hole-board response of mice at 1.5 and 3 mg / kg. After an acute
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Combining all the data depicted in Figs. 2 and 3 into a factorial ANOVA revealed significant differences in the pretreatment effects of caffeine and pentoxifylline on the diazepam dose-response curve [F(24,150) = 13.296 P < 0.0001]. Follow-up analysis indicated significant differences (i) between the groups that received 1.5 mg/ kg diazepam alone and acute caffeine +1.5 mg/kg diazepam treatments [F = 1.73, P < 0.05] or acute pentoxifylline + 1.5 mg/ kg diazepam treatments [F = 3.39, P < 0.05]; (ii) between the groups that received acute caffeine + 1.5 mg / kg diazepam and repeated caffeine + 1.5 mg / kg diazepam [F = 1.73, P < 0.05] and (iii) between the groups that received acute pentoxifylline + 1.5 mg/ kg diazepam and repeated pentoxifylline + 1.5 mg / kg diazepam [F = 1.61, P < 0.05]. Thus the acute pretreatments with methylxanthines caused a rightward shift in the dose-response curve of diazepam which was not seen after repeated therapy. Although statistically not significant, the potency of repeated pentoxifylline in causing reduction of diazepam-induced head dipping was not marked when compared to repeated caffeine (Figs. 2 and 3).
Discussion
Fig. 3 Effects of acute and repeated administration of pentoxifylline (20 mg/ kg) on diazepam-induced anxiety-like behavior of mice in the hole-board test. Data are presented as mean values ± SEM. VEH Vehicle, clear bars; CON control, vehicle-treated animals that received acute single dose of pentoxifylline but no diazepam, shaded bars; or repeated dose of pentoxifylline but no diazepam, striped bars; dark bars, different doses of diazepam alone, (0.375, 0.75, 1.5 and 3 mg/ kg, upper panel) or in association with acute pentoxifylline (middle panel) or repeated pentoxifylline (lower panel ). The data for VEH, CON (acute or repeated pentoxifylline) and for different doses of diazepam are the same as in Fig. 2 (middle and lower panels). *Different from VEH group, P < 0.05; # different from 1.5 mg/ kg diazepam alone (middle panel ), P < 0.05; @ different from acute pentoxifylline + 1.5 mg /kg diazepam (lower panel), P < 0.05
P < 0.09], but also abolished the increased head dipping effect of diazepam observed at 0.375 and 0.75 mg /kg. Diazepam (1.5 mg / kg)-induced head dips were significantly higher in the group of mice that received a single dose of pentoxifylline (20 mg /kg) [F(5,34) = 29.08, P < 0.0001], but this acute effect was greatly diminished in mice after repeated administration of pentoxifylline (Fig. 3, middle and lower panels).
The hole-board test is a measure of exploratory behaviour in rodents (File and Wardill 1975). In addition, head dips of mice observed in the hole-board experiments have been accepted as a parameter for the evaluation of anxiety conditions in animals. In this model, non-sedative doses of benzodiazepines and other anxiolytic drugs, have been reported to increase head dips in mice while their antagonists decrease it (Crawley 1985). The present study examined the effects of the methylxanthines, caffeine and pentoxifylline, each at a dose of 20 mg/ kg, in the hole-board apparatus. Doses higher than 20 mg/ kg caffeine and 40 mg / kg pentoxifylline were not tested because such doses are likely to cause locomotor, respiratory and cardiovascular alterations (Glowa and Spealman 1984; Coffin and Spealman 1987; Nikodijevic et al. 1993; Howell et al. 1997). Methylxanthines, when given acutely to mice, caused a significant increase in head dips and demonstrated anxiolytic-like activity which was, however, attenuated after repeated administration. This finding may be in agreement with previous studies suggesting that moderate intake of caffeine produces beneficial effects on mood /anxiety, whereas an excessive intake causes an increase in anxiety (Glowa and Spealman 1984; Zahn and Rapoport 1987; Quinlan et al. 1997). The behavioral stimulant effects of these methyl xanthines appear to be mediated principally by two mechanisms, phosphodiesterase inhibition and adenosine receptor antagonism (Daly 1982; Choi et al. 1988). Doses of xanthines higher than those used in this study are required to inhibit PDE activity compared to doses
