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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

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

Published: March 19th, 2021

DOI:

10.3791/62308

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

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.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Dendrites of retinal neurons form intricate, yet specific, patterns that influence their function within neural circuits. In the vertebrate retina, diverse types of retinal ganglion cells (RGCs) and amacrine cell interneurons bear unique dendritic morphologies that differ in arbor size, location, branch length, and density1. During postnatal development, RGCs and amacrine cells extend exuberant dendritic processes into a neuropil called the inner plexiform layer (IPL), where they receive bipolar cell inputs transmitting photoreceptor signals2. As captured by time-lapse imaging of fluorescently labelled retinal population....

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NOTE: This protocol spans 2 days with a minimum period of 4-5 days for viral transduction between experimental days (Figure 1A). Animal experiments are performed in accordance with the Canadian Council on Animal Care Guidelines for Use of Animals in Research and Laboratory Animal Care under protocols approved by the Laboratory of Animal Services Animal Use and Care Committee at the Hospital for Sick Children (Toronto, Canada).

1. Preparations for the neonatal AAV in.......

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Using the above protocol, a high-resolution 3D video of developing starburst cell dendrites was acquired, deconvolved, and corrected for 3D drift. Z-plane maximum projections were produced to make 2D videos for analysis (Supplementary Video 1, Figure 5A). 3D deconvolution of each time point increased the resolution of fine filopodia projections (Figure 5B,C). Fine filopodia protrusions are a feature of developing retinal dendrit.......

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This video demonstrates an experimental pipeline that utilizes existing genetic tools to image dendrite dynamics of developing retinal neurons with confocal live-imaging. Also demonstrated are intraocular injections of Cre-dependent AAVs encoding membrane-targeted fluorescent proteins into neonatal mice. Single cells of genetically targeted populations are brightly labelled as early as 4-5 dpi. Retinal flat-mounts were prepared for standard imaging chambers to perform live-cell confocal imaging. This method produces high.......

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We thank Madison Gray for giving me a hand when I didn't have any. This research was supported by an NSERC Discovery Grant (RGPIN-2016-06128), a Sloan Fellowship in Neuroscience and a Canada Research Chair Tier 2 (to J.L.L). S. Ing-Esteves was supported by the Vision Science Research Program and NSERC Postgraduate Scholarships-Doctoral.

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Name Company Catalog Number Comments
Addgene viral prep #45185-AAV9
Addgene viral prep #45186-AAV9
Dissection tools
Cellulose filter paper Whatman 1001-070
Dumont #5 fine forceps FST 11252-20 Two Dumont #5 forceps are required for retinal micro-dissection
Dumont forceps VWR 82027-426
Fine Scissors FST 14058-09
Mixed cellulose ester membrane (MCE) filter papers, hydrophilic, 0.45 µm pore size Millipore HABG01 300
Petri Dish, 50 × 15 mm VWR 470313-352
Polyethylene disposable transfer pipette VWR 470225-034
Round tip paint brush, size 3/0 Conventional art supply store Two size 3/0 paint brushes (or smaller) are required for retinal flat-mounting
Surgical Scissors FST 14007-14
Vannas Spring Scissors - 2.5 mm Cutting Edge FST 15000-08
Live-imaging incubation system
Chamber polyethylene tubing, PE-160 10' Warner Instruments 64-0755
Dual channel heater controller, Model TC-344C Warner Instruments 64-2401
HC FLUOTAR L 25x/0.95 W VISIR dipping objective Leica 15506374
Heater controller cable Warner Instruments CC-28
Large bath incubation chamber with slice support Warner Instruments RC-27L
MPII Mini-Peristaltic Pump Harvard Apparatus 70-2027
PM-6D Magnetic Heated Platform (incubation chamber heater) Warner Instruments PM-6D
Pump Head Tubing Pieces For MPII Mini-Peristaltic Pump Harvard Apparatus 55-4148
Sample anchor (Harps) Warner Instruments 64-0260 Sample anchor must be compatible with incubation chamber
Sloflo In-line Solution Heater Warner Instruments SF-28
Neonatal Injections
10 µL Microliter Syringe Series 700, Removable Needle Hamilton Company 80314
30 G Hypodermic Needles (0.5 inch) BD PrecisionGlide 305106
4 inch thinwall glass capillary, no filament (1.0 mm outer diameter/0.75 mm)  WPI World Precision Instruments TW100-4
Ethanol 99.8% (to dilute to 70% with double-distilled water [ddH2O]) Sigma-Aldrich V001229 
AAV9.hEF1a.lox.TagBFP. lox.eYFP.lox.WPRE.hGH-InvBYF Penn Vector Core AV-9-PV2453 Addgene Plasmid #45185 
AAV9.hEF1a.lox.mCherry.lox.mTFP
1.lox.WPRE.hGH-InvCheTF
Penn Vector Core AV-9-PV2454 Addgene Plasmid #45186
ChAT-IRES-Cre knock-in transgenic mouse line The Jackson Laboratory 6410
Fast Green FCF Dye content ≥85 % Sigma-Aldrich F7252-25G
Flaming/Brown Micropipette Puller, model P-97 Sutter Instrument Co. P-97
Green tattoo paste Ketchum MFG Co 329A
Phosphate-Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture Sigma-Aldrich 806552
Pneumatic PicoPump WPI World Precision Instruments PV-820
Oxygenated artifiial cerebrospinal fluid (aCSF) Reagents
Calcium chloride dihydrate (CaCl2·2H2O) Sigma-Aldrich C7902
Carbogen (5% CO2, 95% O2) AirGas X02OX95C2003102 Supplier may vary depending on region
D-(+)-Glucose Sigma-Aldrich G7021
HEPES, Free Acid Bio Basic HB0264
Hydrochloric acid solution, 1 N Sigma-Aldrich H9892
Magnesium chloride hexahydrate (MgCl2·6H2O) Sigma-Aldrich M2670
pH-Test strips (6.0-7.7) VWR BDH35317.604
Potassium chloride (KCl) Sigma-Aldrich P9541
Sodium chloride (NaCl) Bio Basic DB0483
Sodium phosphate monobasic (NaH2PO4) Sigma-Aldrich RDD007
Software
ImageJ National Institutes of Health (NIH) Open source

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