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Adenosine A2A receptor antagonists are psychomotor stimulants that also hold therapeutic promise for movement disorders. However, the molecular mechanisms underlying their stimulant properties are not well understood. Here we show that the robust increase in locomotor activity induced by an A2A antagonist in vivo is greatly attenuated by antagonizing cannabinoid CB1 receptor signaling or by administration to CB1−/− mice. To determine the locus of increased endocannabinoid signaling, we measured the amount of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in brain tissue from striatum and cortex. We find that 2-AG is selectively increased in striatum after acute blockade of A2A receptors, which are highly expressed by striatal indirect-pathway medium spiny neurons (MSNs). Using targeted whole-cell recordings from direct- and indirect-pathway MSNs, we demonstrate that A2A receptor antagonists potentiate 2-AG release and induction of long-term depression (LTD) at indirect-pathway MSNs, but not direct-pathway MSNs. Together these data outline a molecular mechanism by which A2A antagonists reduce excitatory synaptic drive on the indirect pathway through CB1 receptor signaling, thus leading to increased psychomotor activation.
The basal ganglia and its primary input nucleus, the striatum, are critical for motivation and motor control (Graybiel et al., 1994; Hikosaka et al., 2000; Yin and Knowlton, 2006). The striatum integrates information from the cortex, thalamus, and midbrain, and sends projections to downstream basal ganglia nuclei that regulate thalamocortical motor circuits (Bolam et al., 2000). Striatal projection neurons, known as MSNs, are GABAergic and can be divided into two sub-classes based on their axonal projections and gene expression patterns. Direct-pathway MSNs, which project directly to basal ganglia output nuclei, express dopamine D1 receptors. Indirect-pathway MSNs, which project to the globus pallidus, express dopamine D2 and adenosine A2A receptors (Gerfen et al., 1990; Schiffmann et al., 1991; Smith et al., 1998). According to classical basal ganglia models, increased direct-pathway activity facilitates movement whereas increased indirect-pathway activity inhibits movement (Albin et al., 1989; DeLong, 1990).
A2A receptors are Gs-coupled metabotropic receptors that are highly expressed in the striatum, and to a lesser extent in other brain regions including the globus pallidus, hippocampus, and cortex (Sebastiao and Ribeiro, 1996). They are enriched in the postsynaptic density of glutamatergic synapses onto striatal indirect-pathway MSNs (Rosin et al., 2003; Schiffmann et al., 2007), although they are also observed in some presynaptic terminals in the striatum and globus pallidus, where they appear to enhance neurotransmitter release (Shindou et al., 2003; Shindou et al., 2008). Behaviorally, A2A receptor agonists decrease movement (Barraco et al., 1993; Hauber and Munkle, 1997), and facilitate the induction of LTP (Flajolet et al., 2008), suggesting that they increase indirect-pathway activity. In contrast, A2A receptor antagonists increase movement and also cause an increase in immediate-early gene expression in the globus pallidus, suggesting that they decrease indirect-pathway activity (Svenningsson et al., 1997; Hauber et al., 1998; Huang et al., 2005; Mingote et al., 2008; Shen et al., 2008a). A2A and D2 receptors have opposing effects on cAMP accumulation in indirect pathway neurons and inhibition of A2A receptors facilitates D2 receptor-mediated processes (Ferre et al., 1997; Stromberg et al., 2000; Tozzi et al., 2007; Kim and Palmiter, 2008). Because of their actions on the indirect pathway, A2A antagonists have been proposed as an adjunct or alternative to dopamine replacement therapy in patients with Parkinson disease (PD) (Schwarzschild et al., 2006; Simola et al., 2008; Jenner et al., 2009). However, despite extensive characterization of the intracellular signaling pathways involved in A2A receptor signaling (for a review, see (Fuxe et al., 2007)), little is known about the effector pathways mediating their inhibition of the indirect pathway.
Endocannabinoid signaling is prominent in the striatum and represents a major downstream target of D2 receptor activation in indirect-pathway MSNs (Giuffrida et al., 1999; Kreitzer and Malenka, 2007; Shen et al., 2008b), raising the possibility that it could be influenced by A2A receptors (Rossi et al., 2009). Two major endocannabinoids have been identified thus far: AEA and 2-AG. These endocannabinoids act as retrograde messengers at synapses, where they are released from postsynaptic dendrites and bind to presynaptic CB1 receptors to depress neurotransmitter release (Chevaleyre et al., 2006; Kano et al., 2009). In the striatum, endocannabinoids underlie both short-term and long-term depression (LTD) of excitatory synapses onto indirect-pathway MSNs (Gerdeman et al., 2002; Narushima et al., 2006a). In this study, we tested the hypothesis that A2A antagonists stimulate motor activity by enhancing endocannabinoid-mediated LTD at striatal glutamatergic indirect-pathway afferents.
