Name: time-lapse imaging of neuronal arborization using sparse adeno-associated virus labeling of genetically targeted retinal cell populations
Uploaded: 2021-03-19T14:00:00
Description: Watch this Scientific Journal Video about Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations at JoVE.com

We thank Madison Gray for giving me a hand when I didn&#39;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.

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...

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...

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 populations in chick or zebrafish larvae, dendrite morphogenesis is highly dynamic3,4,5. Within days, dendritic arbors expand, remodel, and ramify to narrow sublayers of the IPL, where they synapse with select partners. The arbors exhibit different structural dynamics over development, with changes in relative rates of branch addition, retraction, and stabilization. Amacrine and RGC dendrites also exhibit different outgrowth and remodeling behaviors that might reflect type-specific arborization. However, these studies tracked broad amacrine or RGC populations and focused on laminar targeting, which is just one aspect of morphology.
The mechanisms that produce the vast morphological diversity observed across retinal subtypes are poorly understood. The objective of this group was to develop a method to capture dendrite dynamics and arbor remodeling of defined retinal subtypes in mice. Identifying cell type-specific mechanisms of dendrite patterning requires methods to visualize and measure dendrite behaviors of cells of interest. Organotypic cultures of mouse retinas are well suited for live-cell imaging studies using confocal or multiphoton microscopy. Developing retinas are dissected and mounted into a flat explant that can be imaged for several hours in a recording chamber or cultured over a few days with limited effects on the circuitry6,7. Live retinal neurons can be labeled by a variety of techniques, including dye-filling by electrodes, electroporation, biolistic delivery of particles coated with lipophilic dyes or plasmids encoding fluorescent proteins (e.g., Gene Gun), as well as genetically encoded cell labels7,8,9,10. However, these approaches are inefficient for imaging dendrite dynamics of specific retinal subtypes. For instance, dye-filling methods are low-throughput and require electrophysiology apparatus and additional genetic labels to reliably target cells of interest. Moreover, the strong fluorescence signals in the soma can obscure nearby dendrites.
Biolistic gene delivery methods can simultaneously label dozens of cells, but steps involving high-pressure particle delivery and overnight incubation of isolated retina can compromise cell physiology and dendritic outgrowth. This paper proposes that recent genetic tools can be employed to capture early dendrite dynamics with cell type and structural resolution, given the following experimental criteria. First, to resolve the fine branches and filopodia that dominate developing arbors, the method should label neurons with bright, fluorescent proteins that fill processes in the entire arbor. The fluorescence labeling should not fade due to photobleaching during the imaging period. A variety of fluorescent protein variants have been generated and compared for suitability for in vivo/ex vivo&#160;imaging11 based on brightness and photostability. Second, the fluorescent proteins (XFPs) must be expressed at sufficiently high levels by the earliest stage of dendrite morphogenesis, so that the narrow developmental window is not missed. In analyses of static timepoints in the mouse retina, dendrite development occurs during the first postnatal week and includes phases of outgrowth, remodeling, and stabilization10,12,13,14,15. Third, the method should lead to selective labelling or to an increased probability of labelling of the neuronal subpopulation of interest.&#160;Fourth, labelling of the target subpopulation must be sufficiently sparse so that the entire neuronal arbor can be identified and traced.&#160;Although RGC and amacrine subtypes can be distinguished by their mature morphological characteristics and IPL stratification patterns16,17,18,19,20, the challenge is to identify subtypes during development based on immature structures. This task is facilitated by the expansion of transgenic tools to label specific retinal cell types during development.
Transgenic and knock-in mouse lines in which cellular and temporal expression of fluorescent proteins or Cre is determined by gene regulatory elements are widely used to study retinal cell types13,21,22,23. Key observations on subtype-specific patterns of dendrite development have come from studies of transgenic mouse retinas at static timepoints10,14,24,25. The Cre-Lox system, in particular, enables exquisite gene manipulation and monitoring of subtypes using a variety of recombinase-dependent reporters, sensors, and optogenetic activators. These tools have led to discoveries of subtype-specific molecular programs and functional properties that underlie retinal circuit assembly26,27,28,29,30. However, they have yet to be leveraged to study subtype-specific dendrite dynamics in the mouse retina. Low-density labeling can be achieved by combining Cre mouse lines with transgenes introduced by electroporation or by recombinant AAVs. If available, tamoxifen-inducible Cre lines or intersectional genetic strategies can also be used. Finally, the cell should be labelled in a minimally invasive manner and imaged using acquisition parameters so as not to compromise the tissue or interfere with cellular function required for dendrite morphogenesis.
Presented here is a method to apply transgenic tools and confocal microscopy to investigate dendrite dynamics in live mouse retinal explants. Cre transgenic mouse lines have been combined with AAV vectors that express fluorescent proteins upon Cre recombination, which allows for sparse labeling of retinal cells of interest. Commercially available AAVs are delivered to neonatal retina by intravitreal injections. This paper demonstrates that AAVs produce significantly high and cell type-specific fluorescent expression by 4 dpi, allowing access to postnatal time points. To illustrate this approach, the cholinergic &#34;starburst&#34; amacrine interneuron was labelled by delivering Brainbow AAV in neonatal mice expressing the choline acetyltransferase (ChAT)-internal ribosome entry site (IRES)-Cre transgene, which is active in the early postnatal retina31,32. Starburst amacrine cells develop a stereotyped and radial arbor morphology that is shaped by dendrite self-avoidance mediated by the clustered protocadherins33,34. This paper shows that the resolution of starburst dendrites and filopodia is significantly improved by XFPs to the plasma membrane with the addition of the CAAX motif which undergoes farnesylation, as used for the Brainbow AAVs31. Finally, time-lapse imaging and post-processing protocols have been determined&#160;that produce high-quality images amenable for dendrite reconstruction and morphometric quantification. This protocol can be used to identify factors controlling dendrite morphogenesis and to capture several cellular behaviors in the intact retina.

