Regulation of Phosphorylation of AMPA Glutamate Receptors by Muscarinic M4 Receptors in the Striatum In vivo

Abstract

The acetylcholine muscarinic 4 (M4) receptor is a principal muscarinic receptor subtype present in the striatum. Notably, G_αi/o-coupled M4 receptors and G_αs/G_olf-coupled dopamine D1 receptors are coexpressed in striatonigral projection neurons and are thought to interact with each other to regulate neuronal excitability, although underlying molecular mechanisms are poorly understood. In this study, we investigated the role of M4 receptors in the regulation of phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the rat normal and dopamine-stimulated striatum in vivo. We found that a systemic injection of a M4 antagonist tropicamide increased AMPA receptor GluA1 subunit phosphorylation at a protein kinase A-dependent site (S845) in the striatum. The tropicamide-induced S845 phosphorylation was rapid, reversible, and dose-dependent and occurred in the two subdivisions of the striatum, i.e., the caudate putamen and nucleus accumbens. Coadministration of subthreshold doses of tropicamide and a D1 agonist SKF81297 induced a significant increase in S845 phosphorylation. Coadministered tropicamide and a dopamine psychostimulant amphetamine at their subthreshold doses also elevated S845 phosphorylation. Tropicamide alone or coinjected with SKF81297 or amphetamine had no effect on GluA1 phosphorylation at S831. Tropicamide did not affect GluA2 phosphorylation at S880. These results reveal a selective inhibitory linkage from M4 receptors to GluA1 in S845 phosphorylation in striatal neurons. Blockade of the M4-mediated inhibition significantly augments constitutive and dopamine-stimulated GluA1 S845 phosphorylation.

Keywords: D1 receptor; D2 receptor; caudate putamen; muscarinic receptor; nucleus accumbens; protein kinase A.

Figure 1. Effects of tropicamide on GluA1 phosphorylation in the rat CPu

(A) Representative immunoblots illustrating effects of tropicamide on GluA1 phosphorylation at 15 min, 1 h, 2 h, and 3 h after injection. (B–D) Quantifications of effects of tropicamide on GluA1 phosphorylation at S845 (B) and S831 (C) and GluA1 expression (D) at the four time points after injection. Note that tropicamide produced a rapid and reversible increase in S845 phosphorylation, while it had no effect on S831 phosphorylation. Rats were given an i.p. injection of vehicle (Veh) or tropicamide (Tro, 10 mg/kg) and were sacrificed at 15 min, 1 h, 2 h, or 3 h after drug injection for immunoblot analysis. Data were analyzed with Student’s t-test: t-values = 3.904 (pS845 15 min), 3.999 (pS845 1 h), 1.961 (pS845 2 h), 0.901 (pS845 3 h), 0.765 (pS831 15 min), 0.072 (pS831 1 h), 0.982 (pS831 2 h), 1.030 (pS831 3 h), 0.456 (GluA1 15 min), 0.906 (GluA1 1 h), 1.841 (GluA1 2 h), and 0.810 (GluA1 3 h). Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus vehicle at the same time points.

Figure 2. Effects of tropicamide on GluA1 phosphorylation in the rat NAc

(A) Representative immunoblots illustrating effects of tropicamide on GluA1 phosphorylation at 15 min, 1 h, 2 h, and 3 h after injection. (B–D) Quantifications of effects of tropicamide on GluA1 phosphorylation at S845 (B) and S831 (C) and GluA1 expression (D) at the four time points after injection. Note that tropicamide produced a rapid and reversible increase in S845 phosphorylation, while it had no effect on S831 phosphorylation. Rats were given an i.p. injection of vehicle (Veh) or tropicamide (Tro, 10 mg/kg) and were sacrificed at 15 min, 1 h, 2 h, or 3 h after drug injection for immunoblot analysis. Data were analyzed with Student’s t-test: t-values = 3.742 (pS845 15 min), 3.875 (pS845 1 h), 1.217 (pS845 2 h), 1.246 (pS845 3 h), 0.059 (pS831 15 min), 0.725 (pS831 1 h), 0.174 (pS831 2 h), 0.636 (pS831 3 h), 1.200 (GluA1 15 min), 1.123 (GluA1 1 h), 1.949 (GluA1 2 h), and 0.844 (GluA1 3 h). Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus vehicle at the same time points.

