Name: two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis
Uploaded: 2021-12-07T13:00:00
Description: Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

This work was supported by NIH Grant EY12155, EY027193, and EY027387 to JC. We are thankful to Dr. Spyridon Michalakis (Caltech, Pasadena, USA) and Natalie Chen (USC, Los Angeles, USA) for proofreading the manuscript. We would also like to thank Dr. Seth Ruffins (USC, Los Angeles, USA) and Dr. Janos Peti-Peterdi (USC, Los Angeles, USA) for providing the necessary equipment to collect the author provided footage. Material from: Kasey Rose et al, Separation of photoreceptor cell compartments in mouse retina for protein analysis, Molecular Neurodegeneration, published [2017], [Springer Nature].

All experiments were performed according to the local institutional guidelines of the committee on research animal care from the University of Southern California (USC).
1. Live cell retinal peeling method
<ol>
	<li>Preparation of Ames&#39; buffers, peeling papers, and dissection dish
	<ol>
		<li>Using blunt tip iris scissors (or equivalent scissor type), cut the cellulose filter paper (Grade 413) into rectangles measuring approximately 5 mm x 2.5 mm. Store the cuttings for ...

<ol>
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L., Dawson, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+light+intensity+threshold+for+retinal+damage+in+albino+rats+raised+under+6+lx.">Cyclic light intensity threshold for retinal damage in albino rats raised under 6 lx.</a> Experimental Eye Research. 44 (5), 643-661 (1987).</li><li>Brann, M. R., Cohen, L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diurnal+expression+of+transducin+mRNA+and+translocation+of+transducin+in+rods+of+rat+retina.">Diurnal expression of transducin mRNA and translocation of transducin in rods of rat retina.</a> Science. 235 (4788), 585-587 (1987).</li><li>Philp, N. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+isolation+of+retinal+outer+segment+fragments.">The isolation of retinal outer segment fragments.</a> The Journal of Cell Biology. 27 (3), 459-473 (1965).</li><li>Tsang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+target+enzyme+in+deactivation+of+photoreceptor+G+protein+in+vivo.">Role for the target enzyme in deactivation of photoreceptor G protein in vivo.</a> Science. 282 (5386), 117-121 (1998).</li><li>Walston, S. T., Chow, R. H., Weiland, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+recordings+of+retinal+bipolar+cells+in+response+to+extracellular+electrical+stimulation+in+wholemount+mouse+retina.">Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina.</a> Annual International Conference of the IEEE Engineering in Medicine and Biology Society. 2015, 3363-3366 (2015).</li><li>Guido, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+obtain+retinal+cell+preparations+highly+enriched+in+specific+cell+types.+Suitability+for+lipid+metabolism+studies.">A simple method to obtain retinal cell preparations highly enriched in specific cell types. Suitability for lipid metabolism studies.</a> Brain Research. Brain Research Protocols. 4 (2), 147-155 (1999).</li><li>Hayashi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+by+cyclin-dependent+protein+kinase+5+of+the+regulatory+subunit+of+retinal+cGMP+phosphodiesterase.+II.+Its+role+in+the+turnoff+of+phosphodiesterase+in+vivo.">Phosphorylation by cyclin-dependent protein kinase 5 of the regulatory subunit of retinal cGMP phosphodiesterase. II. Its role in the turnoff of phosphodiesterase in vivo.</a> The Journal of Biological Chemistry. 275 (42), 32958-32965 (2000).</li><li>Wolbring, G., Cook, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+and+characterization+of+protein+kinase+C+from+bovine+retinal+rod+outer+segments.">Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments.