Articles in PresS. J Neurophysiol (August 30, 2017). doi:10.1152/jn.00578.2017
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Title Page
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Title:
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Method to Remove Photoreceptors from Wholemount Retina in vitro
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Authors:
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Steven T. Walston1, Yao-Chuan Chang1, James D. Weiland1,2,3, Robert H. Chow1,3,4
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STW performed experiments, analyzed data, and wrote the manuscript. YCC performed experiments, JDW and RHC guided experimental design and analysis, and edited the manuscript.
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Affiliations:
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Running Head:
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“Method to Remove Photoreceptors from Wholemount Retina”
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Correspondence:
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Robert. H. Chow, University of Southern California, 1501 San Pablo St., #323, Los Angeles, CA 90033 (e-mail: [email protected]).
Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089 2 USC Roski Eye Institute, University of Southern California, Los Angeles, CA 90033 3 Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033 4 Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90033
James D. Weiland, University of Michigan, 2800 Plymouth Road, Ann Arbor, MI, 48105 ([email protected])
1 Copyright © 2017 by the American Physiological Society.
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Abstract
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Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct
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in a wholemount retina preparation because surrounding neurons block the path of the patch
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pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at
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the cost of severing lateral connection between neurons. We have developed a technique to
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remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing
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access for patch clamp recording. Repeated application and removal of filter paper to the
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photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells
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and, in degenerate retina, hypertrophied Müller cell endfeet. Live-dead assays applied to neurons
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remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings
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from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated
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cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner
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retinal neurons in a wholemount retina preparation for investigation of signal processing. A
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disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The
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technique may be useful for investigations of extracellular electrical stimulation, photoreceptor
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DNA analysis, and non-pharmacological removal of light input.
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News and Noteworthy
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This study reports a method for removing photoreceptors from rodent wholemount retina while
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preserving the architecture of the inner retina. The method enables easier access to the inner
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retina for studies of neural processing, such as by patch clamp recording.
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Keywords
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retina wholemount, photoreceptor removal, patch clamp, bipolar cell
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Glossary
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(none)
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Main Text
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Introduction
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Patch clamp recordings of rodent retinal bipolar cell somas from in vitro wholemount retina
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preparations are difficult to obtain because bipolar cells are located between several layers of
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retinal neurons. In the normal mouse retina, a pipette approaching from the photoreceptor side
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would have to pass through 23 µm-long (approximate), densely packed outer segments, 10-14
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layers of 4-7 µm-diameter photoreceptors somas, and the outer plexiform layer before reaching
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bipolar cell somas (Carter-Dawson and LaVail 1979; Barhoum et al. 2008). From the ganglion
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cell side, the pipette would have to traverse the inner limiting membrane, the ganglion cell layer
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and a 60-µm (approximate) thick inner plexiform layer (IPL) (Fisher 1979). Each approach is
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technically difficult because debris may impair visualization of the neurons, and the pipette tip
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may become soiled, preventing the giga-seal required for electrical recording. Assuming that a
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successful giga-seal is obtained on a neuron in the inner nuclear layer (INL), there is a possibility
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that the neuron will be a horizontal cell, amacrine cell or Müller cell instead of a bipolar cell,
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further reducing the efficiency of the intended experiment.
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Vertical slice or retinal dissociation techniques are commonly used for patch clamp recording
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from inner retinal neurons. The vertical slice preparation provides ready access to retinal neurons
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and is useful for interrogating the vertical phototransduction pathway through the retina
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(Edwards et al. 1989; Boos et al. 1993; Van Hook and Thoreson 2013). However, the lateral
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processes of horizontal and amacrine cells are severed. Dissociated cell preparations are prepared
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by enzymatic digestion followed by mechanical trituration of the whole retina to prepare a
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primary culture (Sarthy and Lam 1979; Yamashita and Wässle 1991; Grozdanov et al. 2010).
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This technique facilitates single cell recordings from any neuron, but the neuron is completely 6
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disconnected from the retinal network and exhibits altered ion channel functionality due to
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enzymatic digestion (Klumpp et al. 1995).
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Although the slice and dissociated cell preparations have demonstrated great utility in retinal
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research, some studies would benefit from bipolar cell recordings conducted in wholemount
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retinas. Retinal prosthesis studies examining the response of INL neurons to electrical
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stimulation will benefit from preparation conditions that better mimic the operational conditions
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of the prosthesis. Prior studies of bipolar cell responses have relied on the vertical slice
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preparation for bipolar cell access, though each study notes that the slice preparation may not be
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the ideal model because there is significant current shunting around the retina, and the laterally
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projecting neurites have been severed (Margalit and Thoreson 2006; Margalit et al. 2011;
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Cameron et al. 2013).
