Involvement of the cerebellar adenosine A₁ receptor in cannabinoid-induced motor incoordination in the acute and tolerant state in mice

Abstract

Cannabinoids are known to impair motor function in humans and laboratory animals. We have demonstrated an accentuation of cannabinoid (CP55,940)-induced motor incoordination in mice by the adenosine A₁ receptor-selective agonist N⁶-cyclohexyladenosine (CHA) (4 ng) using an intracerebellar (ICB) microinjection method. This effect was mediated by the A₁ receptor because pre-treatment with ICB 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (100 ng), an adenosine A₁ receptor selective antagonist, completely abolished the accentuation. Furthermore, ICB pre-treatment with DPCPX (100 ng) before ICB CP55,940 (15 μg) attenuated the motor incoordination suggesting a modulation by an endogenous adenosine A₁ system. ICB microinjection of CHA or DPCPX prior to ICB vehicle had no effect on normal motor coordination. ICB microinjection of dipyridamole (25 μg), an adenosine transport inhibitor, significantly accentuated the motor incoordination by ICB CP55,940 (15 μg), providing further support for the involvement of endogenous adenosine in the action of CP55,940. Tolerance to the motor incoordinating effect of ICB CP55,940 was demonstrated following 3 days of i.p. CP55,940 (0.1, 1 or 2 mg/kg every 12 or 24 h; total of six or three injections, respectively). Interestingly, animals which exhibited tolerance to ICB CP55,940 also demonstrated tolerance to the accentuating effect of ICB CHA suggesting cross-tolerance between adenosine agonists and cannabinoids. Cross-tolerance was also demonstrated following 3 days of i.p. CHA (0.25 or 1 mg/kg every 24 h; total of three injections) as further evidence of the modulatory role of the cerebellar adenosine system in the acute manifestation of CP55,940-induced motor incoordination. The involvement of cerebellar adenosine and the A₁ receptor in cannabinoid actions is circumstantially supported by previous evidence that CB₁ receptors and A₁ receptors are both localized on cerebellar granule cell parallel fiber terminals and basket cell neurons where they serve to inhibit the release of neurotransmitters.

Introduction

Cannabinoids are known to impair motor function in humans and laboratory animals [1]. We have previously demonstrated that Δ⁹-tetrahydrocannabinol (Δ⁹-THC), the main psychoactive component of marijuana, impairs motor coordination through a cerebellar CB₁ receptor mechanism in mice [7]. This behavioral effect of Δ⁹-THC was not surprising due to previous evidence of dense CB₁ receptor localization in the cerebellum and the well-known function of the cerebellum in coordinating movements [19].

Autoradiographical studies have demonstrated a presynaptic localization of CB₁ receptors on cerebellar granule cell axonal terminals in the molecular layer of the cerebellum [19], [23]. It is likely that these receptors inhibit glutamate release from the neuronal terminal upon activation by cannabinoid agonists such as CP55,940 and anandamide [19]. There is also evidence for localization of CB₁ receptors on the inhibitory basket cells and excitatory climbing fiber terminals which synapse onto Purkinje cell bodies [23], [33]. Several inhibitory presynaptic receptors appear to be co-localized with CB₁ receptors such as the GABA_B, adenosine A₁ and κ opioid. As for these receptors, the CB₁ receptors are G_i/o protein-coupled to inhibit adenylate cyclase and modulate the conductance of Ca²⁺ and K⁺ channels [1], [12], [17], [20], [21]. In vitro studies have demonstrated additive GTPase activity when GABA_B and A₁ agonists are co-incubated [35] and also when CB₁ and GABA_B agonists are co-incubated [26]. It was concluded that these receptors may couple to their own pool of G-proteins but converge to inhibit common adenylate cyclase enzymes [26]. Such an interaction is plausible between CB₁ and A₁ receptors.

The purpose of our study was to expand on our previous work which demonstrated a functional interaction between CB₁ and A₁ receptors in the cerebellum of male CD-1 mice using motor incoordination as the behavioral test. We have recently demonstrated accentuation of Δ⁹-THC-induced motor incoordination by the adenosine A₁ selective agonist, N⁶-cyclohexyladenosine (CHA), through a cerebellar mechanism [7]. The dose of CHA utilized was too low by itself to induce motor incoordination but significantly accentuated the motor incoordination by intracerebellar (ICB) Δ⁹-THC when co-administered. Furthermore, ICB administration of the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), significantly attenuated the motor incoordination induced by Δ⁹-THC.

The purpose of the present investigation was to establish whether the synthetic cannabinoid agonist, CP55,940, shares the same interaction with the adenosine A₁ receptor system when microinjected directly into the mouse cerebellum. We also set out to extend present and previous research by hypothesizing that tolerance to intracerebellar cannabinoid-induced motor incoordination occurs following repeated systemic administration. As further support of the adenosine A₁ receptor involvement in cannabinoid-induced motor incoordination, we tested for a cross-tolerance effect with the A₁ selective agonist, CHA, by daily systemic dosing for 3 days with CHA followed by an ICB CP55,940 challenge. We suggest that the acute and chronic interactions between drugs acting through CB₁ and A₁ receptors may result from anatomical co-localization and overlap in their signal transduction cascade.