</a> European Journal of Biochemistry. 201 (3), 601-606 (1991).</li><li>Williams, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+protein+kinase+C+in+photoreceptor+outer+segments.">Characterization of protein kinase C in photoreceptor outer segments.</a> Journal of Neurochemistry. 69 (4), 1693-1702 (1997).</li><li>Watson, A. J., Aragay, A. M., Slepak, V. Z., Simon, M. 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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GTPase+activating+factor+for+transducin+in+rod+photoreceptors+is+the+complex+between+RGS9+and+type+5+G+protein+beta+subunit.">The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein beta subunit.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (5), 1947-1952 (1999).</li><li>Chen, J., Shi, G., Concepcion, F. A., Xie, G., Oprian, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+rhodopsin/arrestin+complex+leads+to+retinal+degeneration+in+a+transgenic+mouse+model+of+autosomal+dominant+retinitis+pigmentosa.">Stable rhodopsin/arrestin complex leads to retinal degeneration in a transgenic mouse model of autosomal dominant retinitis pigmentosa.</a> The Journal of Neuroscience. 26 (46), 11929-11937 (2006).</li><li>Chen, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+photoresponses+and+light-induced+apoptosis+in+rods+lacking+rhodopsin+kinase.">Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3718-3722 (1999).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mistrafficking+of+prenylated+proteins+causes+retinitis+pigmentosa+2.">Mistrafficking of prenylated proteins causes retinitis pigmentosa 2.</a> FASEB Journal. 29 (3), 932-942 (2015).</li><li>Weiss, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+differences+in+expression+of+G-protein-coupled+receptor+kinase+(GRK)+7+and+GRK1+in+mammalian+cone+photoreceptor+cells:+implications+for+cone+cell+phototransduction.">Species-specific differences in expression of G-protein-coupled receptor kinase (GRK) 7 and GRK1 in mammalian cone photoreceptor cells: implications for cone cell phototransduction.</a> The Journal of Neuroscience. 21 (23), 9175-9184 (2001).</li><li>Zhao, X., Huang, J., Khani, S. C., Palczewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+forms+of+human+rhodopsin+kinase+(GRK1).">Molecular forms of human rhodopsin kinase (GRK1).</a> The Journal of Biological Chemistry. 273 (9), 5124-5131 (1998).</li><li>Newton, A. C., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+protein+kinase+C+in+the+phosphorylation+of+rhodopsin.">Involvement of protein kinase C in the phosphorylation of rhodopsin.</a> The Journal of Biological Chemistry. 266 (27), 17725-17728 (1991).</li><li>Pinzon-Guzman, C., Zhang, S. S., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+protein+kinase+C+isoforms+are+required+for+rod+photoreceptor+differentiation.">Specific protein kinase C isoforms are required for rod photoreceptor differentiation.</a> The Journal of Neuroscience. 31 (50), 18606-18617 (2011).</li><li>Sokal, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+kinase+C+isozymes+responsible+for+the+phosphorylation+of+photoreceptor-specific+RGS9-1+at+Ser475.">Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at Ser475.</a> The Journal of Biological Chemistry. 278 (10), 8316-8325 (2003).</li><li>Rose, K., Walston, S. 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Many retinal diseases affect rod photoreceptor cells, leading to rod death and, ultimately, complete vision loss37. A significant portion of the genetic and mechanistic origins of human retinal degeneration have been successfully recapitulated in numerous mouse models over the years. In that context, the ability to easily and selectively separate individual rod subcellular compartments from the small mouse retina would greatly enhance our understanding of the localized biochemical and molecular un...