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			<td>Addgene viral prep #45185-AAV9</td>
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			<td>Addgene viral prep #45186-AAV9</td>
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			<td>Dissection tools</td>
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			<td>Cellulose filter paper</td>
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			<td>Dumont #5 fine forceps</td>
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			<td>Two Dumont #5 forceps are required for retinal micro-dissection</td>
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			<td>Dumont forceps</td>
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			<td>Round tip paint brush, size 3/0</td>
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			<td>Live-imaging incubation system</td>
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			<td>Chamber polyethylene tubing, PE-160 10&#39;</td>
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			<td>Dual channel heater controller, Model TC-344C</td>
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			<td>HC FLUOTAR L 25x/0.95 W VISIR dipping objective</td>
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			<td>Heater controller cable</td>
			<td>Warner Instruments</td>
			<td>CC-28</td>
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			<td>Large bath incubation chamber with slice support</td>
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			<td>MPII Mini-Peristaltic Pump</td>
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			<td>PM-6D Magnetic Heated Platform (incubation chamber heater)</td>
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			<td>PM-6D</td>
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			<td>Pump Head Tubing Pieces For MPII Mini-Peristaltic Pump</td>
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			<td>Sample anchor (Harps)</td>
			<td>Warner Instruments</td>
			<td>64-0260</td>
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			<td>Sloflo In-line Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SF-28</td>
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			<td>Neonatal Injections</td>
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			<td>10 &#181;L Microliter Syringe Series 700, Removable Needle</td>
			<td>Hamilton Company</td>
			<td>80314</td>
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			<td>30 G Hypodermic Needles (0.5 inch)</td>
			<td>BD PrecisionGlide</td>
			<td>305106</td>
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			<td>4 inch thinwall glass capillary, no filament (1.0 mm outer diameter/0.75 mm)&#160;</td>
			<td>WPI World Precision Instruments</td>
			<td>TW100-4</td>
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			<td>Ethanol 99.8% (to dilute to 70% with double-distilled water [ddH2O])</td>
			<td>Sigma-Aldrich</td>
			<td>V001229&#160;</td>
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			<td>AAV9.hEF1a.lox.TagBFP. lox.eYFP.lox.WPRE.hGH-InvBYF</td>
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			<td>AV-9-PV2453</td>
			<td>Addgene Plasmid #45185&#160;</td>
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			<td>AAV9.hEF1a.lox.mCherry.lox.mTFP 
			1.lox.WPRE.hGH-InvCheTF</td>
			<td>Penn Vector Core</td>
			<td>AV-9-PV2454</td>
			<td>Addgene Plasmid #45186</td>
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			<td>ChAT-IRES-Cre knock-in transgenic mouse line</td>
			<td>The Jackson Laboratory</td>
			<td>6410</td>
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			<td>Fast Green FCF Dye content &#8805;85 %</td>
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			<td>F7252-25G</td>
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			<td>Flaming/Brown Micropipette Puller, model P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
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			<td>Green tattoo paste</td>
			<td>Ketchum MFG Co</td>
			<td>329A</td>
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			<td>Phosphate-Buffered Saline, pH 7.4, liquid, sterile-filtered, suitable for cell culture</td>
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			<td>Oxygenated artifiial cerebrospinal fluid (aCSF) Reagents</td>
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			<td>Calcium chloride dihydrate (CaCl2&#183;2H2O)</td>
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			<td>Hydrochloric acid solution, 1 N</td>
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			<td>Magnesium chloride hexahydrate (MgCl2&#183;6H2O)</td>
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			<td>pH-Test strips (6.0-7.7)</td>
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time-lapse imaging of neuronal arborization using sparse adeno-associated virus labeling of genetically targeted retinal cell populations

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.

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...

Watch this Scientific Journal Video about Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations at JoVE.com

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

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 ...

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