Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Moye, Stefanie; Díaz-Castro, Blanca; Gangwani, Mohitkumar R.; Khakh, Baljit S.

doi:10.3791/60225

Cited by 15 publications

(15 citation statements)

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“…In the NGV circuit, the number of perivascular processes was constrained for juvenile rodents at 2 ± 1 processes. These numbers are in accordance with literature measurements (Calì et al, 2019;Moye et al, 2019). Furthermore, the number of primary processes in the NGV was measured as 8 ± 1 processes ( Figure 7 M).…”

Section: Data Validation: Population-levelsupporting

confidence: 91%

“…To establish the connectivity between astrocytes and the vasculature, we first distributed potential targets on the vasculature skeleton graph with a frequency of 0.17 μm -1 (McCaslin et al, 2011) and then determined which fraction of the resulting point cloud was included in each microdomain boundary polygon. Based on literature data, each astrocyte was assigned a number of endfeet, ranging from 1 to 5 (Moye et al, 2019) and the endfeet sites were sequentially selected according to the following observations and experimental astrocyte reconstructions: endfeet processes minimize their distance to the vascular site (Kacem et al, 1998), maximize the distance to nearby endfeet sites and target different branches (Calì et al, 2019).…”

Section: Reconstructing the Ngv Connectomementioning

confidence: 99%

“…Protoplasmic astrocytes radially extend between five and ten primary processes (Calì et al, 2019;Damoiseaux & Greicius, 2009Di Benedetto et al, 2016;Moye et al, 2019), which ramify progressively into finer and finer branches, filling up their entire spatial extent (Bushong et al, 2002). Their fine leaflet-like processes wrap around the axon-spine interface (Genoud et al, 2006;Ventura & Harris, 1999) forming tripartite units (Araque et al, 1999;Santello et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

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Architecture of the Neuro-Glia-Vascular System

Zisis

Keller

Kanari

et al. 2021

Preprint

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Astrocytes connect the vasculature to neurons and mediate the supply of nutrients and biochemicals. They also remove metabolites from the neurons and extracellular environment. They are involved in a growing number of physiological and pathophysiological processes. Understanding the biophysical, physiological, and molecular interactions in this neuro-glia-vascular ensemble (NGV) and how they support brain function is severely restricted by the lack of detailed cytoarchitecture. To address this problem, we used data from multiple sources to create a data-driven digital reconstruction of the NGV at micrometer anatomical resolution. We reconstructed 0.2 mm3 of rat somatosensory cortical tissue with approximately 16000 morphologically detailed neurons, its microvasculature, and approximately 2500 morphologically detailed protoplasmic astrocytes. The consistency of the reconstruction with a wide array of experimental measurements allows novel predictions of the numbers and locations of astrocytes and astrocytic processes that support different types of neurons. This allows anatomical reconstruction of the spatial microdomains of astrocytes and their overlapping regions. The number and locations of end-feet connecting each astrocyte to the vasculature can be determined as well as the extent to which they cover the microvasculature. The structural analysis of the NGV circuit showed that astrocytic shape and numbers are constrained by vasculature’s spatial occupancy and their functional role to form NGV connections. The digital reconstruction of the NGV is a resource that will enable a better understanding of the anatomical principles and geometric constraints which govern how astrocytes support brain function.Table of contentsMain pointsThe Blue Brain Project digitally reconstructs a part of neocortical Neuro-Glia-Vascular organizationInterdependencies and topological methods allow dense in silico reconstruction from sparse experimental dataThe polarized role of protoplasmic astrocytes constrains their shapes and numbersTable of contents image

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Section: Data Validation: Population-levelsupporting

confidence: 91%

Section: Reconstructing the Ngv Connectomementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Architecture of the Neuro-Glia-Vascular System

