Involvement of Adenosinergic Receptors in Anxiety Related

Indian Journal of Experimental Biology Vol. 45, May 2007, pp. 439-443

Involvement of adenosinergic receptors in anxiety related behaviours Shrinivas K Kulkarni*ª, Kulwinder Singhª & Mahendra Bishnoi* *Centre with Potential for Excellence in Biomedical Sciences (CPEBS), Panjab University, Chandigarh 160 014, India ªPharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160 014, India Received 14 September 2006; revised 22 November 2006

In the present study, the effect of adenosine (A1 and A2 receptor agonist), caffeine (A2A receptor antagonist), theophylline (A2A receptor antagonist) and their combination was studied in anxiety related behaviours using elevated zero maze and elevated plus maze paradigms and compared their various behavioural profiles. Adenosine (10, 25, 50,100 mg/kg) significantly showed anxiolytic effect at all the doses, whereas caffeine (8, 15, 30, 60 mg/kg) and theophylline (30, 60 mg/kg) showed psychostimulatory action at lower doses and anxiogenic effect at higher doses. Pretreatment with caffeine (8, 15, 30 mg/kg) and theophylline (30 mg/kg) reversed the anxiolytic effect of adenosine. The study suggested the involvement of adenosinergic receptor system in anxiety related behaviours. Keywords: Adenosine, Caffeine, Zero maze

Adenosine is now generally accepted as the major Material and Methods inhibitory neurotransmitter other than GABA in the Animals — Laca mice of either sex bred at Central mammalian central nervous system1,2. In the central Animal House (CAH) Panjab University, Chandigarh nervous system, its effects are believed to be mainly weighing 25-30 grams were used. The animals were 3 mediated by two receptors, A1 and A2A . Adenosine is housed under standard laboratory conditions, reported to produce locomotor activity, depressant, maintained on a 12 hour light and dark cycle, with anticonvulsant, hypnotic, anxiolytic and analgesic free access to standard food and water. Animals were effects4,5. Caffeine and theophylline, the antagonists acclimatized to laboratory conditions before the test. of adenosinergic receptors3,6, are consumed All the experiments were carried out between 0900 to throughhout the world in the form of coffee, tea, 1500 hrs. The experimental protocols were approved chocolate and soft drinks, as well as over-the-counter by the Institutional Animal Ethical Committee and or prescription drugs2. The popularity of caffeine is conducted according to the CPCSEA guidelines on thought to be related to its subjective effects, which the use and care of experimental animals. include increased alertness and CNS stimulation7. Drug treatment and Experimental procedures — However, variations in acute responses to caffeine Mice were treated with different drugs as caffeine (8, have been reported, especially in anxiety-inducing 15, 30, 60 mg/kg), theophylline (30, 60 mg/kg), effects of the drug. Controlled studies have confirmed adenosine (10, 25, 50,100 mg/kg), caffeine (8 mg/kg) that caffeine produces positive effects such as + adenosine (25 mg/kg), caffeine (15 mg/kg) + increased stimulation in many, while others adenosine (25 mg/kg), caffeine (30 mg/kg) + experience negative effects such as increased adenosine (25 mg/kg), theophylline (15 mg/kg) + anxiety8,9. There are certain variations in the effect of adenosine (15 mg/kg). All the drugs were dissolved in theophylline also. The exact role of different distilled water and were administered 30 min prior to adenosinergic receptor modulators in anxiety remains the experimentation. Elevated zero maze and plus unclear, therefore the effect of adenosine (A1 and A2 maze test were carried out simultaneously in each of receptor agonist), caffeine (A2A receptor antagonist), the drug treatment groups. theophylline (A2A receptor antagonist) and their Elevated zero maze — All the animals were combination on animal behaviours elevated zero maze analyzed for anxiety levels by using elevated zero and elevated plus maze paradigms have been maze. This test is a pharmacologically validated assay investigated. of anxiety in animal models that is based on the ______natural aversion of mice to elevated zero maze. It is E-mail: [email protected] composed of a 6 cm wide ring with outer diameter of 440 INDIAN J EXP BIOL, MAY 2007

