Name: preparing fresh retinal slices from adult zebrafish for ex vivo imaging experiments
Uploaded: 2018-05-09T12:30:00
Description: Watch this Scientific Journal Video about Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments at JoVE.com

We thank Ralph Nelson and Daniel Possin for thoughtful guidance while developing this protocol, and Eva Ma, Ashley George and Gail Stanton for generation of stable transgenic zebrafish lines. The work was supported by NSF GRFP 2013158531 to M.G., NIH NEI 5T32EY007031 to W.C. and M.G., and EY026020 to J.H. and S.B.

All animal experiments were approved by the University of Washington Institutional Animal Care and Use Committee.
1. Preparing Animals and Equipment
NOTE: The retinal pigment epithelium (RPE) is a dark sheet of tissue surrounding the outside of the retina whose pigmentation can obscure retinal features and damage the tissue when confocal imaging ex vivo. In darkness, the RPE of zebrafish is retracted away from the retina; dark adapt fish to facilitate future ...

<ol>
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Ex vivo imaging of fresh zebrafish retinal slices has proven to be a versatile tool for studying photoreceptor biology20,21,22, and is unique in that it enables analysis of single cells in a mature, fully differentiated retina. With practice, it is possible to conduct multiple experiments with tissue from a single fish, even using serial slices from the same part of the retina. In addition to the challenges and suggesti...

The zebrafish (Danio rerio) has become widely used in medical and basic scientific research1, owing to its small size, rapid development and vertebrate organ systems. The natural transparency of zebrafish larvae combined with established methods for transgenesis have enabled detailed visualization of cellular processes in a living animal. A number of genetically encoded fluorescent biosensors have been targeted to specific zebrafish cells to detect Ca2+ 2, hydrogen peroxide3, apoptotic activation4 and ATP5.
In vivo imaging of zebrafish larvae has led to breakthroughs in the field of neuroscience, including mapping of brain circuitry6 and drug development for central nervous system disorders7. Zebrafish are well suited for vision research because their retinas feature the laminar structure and neuron types of higher vertebrates, and they display robust visual behaviors8,9. Several types of retinal degenerations analogous to human disease have been modeled successfully and studied in zebrafish10,11, including live imaging of individual photoreceptors degenerating within a retina2,12.
While in vivo larval zebrafish imaging is a valuable tool, it becomes more challenging as fish grow and develop pigmentation, and some pharmacological treatments cannot permeate an entire animal. Further, certain cellular processes change with development and age, making later time points critical for understanding function and the progression of disease in adult animals. Biochemical methods such as immunoblot, quantitiative PCR, O2 consumption, and metabolomic analyses can provide important clues about biology of the retina as a whole, but it is difficult to discern contributions of individual cell types affected by disease. Imaging isolated retinal tissue ex vivo bypasses these issues, and while imaging flat mounted retinas affords a view of the outer retina13, deeper inner retinal features are obscured. Transverse retinal slices, such as those presented in fixed immunohistochemical analyses, enable a clear view of all layers and cell types but only offer a single snapshot of the dynamic processes involved in normal function and disease.
Here, we present a method for generating ex vivo transverse retinal slices from adult zebrafish for imaging. It is similar to methods for preparing amphibian and zebrafish retinal slices for electrophysiological and morphological studies14,15, with important modifications for time lapse imaging ex vivo using confocal microscopy. Fluorescence responses of biosensors or dyes in slices are monitored in real time with a confocal microscope while delivering pharmacological agents using perfusion. While the method was developed for imaging photoreceptors, it may be feasible to use it for visualizing M&#252;ller cells, bipolar cells, horizontal cells, amacrine cells, or retinal ganglion cells with appropriate fluorescent markers. Additionally, slices can be loaded with fluorescent cell-permeable dyes to report cell viability, vesicular transport, mitochondrial function, or redox state. This versatile preparation allows visualization of a wide range of subcellular processes throughout the retina, including Ca2+ dynamics, signal transduction and metabolic state.