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To overcome the difficulty of obtaining patch clamp recordings from INL neurons in
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wholemount retina, we have developed a method to mechanically remove photoreceptors from
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the retina by repeated application and peeling away of filter paper adhering to tissue on the
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photoreceptor side of an isolated retina. The photoreceptor peel technique described exposes the
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INL for single cell recording while maintaining cell viability. A subset of these results has been
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previously reported (Walston et al. 2015).
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Methods
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Transgenic
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EGFP)GI206Gsat (PD > 300) mice with a C57BL/6J background were used (Gong et al. 2003;
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Huang et al. 2003). These transgenic mice express enhanced green fluorescent protein (EGFP) in
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ON-type bipolar cells. Normal mice were gifts from A. Sampath (University of Southern
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California, Los Angeles). Normal mice were crossed with rd10 mice to produce rd10 mice
normal
Tg(Gng13-EGFP)GI206Gsat
(PD
>
60)
and
rd10
Tg(Gng13-
7
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containing EGFP. Mice were anaesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg)
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followed by cervical dislocation. Eyes were enucleated and placed on moist filter paper. A small
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slit was made across the cornea with a razor blade. The eye was then placed into 30 °C Ames’
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media (#A1420, Sigma Aldrich, St. Louis, MO) buffered with sodium bicarbonate and penicillin-
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streptomycin (#P4333, Sigma-Aldrich) (280mOsm, pH 7.3). The cornea was excised above the
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ora serrata followed by removal of the lens. The vitreous was removed with forceps and four
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radial relief cuts were made around the eyecup. The retina was severed from the optic nerve and
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removed from the eyecup. A quarter of the retina was isolated such that it contained the optic
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disc. All procedures were approved by the Institutional Animal Care and Use Committee of the
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University of Southern California.
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Photoreceptor Peel Technique
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To remove photoreceptor outer segments, inner segments, and somas, the photoreceptor peel
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technique was performed as depicted in Figure 1. The quarter of retina was placed
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photoreceptor-side down on a piece of VWR 415 Grade Qualitative Filter Paper (#28320, VWR
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International, Radnor, PA) bathed in oxygenated solution. The size of the filter paper piece was
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5x5 mm (approximately 3 times larger than the quarter of retina). The coarse filter paper is made
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of pure cellulose with 25-µm particle retention. The filter paper-containing retina was removed
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from the solution and placed filter paper-side down on a paper towel. Drops of solution were
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applied atop the retina with a transfer pipette. The solution was allowed to move through the
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retina and filter paper. Care was taken to keep the retina hydrated at all times. Additionally, the
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retina was outside of the oxygenated solution for no more than 10 seconds at any time. The retina
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was placed back into the medium and gently removed from the filter paper using forceps to lift
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up the retina by grasping at the optic disc. When necessary, the sides of the retina were also lifted
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with forceps. This process was repeated approximately 10-15 times to remove the majority of
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photoreceptor outer segments, inner segments and somas. The photoreceptor peel technique was
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performed only 1-2 times on the remaining outer nuclear layer of the rd10 retina, leaving out
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Step 5 and medium application in Step 4 shown in Figure 1. Only a quarter of the retina was used
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because it was easier to lay flat and because grasping at the optic disc (at the corner of the
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quarter) reduced retinal tears. The VWR 415 filter paper was used because its surface roughness
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was sufficiently coarse to remove layers of neurons yet fine enough to avoid tearing the retina in
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its entirety during the peeling process, but filter paper properties were not investigated
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systematically.
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Fluorescence imaging was used to determine the thoroughness of the photoreceptor peel process.
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The retina was mounted ganglion cell-side down on a glass-bottom petri dish, held in place with
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a PTFE membrane (JVWP01300, Millipore) weighted by a titanium ring, and viewed with an
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upright microscope. Photoreceptor removal was judged adequate by the clarity of EGFP
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fluorescence arising from bipolar cells and by presence of three or fewer layers of photoreceptor
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somas. The retina was removed from the microscope stage and the peeling process was resumed
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if necessary.