Figure 3. Effects of tropicamide at different doses on GluA1 phosphorylation in the rat CPu and NAc

(A) Effects of tropicamide on GluA1 phosphorylation at S845 and S831 and GluA1 expression after injection at 2, 10, and 20 mg/kg in the CPu. (B) Effects of tropicamide on GluA1 phosphorylation at S845 and S831 and GluA1 expression after injection at 2, 10, and 20 mg/kg in the NAc. Note that tropicamide produced a dose-dependent increase in S845 phosphorylation in both the CPu and NAc, while it had no effect on S831 phosphorylation. Representative immunoblots are shown to the left of the quantified data. Rats were given an i.p. injection of tropicamide at different doses (2, 10, or 20 mg/kg) or vehicle (0 mg/kg) and were sacrificed 1 h after drug injection for immunoblot analysis. Data were analyzed with one-way ANOVA: CPu pS845: F(3,12) = 11.62, n = 16; CPu pS831, F(3,12) = 0.56, n = 16; CPu GluA1, F(3,12) = 0.01, n = 16; NAc pS845, F(3,12) = 8.34, n = 16; NAc pS831, F(3,12) = 0.19, n = 16; and NAc GluA1, F(3,12) = 0.16, n = 16. Data are presented as means ± SEM. *P < 0.05 versus vehicle.

Figure 4. Effects of tropicamide on GluA2 phosphorylation in the rat CPu and NAc

(A) Effects of tropicamide on GluA2 S880 phosphorylation in the CPu. (B) Effects of tropicamide on GluA2 S880 phosphorylation in the NAc. Representative immunoblots are shown to the left of the quantified data. Rats were given an i.p. injection of tropicamide at different doses (2, 10, or 20 mg/kg) or vehicle (0 mg/kg) and were sacrificed 1 h after drug injection for immunoblot analysis. Data were analyzed with one-way ANOVA: CPu pS880: F(3,12) = 0.62, n = 16; CPu GluA2, F(3,12) = 0.47, n = 16; NAc pS880, F(3,12) = 0.32, n = 16; and NAc GluA2, F(3,12) = 0.50, n = 16. Data are presented as means ± SEM.

Figure 5. Effects of coadministration of tropicamide and SKF81297 on GluA1 phosphorylation in the rat striatum

(A) Effects of coadministration of tropicamide and SKF81297 on GluA1 phosphorylation at S845 and S831 and GluA1 expression in the CPu. (B) Effects of coadministration of tropicamide and SKF81297 on GluA1 phosphorylation at S845 and S831 and GluA1 expression in the NAc. Note that tropicamide and SKF81297 when coadministered at subthreshold doses produced an increase in S845 phosphorylation, while either drug alone did not alter S845 phosphorylation. Representative immunoblots are shown to the left of the quantified data. Rats were given an i.p. injection of vehicle (Veh) or tropicamide (Tro, 2 mg/kg) 15 min prior to SKF81297 (SKF, 0.5 mg/kg) and were sacrificed 20 min after SKF81297 injection for immunoblot analysis. Data were analyzed with one-way ANOVA: CPu pS845: F(3,12) = 7.27, n = 16; CPu pS831, F(3,12) = 0.44, n = 16; CPu GluA1, F(3,12) = 0.59, n = 16; NAc pS845, F(3,12) = 7.98, n = 16; NAc pS831, F(3,12) = 0.05, n = 16; and NAc GluA1, F(3,12) = 0.15, n = 16. Data are presented as means ± SEM. *P < 0.05 versus vehicle + saline.

Figure 6. Effects of coadministration of tropicamide and AMPH on GluA1 phosphorylation in the rat striatum

(A) Effects of coadministration of tropicamide and AMPH on GluA1 phosphorylation at S845 and S831 and GluA1 expression in the CPu. (B) Effects of coadministration of tropicamide and AMPH on GluA1 phosphorylation at S845 and S831 and GluA1 expression in the NAc. Note that tropicamide and AMPH when coadministered at subthreshold doses produced an increase in S845 phosphorylation, while either drug alone did not alter S845 phosphorylation. Representative immunoblots are shown to the left of the quantified data. Rats were given an i.p. injection of vehicle (Veh) or tropicamide (Tro, 2 mg/kg) 15 min prior to AMPH (0.5 mg/kg) and were sacrificed 15 min after AMPH injection for immunoblot analysis. Data were analyzed with one-way ANOVA: CPu pS845: F(3,12) = 6.15, n = 16; CPu pS831, F(3,12) = 0.35, n = 16; CPu GluA1, F(3,12) = 0.32, n = 16; NAc pS845, F(3,12) = 9.25, n = 16; NAc pS831, F(3,12) = 0.64, n = 16; and NAc GluA1, F(3,12) = 0.41, n = 16. Data are presented as means ± SEM. *P < 0.05 versus vehicle + saline.