Fetal and Postnatal Development of Visual Connections

描述(由申请人提供)：是什么使婴儿的大脑在早期发育关键期如此迅速地学习？是什么细胞和分子机制导致成年后广泛的可塑性下降？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。具体地说，操纵神经元受体PirB(配对的免疫球蛋白样受体B；在人类中为Lilrb3)能否对视觉皮质中的一种依赖经验的突触可塑性--眼优势(OD)可塑性--“松开刹车”？在免疫系统中，PirB是主要组织相容性I类分子的受体，这些分子是著名的T细胞受体的配体。这个实验室有一个意想不到的发现，神经元在突触上表达PirB和MHCI分子。PirB基因胚系缺失的小鼠视皮层外径可塑性增强，这与PirB抑制突触可塑性的作用一致。提出了三个特定的目标：1)确定PirB的急性缺失是否会在出生后增强OD可塑性：已经制造了PirB的条件等位基因(PirB Flox/Flox)，通过将小鼠与他莫昔芬诱导的Cre转基因系杂交，允许PirB在时间和细胞类型上的急性破坏。用重组可溶性截短的PirB蛋白或功能阻断抗体直接阻断PirB也将被使用。这些实验应该会揭示PirB在什么时候以及在什么细胞类型中起作用。2)PirB-/-小鼠增强的OD可塑性与突触可塑性的细胞机制有关。长时程增强(LTP)和长时程增强(LTD)将在体外用生理学方法在视皮层脑片上进行研究。YFP标记的第5层锥体神经元的树突密度将在PirB-/-VS WT小鼠中进行测量，这些小鼠在正常视觉体验或单眼闭眼情况下饲养；脊椎稳定性将使用双光子显微镜进行检查。这些实验应该会扩大对PirB如何在突触和结构水平上抑制可塑性的理解。3)确定PirB在小鼠视皮质中的信号转导途径：通过比较WT和胚系PirB-/-小鼠视皮层中的视觉驱动信号在关键期和之后的变化来确定和评估PirB下游的候选信号通路。表达和磷酸化水平的变化将在候选通路中进行评估，包括MAP激酶、AKT和mTOR信号。这项研究将利用遗传学、生化、电生理、成像和解剖学方法在系统水平上评估外径可塑性，并了解PirB功能的细胞和分子机制。总之，实验应该阐明PirB在关键期内和之后如何在神经元中正常作用以抑制突触可塑性信号通路，并测试通过急性PirB阻断恢复外径可塑性的可行性。它们代表了理解发育关键期机制的关键步骤，以及设计通过利用大脑固有的神经可塑性来增强中枢神经系统功能和修复的新方法。 与公共健康相关：通过对内源性作用于抑制可塑性和回路改变的机制“松开刹车”，大脑可能会恢复广泛的突触可塑性，这一想法具有重要的临床意义。如果大脑回路能够恢复到更不成熟的状态，在这种状态下突触的可塑性可以很容易地进行，那么在中风、阿尔茨海默氏症或其他与衰老有关的神经退行性疾病造成的损伤或记忆丧失后，可能会促进恢复。大脑再培训也可能用于儿童学习障碍，包括阅读障碍或发育中的视觉系统的弱视，甚至可能用于自闭症的治疗。

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. PUBLIC HEALTH RELEVANCE: The idea that extensive synaptic plasticity might be restored to the brain by "releasing the brake" on mechanisms that act endogenously to suppress plasticity and circuit change has important clinical implications. If brain circuits can be restore to a more immature state in which synaptic plasticity can be easily engaged, it might be possible to facilitate recovery following damage or memory loss associated with Stroke, Alzheimer's or other neurodegenerative disorders of aging. Retraining the brain might also be applied post hoc for childhood learning disabilities including Dyslexia or Amblyopia in the developing visual system, and possibly even for treatment of Autism.