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Live-Dead Assay
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We assessed retinal health 45 minutes after performing the photoreceptor peel technique using a
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live-dead assay. The free-floating retina was incubated in Ames’ media supplemented with 4 µM
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calcein-blue AM (#C1429, Invitrogen, Grand Island, NY) and 1 µM ethidium homodimer-1
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(#E1169, Invitrogen, Grand Island, NY) for the live and dead assay, respectively. The solution
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was continuously oxygenated and maintained at 30 °C for 25 minutes. After incubation, the
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retina was mounted ganglion cell side-down on glass-bottom petri dish and perfused in Ames’ 9
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media for 30 minutes while being gently held down with the weighted membrane. Fluorescence
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imaging was performed at multiple regions across the retina under 60x magnification. Images
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were manually post-processed in ImageJ with the Cell Counter plugin to quantify the number of
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cells labeled with EGFP, calcein-blue, and ethidium homodimer-1, and to determine
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fluorescence colocalization.
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Imaging of Vertical Slices
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Retinas with and without photoreceptors removed were prepared for imaging in vertical slices.
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After dissections, retinas were fixed in 4% paraformaldehyde for 2 hours. They were incubated
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in 0.01M phosphate buffered saline 3 times for 5 minutes on a shaker. The retinas were mounted
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flat in blocks of 4% agarose. Each agarose block was prepared on a vibratome and sectioned at
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50-µm increments to obtain vertical retina slices. Retinal slices were mounted on microscope
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slides, covered with an ethanol-based mounting solution supplemented with 1 µM ethidium
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homodimer-1, covered with glass cover slips, and imaged.
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Patch Clamp Electrophysiology
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After performing the photoreceptor peel technique, the retina was mounted ganglion cell-side
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down on a glass-bottom petri dish. The retina was held in place with a PTFE membrane
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containing manually perforated 1 mm-diameter access holes and weighted by a titanium
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horseshoe. The retina was perfused with buffered Ames’ media at 30 °C at a flow rate of 4-
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6mL/min. A silver-silver/chloride pellet (#64-1305, Warner Instruments, Hamden, CT) reference
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electrode was placed in solution. Borosilicate glass (outer diameter=1.2 mm, inner diameter=1.0
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mm, Sutter Instruments, Novato, CA, #B120-69-10) was pulled to obtain resistances of 7-14MΩ
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(when pipette was filled with internal solution described below) using a laser pipette puller
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(Sutter Instruments, Novato, CA, #P-2000). Pipettes were filled with internal solution containing
10
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the following components (mM): 120 K-Gluconate, 10 NaCl, 3 ATP-Mg, 0.3 GTP-Na, 10
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HEPES, 0.5 EGTA, 10 Phosphocreatine Disodium Hydrate, adjusted to pH=7.3 with 1M KOH
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(Sigma, Louis, MO) and an osmolality of 270 mOsm. K-gluconate was substituted for 140 mM
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Cs-gluconate, and 20 mM tetraethylammonium (TEA) was added to the external solution for
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tests blocking potassium channels. The junction potential of 14 mV and 15.2 mV was corrected
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in control and blocker solutions, respectively (Neher 1992). An EPC9 amplifier (HEKA,
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Bellmore, New York) under control of PatchMaster software was used for data acquisition. Input
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resistance from recorded neurons averaged 2.78±1.31 GΩ, and membrane capacitance averaged
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4.59±1.55 pF (mean±std).
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Whole-cell voltage-clamp recordings were collected from ON-type bipolar cell somas. Cells
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were held at -80 mV and stepped in increments of 10 mV from -80 mV to 60 mV. Each step was
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held for 100ms. Current-voltage (I-V) curves were obtained from the sustained current in
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response to each voltage step. In current clamp, the holding current was 0 pA, current injections
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of -10 pA to 20 pA in steps of 2 pA were delivered through the pipette for 6 or 8 seconds.
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Membrane potentials were estimated from the median membrane potential and oscillation
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amplitudes were estimated from the standard deviation of the membrane potential during the
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current step.
