How do neurons in the brain decide to refine their synaptic connections in vivo?

功能神经回路的形成对大脑的正常运作至关重要。要建立最多的 高效的突触电路，突触连接必须在发育过程中通过神经活动来完善。在这 提议，我们将确定神经建立功能电路的分子和方式 活动，集中在边缘系统(包括海马体和扣带回皮质)，这与 情绪处理、记忆形成和社会行为。使用一种老鼠遗传系统，其中 限制群体的海马神经元可以有条件地灭活，我们发现海马区 轴突通过活动依赖的竞争得到提炼，活跃的神经元连接停留(维持)。 而不活跃的人则离开(消除)。我们进一步发现，细胞黏附分子Sirp来自 突触后神经元通过其突触前受体CD47来稳定活跃的突触，起到“停留”的作用 信号。为了确定在非活动轴突消除(“GO”信号)中起关键作用的信号分子，我们 创造了一种新的系统，在其中神经活动和基因表达可以在体内有条件地控制， 宫内使用电穿孔。当扣带回部分神经元的神经递质释放受阻时 大脑皮层，他们的胼胝体投射(大脑半球之间的主要联系)被消除。 在开发过程中。使用这个系统，我们筛选了在非活动状态下上调的信号分子 就在轴突开始离开之前的神经元，发现了钙依赖的酪氨酸激酶PYK2。非活动 当Pyk2的激酶死亡突变体被表达时，轴突没有被消除，这表明Pyk2的活性是 是不活跃的轴突离开所必需的。我们进一步鉴定出，一个与Pyk2相互作用的激酶JAK2也是 对于消除不活跃的轴突来说是必要的。一直以来，在不活跃的神经元中，Pyk2和JAK2被激活。最后， 我们发现，即使在轴突活跃的情况下，Pyk2或JAK2的过表达也会诱导轴突消除。我们 提出PYK2-JAK2通路是“GO”信号，是轴突细化的决定因素。至 进一步描述这一通路，并了解“去”和“留”通路如何调节活动-- 依赖轴突/突触细化，我们建议：目标1：研究Pyk2和JAK2在 生理条件下轴突/突触的细化。目的2：分析电生理效应 使用条件性KO小鼠在突触精化过程中的PYK2/JAK2失活。目标3：检查是否 PYK2-JAK2通路为体内小胶质细胞清除非活动轴突提供了线索。目标4：调查 轴突/突触细化过程中Stay(Sirp-CD47)和Go(PYK2-JAK2)通路的相互作用 活着。我们的项目将从分子上描绘神经元如何决定在脑内建立功能性突触连接 哺乳动物的大脑。PYK2和JAK2与多种神经精神障碍有关。多种形式的 包括自闭症和精神分裂症在内的精神疾病与边缘的异常改变有关 电路。因此，我们的研究也应该对这类疾病的病因和治疗产生新的见解。

Formation of functional neural circuits is critical for proper functioning of the brain. To establish the most efficient synaptic circuits, synaptic connections must be refined by neural activity during development. In this proposal, we will determine the molecules and manner by which functional circuits are established by neural activity, focusing on the limbic system (including the hippocampus and cingulate cortex), which is implicated in emotional processing, memory formation and social behavior. Using a mouse genetic system in which restricted populations of hippocampal neurons can be conditionally inactivated, we found that hippocampal axons are refined through activity-dependent competition, where active neuronal connections stay (maintained) while inactive ones leave (eliminated). We further found that a cell adhesion molecule SIRP from postsynaptic neurons stabilizes active synapses through its presynaptic receptor CD47, serving as a "Stay" signal. To identify the signaling molecules that play critical roles in inactive axon elimination ("Go" signal), we generated a new system in which neural activity and gene expression can be conditionally controlled in vivo, using in utero electroporation. When neurotransmitter release is blocked in a subset of neurons in the cingulate cortex, their callosal projections (the major connections between the cerebral hemispheres) are eliminated during development. Using this system, we screened for signaling molecules that are upregulated in inactive neurons right before their axons start to leave and identified the Ca2+-dependent tyrosine kinase Pyk2. Inactive axons were not eliminated when a kinase-dead mutant of Pyk2 was expressed, indicating that Pyk2 activity is necessary for inactive axons to leave. We further identified that a Pyk2-interacting kinase, JAK2, is also necessary for inactive axon elimination. Consistently, Pyk2 and JAK2 are activated in inactive neurons. Finally, we found that overexpression of Pyk2 or JAK2 induces axonal elimination even when axons are active. We propose that the Pyk2-JAK2 pathway is the "Go" signal and serves as the determinant of axon refinement. To further characterize this pathway and to understand how the "Go" and "Stay" pathways regulate activity- dependent axon/synapse refinement, we propose to: Aim 1: Investigate the role of Pyk2 and JAK2 for axon/synapse refinement in physiological conditions. Aim 2: Analyze the electrophysiological consequences of Pyk2/JAK2 inactivation during synapse refinement using conditional KO mice. Aim 3: Examine whether the Pyk2-JAK2 pathway provides cues for microglial clearance of inactive axons in vivo. Aim 4: Investigate the interaction between the Stay (SIRP-CD47) and Go (Pyk2-JAK2) pathways in axon/synapse refinement in vivo. Our project will molecularly delineate how neurons decide to establish functional synaptic connections in the mammalian brain. Pyk2 and JAK2 are associated with various neuropsychiatric disorders. Many forms of mental illness including autism and schizophrenia are associated with abnormal alterations in the limbic circuitry. Thus, our studies should also yield novel insights into the etiology and treatment of such disorders.