Antipsychotic-like Effects of M4 Positive Allosteric Modulators Are Mediated by CB2 Receptor-Dependent Inhibition of Dopamine Release

Abstract

Muscarinic receptors represent a promising therapeutic target for schizophrenia, but the mechanisms underlying the antipsychotic efficacy of muscarinic modulators are not well understood. Here, we report that activation of M4 receptors on striatal spiny projection neurons results in a novel form of dopaminergic regulation resulting in a sustained depression of striatal dopamine release that is observed more than 30 min after removal of the muscarinic receptor agonist. Furthermore, both the M4-mediated sustained inhibition of dopamine release and the antipsychotic-like efficacy of M4 activators were found to require intact signaling through CB2 cannabinoid receptors. These findings highlight a novel mechanism by which striatal cholinergic and cannabinoid signaling leads to sustained reductions in dopaminergic transmission and concurrent behavioral effects predictive of antipsychotic efficacy.

Figure 1

mAChR activation induces a sustained reduction in DA release that is independent of nAChR signaling. 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. (E,F) Box plot summaries depicting the % inhibition of DA release observed under different conditions at acute (15 min) or sustained (30 min) time points (n=5–7; * Significant difference from 30µM Oxo-M, p<0.05; one-way ANOVA with a post hoc Dunnett’s test).

Figure 2

M₄ expressed on spiny projection neurons mediate the muscarinic-induced sustained suppression of DA release. (A) Cartoon depicting the anatomical location of M₄ in the striatum. (B,C) Time-courses and (D) box plot summaries of DA release following bath application of 10µM Oxo-M +/− 3µM VU0467154 (VU’154) in D₁-M₄ KO mice and control littermates (M₄ fl/fl; n=5–6; * Significant difference from 10µM Oxo-M, p<0.05; one-way ANOVA with a post hoc Bonferroni test). (E) Time-course showing the effect of 30µM Oxo-M +/− 3µM VU0467154 in D₁-M₄ KO mice. Time-course (F) and box plot summaries (G) depicting the effects of 30µM Oxo-M following pretreatment with DHβE (n=5, # Significant difference from control littermate; p<0.05; two-tailed Mann-Whitney test).

Figure 3

VU0467154-mediated effects on DA release are CB₂-dependent. (A,B,C) Time-courses and (D) box plot summaries showing that the effects of VU0467154 (VU’154) on DA release are blocked by pretreatment with the CB₂ antagonist AM630 (3µM) and absent in CB₂ KO mice (n=5–6; * Significant difference from 10µM Oxo-M, p<0.05; one-way ANOVA with a post hoc Bonferroni test). (E) Time-course and (F) box plot summaries showing that VU’154-mediated effects on sustained dopamine release are blocked by pretreating with DAG-Lipase inhibitor DO34 (1µM) but unaffected by treatment with 2-APB or thapsigargin (n=5–6; * Significant difference from VU’154 + Oxo-M, p<0.05; one-way ANOVA with a post hoc Dunnett’s test).

Figure 4

VU0467154 reverses disrupted prepulse inhibition (PPI) via a mechanism requiring activation of M₄ on D₁-expressing neurons and subsequent CB₂ receptor activation. (A) Experimental paradigm for monitoring PPI (B) Averaged data depicting % PPI observed in the absence or presence of VU0467154 (VU’154; 10mg/kg), amphetamine (Amp; 4mg/kg) or vehicle in D₁-M₄ KO mice and control littermates (M₄ fl/fl; n=11–21; * Significant difference from Amp, p<0.05). (C) Averaged data depicting the % PPI observed in WT mice dosed with VU’154, AMP, and/or 10mg/kg AM630 (n=11–21; * Significant difference from Amp, p<0.05; one-way ANOVA with a post hoc Dunnett’s test). (D) Cartoon depicting mechanistic model for how striatal M₄ receptors mediate sustained reductions in DA release and antipsychotic-like efficacy.