Developing a Strategy for 4-Color in Vivo Two-Photon Imaging
开发体内四色双光子成像策略
基本信息
- 批准号:10577846
- 负责人:
- 金额:$ 19.37万
- 依托单位:
- 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:2022-03-01 至 2024-02-28
- 项目状态:已结题
- 来源:
- 关键词:AMPA ReceptorsAddressAffectBiteBrainBrain DiseasesCalciumCategoriesCell Surface ReceptorsCell surfaceCellsColorCommunicationComplexCre driverDLG4 geneDetectionDevelopmentDiseaseDisease modelEnvironmentErythrocytesExcitatory SynapseFamilyGABA ReceptorGeneticGlutamate ReceptorGoalsImageImpairmentIndividualKnockout MiceLabelMediatingMicroscopeMicroscopicMicroscopyMolecularMonitorMorphologyMusN-Methyl-D-Aspartate ReceptorsNerve DegenerationNeuronsPHluorinProteinsPyramidal CellsReporterSideSiteSourceStructureSynapsesSynaptic CleftSynaptic ReceptorsSynaptophysinTestingThalamic structureTimeTransgenic OrganismsVertebral columnVisualizationcell typedesignexperiencefluorophorehippocampal pyramidal neuronin vivoin vivo monitoringin vivo two-photon imagingmutantnerve supplyoptical spectrapostsynapticpresynapticpupreceptorscaffoldsensorsynaptogenesistooltwo photon microscopytwo-photon
项目摘要
Many neurodevelopmental and neurodegenerative brain disorders manifest impaired synaptic integrity, stability,
and experience-dependent selection, resulting in wiring deficits and perturbed function. Yet our ability to
investigate how such disorders affect synaptic structure and function is severely limited by the difficulty of
visualizing synapses in the living brain and tracking their varied protein components. We propose developing
and testing new labeling and microscope configurations that would enable simultaneous live tracking of up to
four cellular proteins in the context of the intact mouse brain. The aims below put forth two 4-color constellations
that are designed to address distinct classes of experimental questions. Adjustments to the two-photon
microscope design that could accommodate both aims would primarily be in the detection path. Aim 1: To
develop and implement spectrally resolved two-photon microscopy for simultaneous tracking of up to
four proteins situated on both sides of the synaptic cleft. The 4-color constellation in this aim is designed to
enable in vivo monitoring of a presynaptic afferent label in addition to two postsynaptic proteins and a
postsynaptic cell fill. We will label two excitatory post-synaptic markers that are considered mutually exclusive,
PSD95 and PSD93, in the context of a thalamic afferent genetic label. In this scenario, tdTomato labels thalamic
afferents, eYFP serves as a cell fill, PSD95-teal labels mature excitatory synapses, and PSD93 fused to a far
red fluorophore (iRFP682) labels immature excitatory synapses. With this labeling one could ask questions such
as, what is the ratio and dynamics of thalamic innervation to mature PSD95 positive spines vs immature PSD93
positive spines? This 4-color combination could also be used to monitor any two postsynaptic labels in
combination with any cell-type specific afferent label. Aim 2: To develop and implement spectrally resolved
two-photon microscopy for simultaneous tracking of up to four postsynaptic fluorophores, one of these
being green. There are several categories of fluorophores in the green range that would be particularly useful
to combine with structural markers. Most notably, the pH-sensitive GFP mutant, Super ecliptic pHluorin (SEP)
used to tag and track synaptic receptors, and the GCaMP family of calcium sensors, broadly used to monitor
neuronal activity. Unfortunately, a green fluorophore is incompatible with most blue and yellow labels due to
overlap of their emission spectra. Here pyramidal neurons would co-express: a red cell fill (mScarlet) to label
dendritic morphology, PSD95-iRFP682 and PSD93-BFP (a short wavelength blue) to label mature and immature
excitatory synapses, respectively, and SEP-GluR1. We could then ask questions such as, what are the dynamics
of AMPA receptor insertion into mature PSD95 positive vs immature PSD93 positive spines? This 4-color
combination could also be used to tag and track NMDA or GABA receptor subunits with SEP. Swapping the
SEP-tagged receptor for a GCaMP calcium sensor would further expand the possibilities of this 4-color
constellation by enabling the integration of an activity reporter with synaptic labeling.