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Results
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Photoreceptor Removal
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The photoreceptor peel technique effectively removes photoreceptor outer segments, inner
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segments, and somas from normal retina. EGFP fluorescence identifies ON-type bipolar cells in
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the INL, but the fluorescence is scattered by photoreceptors when imaging from the subretinal
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side (Figure 2A). After removing photoreceptors, the contours of bipolar cells are readily
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visualized (Figure 2B). The majority of photoreceptor outer segments were removed from the
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retina after the first few peels, however 10-15 cycles of the process were necessary to provide
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adequate bipolar cell visualization and access. Remaining photoreceptor soma layers ranged
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from 0-3 depending on the location. In the rd10 retina, the objective of the photoreceptor peel
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process is to loosen the hypertrophied glial seal covering the INL. Only 1-2 gentle peels were
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necessary. In rd10 retina, the fluorescence from the ON-type bipolar cells is visible without the
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photoreceptor peel technique, but we found that successful “giga-seal” recordings were easier to
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obtain, due to less soiling of the patch pipette tip from the glial cell contact.
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After mechanically removing photoreceptors in three retina preparations, we performed a live-
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dead assay to assess the health of bipolar cells. Calcein blue-AM and ethidium homodimer-1
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served as the live and dead assay, respectively. Figure 3 illustrates representative images of how
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the assays labeled the retina. The ON-type bipolar cells are labeled with EGFP (Figure 3A).
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Figure 3D illustrates the cell contours in the INL. The live assay stains cells in the INL including
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the ON-type bipolar cells (Figure 3B). The labeling of the live assay appears to be limited to the
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outer surface of the retina which restricts our evaluation to the most exposed neurons. From 34
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image regions, we identified 886 ON-type bipolar cells, 691 (77.9%) of which were co-labeled
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by the live assay. In the same image regions, 151 cells were labeled by the dead assay in total.
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Though these cells were labeled by the dead assay, none were colocalized with the EGFP-labeled
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bipolar cells (Figure 3C). This result demonstrates that the majority of labeled ON-type bipolar
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cells have intact cell membranes, suggesting healthy cells. The same live-dead assay procedure
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was applied to two rd10 retina preparations after applying one or two peels with the filter paper.
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From 23 image regions, we identified 868 ON-type bipolar cells (Figure 3E), 679 (78.2%) of
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which were labeled with the live assay (Figure 3F). The contours of cell somas are illustrated in
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the infrared image (Figure 3H). 103 cells were labeled with the dead assay (Figure 3G), but none
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were colocalized with the EGFP-labeled bipolar cells. 0.3% of cells labeled by the live assay
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were also labeled by the dead assay. The live-dead assay was also performed on 4 rd10 retinas
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without applying the peel technique in order to assess potential effects of the peel technique. The
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staining performance of the live assay was heterogeneous and less robust, which may be due to
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the glial seal. In 11 image areas with discernible labeling, 236 (73%) of EGFP labeled bipolar
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cells were colocalized with the live assay. None were colocalized with the dead assay.
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Vibratome sectioning of the retina reveals that the retina is still morphologically intact. The
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normal retina without photoreceptor peeling contains 10-14 layers of photoreceptors somas
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above the INL (Figure 4A). ON-type bipolar cells are identified in the INL by EGFP
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fluorescence. Neurons not labeled with EGFP in the INL are OFF-type bipolar cells, horizontal
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cells, amacrine cells, and Müller cells. ON-type bipolar cell terminals are also observed in the
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lower strata of the IPL. Ganglion cells and displaced amacrine cells in the ganglion cell layer are
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also labeled by ethidium homodimer-1. Figure 4B depicts the retina after the photoreceptor peel
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technique has been performed. The majority of photoreceptor somas in the outer nuclear layer
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have been removed. The number of somas removed is largely determined by the number of peel
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cycles. The remaining inner retina appears intact, with a similar structural morphology to the
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control retina.
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Electrophysiological Measurements
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Whole-cell patch clamp recordings were performed on ON-type bipolar cells in wholemount
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mouse retina after photoreceptor removal. The transmembrane current was recorded in response
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to voltage steps from a holding potential of -80 mV (Figure 5A). Membrane voltage was stepped 13
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from -80 mV to +60 mV in steps of 10 mV. Outward currents are observed above approximately
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-40 mV and increase above -10 mV. Steady-state current responses are plotted as a function of
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step potential in Figure 5B. Recordings with 140 mM Cs-gluconate internal and 20 mM TEA in
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the external solution significantly reduced the outward currents indicating that they are largely
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dependent on potassium channels (Figure 5B). Though reduced, the potassium current at steady-
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state increases approximately linearly with membrane potential for step potentials greater than
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approximately -10 mV. The addition of potassium channel blockers also revealed small inward
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currents when voltage was stepped to -30 and -40 mV.
