Function and Structure Adaptations in Forebrain Development

前脑发育中的功能和结构适应

• 批准号: 9925833
• 负责人: PAT LEVITT
• 金额: $ 77.98万
• 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/9925833
• 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.

电路的发展依赖于基因和经验的正确组装和成熟。定义 在可塑性增强期间驱动电路特异性适应的机制是 了解典型和非典型的发展。我们假设，调节细胞周期的分子 成熟调节经验依赖的发展和差异电路的脆弱性， 发育障碍（NDD）。先进的分子和连接组学技术提供了一个更 成熟大脑中神经元和回路多样性程度的完整观点，但有一个 知识缺口为塑期的成长和完善。对这一差距的洞察力已经从研究中显现出来 在目前的资助期间，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）的离散亚群中表达， 皮质-丘脑（CT）神经元;和3）失调的MET信号传导改变关键期（CP）的时间 双眼的可塑性并破坏恐惧学习。发展分子生物学的基础研究 连接亚型及其在皮层中的功能包括三个特定目标。在目标1中， 将使用连接组学分析前额叶皮层（mPFC）和初级视觉皮层（V1）MET+神经元， 转录组学方法。一个新衍生的转基因小鼠系，MetGFP，将与特异性示踪相结合， 使用病毒转导的split-Cre技术的MET+连接性， GFP+（MET+）神经元及其轴突投射的稳定标记。荧光逆行注射 mPFC和V1靶中的示踪剂将标记IT或CT神经元。将对标记的神经元进行FACS分选和分析 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， 以及用于验证发现并确定性别和发育时间是否 子类型的变量。目的2将检验Met下调是结构和功能所需的假设。 在V1的CP期间的功能可塑性。一种新型、可控的转基因小鼠（ctg-Met），可维持MET 超过其内源性表达期的信号传导将与双光子树突棘结合使用 成像以量化V1 CP期间的脊柱发生和修剪。功能电路连接将 通过激光扫描光刺激结合谷氨酸盐打开来评估。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测量。在目标3中， MET+ mPFC神经元在介导背景恐惧记忆持久性的CP中的作用将是 使用split-Cre方法，使用GFP+神经元中DREADD的选择性表达来确定。的影响 将检查Met缺失或ctg-Met介导的延伸表达的发育出现 条件性恐惧记忆的持续性这些研究对于确定 在与NDD相关的新皮层中，典型和非典型回路发育和可塑性的基础。