<table border="0" cellpadding="0" cellspacing="0" width="1153">
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		<tr height="40">
			<td height="40" style="height:40px;width:332px;">zebrafish</td>
			<td style="width:239px;">Univeristy of Washington South Lake Union Aquatics Facility</td>
			<td style="width:181px;"></td>
			<td style="width:401px;">stocks maintained in-house as stable transgenic lines</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">petroleum jelly</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">19-090-843</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">3-mL slip tip syringe</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">14-823-436</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">20g 3.8 cm slip tip needle</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">14-826-5B</td>
			<td style="width:401px;">for petroleum jelly syringe</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">plain 7 cm X 2.5 cm microscope slide</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">12-550-A3</td>
			<td style="width:401px;">for eyecup dissection, slicing chamber</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Seche Vite clear nail polish</td>
			<td style="width:239px;">Amazon</td>
			<td style="width:181px;">B00150LT40</td>
			<td style="width:401px;">for slicing chamber</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">18 mm X 18 mm #1 glass coverslips</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td>12-542A</td>
			<td style="width:401px;">for imaging ladders</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">unflavored dental wax</td>
			<td style="width:239px;">Amazon</td>
			<td style="width:181px;">B01K8WNL5A</td>
			<td style="width:401px;">for imaging ladders</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">double edge razor blades</td>
			<td style="width:239px;">Stoelting</td>
			<td style="width:181px;">51427</td>
			<td style="width:401px;">for tissue slicing</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">tissue slicer with digital micrometer</td>
			<td style="width:239px;">Stoelting</td>
			<td style="width:181px;">51415</td>
			<td style="width:401px;">for tissue slicing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">filter paper - white gridded mixed cellulose, 13 mm diameter, 0.45 &#181;m pore size</td>
			<td style="width:239px;">EMD Millipore</td>
			<td style="width:181px;">HAWG01300</td>
			<td style="width:401px;">filter paper for mounting retinas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">10 cm petri dish</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">FB0875712</td>
			<td style="width:401px;">for fish euthanasia, dissection, imaging ladder assembly</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">15 cm plain-tipped wood applicator stick</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">23-400-112</td>
			<td style="width:401px;">for wire eye loop tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">30g (0.25 mm diameter) tungsten wire</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">AA10408G6</td>
			<td style="width:401px;">for wire eye loop tool</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-glucose</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G8270</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium L-lactate</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">L7022</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sodium pyruvate</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P2256</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-glutamine</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G3126</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;L-glutathione, reduced</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">G4251</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-ascorbic acid</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">A5960</td>
			<td style="width:401px;">component of supplement stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">S7653</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P9333</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2 &#183; 2H2O</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">C3881</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaH2PO4</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">S8282</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2 &#183; 6H2O</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">M0250</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">H3375</td>
			<td style="width:401px;">component of Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris base</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">BP152</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 N HCl</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">02-003-063</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KH2PO4</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">P5655</td>
			<td style="width:401px;">component of Na+-free Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">50 mL conical centrifuge tube</td>
			<td style="width:239px;">Denville Scientific</td>
			<td style="width:181px;">C1062-P</td>
			<td style="width:401px;">container for Ringer&#39;s solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">Vannas scissors - 8 cm, angled 5 mm blades</td>
			<td style="width:239px;">World Precision Instruments</td>
			<td style="width:181px;">501790</td>
			<td style="width:401px;">micro-scissors for eyecup dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Swiss tweezers - #5, 11 cm, straight, 0.06 X 0.07 mm tips</td>
			<td style="width:239px;">World Precision Instruments</td>
			<td style="width:181px;">504510</td>
			<td style="width:401px;">fine forceps for eyecup dissection and slice manipulation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">single edge razor blades</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">12-640</td>
			<td style="width:401px;">for eyecup dissection and trimming filter paper</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">EMD Millipore filter forceps</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">XX6200006P</td>
			<td style="width:401px;">flat forceps for handling wet filter paper</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">C12 558/568 BODIPY</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">D3835</td>
			<td style="width:401px;">stains live cell nuclei; incubate 5 &#181;g/mL for 15 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">propidium iodide (PI)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">P3566</td>
			<td style="width:401px;">stains dead cell nuclei; incubate 5 &#181;g/mL for 20 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Hoechst 33342</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">62249</td>
			<td style="width:401px;">stains live cell nuclei; incubate 5 &#181;g/mL for 20 min at room temperature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Tetramethylrhodamine, methyl ester (TMRM)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">T668</td>
			<td style="width:401px;">stains functional, negatively-charged mitochondria; incubate 1 nM for 30 min at room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">tissue perfusion chamber</td>
			<td style="width:239px;">Cell MicroControls</td>
			<td style="width:181px;">BT-1-18/BT-1-18BV [-SY]</td>
			<td style="width:401px;">imaging chamber for injection or perfusion</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:332px;">2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (NBDG)</td>
			<td style="width:239px;">Fisher Scientific</td>
			<td style="width:181px;">N13195</td>
			<td style="width:401px;">fluorescent glucose analog adminitered orally to zebrafish 30 min prior to euthanasia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Olympus laser scanning confocal microscope</td>
			<td style="width:239px;">Olympus</td>
			<td style="width:181px;">FV1000</td>
			<td style="width:401px;">confocal microscope for visualizing fluorescence of slices at single-cell resolution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)</td>
			<td style="width:239px;">Sigma Aldrich</td>
			<td style="width:181px;">C2759</td>
			<td style="width:401px;">experimental reagent which ablates mitochondrial respiration; treat slices to a final concentration of 1 &#181;M</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:332px;">miniature aspirator positioner</td>
			<td style="width:239px;">Cell MicroControls</td>
			<td style="width:181px;">FL-1</td>
			<td style="width:401px;">for perfusion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:332px;">perfusion manifold, gas bubbler manifold, flow valve, 60cc syringe holder</td>
			<td style="width:239px;">Warner Instruments</td>
			<td style="width:181px;">various</td>
			<td style="width:401px;">for perfusion</td>
		</tr>
	</tbody>
</table>