Spontaneous locomotor activity was measured in an automated Flex-Field/Open Field Photobeam Activity System (San Diego Instruments, San Diego, CA). Male wildtype C57BL/6 mice (Charles River) and CB1−/− mice on the C57BL/6 background (Marsicano et al., 2002) aged 7–11 weeks old, were used for behavioral testing. Mice were acclimated to the testing room for at least 30 minutes. Each mouse was injected with 5 µl/g of either AM251 (5 mg/kg, i.p.) or its vehicle, a solution of 50% polyethylene glycol (PEG) and 50% saline (0.9% NaCl) immediately before being placed in the center of the test chamber. Following a 15-minute habituation period, baseline locomotor activity was monitored for 15 minutes. Then, mice were injected with 5 µl/g of SCH442416 (3 mg/kg, i.p., also in 50% PEG vehicle) and monitored for another 30 minutes. Beam breaks were recorded and binned in 3 minute intervals. The test chamber was cleaned with 70% ethanol between testing of each mouse. Mice were excluded from analysis if their average number of beam breaks per 3 minute period during baseline was less than 5 or more than 150 (to eliminate unusually hypo- or hyperactive mice), or if the standard deviation of baseline values was greater than 80 (to eliminate mice that exhibited highly variable activity). Statistical significance vs. baseline within each group was evaluated by a paired t-test. Statistical significance between groups was evaluated by one-way ANOVA with Tukey’s HSD test.
Mice (C57BL/6, aged 4–5 weeks old) were injected i.p. with 5 µl/g of either SCH442416 (3 mg/kg) or its vehicle (50% PEG). Five to seven minutes after injection, mice were sacrificed, and their right and left striatum, as well as their right and left cortex were dissected within 1 minute. Each tissue sample (4 per mouse) was immediately placed in a 1.5 mL tube, frozen in liquid nitrogen, and stored at −80°C. To reliably quantify the amount of AEA and 2-AG in tissue samples by CI-GC-MS, 3 samples of striatum were combined according to each treatment and their total mass determined. Because of their larger mass, individual cortical samples were analyzed. All samples were placed in 10 mL CHCl3 and homogenized for 1 minute at 10,000 rpm using a PRO 200 homogenizer (Pro Scientific Inc.; Oxford, CT). The following deuterated standards were added to each homogenate: 150 pmole of d5-2-AG and 50 pmole of 3[H]-AEA (Cayman Chemical Co.; Ann Arbor, MI). Lipids were then extracted, purified and derivatized as described by Muccioli and Stella (Muccioli and Stella, 2008). Three µL of each sample (corresponding to 4–8.5 mg tissue/injection) was then injected by a CP-8400 autosampler into a Varian CP3800 Gas Chromatogram. The temperature elution protocol, chemical ionization parameters and isotope dilution quantification were as described in Muccioli and Stella (Muccioli and Stella, 2008). The total ion currents were recorded for each sample and the individual endocannabinoids were identified by their diagnostic peaks (2-AG: 433 m/z; AEA 330 + 420 + 493m/z) using MSDATA Review (Varian). Statistical significance was evaluated using a two-tailed unpaired t-test.
Coronal brain slices (300 µm) were prepared from Drd2-GFP heterozygotic BAC transgenic mice on the C57BL/6 background (postnatal days 21–35). Slices were superfused with an external solution containing (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 26 NaHCO3, 1.25 NaH2PO4-H2O and 12.5 glucose, bubbled with 95% O2/5% CO2. Slices were allowed to recover for at least 1 hour before recording. Whole-cell voltage-clamp recordings were obtained from visually-identified GFP-positive or GFP-negative MSNs in dorsolateral striatum at a temperature of 30–32°C, with picrotoxin (50 µM) present to suppress GABAA-mediated currents. Resistance of the patch pipettes was 2.5–4MΩ when filled with intracellular solution containing (in mM): 120 CsMeSO3, 15 CsCl, 8 NaCl, 0.2 EGTA, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, 10 TEA, 5 QX-314, adjusted to pH 7.3 with CsOH. MSNs were held at −70 mV and excitatory synaptic currents were evoked by intrastriatal microstimulation with a saline-filled glass pipette placed 50–100 µm dorsolateral of the recorded neuron. Test pulses, which consisted of 2 stimuli 50 ms apart, were given every 20 seconds. To evoke LTD, MSNs were stimulated at 20Hz or 100Hz for 1s, paired with postsynaptic depolarization to −10 mV. THL was purchased from Sigma-Aldrich and all other drugs were from Tocris Bioscience. THL was dissolved in DMSO at 10 mM, and used at 10 µM, yielding 0.1% DMSO, a concentration that does not affect striatal LTD (Gerdeman et al., 2002). All data acquisition and analysis was performed online with custom Igor Pro software. Statistical significance was evaluated using a two-tailed unpaired t-test.