Rod photoreceptor cells, tightly packed in the outermost layer of the neural retina, are an integral part of dim light vision. To function as faithful photon counters, rods utilize a G-protein-based signaling pathway, termed phototransduction, to generate rapid, amplified, and reproducible responses to single photon capture. This response to light ultimately triggers a change in the current at the plasma membrane and is subsequently signaled to the rest of the visual system1. As their name implies, each rod cell has a distinct rod-like shape and exhibits a highly polarized cellular morphology, consisting of an outer segment (OS), inner segment (IS), cell body (CB), and synaptic terminal (ST). Each subcellular compartment has specific protein machinery (membrane-bound and soluble), biomolecular features, and protein complexes that play crucial roles such as visual phototransduction, general housekeeping and protein synthesis, and synaptic transmission2,3.
Over 30 years ago, the light-dependent reciprocal movement of subcellular proteins, specifically transducin (away from the OS) and arrestin (towards the OS), was first observed4,5,6,7. Early on, this observed phenomenon was received with skepticism, due in part to immunohistochemistry&#39;s vulnerability to epitope masking8. In the early 2000s, stimulus-dependent protein translocation was confirmed by using a rigorous and arduous physical sectioning technique9. Serial tangential sectioning of the frozen flat-mounted rodent retinae followed by immunoblotting revealed that transducin9,10, arrestin11,12, and recoverin13 all undergo subcellular redistribution in response to light. It is believed that light-driven translocation of these key signaling proteins not only regulates the sensitivity of the phototransduction cascade9,14,15, but may also be neuroprotective against light damage16,17,18. Because light-driven protein transport in rods appears to be very significant to rod cell biology and physiology, techniques that permit the isolation of different subcellular compartments to determine protein distribution are valuable research tools.
Currently, there are a few methods aimed at isolating the rod subcellular compartments. However, these methods can be lengthy and difficult to reproduce, or require a sizable amount of retinal isolate. Rod outer segment (ROS) preparations via density gradient centrifugation19, for example, is commonly used to separate the ROS from retinal homogenate. This method is widely used for western blot, but the procedure is very time consuming and requires a minimum of 8-12 murine retinae20. On the other hand, serial tangential sectioning of frozen murine and rat retinae has been successfully implemented in isolating the OS, IS, CB, and ST9,11,13. However, this method is technically challenging due to the necessity of fully flattening the small and highly curved murine retina to align the retinal layers prior to tangential sectioning. Since there are a plethora of mouse models and transgenic mice recapitulating diseases of the visual system, the creation of a technique that reliably, quickly, and easily separates individual rod compartments holds promise in revealing the physiologic processes that occur in each specialized compartment and the mechanisms that underlie visual processes in health and disease.
To facilitate these investigations, we describe two peeling methods that isolate rod subcellular compartments more easily than current protocols. The first peeling method, adapted from a technique to expose fluorescently labeled bipolar cells for patch clamp recording21, employs cellulose filter paper to sequentially remove the ROS from a live, isolated murine retina (Figure 1). The second method, adapted from a procedure that isolates the three primary retinal cell layers from a chick22 and frog23 retina, utilizes adhesive tape to remove the ROS and rod inner segment (RIS) from a lyophilized retina (Figure 2). Both procedures can be completed in 1 h and are considerably user-friendly. We provide validation of the effectiveness of these two separation protocols for western blot by utilizing dark-adapted and light-exposed retinae from C57BL/6J mice to demonstrate light-induced translocation of rod transducin (GNAT1) and arrestin (ARR1). Moreover, using the tape peeling method, we provide additional evidence that our technique can be used to examine and address inconsistencies between protein localization data acquired by immunocytochemistry (ICC) and western blots. Specifically, our technique showed that: 1) the protein kinase C-alpha (PKC&#945;) isoform is present not only in bipolar cells, but also in murine ROS and RIS, albeit in low concentrations24,25, and 2) rhodopsin kinase (GRK1) is present predominantly in the isolated OS sample. These data demonstrate the effectiveness of our two peeling techniques for separating and quantifying specific rod and retinal proteins.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mL laboratory or media bottle equipped with a tubing cap adapter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>100% O2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>1000mL Bottle Top Filter, PES Filter Material, 0.22 &#956;m</td>
			<td>Genesee Scientific</td>
			<td>25-235</td>
			<td></td>
		</tr>
		<tr>
			<td>4X SDS Sample Buffer</td>
			<td>Millipore Sigma</td>
			<td>70607-3</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>95% O2 and 5% CO2 tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ames&#8217; Medium with L-glutamine, without bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A1420</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2 (99%, dihydrate)</td>
			<td>Sigma</td>
			<td>C-3881</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite (Anhydrous calcium sulfate, &#62;98% CaSO4, &#62;2% CoCl2)</td>
			<td>WA Hammond Drierite Co LTD</td>
			<td>21005</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Petri Dish (polystyrene, 35 x 10 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-757-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Easy-Grip Tissue Culture Dish (60 x 15 mm)</td>
			<td>Falcon-Corning</td>
			<td>08-772F</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 10, 40 mm)</td>
			<td>VWR</td>
			<td>100499-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Feather Scalpel (No. 11, 40 mm)</td>
			<td>VWT</td>
			<td>100499-580</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl (99%)</td>
			<td>Sigma</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle</td>
			<td>Sigma</td>
			<td>z359971</td>
			<td></td>
		</tr>
		<tr>
			<td>Labconco Fast-Freeze Flasks</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>LN2 (liquid nitrogen) + Dewar flask or similar vacuum flask</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M-9272</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q/de-ionized water</td>
			<td>EMD Millipore</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4 (powder)</td>
			<td>J.T. Baker</td>
			<td>4062-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (crystal)</td>
			<td>EMD Millipore</td>
			<td>Sx0420-3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Amresco</td>
			<td>0865</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur EDTA</td>
			<td>EMD</td>
			<td>4005</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniPur HEPES, Free Acid</td>
			<td>EMD</td>
			<td>5320</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm M</td>
			<td>Sigma-Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Reynolds Wrap Aluminum Foil</td>
			<td>Reynolds Brands</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Scotch Magic Tape (12.7 mm x 32.9 m)</td>
			<td>Scotch-3M</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D67501</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrafuge mini centrifuge</td>
			<td>Labnet International, Inc</td>
			<td>C1301</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue incubation chamber (purchased or custom made)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>J.T.Baker</td>
			<td>4103-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Signma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>VirTis Benchtop 2K Lyophilizer or equivalent machine</td>
			<td>SP Scientific</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR Grade 413 Filer Paper (diameter 5.5 cm, pore size 5 &#956;m)</td>
			<td>VWR</td>
			<td>28310-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 Qualitative Filter Paper (diameter 9 cm, pore size 11 &#956;m)</td>
			<td>Whatman/GE Healthcare</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide bore transfer pipet, Global Scientific</td>
			<td>VWR</td>
			<td>76285-362</td>
			<td></td>
		</tr>
	</tbody>
</table>

