Signaling Mechanisms Underlying Neuroleptic Drug Actions

项目1将探讨典型和非典型抗精神病药物如何影响纹状体和前额皮质的蛋白质磷酸化途径。预期典型和非典型抗精神病药共享一些，但不是全部，细胞内信号传导靶点。相似之处可能反映了这些化合物抵消阳性症状的共同能力，而差异可能反映了它们对阴性症状和锥体外系副作用的不同作用。目的全面研究抗精神病药在皮层和纹状体中调控的蛋白磷酸化通路。先前的研究表明，DARPP-32与抗精神病药的作用有关。到目前为止，还无法区分DARPP-32在纹状体特定神经元细胞类型中的生化调节。这些神经元在形态学上难以区分，在解剖学上是混杂的。Aim II的目标是更详细地了解抗精神病药起作用的特定细胞群。新的BAC转基因小鼠技术将用于在纹状体、纹状顶或皮质神经元中过表达表位标记的DARPP-32、RCS和mGluR5。拉下实验之后，免疫印迹或质谱从这些动物将允许磷酸化事件的细胞特异性分析。DARPP-32 KO小鼠对神经阻滞剂和拟精神药物的反应发生改变。Aim III的目标是确定DARPP-32介导神经阻滞剂作用的神经回路。Cre/loxP技术将用于生成纹状体、纹状顶或皮质神经元缺乏DARPP-32的小鼠。小鼠系将在生化，行为和电生理分析方面研究其对神经抑制剂的反应，最后，在Aim IV中，将研究与代谢性谷氨酸，5 -羟色胺和毒蕈碱受体相互作用的新蛋白质的细胞生物学。因此，拟议的研究应该提供关于神经阻滞剂如何影响特定神经元回路中的蛋白质磷酸化途径的详细知识。

DESCRIPTION (provided by applicant): Project 1 will explore how typical and atypical neuroleptic drugs affect protein phosphorylation pathways in striatum and prefrontal cortex. It is anticipated that typical and atypical neuroleptics share some, but not all, intracellular signaling targets. The similarities could reflect the common ability of these compounds to counteract positive symptoms, whereas the differences could reflect their differential actions on negative symptoms and extrapyramidal side effects. Aim I will establish a comprehensive survey of protein phosphorylation pathways regulated by neuroleptics in cortex and striatum. Previous work has shown that DARPP-32 is implicated in the actions of neuroleptics. Up until the present time, it has not been possible to distinguish between the biochemical regulation of DARPP-32 in specific neuronal cell types of striatum. These neurons are morphologically indistinguishable and are intermixed anatomically. The goal of Aim II is to obtain a more detailed understanding of the specific cell population where neuroleptics act. Novel BAC transgenic mouse technology will be used to overexpress epitope-tagged DARPP-32, RCS and mGluR5 in striatonigral, striatopallidal or cortical neurons. Pull down experiments followed by immunoblotting or mass spectrometry from these animals will allow cell-specific analyses of phosphorylation events. DARPP-32 KO mice show altered responses to neuroleptics and psychotomimetics. The goal of Aim III is to define neuronal circuitries in which DARPP-32 mediates actions of neuroleptics. The Cre/loxP technology will be used to generate mice lacking DARPP-32 in striatonigral, striatopallidal or cortical neurons. The mouse lines will be studied in terms of their responsivity to neuroleptics in biochemical, behavioural and electrophysiological assays, finally, in Aim IV, studies on the cellular biology of novel proteins interacting with metabotropic glutamate, serotonin and muscarinic receptors will be conducted. Thus, the proposed studies should provide a detailed knowledge on how neuroleptics affect protein phosphorylation pathways in defined neuronal circuitries.