To determine whether psychomotor stimulation by A2A antagonists requires endocannabinoid signaling, the selective A2A antagonist SCH442416 (3 mg/kg i.p.) was administered to three groups of mice: strain-matched wildtype controls, mice pre-treated with the CB1 receptor antagonist AM251 (5 mg/kg, i.p.), and mice lacking CB1 receptors (CB1−/− mice) (Marsicano et al., 2002). A2A antagonist treatment significantly increased ambulatory activity in wildtype mice (Figure 1A–B, Table 1). Pre-treatment of wildtype mice with AM251 (5 mg/kg i.p.) had little effect on baseline ambulatory activity (105±14% of wildtype baseline, p>0.05, n=14), but significantly attenuated the effects of SCH442416 (Figure 1A–B, Table 1) indicating that functional CB1 receptors are required for mice to fully increase their ambulatory activity in response to an A2A antagonist. Similar results were found when we tested the effects of SCH442416 on CB1−/− mice. CB1−/− mice exhibited slightly higher baseline ambulatory activity compared to strain-matched controls of similar age (161±12% of wildtype baseline, p<0.05, n=14). However, similar to AM251-pre-treated mice, the effects of SCH442416 treatment on ambulatory activity were greatly attenuated in CB1−/− mice compared to wildtype vehicle-pretreated controls (Figure 1A–B, Table 1). SCH442416 also induced a small but significant increase in fine movements (Figure 1C–D, Table 1). However, the increase in fine movements was not altered in AM251-treated mice or CB1−/− mice (Figure 1C–D, Table 1), indicating that this feature of psychomotor activation by SCH442416 does not require endocannabinoid signaling. To verify that the increases in both ambulatory activity and fine movements that we observed were caused by SCH442416 and not by the injection procedure itself, we injected vehicle instead of SCH442416 into a subset of mice. Injection of vehicle had no effects on either type of locomotor activity (Table 1).
Our behavioral data indicate that the psychomotor effects of A2A receptor antagonists are mediated at least in part by activation of CB1 receptors. To test whether A2A antagonists alter endocannabinoid levels in the striatum or cortex, we measured the amount of 2-AG and AEA in striatal and cortical tissue samples, 5–7 minutes after injection of SCH442416 (3 mg/kg, i.p.) or vehicle solution. Both endocannabinoids were detectable in striatal and cortical samples from mice injected with vehicle (Figure 2 A–D). In mice injected with SCH442416, 2-AG was increased in striatum (Figure 2A), but not cortex (Figure 2B). No significant differences in AEA levels were observed in striatal or cortical samples from mice injected with SCH442416 vs. vehicle. (Figure 2 C–D). Together, these results shown that A2A antagonists specifically increase the amount of striatal 2-AG.
Within the striatum, A2A receptors are highly enriched at excitatory synapses onto indirect-pathway MSNs (Rosin et al., 2003), and decreasing striatal indirect pathway function increases ambulatory activity (Durieux et al., 2009). Because the effects of SCH442416 depend on CB1 receptor activation, we tested whether it induced the release of endocannabinoids from indirect-pathway MSNs. However, application of SCH442416 (1 µM) did not alter baseline excitatory synaptic responses in indirect-pathway MSNs (Supp. Fig. 1A). We next tested whether SCH442416 could potentiate endocannabinoid-mediated LTD in indirect-pathway MSNs. First, we elicited LTD using high-frequency stimulation (100 Hz), paired with postsynaptic depolarization. Although this protocol elicited robust LTD, as previously reported (Gerdeman et al., 2002; Kreitzer and Malenka, 2007), the magnitude of LTD was not potentiated by SCH442416 (1 µM) (Supp. Fig. 1B). However, a moderate-frequency (20 Hz) stimulation protocol that elicited a small amount of LTD in control conditions gave rise to robust LTD in the presence of SCH442416 (1 µM) (88 ± 7% of baseline at 30–40 min in control conditions; 61 ± 8% of baseline at 30–40 min in SCH442416; p<0.05; Fig. 3A). Furthermore, in the presence of SCH442416, this form of LTD was blocked (102 ± 9% of baseline at 30–40 min; Fig. 3B) by tetrahydrolipstatin (THL; 10 µM), an inhibitor of the 2-AG synthetic enzyme diacylglycerol lipase. This enhancement of 2-AG release was pathway specific, because when we delivered 20 Hz stimulation paired with postsynaptic depolarization to direct-pathway MSNs in SCH442416, no enhancement of LTD was observed (86 ± 6% of baseline at 30–40 min in control conditions; 97 ± 10% of baseline at 30–40 min in SCH442416; p>0.05; Fig. 3C). Therefore, SCH442416 selectively enhances 2-AG release and LTD induction in indirect-pathway MSNs.
