Dopamine Receptor Blockade Attenuates Purinergic P2X4 Receptor-Mediated Prepulse Inhibition Deficits and Underlying Molecular Mechanisms

Abstract

Sensorimotor gating refers to the ability to filter incoming sensory information in a stimulus-laden environment and disruption of this physiological process has been documented in psychiatric disorders characterized by cognitive aberrations. The effectiveness of current pharmacotherapies for treatment of sensorimotor gating deficits in the patient population still remains controversial. These challenges emphasize the need to better understand the biological underpinnings of sensorimotor gating which could lead to discovery of novel drug targets for therapeutic intervention. Notably, we recently reported a role for purinergic P2X4 receptors (P2X4Rs) in regulation of sensorimotor gating using prepulse inhibition (PPI) of acoustic startle reflex. P2X4Rs are ion channels gated by adenosine-5'-triphosphate (ATP). Ivermectin (IVM) induced PPI deficits in C57BL/6J mice in a P2X4R-specific manner. Furthermore, mice deficient in P2X4Rs [P2X4R knockout (KO)] exhibited PPI deficits that were alleviated by dopamine (DA) receptor antagonists demonstrating an interaction between P2X4Rs and DA receptors in PPI regulation. On the basis of these findings, we hypothesized that increased DA neurotransmission underlies IVM-mediated PPI deficits. To test this hypothesis, we measured the effects of D1 and D2 receptor antagonists, SCH 23390 and raclopride respectively and D1 agonist, SKF 82958 on IVM-mediated PPI deficits. To gain mechanistic insights, we investigated the interaction between IVM and dopaminergic drugs on signaling molecules linked to PPI regulation in the ventral striatum. SCH 23390 significantly attenuated the PPI disruptive effects of IVM to a much greater degree than that of raclopride. SKF 82958 failed to potentiate IVM-mediated PPI disruption. At the molecular level, modulation of D1 receptors altered IVM's effects on dopamine and cyclic-AMP regulated phosphoprotein of 32 kDa (DARPP-32) phosphorylation. Additionally, IVM interacted with the DA receptors antagonists and SKF 82958 in phosphorylation of Ca²⁺/calmodulin kinase IIα (CaMKIIα) and its downstream target, neuronal nitric oxide synthase (nNOS). Current findings suggest an involvement for D1 and D2 receptors in IVM-mediated PPI disruption via modulation of DARPP-32, CaMKIIα and nNOS. Taken together, the findings suggest that stimulation of P2X4Rs can lead to DA hyperactivity and disruption of information processing, implicating P2X4Rs as a novel drug target for treatment of psychiatric disorders characterized by sensorimotor gating deficits.

Keywords: DARPP-32; P2X4 receptors; dopamine receptors; ivermectin; prepulse inhibition; schizophrenia.

FIGURE 1

Ivermectin (10 mg/kg) significantly disrupted PPI function in mice at PPI 6 and PPI 12. Co-treatment with SCH 23390 (1 mg/kg) significantly reversed IVM-mediated PPI disruption at both these prepulse intensities. Co-treatment with raclopride (3 mg/kg) reversed IVM-mediated PPI deficits at PPI 6, but not at PPI 12 (A). Neither of the DA receptor antagonists significantly impacted IVM-mediated effects on startle magnitude (B). IVM significantly affected startle magnitude. Values represent mean PPI % or startle amplitude ± SEM from 18 to 22 mice per treatment group. ^∗∗∗P < 0.001 versus control group. ^#P < 0.05, ^##P < 0.01 versus IVM-treated mice. ⁺⁺⁺P < 0.001 versus SCH 23390-treated mice and ^$P < 0.05 versus raclopride-treated mice, Tukey’s multiple comparison test.

FIGURE 2

D1 receptor stimulation by SKF 82958 (0.1 mg/kg) failed to potentiate IVM (5 mg/kg)-mediated PPI disruption. (A) There was no significant interaction between IVM and SKF 82958 in regulation of startle magnitude. (B) Values represent mean PPI % or startle amplitude ± SEM from 15 to 16 mice per treatment group.

