Function and Structure Adaptations in Forebrain Development

前脑发育中的功能和结构适应

• 批准号: 10368094
• 负责人: PAT LEVITT
• 金额: $ 72.02万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10368094
• 关键词: Address; Adult; Anatomy; Appearance; Axon; Biological Assay; Brain; Cells; Cerebral cortex; Cluster Analysis; Complex; Data; Data Analyses; Dendritic Spines; Development; Dissection; Dorsal; Down-Regulation; Excitatory Synapse; Forebrain Development; Foundations; Fright; Gene Combinations; Genes; Genetic Recombination; Glutamates; Goals; Grant; Growth; Hippocampus (Brain); Human; Image; Injections; Interdisciplinary Study; Intervention; Knowledge; Label; Lasers; Learning; LoxP-flanked allele; Measures; Medial; Mediating; Memory; Methods; Molecular; Molecular Profiling; Mus; Neocortex; Neurodevelopmental Disorder; Neurons; Ocular Dominance; Pattern; Predisposition; Prefrontal Cortex; Primates; Process; Protein Tyrosine Kinase; Proteins; Receptor Protein-Tyrosine Kinases; Role; Scanning; Signal Pathway; Signal Transduction; Somatosensory Cortex; Structure; Subgroup; Symptoms; Synapses; Synaptic plasticity; Technology; Testing; Thalamic structure; Time; Tracer; Transgenic Mice; V1 neuron; Variant; Vertebral column; Viral; area striata; base; cellular imaging; conditioned fear; cortex mapping; critical developmental period; critical period; designer receptors exclusively activated by designer drugs; early life stress; experience; experimental study; fear memory; functional plasticity; imaging modality; improved; insight; methionylmethionine; molecular imaging; monocular deprivation; neuronal circuitry; postnatal; postnatal development; postnatal period; receptor expression; response; selective expression; sex; single-cell RNA sequencing; synaptic pruning; synaptogenesis; transcriptome; transcriptomics; two-photon

Circuit development relies on genes and experience for proper assembly and maturation. Defining the mechanisms that drive circuit-specific adaptations during heightened periods of plasticity are key for understanding typical and atypical development. We hypothesize that molecules that regulate timing of maturation modulate experience-dependent development and differential circuit vulnerabilities in neuro- developmental disorders (NDDs). Advanced molecular and connectomics technologies have provided a more complete perspective on the extent of neuronal and circuit diversity in the mature brain, but there is a knowledge gap for the plastic periods of growth and refinement. Insight into this gap has emerged from studies during the current grant period showing that 1) the c-MET receptor tyrosine kinase (MET) regulates timing of excitatory synapse maturation; 2) MET is expressed in discrete subpopulations of intra-telencephalic (IT) and cortico-thalamic (CT) neurons; and 3) dysregulated MET signaling alters the timing of critical period (CP) plasticity for binocularity and disrupts fear learning. Foundational studies of developing molecular and connectivity subtypes and their function in the cortex comprise three specific aims. In Aim 1, developing medial prefrontal cortex (mPFC) and primary visual cortex (V1) MET+ neurons will be profiled using connectomics and transcriptomics methods. A newly derived transgenic mouse line, MetGFP, will be combined with specific tracing of MET+ connectivity using virally-transduced split-Cre technology that produces Cre-mediated, temporally stable labeling of GFP+ (MET+) neurons and their axonal projections. Injections of fluorescent retrograde tracers in mPFC and V1 targets will label IT or CT neurons. Labeled neurons will be FACS-sorted and profiled by single cell RNA sequencing. Transcriptome data analysis will delineate subtypes of Met+ and Met-neurons, with additional methods used to validate discoveries and determine whether sex and developmental timing are variables for the subtypes. Aim 2 will test the hypothesis that Met down-regulation is required for structural and functional plasticity during the CP in V1. A new, controllable transgenic mouse (ctg-Met) that sustains MET signaling beyond its endogenous expression period will be used in combination with two-photon dendritic spine imaging to quantify spine genesis and pruning during the V1 CP. Functional circuit connectivity will be assessed by laser scanning photostimulation combined with glutamate uncaging. V1 plasticity will be measured using a classic paradigm of monocular deprivation-induced ocular dominance plasticity. In Aim 3, the role of MET+ mPFC neurons in mediating the CP for contextual fear memory persistence will be determined using selective expression of DREADDs in GFP+ neurons using the split-Cre approach. The impact of Met deletion or ctg-Met-mediated extended expression will be examined for the developmental emergence of conditioned fear memory persistence. The studies have high impact for determining mechanisms that underlie typical and atypical circuit development and plasticity in the neocortex related to NDDs.

电路的发育依赖于适当组装和成熟的基因和经验。定义 在可塑性高发期驱动电路特异性适应的机制是 了解典型和非典型发展。我们假设调节时间的分子 成熟调节经验依赖的发育和神经元的差异电路脆弱性 发育障碍(NDDS)。先进的分子和连接学技术提供了更多 对成熟大脑中神经元和回路多样性程度的完整透视，但有一个 知识鸿沟为塑胶期的成长和完善。对这一差距的洞察是通过研究得出的 在当前的授权期内，显示1)c-met受体酪氨酸激酶(MET)调节 兴奋性突触成熟；2)MET在端脑内(IT)和 皮层-丘脑(CT)神经元；3)MET信号异常改变关键期(CP)的时间。 双目视觉的可塑性会扰乱恐惧学习。发展中的分子和基础研究 连接亚型及其在大脑皮层中的功能包括三个特定的目标。在目标1中，发展医疗 前额叶皮质(MPFC)和初级视觉皮质(V1)MET+神经元将使用连接和 转录组学方法。一种新的转基因小鼠品系MetGFP将与特定的追踪相结合 使用病毒转导的Split-Cre技术暂时产生Cre中介的MET+连接 GFP+(MET+)神经元及其轴突投射的稳定标记。逆行注射荧光 MPFC和V1靶点中的示踪剂将标记IT或CT神经元。标记的神经元将被FACS分类和剖析 通过单细胞RNA测序。转录组数据分析将描绘Met+和Met-神经元的亚型， 使用其他方法来验证发现，并确定性行为和发育时间是否 子类型的变量。目标2将检验这样的假设，即Met下调对于结构和 V1在CP过程中的功能可塑性。一种新的可控制的维持MET的转基因小鼠(CTG-Met) 超出其内源性表达周期的信号将与双光子树突棘结合使用 在V1CP期间进行影像检查以量化脊柱的形成和修剪。功能电路连接将是 采用激光扫描光刺激结合谷氨酸去除的方法进行评估。V1可塑性将是 使用单眼剥夺诱导的眼优势可塑性的经典范例进行测量。在《目标3》中， Met+mPFC神经元在上下文恐惧记忆持续性CP中的作用将是 使用Split-Cre方法在GFP+神经元中选择性表达DREADD来确定。其影响 Met缺失或CTG-Met介导的扩展表达将被检测为发育出现 条件性恐惧记忆的持久性。这些研究对确定 与NDDS相关的新皮质中典型和非典型的回路发育和可塑性是其基础。