In this study, we identify a molecular mechanism underlying psychomotor activation by A2A antagonists. Specifically, we show that A2A antagonists increase striatal 2-AG and potentiate 2-AG-mediated LTD of excitatory afferents on indirect-pathway MSNs. Furthermore, blocking CB1 receptor function in vivo greatly attenuates the psychomotor stimulating effects of A2A antagonists. Our data are consistent with a model in which endocannabinoid-mediated inhibition of the indirect pathway increases movement.
These findings provide insight into a molecular mechanism for psychomotor stimulation by A2A antagonists. However, A2A receptor antagonists are certain to exhibit complex effects across numerous brain regions. In addition to the striatum, there are other potential sites of interaction between A2A receptors and endocannabinoid signaling, including the cortex and the globus pallidus. However, we don’t observe any change in cortical endocannabinoid levels after A2A antagonist treatment, and in the globus pallidus, A2A transcript is not observed postsynaptically (Rosin et al., 2003), where endocannabinoids are produced. Although presynaptic interactions between A2A and CB1 receptors are possible in the globus pallidus, CB1 receptor-mediated inhibition of IPSCs is reportedly mediated by suppression of calcium influx (Engler et al., 2006), whereas A2A receptor mediated enhancement of IPSCs is independent of calcium (Shindou et al., 2002), suggesting that these pathways act independently of each other. Furthermore, although CB1 receptors were required for the bulk of psychomotor activation by SCH442416, some psychomotor stimulation still occurred when CB1 receptor signaling was blocked (Figure 1A–B). This remaining stimulation was likely due to parallel signaling pathways initiated by A2A receptor inhibition, such as decreased release of GABA in the globus pallidus (Shindou et al., 2003), which would act synergistically with striatal 2-AG-mediated inhibition to reduce the efficacy of the indirect pathway.
We also revealed two interesting features of striatal LTD by using a moderate-frequency (20 Hz) induction protocol. First, we observed a small amount of LTD in both direct- and indirect-pathway MSNs (10–15%), consistent with the idea that endocannabinoids can be produced in both types of MSN under some experimental conditions (Narushima et al., 2006b; Shen et al., 2008b). Second, we found that indirect-pathway LTD in SCH442416 is mediated by 2-AG, whereas LTD elicited by 100-Hz stimulation is reportedly mediated by anandamide (Ade and Lovinger, 2007). Consistent with that study, we also found that striatal LTD elicited by 100-Hz stimulation is not blocked by THL (Supp. Fig. 1C). This suggests that the identity of the endocannabinoid that mediates striatal LTD can vary, depending on the experimental conditions.
A link between A2A antagonists and endocannabinoids has implications for the use of A2A antagonists to treat Parkinson’s disease (PD). In mouse models of PD, dopamine depletion causes a loss of endocannabinoid-dependent long-term depression (LTD) at excitatory synapses onto indirect-pathway MSNs (Kreitzer and Malenka, 2007; Shen et al., 2008b). Our findings raise the possibility that A2A antagonists can help counter the effects of dopamine depletion by increasing endocannabinoid signaling at these synapses. Although A2A antagonists are already being investigated as PD therapeutics, our findings suggest that the efficacy of these drugs may be increased by developing compounds for human consumption that inhibit endocannabinoid degradation.
A, SCH442415 (1 µM) was washed onto the slice at the time indicated by the black bar (n=6). In this and subsequent panels, normalized EPSC amplitudes are plotted over time. B, 100 Hz stimulation of afferents to indirect-pathway MSNs in control solution (n=6) and in 1 µM SCH442416 (n=5). In both B and C, the arrow indicates the time of 100 Hz stimulation, which was paired with postsynaptic depolarization to −10 mV. C, 100 Hz stimulation of afferents to indirect-pathway MSNs in 1 µM SCH442416 and 10 µM THL (n=8).
We would like to thank Beat Lutz for kindly providing CB1−/− mice, Nino Devidze, Iris Lo, Tracy Hamto, Lex Kravitz, and Philip Parker for their assistance with the behavioral experiments, and Sergi Ferre for helpful discussion. This work was supported by NIH grant R01 NS064984, the Pew Biomedical Scholars Program, the W.M. Keck Foundation, and the Wayne and Gladys Valley Foundation.