An Adenosine A2A Receptor Antagonist Improves Multiple Symptoms of Repeated Quinpirole-Induced Psychosis

Abstract

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder characterized by the repeated rise of concerns (obsessions) and repetitive unwanted behavior (compulsions). Although selective serotonin reuptake inhibitors (SSRIs) is the first-choice drug, response rates to SSRI treatment vary between symptom dimensions. In this study, to find a therapeutic target for SSRI-resilient OCD symptoms, we evaluated treatment responses of quinpirole (QNP) sensitization-induced OCD-related behaviors in mice. SSRI administration rescued the cognitive inflexibility, as well as hyperactivity in the lateral orbitofrontal cortex (lOFC), while no improvement was observed for the repetitive behavior. D₂ receptor signaling in the central striatum (CS) was involved in SSRI-resistant repetitive behavior. An adenosine A_2A antagonist, istradefylline, which rescued abnormal excitatory synaptic function in the CS indirect pathway medium spiny neurons (MSNs) of sensitized mice, alleviated both of the QNP-induced abnormal behaviors with only short-term administration. These results provide a new insight into therapeutic strategies for SSRI-resistant OCD symptoms and indicate the potential of A_2A antagonists as a rapid-acting anti-OCD drug.

Keywords: D2 receptor; obsessive-compulsive disorder.

Figure 1.

Electrophysiological characteristics of lOFC pyramidal neurons and fast-spiking interneurons (A–C) Representative firing activity recorded from a pyramidal neuron (A; 200-pA injection) and fast-spiking interneuron (B; 100-pA injection, C; 200-pA injection). D, Current injection-induced firing activity of pyramidal neurons and fast-spiking interneurons. Please note that the data set for pyramidal neurons is same as that in Figure 4B (saline group). (Pyramidal neurons; n = 10 from 3 mice, fast-spiking interneurons; n = 5 from 3 mice.)

Figure 2.

Representative single-cell PCR from a CS dMSN and iMSN. Representative image of single-cell PCR from CS MSNs. Pdyn-positive neurons were considered direct-pathway MSNs (dMSNs; A), while Pdyn-negative and Penk-positive neurons were considered indirect-pathway MSNs (iMSNs; B).

Figure 3.

Repeated injection of QNP elicited multiple OCD-related symptoms. A, Time course of the elevated plus maze test and open field test. B, C, Time spent in the closed arm (B) and the open arm (C) in an elevated plus maze test. D, Total travel distance in the open field test. (Saline; n = 5, QNP; n = 5, B: Student’s t test; t₍₈₎ = 2.178, p = 0.0610, C: Student’s t test; t₍₈₎ = 0.8863, p = 0.4013, D: Student’s t test; t₍₈₎ = 4.343, **p = 0.0025.). E, Time course of recording of QNP-induced repetitive behavior. F, G, Time spent chewing during the 20–30 min after the 1st–8th QNP injection (F) and before and after the 8th QNP injection (G). [F: saline; n = 6, QNP; n = 7, two-way repeated measures ANOVA; drug (F_(1,24.32) = 37.18, p < 0.0001), injection number (F_(2.21,53.75) = 22.41, p < 0.0001), interaction (F_(2.21,53.75) = 20.95, p < 0.0001), Bonferroni post hoc test; *p < 0.05 and ***p < 0.001, G: n = 4, one-way repeated measures ANOVA; F_(6,18) = 38.61, p < 0.0001, Tukey’s multiple comparison test; **p < 0.01, **p < 0.001 vs Pre.] H, Time course of recording for QNP-induced repetitive behavior combined with short-term administration of diazepam and citalopram. I, Effects of the short-term administration of an antianxiety agent, diazepam (Dzp; 0.3 mg/kg) and an antidepressant, citalopram (Cit; 10 mg/kg) on repetitive behavior in QNP-treated mice. [QNP+saline; n = 4, QNP+Dzp; n = 4, QNP+Cit; n = 4, two-way repeated measures ANOVA; drug (F_(2,36) = 0.41, p = 0.6748), injection number (F_(2,36) = 0.56, p = 0.5786), interaction (F_(2,36) = 0.42, p = 0.7918).] J, K, Protocols and percentage of correct choice during training with daily injection of QNP. (Saline; n = 4, QNP; n = 4, K: two-way repeated measures ANOVA; drug (F_(1,20.23) = 0.01, p = 0.9224), injection number (F_(3.37,68.17) = 9.04, p = 0.0004), interaction (F_(3.37,68.17) = 1.66, p = 0.2037).] L, Protocols of a spatial discrimination task and a reversal learning test. M, N, Percentage of correct choice during overtraining (M) and reversal learning (N). [Saline; n = 6, QNP; n = 5, M: two-way repeated measures ANOVA; drug (F_(1,25.63) = 0.07, p = 0.8025), session number (F_(2.85,73.04) = 1.93, p = 0.1519), interaction (F_(2.85,73.04) = 1.58, p = 0.2201), N: two-way repeated measures ANOVA; drug (F_(1,36) = 10.46, p = 0.0102), session number (F_(4,144) = 34.86, p < 0.0001), interaction (F_(4,144) = 4.29, p = 0.0061), Bonferroni post hoc test; **p < 0.01.]