preparing fresh retinal slices from adult zebrafish for ex vivo imaging experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Stable positioning and transverse orientation of slices are key to successful imaging with injection or perfusion of pharmacological agents. Carefully examine and reposition slices prior to confocal imaging as needed to ensure all retinal layers are visible (Figure 2A, slice ii). If a slice is rotated slightly forward (Figure 2A, slice iii), bundles of outer segments will be visible and small adjustments can be made with forceps ...

Watch this Scientific Journal Video about Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments at JoVE.com

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

The overall goal of this procedure is to prepare fresh slices of Zebrafish retinas for experiments that require live imaging of fluorescent proteins or biosensors. This method can help answer key questions in the field of vision research, such as the physiological and metabolic roles of calcium ions in specific cell types and cellular compartments within a retina. The main advantage of this technique is that it provides spatial and temporal information about key metabolites and signaling molecules in live tissues.To make it possible to remove the retinal pigment epithelium, or RPE, from the Zebrafish, dark adapt the fish for one hour prior to making tissue slices. For a slicing chamber, paint lines of nail polish onto a slide. Using clear nail polish, paint narrow lines to create a rectangle.Repaint the polish once when dry. If the chamber will be used for static imaging, or injection experiments with minimal solution flow, then use a syringe to apply two parallel strips of petroleum jelly to a coverslip, one centimeter long and 0.5 centimeters apart. Now prepare the blades.First, clean a double-edged razor with ethanol and allow it to air-dry. Then use scissors to cut it into approximately one centimeter pieces. Next, place the slicing chamber on the stage of the tissue slicer, center it horizontally on the stage, and mark the long edge with permanent marker for alignment.Then, load a piece of blade onto the slicer arm. Secure the blade, so the edge is over the middle of the slide and runs parallel with the slide surface, but does not touch the nail polish. Then lower the blade arm using a one quarter turn.Now, try slicing filter paper. Remount the blade if needed. To proceed, using the syringe deposit a single, small dot of jelly in a 10 centimeter dish, about 1.5 centimeters from the midpoint.Then press the dry side of a coverslip into the jelly using forceps. Next, put another small dot of jelly about one centimeter from the inlet edge of the imaging chamber. Now, use a 20 gauge needle to make two one centimeter long thin parallel strips of petroleum jelly lengthwise along the center of the slicing chamber about one centimeter apart.Then transfer the Zebrafish to a dish and cervically dislocate the fish without decapitation. Now, use a wire loop to loosen the connective tissue around one eye and then pull the eye forward gently. Next, carefully cut the white optic nerve under the eye using microscissors, but do not cut the back of the eye.To proceed, transfer the eye to a dish of cold Ringer's solution on ice. Then harvest the other eye. Continue working under red light until all of the RPE is removed.Next, transfer one eye into a drop of cold Ringer's solution on a glass slide on a petri dish. Then place the dish under a low-powered dissection microscope to dissect the eye cup under red light. First, pierce the cornea with fine forceps.Next, use forceps and scissors to gently remove pieces of clear brittle cornea and silvery sclera. Then remove and discard the lens, and most of the sclera to isolate the eye cup. With minimal handling remove any pieces of fat and sclera that remain attached to the eye cup without removing the retina.If the retina separates from the RPE, then proceed with extra caution to avoid damaging the delicate retina. Now, reposition the eye cup with open side down and cut it into thirds or quarters with a new blade. Discard the pieces of eye cup that are too curved to flatten out.These pieces are often too small to be useful in any case. To mount the retina sections, wet a piece of filter paper with Ringer's solution and use flat forceps to place it next to the eye cup pieces. Then immerse the filter paper and tissues in cold Ringer's solution.Next, use forceps to position retina pieces on top of filter paper with the RPE