Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Samantha Ing-Esteves; Julie L. Lefebvre

doi:10.3791/62308

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genet...

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Neuroscience

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JoVE Journal Neuroscience

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

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13:13 min

March 19, 2021

DOI: 10.3791/62308-v

Samantha Ing-Esteves^1,2, Julie L. Lefebvre^1,2

¹Program for Neuroscience and Mental Health,Hospital for Sick Children, ²Department of Molecular Genetics,University of Toronto

Overview

This study presents a method for investigating neurite morphogenesis in postnatal mouse retinal explants using time-lapse confocal microscopy. The approach involves sparse labeling of retinal cell types through recombinant adeno-associated virus vectors expressing membrane-targeted fluorescent proteins in a Cre-dependent manner.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Imaging Techniques

Background

Investigating neuronal morphogenesis requires single-cell labeling of dendritic or axonal arbors.
The method preserves retinal tissue health for prolonged live imaging.
The ability to visualize arbors in multiple colors accelerates understanding of neonatal morphology.

Purpose of Study

To develop a minimally-invasive labeling technique for live imaging of retinal neurons.
To facilitate the study of neonatal retinal arbor morphologies in vitro.
To provide a protocol applicable for studying neuronal morphologies across the central nervous system.

Methods Used

Utilizes a confocal microscopy platform for imaging retinal explants.
Employs recombinant adeno-associated virus (AAV) vectors for labeling retinal cell types.
The method involves injections into the intravitreal space and careful preparation of retinal aCSF.
Critical steps include selecting appropriate Cre lines and optimizing injection procedures.

Main Results

The method allows for the visualization of individual dendritic and axonal arbors within four days post-injection.
Retinal explants exhibit preserved health, enabling effective imaging for studying morphogenetic changes.
Confirmatory steps include visualization of injected dye for successful AAV delivery.

Conclusions

This study demonstrates a viable protocol for imaging neurite morphogenesis in retinal neurons.
The approach enhances understanding of neuronal plasticity and development mechanisms in vitro.
Implications extend to studying similar processes across diverse neuronal populations in the central nervous system.

Frequently Asked Questions

What are the advantages of the time-lapse confocal microscopy method?

This method allows for real-time imaging of neurite morphogenesis and preserves retinal tissue health for extended periods, making it suitable for detailed studies.

How is the main biological model implemented?

The biological model involves postnatal mouse retinal explants injected with AAV vectors to label specific retinal neuron types for imaging.

What types of data are obtained using this method?

The method provides high-resolution imaging data on the growth and branching patterns of dendritic and axonal arbors.

How can this method be adapted for other models?

The protocol can be modified by selecting different Cre lines or using different target areas within the central nervous system for neuronal imaging.

What are the key considerations when implementing this protocol?

Careful selection of the injection site and optimal dilution of AAV are crucial to ensure successful labeling and minimize tissue damage.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Single-cell labeling and visualization of dendritic or axonal arbors is required to study neuronal morphogenesis. By labeling developing arbors in a minimally-invasive manner, retinal tissue remains healthy and suitable for prolonged confocal live imaging. The main advantage of this method is the ability to visualize individual arbors with multi-color fluorescent proteins within four days post-injection.

This makes studying neonatal arbor morphologies possible. This injection imaging and analysis protocol can be used to study neuronal morphologies across the central nervous system. Appropriate Cre selection and injection location must be determined to target the cell population of interest.

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