Figure 4.

Hyperactivity of lOFC pyramidal neurons in QNP-treated mice. A, Time course of electrophysiological recordings. B, C, Current injection induced firing activity of lOFC pyramidal neurons in the absence (B) and presence (C) of AMPA/NMDA antagonists. [B: saline; n = 10 from 3 mice, QNP; n = 10 from 3 mice, two-way repeated measures ANOVA; drug (F_(1,36.27) = 6.15, p = 0.0227), current (F_(1.91,69.27) = 324.00, p < 0.0001), interaction (F_(1.91,69.27) = 4.07, p = 0.0270), Bonferroni post hoc test; *p < 0.05 and **p < 0.01, C: saline; n = 11 from 3 mice, QNP; n = 10 from 3 mice, two-way repeated measures ANOVA; drug (F_(1,38.97) = 0.03, p = 0.8743), current (F_(2.29,89.24) = 209.69, p < 0.0001), interaction (F_(2.29,89.24) = 0.45, p = 0.6660).] D, Representative traces of spontaneous EPSCs (sEPSCs) and miniature EPSCs (mEPSCs). E, F, sEPSC frequency (E) and amplitude (F) in lOFC pyramidal neurons. (Saline; n = 8 from 3 mice, QNP; n = 15 from 3 mice, E: unpaired t test with Welch’s correction; t₍₂₁₎ = 2.632, *p = 0.0160, F: Student’s t test; t₍₂₁₎ = 0.7675, p = 0.4513.) G, H, mEPSC frequency (G) and amplitude (H) in lOFC pyramidal neurons. (Saline; n = 7 from 3 mice, QNP; n = 9 from 3 mice, G: Student’s t test; t₍₁₄₎ = 2.740, *p = 0.0160, H: Student’s t test; t₍₁₄₎ = 1.277, p = 0.2223.).

Figure 5.

Chronic SSRI treatment rescued cognitive inflexibility in QNP-treated mice but not the abnormal repetitive behavior. A, Time course of recording for QNP-induced repetitive behavior combined with chronic SSRI administration. B, Time spent chewing during the 20–30 min after the 8th QNP injection. [Water+saline; n = 7, Cit+saline; n = 6, water+QNP; n = 7, Cit+QNP; n = 6, two-way ANOVA; p.o. administration (F_(1,22) = 0.56, p = 0.4616), i.p. injection (F_(1,22) = 727.10, p < 0.0001), interaction (F_(1,22) = 0.11, p = 0.7440), Bonferroni post hoc test; not significant (n.s.).] C, Time course of a spatial discrimination task and a reversal learning test combined with chronic SSRI administration. D, E, Percentage of correct choices during overtraining (D) and reversal learning (E). [Water+QNP; n = 5, Cit+QNP; n = 5, D: two-way repeated measures ANOVA; drug (F_(1,26.9) = 2.47, p = 0.1547), session number (F_(3.36,90.38) = 1.27, p = 0.3040), interaction (F_(3.36,90.38) = 0.62, p = 0.6233), E: two-way repeated measures ANOVA; drug (F_(1,32) = 9.71, p = 0.0143), session number (F_(4,128) = 48.66, p < 0.0001), interaction (F_(4,128) = 1.63, p = 0.1920), Bonferroni post hoc test; *p < 0.05.).]