Zisis

Keller

Kanari

et al. 2021

Preprint

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“…The same caveat applies for GFAP, the most widely used structural astrocyte marker, and for the newest small‐molecule astrocyte markers, like the cationic‐pyridinium family of markers (Preston et al., 2018; Preston, Cervasio, & Laughlin, 2019). The majority of structural astrocyte analyses are performed using GFAP exclusively (SheikhBahaei et al., 2018; Viola et al., 2009) or injectable intracellular fluorescent dyes (Butt & Ransom, 1989; Moye, Diaz‐Castro, Gangwani, & Khakh, 2019). GFAP is found in primary, secondary, and some tertiary astrocyte branches, but not in higher‐order processes, excluding significant portions of the astrocyte from analysis.…”

Section: Commentarymentioning

confidence: 99%

“…Beyond use of Lck-GFP, other studies have utilized astrocyte reporter mouse lines or fluorescent dyes to label and identify astrocytes in greater detail than afforded by GFAP IHC (Morel et al, 2017(Morel et al, , 2019Moye et al, 2019). Moreover, a companion article provided in this same issue (see Current Protocols article; Badia-Soteras, Octeau, Verheijen, & Khakh, 2020) details use of live-cell fluorescence resonance energy transfer (FRET) to quantify proximal interactions between neurons and astrocytes (Octeau et al, 2018).…”

Section: Background Informationmentioning

confidence: 99%

High‐Resolution Three‐Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy

2020

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Astrocytes play numerous vital roles in the central nervous system. Accordingly, it is of merit to identify structural and functional properties of astrocytes in both health and disease. The majority of studies examining the morphology of astrocytes have employed immunoassays for markers such as glial fibrillary acidic protein, which are insufficient to encapsulate the considerable structural complexity of these cells. Herein, we describe a method utilizing a commercially available and validated, genetically encoded membrane‐associated fluorescent marker of astrocytes, AAV5‐GfaABC1D‐Lck‐GFP. This tool and approach allow for visualization of a single isolated astrocyte in its entirety, including fine peripheral processes. Astrocytes are imaged using confocal microscopy and reconstructed in three dimensions to obtain detailed morphometric data. We further provide an immunohistochemistry procedure to assess colocalization of isolated astrocytes with synaptic markers throughout the z‐plane. This technique, which can be utilized via a standard laboratory confocal microscope and Imaris software, allows for detailed analysis of the morphology and synaptic colocalization of astrocytes in fixed tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Microinjection of AAV5‐GfaABC1D‐Lck‐GFP into the nucleus accumbens of rats Basic Protocol 2: Tissue processing and immunohistochemistry for post‐synaptic density‐95 Basic Protocol 3: Single‐cell image acquisition Basic Protocol 4: Three‐dimensional reconstruction of single cells Basic Protocol 5: Three‐dimensional colocalization analysis

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Morphometric analysis of astrocytes in vocal production circuits of common marmoset (Callithrix jacchus)

Turk

SheikhBahaei

2021

J of Comparative Neurology

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Astrocytes, the star-shaped glial cells, are the most abundant non-neuronal cell population in the central nervous system. They play a key role in modulating activities of neural networks, including those involved in complex motor behaviors. Common marmosets (Callithrix jacchus), the most vocal non-human primate (NHP), have been usedto study the physiology of vocalization and social vocal production. However, the neural circuitry involved in vocal production is not fully understood. In addition, even less is known about the involvement of astrocytes in this circuit. To understand the role, that astrocytes may play in the complex behavior of vocalization, the initial step may be to study their structural properties in the cortical and subcortical regions that are known to be involved in vocalization. Here, in the common marmoset, we identify all astrocytic subtypes seen in other primate's brains, including intralaminar astrocytes. In addition, we reveal detailed structural characteristics of astrocytes and perform morphometric analysis of astrocytes residing in the cortex and midbrain regions that are associated with vocal production. We found that cortical astrocytes in these regions illustrate a higher level of complexity when compared to those in the midbrain. We hypothesize that this complexity that is expressed in cortical astrocytes may reflect their functions to meet the metabolic/structural needs of these regions.

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Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Cited by 15 publications

References 0 publications

Architecture of the Neuro-Glia-Vascular System

Architecture of the Neuro-Glia-Vascular System

High‐Resolution Three‐Dimensional Imaging of Individual Astrocytes Using Confocal Microscopy

Morphometric analysis of astrocytes in vocal production circuits of common marmoset (Callithrix jacchus)

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