Integrated regulation of AMPA glutamate receptor phosphorylation in the striatum by dopamine and acetylcholine

Abstract

Dopamine (DA) and acetylcholine (ACh) signals converge onto protein kinase A (PKA) in medium spiny neurons of the striatum to control cellular and synaptic activities of these neurons, although underlying molecular mechanisms are less clear. Here we measured phosphorylation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) at a PKA site (S845) as an indicator of AMPAR responses in adult rat brains in vivo to explore how DA and ACh interact to modulate AMPARs. We found that subtype-selective activation of DA D1 receptors (D1Rs), D2 receptors (D2Rs), or muscarinic M4 receptors (M4Rs) induced specific patterns of GluA1 S845 responses in the striatum. These defined patterns support a local multitransmitter interaction model in which D2Rs inhibited an intrinsic inhibitory element mediated by M4Rs to enhance the D1R efficacy in modulating AMPARs. Consistent with this, selective enhancement of M4R activity by a positive allosteric modulator resumed the cholinergic inhibition of D1Rs. In addition, D1R and D2R coactivation recruited GluA1 and PKA preferentially to extrasynaptic sites. In sum, our in vivo data support an existence of a dynamic DA-ACh balance in the striatum which actively modulates GluA1 AMPAR phosphorylation and trafficking. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Keywords: (-)-quinpirole hydrochloride (PubChem CID:55397); (-)-scopolamine hydrobromide (PubChem CID:517999); Caudate putamen; D1; D2; Muscarinic receptor; Nucleus accumbens; Positive allosteric modulator; Protein kinase A; S-(-)-eticlopride hydrochloride (PubChem CID:11973707); S845; SCH23390 (PubChem CID:5018); SKF81297 (PubChem CID:1218); VU0152100 (PubChem CID:864492).

Figure 1. Effects of the D1R and D2R agonists on basal GluA1 phosphorylation in the rat striatum

(A) Representative immunoblots illustrating effects of SKF81297 and quinpirole on GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of SKF81297 and quinpirole on GluA1 phosphorylation at S845 (B) and S831 (C) and total GluA1 expression (D). Note that SKF81297 increased S845 phosphorylation, while quinpirole did not. Co-administration of the two agonists induced a greater increase in pS845 levels than that induced by the D1R agonist alone. Rats were given an i.p. injection of SKF81297 (SKF, 3 mg/kg), quinpirole (Quin, 3 mg/kg) or both drugs and were sacrificed 20 min after drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus saline.

Figure 2. Effects of the D1R and D2R antagonists on basal and SKF81297/quinpirole-stimulated GluA1 phosphorylation in the rat striatum

(A) Representative immunoblots showing effects of SCH23390 and eticlopride on basal and stimulated GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of SCH23390 and eticlopride on basal and stimulated GluA1 phosphorylation at S845 (B) and S831 (C) and total GluA1 expression (D). Note that eticlopride elevated pS845 levels and did not affect the S845 phosphorylation induced by coinjected SKF81297 and quinpirole. Rats were given an i.p. injection of SCH23390 (SCH, 0.1-0.5 mg/kg) or eticlopride (Eti, 0.5 mg/kg) 15 min prior to saline (sal) or SKF81297 (SKF)/quinpirole (Quin) at 3 mg/kg and were sacrificed 20 min after the final drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus saline + saline. +P < 0.05 versus saline + SKF81297/quinpirole.

Figure 3. Effects of the M4R PAM on basal GluA1 phosphorylation in the rat striatum

(A) Representative immunoblots showing effects of VU0152100 on basal GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of VU0152100 on basal GluA1 phosphorylation at S845 (B) and S831 (C) and total GluA1 expression (D). Rats were given an i.p. injection of vehicle or VU0152100 (VU) at 6 or 60 mg/kg and were sacrificed 20 min after drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 4 per group).

Figure 4. Effects of the M4R PAM on the D1R agonist-stimulated GluA1 phosphorylation in the rat striatum

(A) Representative immunoblots showing effects of VU0152100 on SKF81297-stimulated GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of VU0152100 on SKF81297-stimulated GluA1 phosphorylation. Note that VU0152100 significantly reduced the SKF81297-stimulated S845 phosphorylation. Rats were given an i.p. injection of vehicle (Veh) or VU0152100 (VU, 60 mg/kg) 20 min prior to saline (sal) or SKF81297 (SKF, 3 mg/kg) and were sacrificed 20 min after the final drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus vehicle + saline. +P < 0.05 versus vehicle + SKF81297.

Figure 5. Effects of the M4R PAM on the D1R/D2R agonists-stimulated GluA1 phosphorylation in the rat striatum

(A) Representative immunoblots showing effects of VU0152100 on SKF81297/quinpirole-stimulated GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of VU0152100 on SKF81297/quinpirole-stimulated GluA1 phosphorylation. Note that VU0152100 markedly reduced the SKF81297/quinpirole-stimulated S845 phosphorylation. Rats were given an i.p. injection of vehicle (Veh) or VU0152100 (VU, 60 mg/kg) 20 min prior to SKF81297 (SKF)/quinpirole (Quin) at 3 mg/kg and were sacrificed 20 min after the final drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 4 per group). *P < 0.05 versus vehicle + saline. +P < 0.05 versus vehicle + SKF81297/quinpirole.

Figure 6. Effects of scopolamine on GluA1 S845 phosphorylation induced by SKF81297 and by co-administration of SKF81297 and quinpirole in the rat striatum

(A) Representative immunoblots showing effects of scopolamine on SKF81297- and SKF81297/quinpirole-stimulated GluA1 phosphorylation in the striatum. (B-D) Quantification of effects of scopolamine on SKF81297- and SKF81297/quinpirole-stimulated GluA1 phosphorylation. Note that either scopolamine or quinpirole significantly augmented the SKF81297-stimulated S845 phosphorylation. Moreover, in the presence of scopolamine, quinpirole did not further augment the response of S845 phosphorylation to SKF81297. Rats were given an i.p. injection of saline (Sal) or scopolamine (Sco, 5 mg/kg) 10 min prior to SKF81297 (SKF, 1 mg/kg) or co-administration of SKF81297 (1 mg/kg) and quinpirole (Quin, 1.5 mg/kg) and were sacrificed 20 min after the final drug injection for immunoblot analysis. Data are presented as means ± SEM (n = 5 per group). *P < 0.05 versus saline + saline. +P < 0.05 versus saline + SKF81297.

Figure 7. Effects of coactivation of D1Rs and D2Rs on subsynaptic redistribution of AMPARs and PKA in the rat striatum

(A) Effects of SKF81297/quinpirole on GluA1 and PKA expression in the synaptic fraction. (B) Effects of SKF81297/quinpirole on GluA1 and PKA expression in the extrasynaptic fraction. Representative immunoblots are shown left to the quantified data. Rats were given an injection of saline or a coinjection of SKF81297 (SKF) and quinpirole (Quin) at 3 mg/kg (i.p.) and were sacrificed 90 min after drug injection for enriching synaptic and extrasynaptic membranes. Note that SKF81297/quinpirole increased the abundance of pS845 proteins in the synaptic fraction and pS845, GluA1, PKA Cα, and PKA RIIβ proteins in the extrasynaptic fraction, while two agonists had no significant effect on GluA2, GluA3, and pS831 levels in either subsynaptic region. Data are presented as means ± SEM (n = 5 per group). *P < 0.05 versus saline.