Astrocyte-microglial communication in developmental synapse formation

项目摘要/摘要 调节突触的动态平衡对于正常的大脑发育和功能是必不可少的，而且 依赖于星形胶质细胞和小胶质细胞--大脑的支持细胞。星形胶质细胞与成千上万的 突触和促进发育突触的形成。小胶质细胞是脑内的免疫细胞 越来越多地与突触形成和修剪有关。我们发现了一种新的信号电路 在发育中的中枢神经系统促进突触消除的星形胶质细胞和小胶质细胞之间 (CNS)。我们发现星形胶质细胞表达免疫信号IL-33，而小胶质细胞表达 IL-33受体(IL1RL1.)我们随后发现，在脊髓中，IL-33从 发育中的星形胶质细胞导致过度突触，而IL-33向小胶质细胞发出信号来驱动突触 吞噬并导致突触枯竭。我们的中心假设是星形胶质细胞表达和释放 IL-33对神经元衍生信号的反应，而IL-33反过来驱动小胶质突触 淘汰赛。我们将在三个不同但相互关联的目标上测试这一假设，重点是一个定义明确的 IL-33高表达的丘脑腹底感觉核(VB)的实验可及环路 在突触细化过程中。在第一个目标中，我们将确定星形细胞IL-33是如何调节丘脑突触的 亚型和电路功能。我们先前的研究表明，IL-33的整体缺失会导致细胞的过度兴奋性 VB回路和过多的突触。在这里，我们将有条件地从星形胶质细胞中删除IL-33，并探索这些 更详细的表型，量化传入兴奋性和抑制性突触的亚型以了解 电路的不同组件如何改变。在第二个目标中，我们将确定分子机制 调节小胶质细胞突触吞噬。我们先前发现IL-33促进突触后吞噬 小胶质细胞的蛋白质。在这里，使用标准和高分辨率技术(膨胀显微镜)，我们 将量化突触前和突触后兴奋性元素以及抑制性突触的吞噬。我们 将测试通过有条件地删除其受体直接向小胶质细胞发送信号的要求。在《目标3》中，我们将 确定诱导星形胶质细胞表达和释放IL-33的神经元分子。我们的初步数据 证明去甲肾上腺素是一种促进灰质IL-33表达的神经元来源的信号 星形胶质细胞。在这里，我们将进一步测试星形胶质细胞上的去甲肾上腺素能受体在体内介导这一效应。我们 也将检验IL-33的细胞外释放依赖于神经元突触活动的假设，通过 在体外和体内调节活性。这三个目标共同探索了一种新的神经胶质细胞的作用。 回路调节突触的动态平衡。我们预测，更广泛地了解胶质细胞是如何通过 调节突触的免疫分子将从根本上影响我们对神经回路如何 在学习和发育以及神经发育疾病方面的变化。

PROJECT SUMMARY/ ABSTRACT Regulation of synapse homeostasis is essential for normal brain development and function, and intimately dependent on astrocytes and microglia -- the support cells of the brain. Astrocytes contact thousands of synapses and promote developmental synapse formation. Microglia are brain-resident immune cells increasingly implicated in both synapse formation and pruning. We have discovered a novel signaling circuit between astrocytes and microglia that promotes synapse elimination in the developing central nervous system (CNS). We found that astrocytes express the immune signal Interleukin-33 (IL-33) whereas microglia express the IL-33 receptor (IL1RL1.) We subsequently showed that in the spinal cord, eliminating IL-33 from developing astrocytes leads to excess synapses, and that IL-33 signals to microglia to drive synapse engulfment and lead to synapse depletion. Our central hypothesis is that astrocytes express and release IL-33 in response to neuron-derived signals, and that IL-33 in turn drives microglial synapse elimination. We will test this hypothesis in three distinct but interrelated aims, focusing on a well-defined and experimentally accessible circuit in the ventrobasal sensory thalamus (VB), where IL-33 is highly expressed during synapse refinement. In Aim One we will determine how astrocytic IL-33 regulates thalamic synapse subtypes and circuit function. We previously showed that global deletion of IL-33 lead to hyperexcitability of the VB circuit and excess synapses. Here we will conditionally delete IL-33 from astrocytes and explore these phenotypes in more detail, quantifying subtypes of afferent excitatory and inhibitory synapses to understand how different components of the circuit are altered. In Aim Two, we will determine the molecular mechanisms regulating microglial synapse engulfment. We previously found that IL-33 promotes engulfment of postsynaptic proteins by microglia. Here, using both standard and high resolution techniques (expansion microscopy), we will quantify engulfment of both pre- and postsynaptic excitatory elements, as well as inhibitory synapses. We will test the requirement for direct signaling to microglia via conditional deletion of its receptor. In Aim 3, we will identify neuronal molecules that induce astrocyte expression and release of IL-33. Our preliminary data demonstrates that norepinephrine is a neuron-derived cue that promotes expression of IL-33 in gray matter astrocytes. Here we will further test which noradrenergic receptors on astrocytes mediate this effect in vivo. We will also test the hypothesis that extracellular release of IL-33 is dependent on neuronal synaptic activity, by modulating activity in vitro and in vivo. Together, these three aims explore the role of a novel glial-neuronal circuit mediating synapse homeostasis. We predict that a broader understanding of how glia communicate via immune molecules to regulate synapses will fundamentally impact our understanding of how neural circuits change in learning and development as well as in neurodevelopmental diseases.