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Characterizing excitatory synapse in vivo structural dynamics

表征兴奋性突触体内结构动力学

基本信息

批准号：
10512611
负责人：
Elly Nedivi
金额：
$ 47.5万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10512611
关键词：
Acids Affect Anatomy Architecture Binding Brain Brain Diseases Cell Nucleus Cells Color Development Elements Event Excitatory Synapse Extracellular Protein Failure Genes Glutamate Receptor Glycosylphosphatidylinositols Goals Imaging technology Impairment Individual Knockout Mice Label Lateral Geniculate Body Lateral posterior nucleus of thalamus Lead Learning Link Maps Mediator of activation protein Memory Methods Microscopy Molecular Monitor Mus Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Physiological Proteins Proteome Proteomics Recording of previous events Resolution Sensory Specificity Synapses Synaptophysin Testing Tissues Transgenic Mice Transmembrane Domain Vertebral column Visual Visual Cortex Visual Perception Visual system structure area striata experience extracellular fluorophore functional adaptation gene product hippocampal pyramidal neuron in utero in vitro Assay in vivo in vivo imaging in vivo monitoring innovation mouse genetics neural circuit operation postsynaptic postsynaptic density protein prevent protein complex pup receptor recruit response stargazin synaptic function synaptogenesis two photon microscopy visual deprivation

项目摘要

Many brain disorders manifest impaired synaptic integrity, stability, and experience-dependent selection, resulting in wiring deficits and perturbed function. Unfortunately, our ability to monitor synaptic or circuit failures as they occur has been hindered by the difficulty of visualizing synapses in vivo. Here we propose in vivo monitoring of the ‘order of operations’ in excitatory synapse formation and elimination, and identifying the steps and molecules controlling experience-dependent synapse selection. We focus on the visual system, where there is a well-characterized toolkit for manipulating experience. We hypothesize that the dynamics of a synapse's assembly and disassembly, and its propensity to remodel, are intimately linked to its connection identity and proteomic content. To test this, we propose the following aims: Aim1: To track the structural remodeling of excitatory synapses and how it relates to their afferent input specificity and proteomic content. We will label LGN or LP thalamic inputs onto the full dendritic arbor of single L2/3 pyramidal neurons in mouse visual cortex, track their daily dynamics and their response to visual deprivation, and analyze their proteomic content in relation to dynamic history and afferent identity. To this purpose, we will implement triple color two-photon microscopy to simultaneously track, in vivo, both pre- and postsynaptic elements of excitatory synapses, followed by Magnified Analysis of Proteome (MAP), a combination of tissue clearing and expansion microscopy, for super resolution analysis of synaptic protein content across the entire neuron. Aim 2: To dissect, at a molecular level, experience-dependent selection and stabilization of excitatory synapses. CPG15/neuritin is an activity-regulated gene product critical for synapse stabilization and maturation. In vivo imaging in WT and CPG15 knockout mice revealed that while spine formation occurs normally in the absence of visual experience or CPG15, in both cases PSD95 recruitment to nascent spines is deficient. CPG15 expression in the absence of activity is sufficient to restore normal PSD95 recruitment and spine stabilization, suggesting it acts as an activity-dependent synapse selector. A puzzling aspect in this scenario is that CPG15 is extracellular while PSD95 is intracellular, and neither has a transmembrane domain. Interestingly, CPG15 was previously identified as part of the AMPA-type glutamate receptor (AMPAR) proteome. Yet, CPG15's mechanism of action remains unclear. To probe CPG15's synaptic function, we will map the minimal CPG15 binding domain on the AMPAR, and test whether preventing its interaction with CPG15 effects AMPAR interaction with stargazin, an adaptor molecule that is essential for delivering, inserting, and retaining functional receptors at the PSD. To probe how CPG15 binding influences AMPAR stability at the synapse and how this, in turn, effects synaptic presence of its downstream interacting proteins, stargazin and PSD95, we will develop an in vitro assay for synaptic AMPAR mobility. Finally, we will ask how loss of CPG15, as a surrogate of experience, impacts the molecular sequence of synapse formation, stabilization, and maturation in vivo, using two photon microscopy followed by MAP.

许多脑部疾病表现为突触完整性、稳定性和经验依赖选择受损，