In Vivo Non-radioactive Assessment of mGlu5 Receptor-Activated Polyphosphoinositide Hydrolysis in Response to Systemic Administration of a Positive Allosteric Modulator.

Anna R Zuena,Rosamaria Orlando,Giuseppe Battaglia,Gabriele Di Cicco,Luisa Di Menna,Giovanni Sebastiano Alemà,Luisa Iacovelli,Ferdinando Nicoletti,Paola Casolini

doi:10.3389/fphar.2018.00804

Anna R Zuena, Rosamaria Orlando + Show 7 more

Open Access

https://doi.org/10.3389/fphar.2018.00804

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Abstract

mGlu5 receptor-mediated polyphosphoinositide (PI) hydrolysis is classically measured by determining the amount of radioactivity incorporated in inositolmonophosphate (InsP) after labeling of membrane phospholipids with radioactive inositol. Although this method is historically linked to the study of mGlu receptors, it is inappropriate for the assessment of mGlu5 receptor signaling in vivo. Using a new ELISA kit we showed that systemic treatment with the selective positive allosteric modulator (PAM) of mGlu5 receptors VU0360172 enhanced InsP formation in different brain regions of CD1 or C57Black mice. The action of VU0360172 was sensitive to the mGlu5 receptor, negative allosteric modulator (NAM), MTEP, and was abolished in mice lacking mGlu5 receptors. In addition, we could demonstrate that endogenous activation of mGlu5 receptors largely accounted for the basal PI hydrolysis particularly in the prefrontal cortex. This method offers opportunity for investigation of mGlu5 receptor signaling in physiology and pathology, and could be used for the functional screening of mGlu5 receptor PAMs in living animals.

Highlights

MGlu5 metabotropic glutamate receptors are coupled to Gq/11, and their activation stimulates phospholipase-C-mediated polyphosphoinositide (PI) hydrolysis with ensuing formation of inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol. mGlu5 receptors play a key role in mechanisms of activity-dependent synaptic plasticity and are promising candidate drug targets for neuroprotective drugs
To examine whether an enhanced formation of InsP could be detected after systemic treatment with a selective mGlu5 receptor positive allosteric modulator (PAM), we used six groups of CD1 mice treated with vehicle, VU0360172 (0.3, 3, or 30 mg/kg), MTEP (10 mg/kg), or VU0360172 (30 mg/kg) + MTEP, respectively
This might reflect a high level of endogenous activation or a constitutive activity of mGlu5 receptors because InsP levels in the prefrontal cortex were halved by treatment with the mGlu5 receptor negative allosteric modulator (NAM), MTEP (Figure 1B)

Summary

Introduction

MGlu metabotropic glutamate receptors are coupled to Gq/11, and their activation stimulates phospholipase-C-mediated polyphosphoinositide (PI) hydrolysis with ensuing formation of inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (reviewed by Nicoletti et al, 2011). mGlu receptors play a key role in mechanisms of activity-dependent synaptic plasticity and are promising candidate drug targets for neuroprotective drugs (reviewed by Bruno et al, 2017). Selective ligands of mGlu receptors (both positive and negative allosteric modulators) are under development for the treatment of numerous CNS disorders, such as unipolar depression, obsessive-compulsive disorder, schizophrenia, autism, chronic pain, and Parkinson’s disease (Vinson and Conn, 2012; Nicoletti et al, 2015; Bruno et al, 2017; Rutrick et al, 2017) This raises the interest on how expression and function of mGlu receptors change during CNS development (Minakami et al, 1995; Casabona et al, 1997), in association with learning and memory processes. (i) tissue from 4 to 10 rats or mice (depending on the brain region) must be pooled for slice preparation; (ii) the uncontrolled extracellular concentrations of endogenous glutamate in brain slice preparations renders this model inappropriate for the study of the action of different classes of positive allosteric modulators (PAMs) on native mGlu receptors; (iii) measurements of radioactive InsP are routinely performed by anion exchange chromatography followed by radioactivity counting with no information on endogenous InsP levels (and, on the specific activity of [3H]-InsP); and, (iv) when applied to in vivo studies the method requires the intracerebral injection of radioactive inositol

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