Do mGluR5 positive allosteric modulators increase NMDA-induced phosphorylation of extracellular signal-regulated kinase (ERK)?

Abstract

One of the suggested hallmarks in Schizophrenia is a hypo-function of the NMDA system. One attempt to overcome the hypofunction of the glutamate system is to modulate the mGluR5 receptors. Since it is quite problematic to activate the glutamatergic system without excitotoxic side effects, it would be desirable to use mGluR5 positive allosteric modulators instead of i.e. NMDA or mGluR5 agonists. We have used murine cortical neurons to establish a model for measuring single cell ERK phosphorylation. Cortical neurons cultured for 7 – 8 days in vitro were stimulated very briefl y with NMDA, following by fi xation with 4% paraformaldehyde. Subsequently, the cells were stained with an anti-phospho-p44/42 MAP kinase (pERK1/2) antibody and then a fl uorescent secondary antibody. The fl uorescent intensity in both the cytoplasm and nucleus of single cells was measured on an ArrayScanner® (Cellomics). Having the method set up, we wanted to address, if the mixed cortical cultures were suitable to study NMDA- and mGluR5 receptor interaction. The cultures expressed NMDA and mGluR5 receptors, and the pERK response was dependent on the concentration of NMDA. The results obtained with single cell pERK measurement were confi rmed by Western blotting and by pharmacological intervention. To examine effect of mGluR5 agonists and allosteric modulators, several compounds were tested: (RS)-2- Chloro-5-hydroxyphenylglycine (CHPG), (RS)-3,5-dihydroxyphenylglycine (DHPG), 3,3’-difl uorobenzaldazine (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3- dihydro-2H-isoindol-2-yl)methyl] phenyl}-2-hydroxybenzamide (CPPHA) and 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (8q). We observed a synergistic effect on the NMDA response and the modulators have no effect on their own. Measurement of ERK phosphorylation is a new way to measure NMDA enhancement by mGluR5 on single cell level in cortical neurons with high throughput and statistical validity.