and photoreceptors up. Gently handle the edges of the eye cup pieces with fine forceps to orient them into a single line at the center of the filter paper. Now flatten the retina by using forceps to lift the filter paper with retina attached and setting it on a dry paper towel.For about three seconds, let the force of liquid wicking into the paper towel flatten the retina. Process all of the retina pieces in this manner, just don't let them dry out. Now, apply a drop of Ringer's solution and use fine forceps to peel away any remaining black sheets of RPE from the tissue.Peel gently, and if the retina lifts off the paper, wick away more solution as before and it should flatten again. Now transfer the paper with retina pieces to a slide and trim the paper into a rectangle leaving about 0.5 centimeters on either side of the row of tissue. Then, move the filter paper to the prepared slicing chamber.Push the long filter paper edges into the thin petroleum jelly lines using forceps, and then immerse the retinas in three to four drops of cold Ringer's solution. First, transfer the preparation to the tissue slicer stage. Position the long edge along the marked line and secure the chamber ends to the stage with laboratory tape.Then, starting at one end, cut the retina and filter paper using firm, gentle pressure on the slicing arm. Check that the first slice was cut fully, and then proceed to use the micrometer to cut slices that are approximately 400 microns thick. Next, transfer the chamber with sliced sections to the petri dish containing the prepared coverslip, which will serve as a imaging ladder.Flood the dish with cold Ringer's solution and place it on the stage of the dissecting microscope. Now, under low magnification, use the forceps to secure a strip and move it onto the imaging ladder by sliding the underlying petri dish. Rotate the slices 90 degrees and bury the filter paper edges into the jelly.It is important to keep the retina submerged and to avoid making any direct contact with the retina. Next, reposition the slices in the ladders so that the retina layers are clearly visible. For the slices at each end of the ladder, make the retina face inward toward the other slices to minimize motion during injection or flow.Discard any slices that do not adhere well to the paper. Now, stain the tissue. Meanwhile, prepare the imaging chamber and injection apparatus.First, flush the tubing with Ringer's solution, until no air bubbles remain. Then, attach the tubing to the imaging chamber inlet and close the stop cock. Then, refill the syringe with the solution for injection and reattach it to the tubing.Now, use forceps to transfer the prepared imaging ladder to the imaging chamber. Press it into the dot of jelly near the inlet edge and fill the chamber with Ringer's solution to cover the slices. Then proceed with imaging.Using the described techniques, perfectly transversed slices can be imaged through all of the retinal layers. When slightly rotated, bundles of the outer segments will be visible. Using PI staining, dead cells can be eliminated from analysis.Healthy PI-negative photoreceptors have a stereotypical polarized elongated morphology. When in oxygenated Ringer's, they can be imaged for up to four hours. One imaging strategy is to visualize the dynamics of cone photoreceptor endoplasmic reticulum relative to the nucleus.Using tissues that express GFP, and the cone ER, while counterstaining with a nuclear dye. A similar strategy can be employed to view the actin cytoskeleton with tissue expressing GFP-fused actin. Using time-lapsed imaging, cellular dynamics such as cytosolic calcium fluctuations in a cone photoreceptor can be observed.For example, while isotonically depleting sodium in the imaging chamber, GCaMP can be used to visualize rising cytosolic calcium concentrations. By quantifying the fluorescence responses from single cone outer segments, cell bodies and synapses, the intracellular calcium dynamics can be determined during pharmacological manipulations and so forth. While attempting this procedure, it's important to remember to use red light before the RPE is removed.Carefully flatten eye cups before slicing, and keep the slices submerged at all times. Once mastered, tissue dissection, slicing, and preparation for imaging can be done in less than 30 minutes. After watching this video, you should have a good understanding of how to prepare retinal slices from Zebrafish to study metabolism, and physiology in specific cell types in cellular compartments.

