Development of Visual Connections

描述（由申请人提供）：是什么让婴儿的大脑在早期发育的关键时期如此迅速地学习？什么细胞和分子机制导致成年期广泛可塑性下降？这里的目标是通过发现并阻断抑制可塑性和电路变化的内源性机制来增强突触的可塑性。具体来说，对神经元受体PirB（配对免疫球蛋白样受体B；人类中的Lilrb3）的操作是否可以“释放制动”眼优势（OD）可塑性，这是视觉皮层中经验依赖的突触可塑性的一种形式？在免疫系统中，PirB是主要组织相容性I类分子的受体，是t细胞受体的著名配体。本实验室意外发现神经元在突触上表达PirB和MHCI分子。PirB种系缺失小鼠视觉皮层OD可塑性增强，与PirB抑制突触可塑性的作用一致。提出了三个具体目标：1)确定PirB的急性缺失是否会增强OD可塑性：通过与他莫昔芬诱导的Cre转基因系杂交，制备了PirB的条件等位基因（PirB flox/flox），允许PirB的急性时间和细胞型破坏。用重组可溶性截断PirB蛋白或功能阻断抗体直接阻断PirB也将被使用。这些实验将揭示PirB何时以及在何种细胞类型中起作用。2) PirB-/-小鼠OD可塑性增强与突触可塑性的细胞机制有关。我们将采用生理方法在体外视觉皮层切片上研究长期增强（LTP）和长期抑制（LTD）。在正常视觉体验或单眼闭眼饲养的PirB-/- vs WT小鼠中，测量yfp标记的第5层锥体神经元的树突棘密度；脊柱稳定性将使用双光子显微镜检查。这些实验应该扩大对PirB如何在突触和结构水平上抑制可塑性的理解。3)识别小鼠视觉皮层PirB信号转导通路：通过比较WT和种系PirB-/-小鼠视觉皮层在关键时期和之后的视觉驱动信号，识别和评估PirB下游的候选信号通路。将评估候选通路的表达和磷酸化水平的变化，包括MAP激酶、AKT和mTOR信号。本研究将采用小鼠遗传、生化、电生理、成像和解剖学方法，在系统水平上评估OD的可塑性，并了解PirB功能的细胞和分子机制。总之，实验应该阐明PirB在关键时期及之后如何在神经元中正常作用，抑制突触-可塑性信号通路，并测试急性PirB阻断恢复OD可塑性的可行性。它们代表了理解发育关键时期机制的关键步骤，以及通过参与大脑固有的神经可塑性能力来设计增强中枢神经系统功能和修复的新方法。

DESCRIPTION (provided by applicant): What enables a baby's brain to learn so rapidly during early developmental critical periods? What cell and molecular mechanisms cause the decline in extensive plasticity by adulthood? The goal here is to enhance synaptic plasticity by discovering and then blocking endogenous mechanisms that function to suppress plasticity and circuit change. Specifically, can manipulations of the neuronal receptor PirB (Paired Immunoglobulin-like receptor B; Lilrb3 in humans) "release the brake" on ocular dominance (OD) plasticity, a form of experience-dependent synaptic plasticity in visual cortex? In the immune system PirB is a receptor for Major Histocompatibility Class I molecules, famous ligands for T-cell receptors. This Lab made the unexpected discovery that neurons express PirB and MHCI molecules at synapses. OD plasticity is enhanced in visual cortex of mice with germline deletion of PirB, consistent with PirB acting to brake synaptic plasticity. Three specific aims are proposed: 1) Determine if acute deletion of PirB postnatally enhances OD plasticity: A conditional allele of PirB (PirB flox/flox) has been made, allowing acute temporal and cell-type disruption of PirB by crossing mice with tamoxifen-inducible Cre transgenic lines. Direct blockade of PirB with recombinant soluble truncated PirB protein or function-blocking antibodies will also be used. These experiments should reveal when and in what cell types PirB acts. 2) Link enhanced OD plasticity in PirB-/- mice to cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) will be studied in vitro in visual cortex slices using physiological methods. Dendritic spine density of YFP-labeled layer 5 pyramidal neurons will be measured in PirB-/- vs WT mice reared with normal visual experience or with monocular eye closure; spine stability will be examined using two-photon microscopy. These experiments should broaden understanding of how PirB acts at synaptic and structural levels to suppress plasticity. 3) Identify PirB signal transduction pathways in mouse visual cortex: Candidate signaling pathways downstream of PirB will be identified and evaluated by comparing visually-driven signaling in WT vs germline PirB-/- mouse visual cortex during and after the critical period. Changes in expression and phosphorylation levels will be assessed in candidate pathways including MAP Kinase, AKT and mTOR signaling. Studies here will employ genetic, biochemical, electrophysiological, imaging and anatomical methods in mice to assess OD plasticity at the systems level and to understand cellular and molecular mechanisms of PirB function. Together, experiments should elucidate how PirB normally acts in neurons to suppress synaptic-plasticity signaling pathways during and beyond the critical period, as well as test feasibility of restoring OD plasticity by acute PirB blockade. They represent key steps in understanding mechanisms of developmental critical periods, as well as for designing new ways to enhance CNS function and repair by engaging the brain's inherent capacity for neural plasticity.