Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine: Involvement of adenosine and dopamine receptors

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Abstract

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A1 agonist, N6-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D1 and D2 receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D1R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D2R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

Highlights

► We administered theacrine to rats to investigate locomotor activity. ► Motor stimulant effects of theacrine are mediated by actions in the NAc. ► Chronic administration didn't result in sensitization or tolerance. ► Theacrine represents a potential novel pharmacotherapeutic agent.

Introduction

Theacrine (1,3,7,9-tetramethyluric acid), the predominant purine alkaloid extracted from the cultivated tea plant, Camellia kucha, has in recent years come under investigation for potential medicinal uses. Caffeine, a widely consumed psychostimulant also derived from kucha, is structurally similar to theacrine but has been more extensively studied and thoroughly defined in terms of neuromodulation and subsequent behavioral outcomes (Fisone et al., 2004, Fredholm et al., 1999, Yacoubi et al., 2000). With few known side effects, these alkaloids harness potential for therapeutic treatments, with caffeine already proven useful in alleviating symptoms of Parkinson's disease (Prediger, 2010, Xu et al., 2010). In mice, theacrine exhibited anti-inflammatory and analgesic properties (Wang et al., 2010), as well as antidepressant effects in an animal model of depression (Xie et al., 2009).

Caffeine and other methylxanthines act as competitive antagonists at adenosine receptors which allow them to modulate attention, vigilance, arousal, and motor activity (Fredholm et al., 1999, Porkka-Heiskanen et al., 2000, Snyder et al., 1981). Of the four identified adenosine receptors, two subtypes (A1 and A2A) are important for mediating (Yacoubi et al., 2000) these behavioral outcomes through multiple mechanisms at both pre- and post-synaptic sites (Daly et al., 1983, Snyder et al., 1981, Svenningsson et al., 1997). A1 and A2A are G-protein-coupled receptors (GPCRs) that are functionally coupled to the pertussis toxin-sensitive family of G proteins (Gi/o and Gs/olf), respectively (Kull et al., 2000, MacDonald et al., 1986, van Calker et al., 1979). The A1 receptors are widely distributed in the brain with high expression in the hippocampus, cortex, and cerebellum (Rivkees et al., 1995), whereas the A2A receptors are expressed in regions rich in dopaminergic projections, such as the striatum, nucleus accumbens (NAc) and olfactory tubercle (Fredholm et al., 2001, Jarvis and Williams, 1989). Caffeine-mediated increases in motor activity are primarily attributed to blockade of both A1 and A2A receptors and the subsequent increase in dopaminergic neurotransmission (Quarta et al., 2004b, Solinas et al., 2002, Solinas et al., 2005).

The enhancement of extracellular concentrations of dopamine (DA) in the NAc produced by caffeine and classical psychostimulants, such as amphetamine, appears to be pivotal in modulating an animal's motivational arousal and motor responses (Wise, 2004). For example, administration of DA receptor antagonists reduces the stimulant-induced hyperlocomotion (Garrett and Holtzman, 1994), as well as the concurrent increase of accumbal DA (Hamamura et al., 1991). Other striatal regions and the basal ganglia circuit also participate in the motor-activating effects of caffeine by complex interactions between adenosine and dopamine receptors (Ferre et al., 1997, Pinna et al., 1996, Pollack and Fink, 1996).

Since little is known concerning the pharmacological and behavioral properties of theacrine, we investigated the locomotor activity of rats following theacrine treatment and carried out experiments to determine the mechanism of action underlying the motor-activating effects of this compound. Although theacrine and caffeine are very similar in structure, it has yet to be confirmed that theacrine is acting as a adenosine receptor antagonist; therefore, theacrine was adminstered to test whether it would counteract the motor-depressant effects of adenosine A1 and A2A receptor agonists. To evaluate the role of DA receptors in theacrine-mediated motor activity, selective D1 and D2 receptor antagonists were injected prior to theacrine or vehicle treatment. In addition, the drug was administered for seven consecutive days to test for behavioral sensitization or tolerance. Finally, to assess the role of the ventral striatum in theacrine activity modifications, the drug was microinfused directly into the NAc.

Section snippets

Drugs and chemicals

Theacrine was extracted from C. kucha (purification > 99%) as previously described in Wang et al. (2010). Theacrine, DA D1R antagonist, SCH23390 (Sigma-Aldrich, St. Louis, MO), and DA D2R antagonist, eticlopride (Sigma-Aldrich, St. Louis, MO), were all dissolved in double de-ionized water immediately before each injection (Fig. 1). Theacrine was injected in a volume of 2 ml/kg while the DA receptor antagonists were injected in a volume of 1 ml/kg. The adenosine A1 receptor agonist N6

Effect of theacrine on locomotor activity

A one-way ANOVA revealed that the highest dose of theacrine (48 mg/kg, i.p.) resulted in significantly greater activity (2-h period following injections) counts compared to vehicle while the low dose (24 mg/kg) did not [ambulatory distance: F(2, 20) = 6.114, p < 0.01; stereotypic counts: F(2, 20) = 13.20, p < 0.001; Fig. 2]. To further analyze this effect, a two-way repeated measures ANOVA (treatment × time) was performed on theacrine (48 mg/kg, i.p.) and vehicle activity counts from the entire 3 h session

Discussion

The present experiments are the first to demonstrate that the purine alkaloid theacrine significantly increases locomotor activity in rats. Following administration, theacrine treated animals showed greater motor activity on both behavioral measures (ambulatory distance and stereotypic counts) compared to the vehicle treated group and this effect lasted throughout the duration of the test session. Administration of adenosine A1 and A2A receptor agonists resulted in motor depression and

Acknowledgments

We would like to thank Jade J. Bito-Onon for her excellent technical assistance with the behavioral experiments. This work was supported by funding from the State of California for Medical Research through UCSF to S.E.B, Department of Defense Grant (W81XWH-07-1-0075) to S.E.B., and the NIH International Coop Biodiversity Groups (U01TW008160) subcontract to S.E.B. The experiments contained herein comply with the current laws of the USA. All procedures were preapproved by the Gallo Center

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