Figure 6.

Chronic SSRI treatment rescued lOFC hyperactivity in QNP-treated mice. A, Time course of electrophysiological recordings combined with chronic SSRI administration. B, C, Current injection induced firing activity of lOFC pyramidal neurons in the absence (B) and presence (C) of GABA_A antagonists. [B: water+QNP; n = 8 from 3 mice, Cit+QNP; n = 12 from 3 mice, two-way repeated measures ANOVA; drug (F_(1,27.96) = 6.96, p = 0.0167), current (F_(1.55,43.34) = 309.92, p < 0.0001), interaction (F_(1.55,43.34) = 4.04, p = 0.0297), Bonferroni post hoc test; *p < 0.05 and **p < 0.01, C: water+QNP; n = 11 from 3 mice, Cit+QNP; n = 11 from 3 mice, two-way repeated measures ANOVA; drug (F_(1,40.58) = 0.00, p = 0.9673), current (F_(2.03,82.38) = 250.93, p < 0.0001), interaction (F_(2.03,82.38) = 0.12, p = 0.8889).] D, Representative traces of sIPSCs and mIPSCs. E, F, sIPSC frequency (E) and amplitude (F) in lOFC pyramidal neurons. (Water+saline; n = 14 from 3 mice, water+QNP; n = 17 from 3 mice, Cit+QNP; n = 15 from 3 mice, E: one-way ANOVA; F_(2,43) = 3.758, *p = 0.0313, Tukey’s multiple comparison test; *p < 0.05, F: one-way ANOVA; F_(2,43) = 0.1188, p = 0.8883.) G, H, mIPSC frequency (G) and amplitude (H) in lOFC pyramidal neurons. (G: water+saline; n = 12 from 3 mice, water+QNP; n = 13 from 3 mice, Cit+QNP; n = 11 from 3 mice, one-way ANOVA; F_(2,33) = 0.2934, p = 0.7476, H: water+saline; n = 12 from 3 mice, water+QNP; n = 11 from 3 mice, Cit+QNP; n = 10 from 3 mice, one-way ANOVA; F_(2,30) = 0.1310, p = 0.8777.).

Figure 7.

D₂-ERK signaling in the CS was required for repetitive behavior in QNP-treated mice. A, B, Representative images from AAV-hSyn-Venus mediated labeling of lOFC neurons. Green fluorescence was observed at both the AAV injection site (A; lOFC) and the striatal projection site (B; CS). Scale bar = 100 μm (B, center) and 20 μm (B, right). C, Time course of stereotaxic surgery and recording of QNP-induced repetitive behavior combined with intra-CS local drug injection. D, E, Effect of intra-CS injection of racroprode (Rac; 1 μg/side) or PD98059 (1 μg/side) on repetitive chewing behavior in QNP-treated mice. (D: n = 5, paired t test; t₍₅₎ = 15.31, ***p < 0.0001, E: n = 5, paired t test; t₍₅₎ = 3.643, **p = 0.0070.) F, Time course of recording of low-dose QNP-induced chewing behavior. G, Time spent chewing during the 20–30 min after the 8th–10th QNP injection (1.0, 0.5, and 0.3 mg/kg, respectively); n = 5. H, Time course of stereotaxic surgery and recording of intra-CS local injection-induced repetitive behavior combined with subthreshold dose of QNP injection. I, Time spent chewing during the 20–30 min after intra-CS injection of CGS 21680A (CGS; 0.3 ng/side) and subthreshold dose of QNP injection (0.3 mg/kg); n = 3, paired t test; t₍₂₎ = 4.395, *p = 0.0481.

Figure 8.

