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

谷氨酸是主要的兴奋性神经递质，可能调节新纹状体中的多个信号级联。DARPP-32在新纹状体中棘神经元的多种神经递质通路的整合中起着中心作用。我们通过分析DARPP-32在Thr34(PKA位点)的磷酸化来研究小鼠新纹状体脑片中谷氨酸依赖的信号级联反应。谷氨酸(5 MM)处理后，Thr34区DARPP-32的磷酸化水平迅速增加(在孵育15天的S时，增加了3.5倍)。Thr34磷酸化水平的增加是一过性的，在1 mM时恢复到基础水平，然后在2和5 mM时下降到低于基础水平。Thr34磷酸化的快速增加依赖于NMDA和AMPA受体，并被nNOS抑制剂和可溶性鸟苷酸环化酶抑制剂所阻断，提示谷氨酸的快速作用是通过激活nNOS/NO/cGMP/PKG信号通路来实现的。在钙调神经磷酸酶抑制剂存在的情况下，Thr34磷酸化的增加持续了至少2分钟。在NMDA和AMPA受体拮抗剂存在的情况下，谷氨酸缓慢增加Thr34的磷酸化(在孵育10min时增加4倍)，而mGluR5拮抗剂、ERK和PLC抑制剂可减弱Thr34的磷酸化。因此，对DARPP-32Thr34磷酸化的分析表明，谷氨酸依次激活了新纹状体中三个不同的信号级联：(1)nNOS/NO/cGMP/PKG信号级联；(2)Ca~(2+)&lt；2+&Gt；(3)mGluR5/PLC/ERK信号级联。

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.