two peeling methods for the isolation of photoreceptor cell compartments in the mouse retina for protein analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

The present strategies were developed to provide relatively rapid and simple methods to isolate and analyze proteins among specific rod subcellular compartments for western blot analysis. We applied two sequential peeling techniques (Figure 1 and Figure 2) followed by immunoblotting to demonstrate that these methods could reliably be used to detect the known distribution of rod transducin (GNAT1) and arrestin (ARR1) in both dark- and light-adapted animals. To va...

Watch this Scientific Journal Video about Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis at JoVE.com

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

The two peeling techniques demonstrated here isolate individual rod subcellular compartments, and can help reveal the important physiologic processes occurring in each specialized compartment in healthy and diseased rods. Isolating different rod cell compartments from the mouse retina can be challenging. These simple techniques use inexpensive and commonplace lab materials to reliably isolate rod subcellular compartments for protein analysis.To begin, place a piece of filter paper into a Petri dish filled with Ames HEPES buffer bubbled with 100%oxygen. Then, using a wide-bore transfer pipette, transfer one retinal rectangle dissected out from a two to three-months-old C57 Black 6J mouse. Orient the partitioned retina concave up and ensure that the photoreceptors are facing down toward the bottom of the dish.Then, using tweezers, lightly grasp the sides of the halved retina and move it on top of the filter paper. Once the retina is centered, move the filter paper upward so the halved retina touches the filter paper to create rod outer segment, or ROS to filter paper adhesion. Carefully lift the filter paper with the attached retina out of the Ames HEPES buffer.Place the bottom side of the filter paper on a paper towel and dab two to three times to dry. Add a drop of Ames HEPES onto the side of the filter paper with the retina and place the filter paper on the paper towel again to dry. Next, place the filter paper with the retina back into the Petri dish, and using tweezers, gently push all edges of the halved retina up and away from the filter paper on all sides.Delicately peel the retina away from the filter paper, touching only the extreme perimeter of the retina to preserve its structural integrity. Then, lift the filter paper from the Petri dish and remove excess liquid on a paper towel, ensuring that the side in contact with the retina does not contact the paper towel. Place the filter paper with the partial ROS layer into a labeled tube on ice, and keep the peeled retina submerged in Ames HEPES buffer.Repeat the peeling process for this halved retina approximately seven to eight times to remove the entire ROS layer. After each peel, place the filter paper into the same tube to combine all ROS isolated from one halved retina. Keep the tube containing all filter paper peels of the isolated ROS on ice for immediate use.Using tweezers, transfer the leftover peeled retina into an appropriately-labeled tube and keep the tube on ice for immediate use. To prepare the retinal sample for lyophilization, place a piece of filter paper and transfer a halved retina into a Petri dish filled with cold Ringer's buffer. Next, using tweezers, carefully flip the retina so it is oriented concave up and move it to rest on top of the filter paper.Lift the filter paper by the edges upward so the halved piece of retina touches the filter paper. Continue to lift the filter paper out of Ringer's solution. Then, place the bottom side of the filter paper on a paper towel and carefully dab two to three times to remove excess liquid.Next, pick up the filter paper with tweezers and add a drop of cold Ringer's onto the side of the filter paper with the retina. Again, place the filter paper on the paper towel to remove excess liquid. Store each adhered retina and filter paper sample in a Petri dish filled with cold Ringer's until ready to freeze all samples.Then, using tweezers, lift each sample out of the cold Ringer's buffer, ensuring that the tweezers contact only the filter paper edges, avoiding