Volume 91, Issue 6, Pages 1244-1252 (September 2016) Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release  Daniel J. Foster, Jermaine M. Wilson, Daniel H. Remke, M. Suhaib Mahmood, M. Jashim Uddin, Jürgen Wess, Sachin Patel, Lawrence J. Marnett, Colleen M. Niswender, Carrie K. Jones, Zixiu Xiang, Craig W. Lindsley, Jerri M. Rook, P. Jeffrey Conn  Neuron  Volume 91, Issue 6, Pages 1244-1252 (September 2016) DOI: 10.1016/j.neuron.2016.08.017 Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 mAChR Activation Induces a Sustained Reduction in DA Release that Is Independent of nAChR Signaling (A–D) Time courses of Oxo-M-induced inhibition of DA release in the presence or absence of the nAChR antagonist DHβE using electrically evoked (A–C) or optically evoked (D) DA release paradigms. All time-course data are depicted as the mean ± SEM. (E and F) Boxplot summaries depicting the percent inhibition of DA release observed under different conditions at acute (15 min) or sustained (30 min) time points (n = 5–7; ∗significant difference from 30 μM Oxo-M; p < 0.05; and one-way ANOVA with a post hoc Dunnett’s test). Neuron 2016 91, 1244-1252DOI: (10.1016/j.neuron.2016.08.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 M4 Expressed on Spiny Projection Neurons Mediates the Muscarinic-Induced Sustained Suppression of DA Release (A) Cartoon depicting the anatomical location of M4 in the striatum. (B–D) Time courses (B and C) and boxplot summaries (D) of DA release following bath application of 10 μM Oxo-M ± 3 μM VU0467154 (VU’154) in D1-M4 KO mice and control littermates (M4 fl/fl; n = 5–6; ∗significant difference from 10 μM Oxo-M; p < 0.05; and one-way ANOVA with a post hoc Bonferroni test). (E) Time course showing the effect of 30 μM Oxo-M ± 3 μM VU0467154 in D1-M4 KO mice. (F and G) Time course (F) and boxplot summaries (G) depicting the effects of 30 μM Oxo-M following pretreatment with DHβE (n = 5, #significant difference from control littermate; p < 0.05; and two-tailed Mann-Whitney test). Boxplot summaries depict the percent inhibition of dopamine release observed at sustained (30 min) time points; all time course data are depicted as the mean ± SEM. Neuron 2016 91, 1244-1252DOI: (10.1016/j.neuron.2016.08.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 VU0467154-Mediated Effects on DA Release Are CB2 Dependent (A–D) Time courses (A–C) and boxplot summaries (D) showing that the effects of VU0467154 (VU’154) on DA release are blocked by pretreatment with the CB2 antagonist AM630 (3 μM) and absent in CB2 KO mice (n = 5–6; ∗significant difference from 10 μM Oxo-M; p < 0.05; and one-way ANOVA with a post hoc Bonferroni test; sustained inhibition of dopamine release depicted at 30 min). (E and F) Time course (E) and boxplot summaries (F) showing that VU’154-mediated effects on sustained DA release are blocked by pretreating with DAG-Lipase inhibitor DO34 (1 μM), but unaffected by treatment with 2-APB or thapsigargin (n = 5–6; ∗significant difference from VU’154 + Oxo-M; p < 0.05; and one-way ANOVA with a post hoc Dunnett’s test; sustained inhibition of dopamine release depicted at 40 min). All time course data are depicted as the mean ± SEM. Neuron 2016 91, 1244-1252DOI: (10.1016/j.neuron.2016.08.017) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 VU0467154 Reverses Disrupted PPI via a Mechanism Requiring Activation of M4 on D1-Expressing Neurons and Subsequent CB2 Receptor Activation (A and B) Experimental paradigm for monitoring PPI (B) averaged data depicting percent PPI observed in the absence or presence of VU0467154 (VU’154; 10 mg/kg), amphetamine (Amp; 4 mg/kg), or vehicle in D1-M4 KO mice and control littermates (M4 fl/fl; n = 11–21; ∗significant difference from Amp; and p < 0.05). (C) Averaged data depicting the percent PPI observed in WT mice dosed with VU’154, Amp, and/or 10 mg/kg AM630 (n = 11–21; ∗significant difference from Amp; p < 0.05; and one-way ANOVA with a post hoc Dunnett’s test). All PPI data are depicted as the mean ± SEM. (D) Cartoon depicting mechanistic model for how striatal M4 receptors mediate sustained reductions in DA release and antipsychotic-like efficacy. Neuron 2016 91, 1244-1252DOI: (10.1016/j.neuron.2016.08.017) Copyright © 2016 Elsevier Inc. Terms and Conditions