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Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.

The use of neural progenitor cells (NPCs) is limited by the incomplete knowledge of the extracellular signals regulating their proliferation and survival. We report that cultured mouse NPCs express functional mGlu3 and mGlu5 metabotropic glutamate receptors. Pharmacological blockade of both receptors reduced NPC proliferation and survival, whereas activation of mGlu5 receptors substantially enhanced cell proliferation. Adult mice lacking mGlu5 receptors or treated with mGlu5 or mGlu3 receptor antagonists showed a dramatic reduction in the number of dividing neuroprogenitors present in the subventricular zone and in the dentate gyrus of the hippocampus. These data disclose a novel function of mGlu receptors and offer new potential strategies for the optimization of cell replacement therapy in neurodegenerative disorders.