Role of purinergic P2X4 receptors in regulating striatal dopamine homeostasis and dependent behaviors

Abstract

Purinergic P2X4 receptors (P2X4Rs) belong to the P2X superfamily of ion channels regulated by ATP. We recently demonstrated that P2X4R knockout (KO) mice exhibited deficits in sensorimotor gating, social interaction, and ethanol drinking behavior. Dopamine (DA) dysfunction may underlie these behavioral changes, but there is no direct evidence for P2X4Rs' role in DA neurotransmission. To test this hypothesis, we measured markers of DA function and dependent behaviors in P2X4R KO mice. P2X4R KO mice exhibited altered density of pre-synaptic markers including tyrosine hydroxylase, dopamine transporter; post-synaptic markers including dopamine receptors and phosphorylation of downstream targets including dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa and cyclic-AMP-response element binding protein in different parts of the striatum. Ivermectin, an allosteric modulator of P2X4Rs, significantly affected dopamine and cyclic AMP regulated phosphoprotein of 32 kDa and extracellular regulated kinase1/2 phosphorylation in the striatum. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Using the 6-hydroxydopamine model of DA depletion, P2X4R KO mice exhibited an attenuated levodopa (L-DOPA)-induced motor behavior, whereas ivermectin enhanced this behavior. Collectively, these findings identified an important role for P2X4Rs in maintaining DA homeostasis and illustrate how this association is important for CNS functions including motor control and sensorimotor gating. We propose that P2X4 receptors (P2X4Rs) regulate dopamine (DA) homeostasis and associated behaviors. Pre-synaptic and post-synaptic DA markers were significantly altered in the dorsal and ventral striatum of P2X4R KO mice, implicating altered DA neurotransmission. Sensorimotor gating deficits in P2X4R KO mice were rescued by DA antagonists. Ivermectin (IVM), a positive modulator of P2X4Rs, enhanced levodopa (L-DOPA)-induced motor behavior. These studies highlight potential interactions between P2X4Rs and DA system.

Keywords: 6-OHDA; Ivermectin; P2X4 receptors; Parkinson's disease; dopamine receptors; schizophrenia.

Figure 1

P2X4R KO mice exhibited significant increases in TH protein density in the dorsal striatum, but no changes in the ventral striatum [A & B (i)]; increased DAT protein density in both parts of the striatum [A & B (ii)]; increased D1R [A &B (iii)] and D2R [A&B (iv)] protein densities in the ventral, but no change in the dorsal striatum. The protein levels of DA markers were normalized to β-actin and expressed as arbitrary units (AU). The average of densitometry value of WT samples was arbitrarily normalized to 1. P2X4R KO samples were normalized by dividing each value by the average of WT samples and presented as fold change of P2X4R KO versus WT in that membrane. Values represent mean ± SEM from 5-8 WT, 7-8 P2X4R KO mice for TH, DAT analyses and 11-12 WT, 12-13 P2X4R KO for D1Rs and D2Rs analyses. 2 representative bands from each genotype from the same membrane are shown. *P <0.05, ** P <0.01 versus WT controls. Unpaired Student's t-test

Figure 2

P2X4R KO mice exhibited increased DARPP-32 phosphorylation in the dorsal striatum, but a decrease in the ventral striatum [A & B (i)]; no changes in ERK 1/2 phosphorylation in dorsal or ventral striatum of P2X4R KO mice [A & B(ii)]; increased phosphorylation of CREB in the ventral, but not in the dorsal striatum [A & B(iii)].. Details of normalization and analyses are presented in Figure 1. Values represent mean ± SEM from 3-6 WT and 4-8 P2X4R KO mice for DARPP-32 analysis; 6-8 WT & P2X4R KO for ERK 1/2 and CREB analyses. 2 representative bands from each genotype from the same membrane are shown. *P <0.05, ***P<0.001 versus WT counterparts. Unpaired Student's t-test.

Figure 3

IVM (5 mg/kg) significantly upregulated DARPP-32 phosphorylation [A & B (i)] via P2X4R potentiation in the dorsal striatum. IVM increased ERK 1/2 phosphorylation independent of P2X4R function [A& B (ii)]. No effect of IVM on CREB phosphorylation [A& B (iii)]. The average of densitometry value of vehicle treated WT samples was arbitrarily normalized to 1. The IVM treated WT mice, vehicle and IVM treated P2X4R KO were normalized by dividing each value by the average of the vehicle treated WT samples. The data is presented as fold change of IVM treated WT, P2X4R KO and vehicle treated P2X4R KO samples versus vehicle treated WT samples in that membrane. Values represent mean ± SEM from 4-6 WT and P2X4R KO mice per treatment group. 2 representative bands from each treatment group are shown. *P<0.05 versus vehicle treated WT group, Bonferroni post hoc test.

Figure 4

IVM (5 mg/kg) significantly affected DARPP-32 [A & B (i)] but not, ERK 1/2 [A & B (ii)] or CREB phosphorylation [A& B (iii)], via P2X4R potentiation in the ventral striatum. Details of normalization and analyses are presented in Figure 3. Values represent mean ± SEM from 4-6 WT and P2X4R KO mice per treatment group. 2 representative bands from each treatment group are shown. *P<0.05 versus vehicle treated WT group, Bonferroni post hoc test.

Figure 5

SCH-23390 (1 mg/kg) and raclopride (3 mg/kg) significantly increased prepulse inhibition of acoustic startle reflex in P2X4R KO mice (A) without any changes in startle amplitude (B). There were no changes in PPI in WT mice upon treatment with both antagonists (A). Values represent the mean of ΔPPI and mean startle amplitude ± SEM from 14 WT (saline), 15 WT (SCH 23390 and raclopride), 14 P2X4R KO (saline) and 16 P2X4R KO (SCH-23390 and raclopride). *P <0.05 versus saline treated WT mice, ## P<0.01, ### P<0.001 versus saline treated P2X4R KO mice, Bonferroni post hoc test.

Figure 6

L-DOPA (5 mg/kg) induced rotational behavior is significantly attenuated in P2X4R KO mice. IVM (5 mg/kg) significantly potentiated L-DOPA's effect on the number of contralateral turns in WT and P2X4R KO mice (A). IVM' ability to enhance L-DOPA induced motor behavior was significantly altered in P2X4R KO mice (B). Values on the y-axis represent the mean of number of contralateral turns per 10 minute interval ± SEM from 14 WT, 8 P2X4R KO. *P<0.05, *** P <0.001 versus L-DOPA treated WT mice, ## P<0.01 versus L-DOPA treated P2X4R KO mice for (A), *P<0.05 versus WT mice for (B), Bonferroni post hoc test.