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that effectively block adenosine receptors (Wu et al. 1982; Marangos et al. 1984; Beavo and Reifsnyder 1990). Therefore it is likely that adenosine antagonism, not phosphodiesterase inhibition, accounts for the observed stimulant effects of methylxanthines on head dipping. Type-4 phosphodiesterase inhibitors (rolipram and Ro 20–174) were initially developed clinically for the treatment of depression and for the respiratory stimulant effects in asthmatic subjects (Bobon et al. 1988; Howell 1993). In this study, pentoxifylline, the PDE4 inhibitor, was able to produce an anxiolytic-like effect similar to caffeine, a non-specific PDE inhibitor. However, its selectivity to adenosine receptor antagonism is yet unknown. Tolerance to anxiolytic-like behavior following repeated caffeine was evident in our experimental conditions, which is consistent with literature observations in humans and rodents (Katz and Goldberg 1987; Ramakumar et al. 1988). It seems likely that the pharmacodynamic factors rather than pharmacokinetic effects contribute greatly to the development of tolerance to methylxanthines (Bonati et al. 1985). Since adenosine functions as a neuromodulator in the mammalian brain and stimulation of adenosinergic pathways can induce sedation (Dunwiddie 1985), many studies suggest that up-regulation of adenosine A1receptors is the basis of caffeine tolerance (Hawkins et al. 1988; Nikodijevic et al. 1993) with the exception of one study that reports no detectable up-regulation of adenosine A1 -receptors (Holtzman et al. 1991). In our studies, unlike caffeine, pentoxifylline showed less tolerance on repeated administration, probably because of its ability to induce both PDE inhibition and adenosine receptor blockade or alternatively due to its less functional significance on adenosine receptor sites. Pentoxifylline may therefore have a clinical advantage over caffeine for the treatment of mood /anxiety disorders. The complex differences in the action of methylxanthines on the central nervous system, when given acutely versus chronically, may be a result of variations in their ability to inhibit cyclic nucleotide phosphodiesterases, the enzymes responsible for the hydrolytic inactivation of cyclic AMP and cyclic GMP (Daly 1977, 1982), sometimes at doses comparable to those capable of blocking adenosine receptors (Smellie et al. 1979; Choi et al. 1988). Unless these are clarified, it would be difficult to offer a satisfactory explanation for the differences in the development of tolerance associated with chronic use of methylxanthines. In the present experiments, compared to controls, diazepam-treated animals manifested significantly increased number of head dips at lower doses (0.375 and 0.75 mg/ kg), but not at highest doses (1.5 and 3 mg/kg) indicating an anxiolytic-like effect. Biochemical and electrophysiological studies show that benzodiazepine anxiolytics facilitate GABAergic transmission in the central nervous system via positive allosteric modulation of the GABAA receptor complex
in a dose-dependent manner. Low doses of benzodiazepines enhance and higher doses antagonize GABAergic transmission (Tallman et al. 1980; Sieghart 1995). Thus the diazepam-induced increases of mouse head dips in hole board test may be a result of facilitatation of GABAergic transmission. A complex interaction seems to exist between adenosine- and benzodiazepine-recognition sites within the GABA /benzodiazepine receptor complex (Phillis and O’Regan 1988). Lack of significant anxiolysis with higher doses of diazepam implies a likely interaction beteween diazepam with endogenous adenosine with a consequential impairment of GABAergic transmission. After an acute dose exposure of mice to caffeine and pentoxifylline, a rightward shift in the dose-response curve of diazepam was observed and particularly at 1.5 mg/ kg dose, the net effect of diazepam was significantly enhanced. In mice, on repeated dose regimen with methylxanthines, not only the net effect of diazepam but also the xanthine-induced per se effect on head dips was greatly impaired, more so with caffeine than with pentoxifylline. It clearly indicates the tolerance inducing effect of methylxanthines for the behavioral effects. They may do so through up-regulation of adenosine receptors and or by reducing diazepam binding to brain receptors (Phillis and Wu 1981; Nikodijevic et al. 1993). In conclusion, the present study demonstrates that xanthine drugs can exert anxiolytic-like activity similar to diazepam in the hole-board test. In addition, they seem to modulate the anxiolytic effects of diazepam after both acute and repeated administration probably involving endogenous adenosinergic mechanism which may have a therapeutic significance. Acknowledgements This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), Brazil.
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