Neuroscience Letters 440 (2008) 181–184 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Caffeine reverses antinociception by amitriptyline in wild type mice but not in those lacking adenosine A 1 receptors Jana Sawynok a,∗ , Allison R. Reid a , Bertil B. Fredholm b a Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 b Section of Molecular Neuropharmacology, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden article info Article history: Received 30 April 2008 Received in revised form 21 May 2008 Accepted 21 May 2008 Keywords: Caffeine Amitriptyline Antinociception Adenosine A1 receptors abstract Amitriptyline is used to treat neuropathic pain in humans. It produces antinociception in several animal models of pain, and this effect is blocked by methylxanthine adenosine receptor antagonists which impli- cates adenosine it its actions. Here, the antinociceptive effect of amitriptyline, and the ability of caffeine to reverse it, were examined using the formalin test (a model of persistent pain) in wild type mice and mice lacking the adenosine A 1 receptor (A1R). Amitriptyline produced dose-related suppression of flinching in wild type mice following both systemic and intraplantar drug administration; both of these effects were unaltered in A1R -/- mice. Following systemic administration, caffeine reversed the systemic effect of amitriptyline in wild type, but not A1R -/- mice; -/+ mice exhibited an intermediate effect. Intraplantar administration of caffeine also reversed the effect of intraplantar amitriptyline in A1R +/+, but not in -/- or +/- mice. These results indicate that adenosine A 1 receptors are not required in order for amitriptyline to cause antinociception in mice, but they are required to see caffeine reversal of this antinociceptive effect. When A1Rs are present, actions of amitriptyline may, however, partly depend on A1Rs. © 2008 Published by Elsevier Ireland Ltd. The tricyclic antidepressant amitriptyline is widely used in the treatment of neuropathic pain in humans [5]. A variety of mechanisms have been suggested to explain its antinocicep- tive actions, including block of uptake of noradrenaline and 5-hydroxytryptamine, engagement of opioid mechanisms, inhibi- tion of ion channel activity, block of NMDA receptors, increased GABA receptor activity and modulation of immune function [13]. Adenosine mechanisms are also implicated because caffeine and other methylxanthine adenosine receptor antagonists inhibit antinociception produced by systemic [3,4,23] and peripherally administered amitriptyline [20,23] in models of chronic pain; this also was demonstrated in an acute pain model [14]. Adeno- sine A 1 receptors (A1Rs) mediate suppression of nociception, and methylxanthines block adenosine analogue effects on pain medi- ated via A1Rs [16,17]. Amitriptyline has been demonstrated to inhibit uptake of adenosine [15,21] and, using microdialysis, to increase extracellular adenosine levels [21]. Adenosine receptor knock-out mice, especially A1R (-/-) and A2AR (-/-) mice, have proven very useful to delineate mechanisms underlying drug effects, including the actions of methylxanthines ∗ Corresponding author. Tel.: +1 902 494 2596; fax: +1 902 494 1388. E-mail address: jana.sawynok@dal.ca (J. Sawynok). [8–12,26]. The use of such mice has revealed a role for A1Rs in mediating antinociception [11,26], and of A2ARs in mediating hyperalgesia [10,12]. In the present study, we used A1R -/- mice to directly examine the involvement of A1Rs in antinociception produced by amitriptyline following both systemic and peripheral administration using the formalin model, a model of persistent or ongoing pain. We also determined whether A1Rs are necessary in order for caffeine to reverse antinociception by amitriptyline. All experiments were approved by the University Committee on Laboratory Animals. Animals used were adenosine A 1 receptor -/-, +/- and +/+ generated by breeding +/- mice on an essen- tially pure C57Bl6 background. Mice of both sexes and between 20 and 30 g were used. They were housed in groups of 2–5 per cage at a temperature of 21 ± 1 ◦ C, with a 12-h/12-h light/dark cycle (light between 7 a.m. and 7 p.m.). The formalin test involves local injection of 20 l of 2% formalin into the plantar surface of the hindpaw, and monitoring of flinch responses (elevation and rapid shaking of the hindpaw) for 60 min following injection. Behaviors were observed in 2min intervals, and two mice were observed at a time in alternating 2 min bins. Phase 1 (0–8 min) and phase 2 (12–60min) behaviors were analysed separately. Amitriptyline and caffeine were administered systemically by intraperitoneal (i.p.) injection (5 ml/kg) 20 min before formalin, or co-administered peripherally with formalin in the 20 l volume. Each mouse was 0304-3940/$ – see front matter © 2008 Published by Elsevier Ireland Ltd. doi:10.1016/j.neulet.2008.05.074