Impact of Nicotinic Signaling through Astrocytes on Glutamate Synapse Formation

描述(申请人提供)：谷氨酸能突触是大脑中主要的兴奋通路。决定谷氨酸能突触强度的一个关键特征是可供激活的突触后AMPA受体的数量。我们有初步证据表明，尼古丁刺激星形胶质细胞会导致它们释放一种成分，这种成分专门将AMPA受体招募到神经元上的突触后位置，使这些以前“沉默的”突触发挥功能。这完全是意想不到的，并提出了一种新的、重要的机制来塑造兴奋电路。在发育过程中，当谷氨酸能突触首次形成时，星形胶质细胞是普遍和丰富的，并具有大量的含？7-烟碱型乙酰胆碱受体(？7-nAChRs)。部分通过7-nAChRs介导的自发烟碱胆碱能活动在发育早期也广泛存在。我们的初步证据表明，正是这些？7-nAChRs负责星形胶质细胞对AMPA受体的影响。鉴于星形胶质细胞和β7-nAChR信号的早期出现和广泛存在，这种机制可能会对网络形成和功能输出产生深远的影响。第一个特定目标将在细胞培养中测试由尼古丁刺激星形胶质细胞释放的独特成分-7-nAChRs的突触生成效应。将检查谷氨酸能突触的突触前和突触后成分，评估突触功能，并开始筛选候选成分。第二个具体目标将在体内验证这一假设，使用病毒构建物和突变小鼠的GFAP-CRE系统来确定星形胶质细胞？7-nAChRs是否在海马神经元上招募突触后AMPA受体方面发挥关键作用。随后的实验将测试星形胶质细胞？7-nAChRs是否也有助于突触的可塑性，重点放在先前研究表明长期增强是由于AMPA受体数量增加所致的部位。除了提供对大脑基本过程的新见解外，这些研究还将具有严重的生物医学意义。发现尼古丁信号影响谷氨酸能突触功能等基本特征，如果在发育早期暴露于尼古丁，如通过二手烟或母乳，过早刺激或脱敏这种调节输入，可能会使系统处于高风险状态。此外，这些结果将直接关系到全球制药公司在药物设计中瞄准7-nAChRs，而没有意识到星形胶质细胞？7-nAChRs可能具有这种独特和关键的作用。这个R21项目将检验中心假设，并为未来的项目理解星形胶质细胞和尼古丁信号在调节神经系统形式和功能中的作用奠定基础。

DESCRIPTION (provided by applicant): Glutamatergic synapses comprise the major excitatory pathways in brain. A key feature determining the strength of a glutamatergic synapse is the number of postsynaptic AMPA receptors available for activation. We have preliminary evidence that nicotinic stimulation of astrocytes causes them to release a component that specifically recruits AMPA receptors to postsynaptic sites on neurons, rendering these previously "silent" synapses functional. This is completely unexpected and suggests a new and important mechanism for shaping excitatory circuits. Astrocytes are pervasive and abundant when glutamatergic synapses are first forming during development, and have significant numbers of ?7-containing nicotinic acetylcholine receptors (?7-nAChRs). Spontaneous nicotinic cholinergic activity mediated in part through ?7-nAChRs is also widespread early in development. Our preliminary evidence suggests that it is these ?7-nAChRs that are responsible for the astrocyte effect on AMPA receptors. In view of the early appearance and widespread occurrence of astrocytes and ?7-nAChR signaling, such a mechanism could have profound consequences for shaping network formation and functional output. The first Specific Aim will test in cell culture the synaptogenic effects of components released uniquely by nicotinic stimulation of astrocyte ?7-nAChRs. Pre- and postsynaptic components at glutamatergic synapses will be examined, synaptic function assessed, and a screen of candidate components initiated. The second Specific Aim will test the hypothesis in vivo, using viral constructs and a GFAP-Cre system in mutant mice to determine if astrocyte ?7-nAChRs play a critical role in recruiting postsynaptic AMPA receptors on neurons in the hippocampus. Subsequent experiments will test whether astrocyte ?7-nAChRs also contribute to synaptic plasticity, focusing on locations where previous studies have shown that long-term potentiation results from increased numbers of AMPA receptors. In addition to providing new insight into fundamental processes in the brain, these studies will have serious biomedical implications. Finding that nicotinic signaling influences such basic features as the functionality of glutamatergic synapses may put the system at high risk during development if exposed early on to nicotine, such as through secondhand smoke or maternal milk, either prematurely stimulating or desensitizing this regulatory input. Further, the results will have immediate relevance for pharmaceutical companies globally targeting ?7-nAChRs in drug design without realizing that astrocyte ?7-nAChRs may have this unique and critical role. This R21 project will test the central hypotheses and lay the groundwork for future projects understanding the role of astrocytes and nicotinic signaling in regulating nervous system form and function.