FIGURE 3

Blockade of D1 receptors by SCH 23390 significantly reversed IVM (10 mg/kg)-mediated increase in DARPP-32 phosphorylation (A,Bi) in the ventral striatum. Blockade of D2 receptors by raclopride antagonized, but did not reverse, IVM (10 mg/kg)-mediated changes in DARPP-32 phosphorylation (A,Bii). Stimulation of D1 receptors by SKF 82958 significantly interacted with IVM (5 mg/kg) in a synergistic manner (A,Biii). The average of densitometry value of control group was arbitrarily normalized to 1. The treatment groups were normalized by dividing each value by the average of the control group. The data is presented as fold change of treatment group versus control group in that membrane. Values represent mean ± SEM from 4 to 10 mice per treatment group. Two representative bands from each treatment group from different gels are shown. IVM alone (or vehicle) images were used twice for the IVM/SCH 23390 and IVM/raclopride blots since both the DA receptor antagonist treatment groups (with or without IVM) ran on the same gel. Black solid lines between the bands indicate that the bands were cropped and spliced together either from different gels or non-consecutive lanes. Full scan of gel images can be found in the Supplementary Figures S1–S3. ^*P < 0.05; ^∗∗P < 0.01 versus control group; ^##P < 0.01 versus IVM –treated mice (5 and 10 mg/kg); ⁺⁺P < 0.01 versus SKF 82958-treated mice, Tukey’s multiple comparison test.

FIGURE 4

IVM (10 mg/kg)-mediated decrease in CaMKIIα phosphorylation was significantly impeded by SCH 23390 (A,Bi) but reversed by raclopride (A,Bii) in the ventral striatum. IVM (5 mg/kg) synergistically interacted with SKF 82958 in regulation of CaMKIIα phosphorylation (A,Biii) in the ventral striatum. Details of normalization and analyses are presented in Figure 3. Values represent mean ± SEM from 3 to 11 mice per treatment group. Two representative bands from each treatment group are shown. IVM alone (or vehicle) images were used twice for the IVM/SCH 23390 and IVM/raclopride blots since both the DA receptor antagonist treatment groups (with or without IVM) ran on the same gel. Black solid lines indicated that bands were cropped and spliced from non-consecutive lanes or different gels. Full scan of gel images can be found in the Supplementary Figures S4, S5. ^*P < 0.05 versus control group; ^##P < 0.01 versus IVM-treated mice (5 and 10 mg/kg), Tukey’s multiple comparison test.

FIGURE 5

SCH 23390 (A,Bi) and raclopride (A,Bii) significantly increased nNOS phosphorylation in presence of IVM (10 mg/kg) in the ventral striatum. IVM (5 mg/kg) significantly attenuated SKF 82958-induced increase in nNOS phosphorylation (A,Biii). Details of normalization and analyses are presented in Figure 3. Values represent mean ± SEM from 4 to 12 mice per treatment group. Two representative bands from each treatment group are shown. IVM alone (or vehicle) images were used twice for the IVM/SCH 23390 and IVM/raclopride blots since both the DA receptor antagonist treatment groups (with or without IVM) ran on the same gel. Black solid lines between bands indicate that bands were cropped and spliced together from non-consecutive lanes or from different gels. Full scan of gel images can be found in the Supplementary Figures S6, S7. ^*P < 0.05 versus control group; ^##P < 0.01, ^###P < 0.001 versus IVM-treated mice (5 and 10 mg/kg); ⁺⁺P < 0.01 versus SKF 82958-treated mice, Tukey’s multiple comparison test.

FIGURE 6

IVM modulated DARPP-32 phosphorylation via P2X4R potentiation in a D1 receptor-dependent manner in the ventral striatum. Inter-dependence between P2X4Rs and D1 receptors in DARPP-32 phosphorylation may be relevant to regulation of transcription factors (e.g., CREB) and subsequent gene expression in the ventral striatum and DA-dependent behaviors such as sensorimotor gating (A). The ability of raclopride to block IVM-mediated changes at behavioral and cellular level indicates a plausible interaction between P2X4Rs and D2 receptors in regulation of DA-dependent behaviors and signaling pathways (B).