Molecular mechanisms of the integration of inhibitory dopaminergic inputs and excitatory glutamatergic inputs in neostriatal neurons

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Glutamate, the major excitatory neurotransmitter, likely regulates multiple signaling cascades in the neostriatum. DARPP-32 plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter pathways. We investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr34 (PKA-site). Treatment with glutamate (5mM) rapidly increased the phosphorylation of DARPP-32 at Thr34 (by 3.5-fold at 15 s of incubation). The increase in Thr34 phosphorylation was transient, returned to the basal level at 1 mm, and was followed by a decrease to below the basal level at 2 and 5 mm. The rapid increase in Thr34 phosphorylation was NMDA and AMPA receptor-dependent, and abolished by an nNOS inhibitor and a soluble guanylyl cyclase inhibitor, suggesting that the rapid effect of glutamate is mediated through activation of the nNOS/NO/cGMP/PKG signaling cascade. In the presence of a calcineurin inhibitor, the increase in Thr34 phosphorylation was sustained for at least 2min. In the presence of both NMDA and AMPA receptor antagonists, glutamate slowly increased Thr34 phosphorylation (by 4-fold at 10 min of incubation), and the increase in Thr34 phosphorylation was attenuated by an mGluR5 antagonist, an ERK, inhibitor and a PLC inhibitor. Thus, analysis of DARPP-32 Thr34 phosphorylation revealed that glutamate sequentially activates three different signaling cascades in the neostriatum: (1) nNOS/NO/cGMP/PKG signaling cascade, (2)Ca^<2+> signaling cascade, (3) mGluR5/PLC/ERK signaling cascade.