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In current clamp, we injected current into ON-type bipolar cells (N=30) in the normal retina and
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measured the voltage response. Injections ranged from -10 pA to 20 pA in steps of 2 pA.
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Negative current steps resulted in hyperpolarization of the membrane potential and reduction in
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the amplitude of membrane oscillations (Figure 6A). Positive current steps elevated the
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membrane potential, which resulted in fast depolarizing transients. Figure 6B shows the relation
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between resting membrane potential and the membrane potential at which oscillations were
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maximal for 30 bipolar cells. The average membrane potential at which the maximum oscillation
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amplitude occurred was -40.6±4.7 mV (mean±std). The average resting membrane potential of
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these cells was -58.8±7.6 mV.
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Discussion
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Mechanically peeling photoreceptors from wholemount retina using filter paper is an effective
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technique to expose neurons in the INL. The removal of retina layers is relatively uniform across
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the treated area (Figure 4), the retina is kept in viable physiological condition, and the progress
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of removing photoreceptors can be monitored. Live-dead assays indicate that majority of
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remaining bipolar cells are still viable after the procedure (Figure 3). In our investigation, we 14
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used a transgenic mouse line expressing EGFP in ON-type bipolar cells to provide a priori
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discrimination of these neurons. This strategy can be adapted with other transgenic lines to allow
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visualization of other retinal cells. Adjusting the number of times the filter paper is applied and
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removed provides some control over the number of photoreceptor somas removed from the
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retina. This feature is beneficial as it allows one to efficiently adjust which layer(s) of neurons
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will be removed and exposed. For example, in this study we determined that only a couple peels
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were necessary to carefully remove the glial seal covering the INL of the rd10 degenerate retina
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in contrast to the 10-15 required to remove the majority of photoreceptor outer segments, inner
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segments, and somas in the normal retina. This investigation did not test the ability of filter paper
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to remove the inner limiting membrane.
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Patch clamp electrophysiology with this preparation provided baseline bipolar cell measurements
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similar to those obtained in traditional slice and dissociated cell preparations (Klumpp et al.
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1995; Zenisek and Matthews 1998; Frech and Backus 2004). I-V curves reveal strong outward
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rectifying potassium currents, initiating at membrane potentials more positive than -40 mV.
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Though complete blockade was not observed, the K+ channel blockers cesium and TEA
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significantly reduced this outward current and revealed small inward currents that are consistent
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with previous reports of calcium channels in bipolar cell terminals (De La Villa 1998). Further,
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application of current steps into bipolar cells revealed that the bipolar cell membrane potentials
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transition into and out of oscillatory behavior peaking at approximately -40 mV. The magnitude
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of the responses showed dependence on membrane potential and appears to be in agreement with
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previously reported bipolar cell calcium spikes (Zenisek and Matthews 1998; Protti et al. 2000;
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Palmer 2006; Baden et al. 2011). The correspondence between the various techniques is
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encouraging because each preparation approach is different. However, the advantage of this
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preparation is that it exposes INL neurons in a wholemount retina, which may preserve certain
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aspects of signal processing.
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Adjusting the number of peel steps makes the photoreceptor removal technique adaptable to
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different types of experiments. Proteomics or DNA analyses can be performed on either
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individual retinal layers that are removed or on the retinal tissue remaining after performing the
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peel technique, and, because no chemicals or enzymes are used during the processing,
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contamination by chemicals and enzymes is minimal. Individual bipolar cell subclass signaling
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pathways can be examined without contamination from broad photoreceptor input to the entire
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retina. Activity could be elicited, for example, by targeting optogenetic channels to single bipolar
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cell subtypes and then measuring the response characteristics of postsynaptic ganglion cells or
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amacrine cells. A few peel steps could be used to remove light input into photoreceptors without
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the use of pharmacological blockers. The technique is also useful for recording INL responses to
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electrical stimulation from microelectrode arrays. A large portion of the retina wholemount
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would remain intact and cover the stimulating electrode. This provides a better model of a human
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implant because electric current paths will be directed through the retina rather than shunting
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around the tissue as in the retinal slice or dissociated cell preparations.
