Published: March 19th, 2021
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....
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.......
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.......
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.......
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.....
|Addgene viral prep #45185-AAV9
|Addgene viral prep #45186-AAV9
|Cellulose filter paper
|Dumont #5 fine forceps
|Two Dumont #5 forceps are required for retinal micro-dissection
|Mixed cellulose ester membrane (MCE) filter papers, hydrophilic, 0.45 µm pore size
|Petri Dish, 50 × 15 mm
|Polyethylene disposable transfer pipette
|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
|Vannas Spring Scissors - 2.5 mm Cutting Edge
|Live-imaging incubation system
|Chamber polyethylene tubing, PE-160 10'
|Dual channel heater controller, Model TC-344C
|HC FLUOTAR L 25x/0.95 W VISIR dipping objective
|Heater controller cable
|Large bath incubation chamber with slice support
|MPII Mini-Peristaltic Pump
|PM-6D Magnetic Heated Platform (incubation chamber heater)
|Pump Head Tubing Pieces For MPII Mini-Peristaltic Pump
|Sample anchor (Harps)
|Sample anchor must be compatible with incubation chamber
|Sloflo In-line Solution Heater
|10 µL Microliter Syringe Series 700, Removable Needle
|30 G Hypodermic Needles (0.5 inch)
|4 inch thinwall glass capillary, no filament (1.0 mm outer diameter/0.75 mm)
|WPI World Precision Instruments
|Ethanol 99.8% (to dilute to 70% with double-distilled water [ddH2O])
|Penn Vector Core
|Addgene Plasmid #45185
|Penn Vector Core
|Addgene Plasmid #45186
|ChAT-IRES-Cre knock-in transgenic mouse line
|The Jackson Laboratory
|Fast Green FCF Dye content ≥85 %
|Flaming/Brown Micropipette Puller, model P-97
|Sutter Instrument Co.
|Green tattoo paste
|Ketchum MFG Co
|Phosphate-Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture
|WPI World Precision Instruments
|Oxygenated artifiial cerebrospinal fluid (aCSF) Reagents
|Calcium chloride dihydrate (CaCl2·2H2O)
|Carbogen (5% CO2, 95% O2)
|Supplier may vary depending on region
|HEPES, Free Acid
|Hydrochloric acid solution, 1 N
|Magnesium chloride hexahydrate (MgCl2·6H2O)
|pH-Test strips (6.0-7.7)
|Potassium chloride (KCl)
|Sodium chloride (NaCl)
|Sodium phosphate monobasic (NaH2PO4)
|National Institutes of Health (NIH)
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