Antipsychotic drug-like effects of the selective M4 muscarinic acetylcholine receptor positive allosteric modulator VU0152100

Abstract

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Figure 1

VU0152100 reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. (a) Amphetamine-induced (1 mg/kg) hyperlocomotion in rats is dose-dependently reversed by VU0152100 (*p<0.05 vs vehicle/amphetamine (Dunnett's test)); (b) VU0152100 reverses amphetamine-induced hyperlocomotion in wild-type mice. (c) In M4 KO mice, amphetamine-induced hyperlocomotion is not blocked by VU0152100 (*p<0.05, vehicle/amphetamine (Bonferroni's test)). Data are mean±SEM of 8 rats or 10–11 mice per group.

Figure 2

VU0152100 reverses sensorimotor gating and contextual fear conditioning deficits elicited by amphetamine. (a) VU0152100 blocks amphetamine-induced (3 mg/kg) disruption of prepulse inhibition (*p<0.05 vs vehicle/vehicle (Dunnett's test)); (b) The disruptive effects of amphetamine (4.8 mg/kg, s.c.) on the acquisition of a context-dependent fear response are dose-dependently reversed by pre-treatment with VU0152100 (**p<0.01, ***p<0.001 vs vehicle/vehicle, ^#p<0.05 vs vehicle/amphetamine (Dunnett's test)). Data are mean±SEM of 7–11 rats per group.

Figure 3

VU0152100 modulates amphetamine-induced cerebral blood volume (CBV) responses. (a) Amphetamine (1 mg/kg, i.p.) elicited CBV increases, reflecting neural activity, while pre-treatment with VU0152100 (56.6 mg/g, i.p.) suppressed this effect in multiple brain areas. (b) Regional CBV time courses for amphetamine and reversal by VU0152100 are shown for the retrosplenial cortex, medial thalamus, hippocampus, NAS, CP, and PFC; note that amphetamine by itself and in combination with VU0152100 did not alter CBV in the PFC. Data are means±SEM of 10–11 animals per group. Functional connectivity analysis of the phMRI data revealed fewer inter-regional correlations in the VU0152100/amphetamine group and the significant ROI–ROI correlation differences between the vehicle/amphetamine group and the VU0152100/amphetamine group (thresholded at p<0.05) are depicted in panel (c). Cells are colored according to the corresponding z-statistic. Abbreviations of regions of interest (number indicates slice): Amyg, amygdala; CP, caudate-putamen; Cg, cingulate; Hipp, hippocampus; MT, medial thalamus; M, motor cortex; NAS, nucleus accumbens; PFC, prefrontal cortex; Pir, piriform cortex; Rs, retrosplenial cortex; S, sensory cortex; SN, substantia nigra; V, visual cortex.

Figure 4

Pre-treatment with VU0152100 (56.6 mg/kg, i.p.) reverses amphetamine-induced (1 mg/kg, s.c.) hyperlocomotion (a, b) and reduces the amphetamine-elicited increase in extracellular dopamine levels in the nucleus accumbens and caudate-putamen (c, d). Data are mean±SEM of 6–8 rats per group; *p<0.05, **p<0.01 vs vehicle/vehicle; ^#p<0.05, ^##p<0.01 vs vehicle/amphetamine (Dunnett's test).

Figure 5

VU0152100 does not cause motor and cardiovascular side effects when given alone, but exacerbates haloperidol-induced catalepsy. (a) VU0152100 (30–100 mg/kg, i.p.) does not induce catalepsy, whereas haloperidol (1.5 mg/kg, s.c.) causes a time-dependent increase in catalepsy (**p<0.01, ***p<0.001 vs vehicle (Bonferroni's test)). (b) VU0152100 (100 mg/kg, i.p.) increases the cataleptogenic effects of submaximal (0.3–0.5 mg/kg, i.p.) doses of haloperidol (**p<0.01, ***p<0.001 vs vehicle/haloperidol (Bonferroni's test)). (c, d) Lack of cardiovascular effects of VU0152100. Rats were injected (arrow) after collecting 30-min baseline data. Following a 15-min post-injection period (acclimation), blood pressure and heart rate data were collected in 10-min intervals. (c) VU0152100 (56.6 mg/kg, i.p.) does not alter blood pressure and (d) heart rate in awake animals. Although two-way ANOVA revealed a significant treatment effect on blood pressure and heart rate there were we no significant differences between vehicle- and VU0152100-treated rats at any time point (Bonferroni test).