许多神经发育和神经退行性脑障碍表现为突触完整性、稳定性
和经验依赖性选择,导致连接缺陷和功能紊乱。然而我们的能力
研究这些疾病如何影响突触结构和功能受到以下困难的严重限制:
可视化活体大脑中的突触并追踪它们的各种蛋白质成分。我们建议发展
并测试新的标签和显微镜配置,以便同时实时跟踪多达
在完整的小鼠大脑中的四种细胞蛋白质。下面的目标提出了两个四色星座
是为了解决不同类别的实验问题而设计的。对双光子的调整
能够适应这两个目标的显微镜设计将主要在检测路径中。
开发和实施光谱分辨双光子显微镜,用于同时跟踪多达
位于突触间隙两侧的四种蛋白质。这个目标中的4色星座被设计成
除了两种突触后蛋白质和一种突触前传入标记外,
突触后细胞填充我们将标记两个被认为是相互排斥的兴奋性突触后标记,
PSD 95和PSD 93,在丘脑传入遗传标记的背景下。在这种情况下,tdTomato标记丘脑
传入,eYFP充当细胞填充物,PSD 95-teal标记成熟的兴奋性突触,PSD 93融合到远侧突触,
红色荧光团(iRFP 682)标记未成熟的兴奋性突触。有了这个标签,人们可以问这样的问题,
成熟PSD 95阳性棘与未成熟PSD 93阳性棘的丘脑神经支配的比率和动力学是什么
积极的脊柱?这种4色组合也可以用于监测任何两个突触后标记,
与任何细胞类型特异性传入标记组合。目标2:开发和实施光谱分辨
双光子显微镜用于同时跟踪多达四个突触后荧光团,其中一个
是绿色。在绿色范围内有几类荧光团特别有用
与结构标记联合收割机。最值得注意的是,pH敏感的GFP突变体,超级黄道pHluorin(SEP)
用于标记和跟踪突触受体,以及广泛用于监测
神经元活动不幸的是,绿色荧光团与大多数蓝色和黄色标记不相容,这是由于
它们的发射光谱重叠。在这里,锥体神经元将共同表达:红细胞填充(mScarlet)以标记
树突状形态、PSD 95-iRFP 682和PSD 93-BFP(短波长蓝色)以标记成熟和未成熟
兴奋性突触,分别,和SEP-GluR 1。我们可以问这样的问题,
AMPA受体插入到成熟的PSD 95阳性和未成熟的PSD 93阳性棘中的比例?这4色
组合也可用于用SEP标记和追踪NMDA或GABA受体亚基。
用于GCaMP钙传感器的SEP标记的受体将进一步扩展这种四色荧光的可能性。
通过使活性报告子与突触标记整合,可以构建一个新的星座。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Elly Nedivi其他文献
Elly Nedivi的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Elly Nedivi', 18)}}的其他基金
Characterizing excitatory synapse in vivo structural dynamics
表征兴奋性突触体内结构动力学
- 批准号:
10708899 - 财政年份:2022
- 资助金额:
$ 19.37万 - 项目类别:
Structured light temporal focusing depth-resolved wide-field FLIM-FRET for in vivo synaptic imaging
用于体内突触成像的结构光时间聚焦深度分辨宽视场 FLIM-FRET
- 批准号:
10570189 - 财政年份:2022
- 资助金额:
$ 19.37万 - 项目类别:
Developing a strategy for 4-color in vivo two-photon imaging
开发 4 色体内双光子成像策略
- 批准号:
10459675 - 财政年份:2022
- 资助金额:
$ 19.37万 - 项目类别:
Characterizing excitatory synapse in vivo structural dynamics
表征兴奋性突触体内结构动力学
- 批准号:
10512611 - 财政年份:2022
- 资助金额:
$ 19.37万 - 项目类别:
Structured light temporal focusing depth-resolved wide-field FLIM-FRET for in vivo synaptic imaging
用于体内突触成像的结构光时间聚焦深度分辨宽视场 FLIM-FRET
- 批准号:
10467534 - 财政年份:2022
- 资助金额:
$ 19.37万 - 项目类别:
in vivo imaging of inhibitory circuit remodeling in mouse visual cortex
小鼠视觉皮层抑制电路重塑的体内成像
- 批准号:
9042367 - 财政年份:2015
- 资助金额:
$ 19.37万 - 项目类别:
New technologies for in vivo spectral resolved high speed multiphoton microscopsy
体内光谱分辨高速多光子显微镜新技术
- 批准号:
9021702 - 财政年份:2015
- 资助金额:
$ 19.37万 - 项目类别:
in vivo imaging of circuit remodeling in mouse visual cortex
小鼠视觉皮层回路重塑的体内成像
- 批准号:
10207000 - 财政年份:2015
- 资助金额:
$ 19.37万 - 项目类别:
in vivo imaging of inhibitory circuit remodeling in mouse visual cortex
小鼠视觉皮层抑制电路重塑的体内成像
- 批准号:
9254550 - 财政年份:2015
- 资助金额:
$ 19.37万 - 项目类别:
