Activation of Muscarinic M4 Receptors Inhibits Dopamine D1Receptor Signalling PathwaysIn-Vivo

The muscarinic M4 receptor is an attractive therapeutic target for schizophrenia as revealed by Xanomeline, a mixed M1/M4 orthosteric agonist (1). Previous studies using transgenic mice have attributed Xanomeline’s antipsychotic effects to M4 receptors located on D1-expressing GABAergic striatal neurones (2). Co-localisation of M4 and D1 receptors has been demonstrated on medium spiny neurons in striatal preparations through measuring the inhibitory effect of the Gi/o-coupled M4 receptor on Gs-coupled D1receptor mediated signalling cascade (3). This resulted in the overall modulation of dopaminergic neurotransmission. Although this interaction between D1 and M4 receptors has been characterised ex-vivo, demonstrating modulation of M4 receptors in-vivo has proven challenging. The aim of this study was to confirm this D1/M4 converging signalling pathway in anin-vivo model.

The D1 agonist, SKF82958 (s.c., dH2O. Rat: 3 mg/kg, 10 mins. Mouse: 1 mg/kg, 15 mins), was used to activate the D1-Gs coupled signalling cascade in maleadultSprague Dawley rats and in-house developed male transgenic mice expressing the human M4 receptor.Following methods to preserve protein phosphorylation (4) increasesin cell signalling markers were detectable in the striatum, withrobust and reproducible cGMP and pCREB ELISA responses. Pre-treatment with Xanomeline (i.p., 0.5% methylcellulose)dose-dependently inhibited SKF82958 induced pCREB in both rat and mouse models (Fig 1A&B) and cGMP in mice (Fig 1C).

This data, in combination with previous studies, suggests that in-vivo activation of the Gi/o-coupled M4 signalling pathwaycan attenuateGs-coupled D1 induced signals. The present findings demonstrate signalling cross-talk between the M4 and D1 receptors in-vivo as verified by the measurement of pCREB and cGMP. The ability to stabilise phosphoproteins in-vivo has allowed the development of an assay that can reliably measure biologically relevant levels of phosphoprotein markers. This approach can confirm hypothesised signalling pathways and provide robust in-vivo assays for pharmacological screening.

(1) Mirza NRet al.(2003).CNS Drug Rev 9: 159-186

(2) Dencker Det al. (2011). J Neurosci 31: 5905-5908

(3) Jeon J et al. (2010). J Neurosci 30: 2396-2405

(4) O’Callaghan and Sriram (2004) J Neurosci Methods 135:159-168