Resting-State fMRI-Based Screening of Deschloroclozapine in Rhesus Macaques Predicts Dosage-Dependent Behavioral Effects

Abstract

Chemogenetic techniques, such as designer receptors exclusively activated by designer drugs (DREADDs), enable transient, reversible, and minimally invasive manipulation of neural activity in vivo Their development in nonhuman primates is essential for uncovering neural circuits contributing to cognitive functions and their translation to humans. One key issue that has delayed the development of chemogenetic techniques in primates is the lack of an accessible drug-screening method. Here, we use resting-state fMRI, a noninvasive neuroimaging tool, to assess the impact of deschloroclozapine (DCZ) on brainwide resting-state functional connectivity in 7 rhesus macaques (6 males and 1 female) without DREADDs. We found that systemic administration of 0.1 mg/kg DCZ did not alter the resting-state functional connectivity. Conversely, 0.3 mg/kg of DCZ was associated with a prominent increase in functional connectivity that was mainly confined to the connections of frontal regions. Additional behavioral tests confirmed a negligible impact of 0.1 mg/kg DCZ on socio-emotional behaviors as well as on reaction time in a probabilistic learning task; 0.3 mg/kg DCZ did, however, slow responses in the probabilistic learning task, suggesting attentional or motivational deficits associated with hyperconnectivity in fronto-temporo-parietal networks. Our study highlights both the excellent selectivity of DCZ as a DREADD actuator, and the side effects of its excess dosage. The results demonstrate the translational value of resting-state fMRI as a drug-screening tool to accelerate the development of chemogenetics in primates.SIGNIFICANCE STATEMENT Chemogenetics, such as designer receptors exclusively activated by designer drugs (DREADDs), can afford control over neural activity with unprecedented spatiotemporal resolution. Accelerating the translation of chemogenetic neuromodulation from rodents to primates requires an approach to screen novel DREADD actuators in vivo Here, we assessed brainwide activity in response to a DREADD actuator deschloroclozapine (DCZ) using resting-state fMRI in macaque monkeys. We demonstrated that low-dose DCZ (0.1 mg/kg) did not change whole-brain functional connectivity or affective behaviors, while a higher dose (0.3 mg/kg) altered frontal functional connectivity and slowed response in a learning task. Our study highlights the excellent selectivity of DCZ at proper dosing, and demonstrates the utility of resting-state fMRI to screen novel chemogenetic actuators in primates.

Keywords: DREADDs; ICA; deschloroclozapine; functional connectivity; macaque monkey; rs-fMRI.

Figure 1.

Schematic of experiments. A, An imaging session. Subjects were lightly anesthetized with isoflurane gas throughout a session. A contrast agent (monocrystalline iron oxide nanoparticle [MION], 10 mg/kg) was administered intravenously before functional scans. Drug (vehicle, 0.1 mg/kg DCZ, or 0.3 mg/kg DCZ) was prepared and systemically administered (i.v.) following baseline scans, and test scans were acquired after 15 min of drug injection. B, Data analysis pipeline. The scan data for each session initially went through preprocessing procedures. Then seed-based analysis was performed using predetermined ROIs based on the CHARM and SARM atlases. The temporally concatenated scan data across all sessions were used for ICA.

Figure 2.

fMRI-based connectome changes after DCZ administration in an example subject. A, CHARM atlas Level 3 visualized on an inflated brain (medial view). B, Brainwide functional connectivity in cortical areas. Confusion matrices represent connectome in baseline scans (left column), test scans (middle column), and the difference between test and baseline (right column) for either in the vehicle (top), low-dose DCZ (middle), and high-dose DCZ condition (bottom), respectively. Color represents z-transformed correlation coefficients. Labels on the right side represent ROIs from CHARM atlas. C, SARM atlas Level 3 visualized on an inflated brain. D, Confusion matrices for subcortical areas with labels for SARM ROIs on the right side. Conventions are as in B.

Figure 3.

Group-level changes in the fMRI-based connectome following DCZ administration. A–C, Functional connectivity changes in cortical areas. A, Confusion matrices represent differences in z value (test – baseline) for vehicle (top), low-dose DCZ (middle), and high-dose DCZ (bottom) conditions. Labels on the right side represent ROIs from CHARM atlas (Level 3). B, Averaged functional connectivity. Bars represent averaged z value differences for vehicle, low-dose DCZ, and high-dose DCZ conditions. Error bars indicate SE. Symbols represent subjects. *p = 0.016, Significant interaction of drug × area category (two-way ANOVA). Numbers on the labels indicate the number of subjects for each condition. C, Functional connectivity in each area. Bars represent averaged z value differences averaged for each area of the CHARM atlas. Colors represent frontal (red), parietal (green), temporal (cyan), and occipital areas (yellow) for low-dose (top) and high-dose (bottom) DCZ conditions. Gray bars represent vehicle condition. Error bars indicate SE. *p < 0.01, Significant difference between vehicle and DCZ conditions (Bonferroni correction, rank-sum test). D–F, Functional connectivity changes in subcortical areas. Conventions are the same as in A–C. F, Colors represent telencephalon (dark red), diencephalon (dark green), mesencephalon (dark blue), metencephalon (dark yellow), and myelencephalon areas (magenta).

Figure 4.

ICA of the effect of high-dose DCZ. A–F, Top panels, ICs as correlation coefficients (r) on inflated brains. Bottom panels, Clusters that showed a significant main effect of drug condition (p < 0.05, two-way ANOVA).

Figure 5.

The impact of systemic DCZ administration on socio-emotional behavior and probabilistic learning. A, The effect of low-dose DCZ in the human intruder task. Bars represent the mean time spent motionless with systemic administration of vehicle (gray) or low-dose DCZ (light blue) in alone condition (left), profile condition (middle), or stare condition (right). Symbols represent individual animals. Error bars indicate SE. B, Dosage-dependent changes in RT in the probabilistic learning task. Bar plots represent the mean RT of 3 subjects in each drug condition (vehicle, low-dose DCZ, high-dose DCZ). Error bars indicate SE. Symbols represent individual subjects. *Significant main effect of drug condition (p = 2.5 × 10⁻⁶, one-way repeated-measures ANOVA). C, Relationship between RT and functional connectivity across conditions in the probabilistic learning task. Scatter plots represent the RT in each behavioral session (y axis) and the change in overall functional connectivity in a corresponding subject (x axis) for cortical areas (left) and for subcortical areas (right), respectively. Individual monkeys completed 6-8 behavioral sessions per each condition. Colors represent drug conditions. Solid and dotted lines indicate linear fitting and CIs, respectively. Red lines indicate significant linear correlation between RT and z value (p = 0.023).