New technologies for in vivo spectral resolved high speed multiphoton microscopsy
体内光谱分辨高速多光子显微镜新技术
- 批准号:
8878595 - 财政年份:2015
- 资助金额:
$ 19.37万 - 项目类别:
相似海外基金
Rational design of rapidly translatable, highly antigenic and novel recombinant immunogens to address deficiencies of current snakebite treatments
合理设计可快速翻译、高抗原性和新型重组免疫原,以解决当前蛇咬伤治疗的缺陷
- 批准号:
MR/S03398X/2 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Fellowship
Re-thinking drug nanocrystals as highly loaded vectors to address key unmet therapeutic challenges
重新思考药物纳米晶体作为高负载载体以解决关键的未满足的治疗挑战
- 批准号:
EP/Y001486/1 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Research Grant
CAREER: FEAST (Food Ecosystems And circularity for Sustainable Transformation) framework to address Hidden Hunger
职业:FEAST(食品生态系统和可持续转型循环)框架解决隐性饥饿
- 批准号:
2338423 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Continuing Grant
Metrology to address ion suppression in multimodal mass spectrometry imaging with application in oncology
计量学解决多模态质谱成像中的离子抑制问题及其在肿瘤学中的应用
- 批准号:
MR/X03657X/1 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Fellowship
CRII: SHF: A Novel Address Translation Architecture for Virtualized Clouds
CRII:SHF:一种用于虚拟化云的新型地址转换架构
- 批准号:
2348066 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Standard Grant
BIORETS: Convergence Research Experiences for Teachers in Synthetic and Systems Biology to Address Challenges in Food, Health, Energy, and Environment
BIORETS:合成和系统生物学教师的融合研究经验,以应对食品、健康、能源和环境方面的挑战
- 批准号:
2341402 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Standard Grant
The Abundance Project: Enhancing Cultural & Green Inclusion in Social Prescribing in Southwest London to Address Ethnic Inequalities in Mental Health
丰富项目:增强文化
- 批准号:
AH/Z505481/1 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Research Grant
ERAMET - Ecosystem for rapid adoption of modelling and simulation METhods to address regulatory needs in the development of orphan and paediatric medicines
ERAMET - 快速采用建模和模拟方法的生态系统,以满足孤儿药和儿科药物开发中的监管需求
- 批准号:
10107647 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
EU-Funded
Ecosystem for rapid adoption of modelling and simulation METhods to address regulatory needs in the development of orphan and paediatric medicines
快速采用建模和模拟方法的生态系统,以满足孤儿药和儿科药物开发中的监管需求
- 批准号:
10106221 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
EU-Funded
Recite: Building Research by Communities to Address Inequities through Expression
背诵:社区开展研究,通过表达解决不平等问题
- 批准号:
AH/Z505341/1 - 财政年份:2024
- 资助金额:
$ 19.37万 - 项目类别:
Research Grant