Istradefylline rescued both the behavioral and cognitive symptoms in QNP-treated mice. A, Time course of recording of QNP-induced repetitive behavior combined with the short-term administration of istradefylline (Ist). B, Time spent chewing during 20–30 min after QNP and Ist injections. [QNP+Veh; n = 5, QNP+Ist; n = 4, two-way repeated measures ANOVA; drug (F_(1,8.05) = 27.48, p = 0.0012), injection number (F_(1.15,9.26) = 17.45, p = 0.0025), interaction (F_(1.15,9.26) = 17.78, p = 0.0024), Bonferroni post hoc test; **p < 0.01, ***p < 0.001.] C, Time course of a spatial discrimination task and a reversal learning test combined with the short-term administration of Ist. D, E, Percentage of correct choices during over training (D) and reversal learning (E). [QNP+Veh; n = 5, QNP+Ist; n = 6, D: two-way repeated measures ANOVA; drug (F_(1,25.01) = 0.72, p = 0.4175), session number (F_(2.78,69.53) = 0.78, p = 0.5068), interaction (F_(2.78,69.53) = 1.04, p = 0.3878), E: two-way repeated measures ANOVA; drug (F_(1,36) = 7.29, p = 0.0244), session number (F_(4,144) = 68.34, p < 0.0001), interaction (F_(4,144) = 1.58, p = 0.2010), *p < 0.05.]

Figure 9.

Altered synaptic functions in the CS iMSNs from QNP-treated mice was rescued by an A_2A antagonist. A, Time course of electrophysiological recordings from CS MSNs. B, Baseline NMDA/AMPA ratios recorded from CS dMSNs. [Control condition: saline; n = 6 from 5 mice, QNP; n = 14 from 5 mice, Student’s t test; t₍₁₈₎ = 0.09770, p = 0.9233, PD98059: saline; n = 12 from 4 mice, QNP; n = 11 from 4 mice, unpaired t test with Welch’s correction; t₍₁₂₎ = 0.7569, p = 0.4637, Ist (istradefylline): saline; n = 4 from 3 mice, QNP; n = 6 from 3 mice, Student’s t test; t₍₈₎ = 0.6724, p = 0.5203.] C, Bath application of QNP-induced changes in the NMDA/AMPA ratio recorded from CS dMSNs. (Control condition: saline; n = 6 from 5 mice, t₍₅₎ = 1.587, p = 0.1735, QNP; n = 14 from 5 mice, t₍₁₃₎ = 0.3219, p = 0.7526, PD98059: saline; n = 12 from 4 mice, t₍₁₁₎ = 0.2176, p = 0.8317, QNP; n = 11 from 4 mice, t₍₁₀₎ = 0.03232, p = 0.9748, Ist: saline; n = 4 from 3 mice, t₍₃₎ = 0.1016, p = 0.9255, QNP; n = 6 from 3 mice, t₍₅₎ = 0.5138, p = 0.6293. One sample t test compared with 100.) D, Baseline NMDA/AMPA ratios recorded from CS iMSNs. (Control condition: saline; n = 7 from 3 mice, QNP; n = 5 from 4 mice, Student’s t test; t₍₁₀₎ = 5.067, p = 0.0005, PD98059: saline; n = 6 from 4 mice, QNP; n = 5 from 3 mice, unpaired t test with Welch’s correction; t₍₆₎ = 2.277, p = 0.0630, Ist: saline; n = 4 from 3 mice, QNP; n = 6 from 3 mice, Student’s t test; t₍₈₎ = 0.3501, ***p = 0.7353.) E, Bath application of QNP-induced changes in the NMDA/AMPA ratio recorded from CS iMSNs. (Control condition: saline; n = 7 from 3 mice, t₍₆₎ = 0.1710, p = 0.8699, QNP; n = 5 from 4 mice, t₍₄₎ = 3.019, *p = 0.0392, PD98059: saline; n = 6 from 4 mice, t₍₅₎ = 0.6388, p = 0.5510, QNP; n = 5 from 3 mice, t₍₄₎ = 0.6333, p = 0.5610, Ist: saline; n = 4 from 3 mice, t₍₃₎ = 1.547, p = 0.2195, QNP; n = 6 from 3 mice, t₍₅₎ = 1.684, p = 0.1529. One sample t test compared with 100.)