45 cm containing 4 equal quadrants of alternating 30 mg/kg) or theophylline (30 mg/kg) signi- walled (closed) or unwalled (open) sections. The ficantly reversed the effect of adenosine (25 mg/kg), entire ring is elevated to the height of 40 cm. Mice (Fig. 1a, b, c) were placed in the walled region at the start of 5 min Elevated plus maze — Caffeine showed stimulant seasons and following parameters were analyzed10. effect at lower doses (8 mg/kg), whereas anxiogenic effect (decrease in time spent in open area, increase in (a) Time spent in open arm; latency to enter in open arm, decrease in number of (b) Latency to enter in open arm; entries in open area) at higher doses (15, 30 and 60 (c) Total number of entries in open arm; mg/kg). Theophylline also showed stimulant effect at lower dose (30 mg/kg), whereas anxiogenic effect (d) Number of stretch attend postures (SAP’s). (decrease in time spent in open area, increase in Elevated plus maze — All the animals were also latency to enter in open arm, decrease in number of analyzed for anxiety levels by using elevated plus entries in open area) at higher dose (60 mg/kg). maze. Plus maze is composed of two open arm Adenosine (10, 25, 50,100 mg/kg) showed increase in time spent in open area, decrease in latency to enter in (16 × 5 cm) and two enclosed arms (16 × 5 × 12 cm) open arm, increase in number of entries in open area with an open roof and is elevated to a height of 25 cm. at initial 3 doses which was decreased at 100 mg/kg. Each animal was placed individually in the centre of In combination, pretreatment with caffeine (8, 15 and the maze facing towards the open arm and recorded 11 30 mg/kg) or theophylline (30 mg/kg) signifi- the following parameters during 5 min session . cantly reversed the effect of adenosine (25 mg/kg), (a) Time spent in open arm; (Fig. 2 a, b, c) (b) Latency to enter in open arm; (c) Total number of entries in open arm; Discussion Adenosine is now generally accepted as a main (d) First preference of the animal (open/closed); inhibitory neurotransmitter other than GABA in the (e) Number of stretching. mammalian central nervous system. It is generated from the adenosine triphosphate (ATP) both Statistical analysis — One specific group of mice intracellularly and extracellularly and transported was assigned to one specific drug treatment condition across the cell membrane by facilitated diffusion and each group comprised six mice (n=6). All the transport1,2. The functional role of adenosine and values are expressed as means ± S.E. The data were related nucleosides and nucleotides in CNS is being analyzed by Student’s t-test. P<0.05 was considered actively pursued. Several drug actions have long been as statistical significant. explained in relation to purinergic substances3.6. In the central nervous system, effects of adenosine are Results believed to be mainly mediated through A1 and A2A Elevated zero maze — Caffeine showed stimulant receptors, both of which are G-protein coupled effect at lower doses (8 mg/kg) where as anxiogenic receptors12. Based on the knowledge of various effect (decrease in time spent in open area, increase in agonists and antagonists of adenosine receptor system latency to enter in open arm, decrease in number of in human as well as behavioural studies in animals the entries in open area) at higher doses (15, 30 and 60 actions of adenosine in CNS have generally been held mg/kg). Theophylline also showed stimulant effect at to comprise locomotor depressant, anticonvulsant, lower dose (30 mg/kg), whereas anxiogenic effect hypnotic, anxiolytics and analgesic effects13,14. Role (decrease in time spent in open area, increase in of adenosine in the modulation of anxiety state is still latency to enter in open arm, decrease in number of under debate. Existence of interaction between entries in open area) at higher dose (60 mg/kg). adenosine and benzodiazepines has been suggested in Adenosine (10, 25, 50,100 mg/kg) showed increase in 1980’s. Down-regulation of the number of adenosine time spent in open area, decrease in latency to enter in A2 receptors in rat forebrain was found following open arm, increase in number of entries in open area chronic treatment with benzodiazepines15. A more at initial 3 doses which was decreased at 100 mg/kg. direct involvement of adenosine in the regulation of In combination, pretreatment with caffeine (8, 15 and anxiety states has been suggested by the fact that KULKARNI et al.: ADENOSINERGIC RECEPTOR AND ANXIETY 441

Fig.1 — Effect of different adenosine receptor agonist and antagonist and their combination on — (a) time spent in open arm (b) latency to enter in open arm (c) number of entries in open in open arm on elevated zero maze paradigm. [Total number of animals in each group is 6 and data is expressed in Mean ± SE. aP ≤ 0.05 as compared to control group, bP ≤ 0.05 as compared to adenosine (25) group.]

caffeine which is an antagonist of A1 and A2A receptor in time spent in open arm, increase in number of is known to increase anxiety in humans as well in entries in open arm and decrease in latency to enter in animals, and papaverine an inhibitor of adenosine open arm in both elevated zero maze and plus maze 15-17 uptake produces anxiolytic effects . Also, the A2A paradigms of anxiety. Caffeine and theophylline, A2A knockout mice are anxious in nature and are receptor antagonists3,6, at lower doses showed considered as a model of anxiety. These animals increase in exploratory behaviour on both the showed higher rates of spontaneous anxiety like paradigms of anxiety suggesting psychomotor responses in two different anxiety like behavioural stimulant action of them. Further, at higher doses they tests, elevated plus maze and light dark box 2,18. showed significant anxiogenic effect as suggested by In the present study, the adenosine significantly decrease in open arm entries and time spent in open showed anxiolytic effects, characterized by increase arm as well as increase in latency to enter in open arm 442 INDIAN J EXP BIOL, MAY 2007

Fig. 2 — Effect of different adenosine receptor agonist and antagonist and their combination on (a) time spent in open arm (b) number of entries in open in open arm (c) latency to enter in open arm on elevated plus maze paradigm. [Total number of animals in each group is 6 and data is expressed in Mean ± SE. aP ≤ 0.05 as compared to control group, bP ≤ 0.05 as compared to adenosine (25) group] in both the mazes. Several studies in human as well as intracellular metabolism of cyclic AMP. Thus, an in animals suggested that the administration of high increase in the intracellular cyclic AMP and produces but not low, doses of caffeine leads to an increase in effects that mimic β-stimulation which is anxiety19. Possibly, at small doses, caffeine binds to characterized by increased alertness and psychomotor 20,21 and inhibits half of the adenosine receptors. But at stimulation . Transgenic mice lacking A2A receptor higher doses there is more pronounced and is abnormally anxious and aggressive and fails to appropriate blockade of A2A and A1 receptor and thus show increased motor activity in response to caffeine produces anxiety state. Methylxanthines (caffeine and suggesting that A2 receptors are involved in anxiety 2 theophylline) induced CNS stimulation at lower states . Also targeted disruption of A1 receptor in doses may be accounted for the inhibition of mice produces increase in anxiety behaviour in phosphodiesterase enzyme, which is responsible for animals20. Further, in combination studies KULKARNI et al.: ADENOSINERGIC RECEPTOR AND ANXIETY 443

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