preparing-fresh-retinal-slices-from-adult-zebrafish-for-ex-vivo

Research

JoVE Journal

Neuroscience

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are optimized to minimize their impact on behavior. This is first achieved by using a holder that minimizes movement hindrances. The back of the fly&#8217;s head is glued to this holder at an angle that allows optical access to the whole brain while retaining the fly&#8217;s ability to walk, groom, smell, taste and see. The back of the head is dissected to remove tissues in the optical path and muscles responsible for head movement artefacts. The fly brain can subsequently be imaged to record brain activity, for instance using calcium or voltage indicators, during specific behaviors such as walking or grooming, and in response to different stimuli. Once the challenging dissection, which requires considerable practice, has been mastered, this technique allows to record rich data sets relating whole brain activity to behavior and stimulus responses.

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

We modified the Marmarou weight drop model for adult zebrafish to examine a breadth of pathologies following blunt-force traumatic brain injury (TBI) and the mechanisms underlying subsequent neuronal regeneration. This blunt-force TBI model is scalable, induces a mild, moderate, or severe TBI, and recapitulates injury heterogeneity observed in human TBI.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

Mouse Retina OCT Fibergram Alignment with Confocal Images

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...

Monitoring Epileptiform Events in &lt;em&gt;Drosophila&lt;/em&gt; via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Ex Vivo Confocal Imaging of Mouse Retina

Enucleate the eyes of a euthanized mouse, marking the temporal side for orientation purposes. Remove the anterior chamber. Then remove the lens and vitreous and place eye cups in paraformaldehyde for 30 minutes.Wash eye cups with PBS for 30 minutes. Replacing the solution three times. Then, wash with 0.5%Triton X-100 for 30 minutes.Incubate eye cups in the blocking buffer for two hours at room temperature. After immunostaining, isolate the retinas from the eye cups using a microscope. Divide the retinas into four leaves and flat mount them with the retinal ganglion cell layer facing up.Ensure the mark on the temporal side remains attached for orientation. Cover the retinas with mounting medium. Place cover slips.And seal the slides using nail polish. Turn on the confocal microscope and under Locate mode, find the area of interest using the eye piece. Switch to Acquisition mode and set up tiles to cover the entire retina, along with Z-Stack slices for all layers of information.Image at least 25 tiles across the whole retina to achieve a total volume. Project the Z-Stack slices to generate two-dimensional on-face confocal microscopy images. The composite vis-OCT fibergram is compared with the corresponding confocal image of flat mounted retina immunostained with TuJ1 for RGC axons.Blood vessels exhibit distinguishable branching structures which can be matched with the ICAM-2 labeled blood vessels on the confocal image. Side-by-side comparison between ex vivo confocal microscopy and in vivo vis-OCT revealed identical RGC axon bundle networks and surrounding retinal vasculature.