the halved retina. Place the bottom side of each filter paper on a paper towel to remove excess liquid. Then, add a drop of cold PBS onto side of the filter paper where the retina is adhered and dab the filter paper on the paper towel again.Place all filter paper pieces into the Petri dish, and using lint-free tissue paper, wick away any excess liquid surrounding the filter paper. Then, tightly wrap the entire dish with aluminum foil and ensure that the edges of the aluminum foil are secured and smoothly pressed into the bottom of the Petri dish. Puncture a handful of 0.1 to 0.2-millimeter holes into the aluminum foil lid.Next, using metal tongs, gradually lower the aluminum foil-covered Petri dish into liquid nitrogen. Keep the samples in liquid nitrogen until ready to lyophilize. For lyophilization, place the aluminum foil-covered Petri dish into a freeze drying flask and attach it to the lyophilizer machine following the manufacturer's protocol.Lyophilize the retinae for 30 minutes. After lyophilization, collect the rod outer and inner segment by carefully laying a small piece of tape on top of the lyophilized retina and applying slight pressure with the tweezers to ensure it bonds with the photoreceptor layer. Slowly peel the tape away and ensure that both the ROS and RIS have adhered to the tape.A thin, white film will be present at the fractured surface. This is the RIS. To separate this RIS, place another piece of tape and push the tape down on the fractured surface.Then, place the piece of tape with only the orange-pink layer into a microcentrifuge tube labeled as ROS, and pieces of tape with the thin, white layer into another tube labeled RIS. Next, using tape, collect the remaining retinal tissue located on the filter paper by peeling off the thick, white layer. Then, place this isolate into a tube labeled OIS.If using the same day, store the isolated layers from the lyophilized retinae at room temperature. In dark-adapted animals, the distribution of GNAT1 and ARR1 closely matched their known dark-state distributions, where GNAT1 signal was visibly strongest in the ROS isolation, and the ARR1 signal was more robust in the ROS isolation. Quantification of the GNAT1 and ARR1 signals showed significant differences between dark-light conditions for GNAT1 and ARR1 in ROS and ROS samples.In both dark and light-adapted samples, G beat 5S, cytochrome C, and actin signals are excluded from the ROS samples, demonstrating an absence of contamination from other cellular layers. Additionally, G beta 5L signal was clearly visible in the ROS samples. Scanning electron microscopy of the surfaces of intact and peeled lyophilized retinae showed that, before peeling with adhesive tape, the surface of intact lyophilized retina closely matched the characteristic cylindrical ROS.After the initial tape peel, the ROS and RIS appeared to be entirely removed, as evidenced by the uniform nuclear layer present on the surface of the leftover, peeled, lyophilized retina. Subsequent tape peels removed RIS from the free surface of the peeled layer. This technique also demonstrated that GRK1 immunoreactivity was most intense in ROS samples in light-exposed retinae, and absent in OIS samples.Conversely, ROS and RIS samples displayed a faint signal for PKC alpha, which is commonly not detected in ROS or RIS by immunofluorescence staining. Finally, suboptimal tape peeling could yield slightly contaminated samples. The RIS sample was contaminated with some OIS sample, producing both G beta 5L and G beta 5S bands, and a higher PKC alpha signal.Care must be taken when lyophilizing the retina. If an improper lyophilization technique is used, the retina may melt, making isolating different rod subcellular layers by tape impossible. Using this tape peeling method in conjunction with qRT-PCR has allowed for the separation of photoreceptor transcripts from the rest of the retina.

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Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

This protocol provides a comprehensive dissection and analysis guide for the use of deep ocular landmarks, s-opsin immunohistochemistry, Retistruct, and custom code to accurately and reliably orient the isolated mouse retina in anatomical space.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...