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Removing retinal layers in rodent wholemount retina has also been reported using other
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methodologies (Shiosaka et al. 1984; Hayashida et al. 2004; Enoki et al. 2006; Feigenspan and
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Babai 2015). One method uses a mounting medium to hold the retina before slicing through the
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retina horizontally with a fine blade (Enoki et al. 2006; Feigenspan and Babai 2015). Drawbacks
304
to this preparation are that it is difficult to align the retina relative to the blade, which is
305
necessary for precise removal of a specific layer. Care must also be taken to prevent exposing the
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retina to high temperatures when covering it in mounting medium and to maintain a healthy 16
307
retinal environment throughout the entire process. Manual horizontal slicing with a blade has
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also been described atop filter paper without mounting media (Hayashida et al. 2004). Another
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method uses enzymatic trypsin digestion and two sets of filter paper attached on both retinal
310
surfaces to remove retinal layers (Shiosaka et al. 1984). Cell exposure to digestion enzymes
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loosens the extracellular matrix holding neurons in place, resulting in as few as 5 applications of
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filter paper to remove the entire ONL and INL from the retina in comparison to the 10-15
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required to remove the ONL without enzymatic digestion in our preparation. However, enzymes
314
must be used with caution because their effect on ion channels may fundamentally change neural
315
activity (Klumpp et al. 1995; Holt et al. 2001).
316
No chemicals or digestion enzymes are used during our photoreceptor peel technique. It does not
317
require the retina to be submerged in a mounting medium. Nor does it require the alignment of a
318
fine blade to cut the retina. The limitation of the photoreceptor peel technique is that it severs the
319
connection between photoreceptors and INL neurons. As such, it is not possible to use this
320
technique and record light-activated responses from the remaining retina. Additionally, the
321
removal of photoreceptors will alter the input to bipolar cells and horizontal cells, which could
322
affect baseline retinal activity. In our study, we did not directly assess the integrity of lateral
323
connections in the INL. Maintenance of such connections is a potential benefit of this approach
324
compared to retinal slice, but must be proven through further experimentation.
325
In conclusion, we have developed and assessed a method for improving the access to bipolar
326
cells of both normal and degenerate mammalian wholemount retina for patch clamp recording.
327
Photoreceptor and glial membranes are removed by repeated application and peeling of
328
commercially available filter paper to the retina. Cell viability and electrophysiological measures
329
suggest
that
the
bipolar
cells
are
viable
after
this
procedure. 17
330
Appendix
331
(none)
18
332
Footnotes
333
(none)
19
334
Acknowledgements
335
(none)
336
Grants
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This research has been supported by the National Eye Institute (Grants RO1 EY022931 and
338
EY022931-S1), National Science Foundation (Grant CBET 1343193), the USC Institute for
339
Biomedical Therapeutics, and a Departmental grant from Research to Prevent Blindness.
340
Disclosures
341
The authors do not have any disclosures with regard to this study.
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20
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Van Hook MJ, Thoreson WB. Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation. J Vis Exp : 1–10, 2013. 21
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Huang L, Max M, Margolskee RF, Su H, Masland RH, Euler T. G protein subunit Gɣ13 is coexpressed with Gαo, Gβ3, and Gβ4 in retinal ON bipolar cells. J Comp Neurol 455: 1–10, 2003.
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Klumpp DJ, Song EJ, Ito S, Sheng MH, Jan LY, Pinto LH. The Shaker-like potassium channels of the mouse rod bipolar cell and their contributions to the membrane current. J Neurosci 15: 5004–5013, 1995.
386 387
De La Villa P. Two types of calcium currents of the mouse bipolar cells recorded in the retinal slice preparation. Eur J Neurosci 10: 317–323, 1998.
388 389
Margalit E, Babai N, Luo J, Thoreson WB. Inner and outer retinal mechanisms engaged by epiretinal stimulation in normal and rd mice. Vis Neurosci 28: 145–154, 2011.
390 391
Margalit E, Thoreson WB. Inner retinal mechanisms engaged by retinal electrical stimulation. Investig Ophthalmol Vis Sci 47: 2606–2612, 2006.
392 393
Neher E. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol 207: 123–131, 1992.
394 395
Palmer MJ. Modulation of Ca(2+)-activated K+ currents and Ca(2+)-dependent action potentials by exocytosis in goldfish bipolar cell terminals. J Physiol 572: 747–762, 2006.
396 397
Protti DA, Flores-Herr N, von Gersdorff H. Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell. Neuron 25: 215–227, 2000.
398
Sarthy PV, Lam DMK. Isolated cells from a mammalian retina. Brain Res 176: 208–212, 1979.
399 400 401
Shiosaka S, Kiyama H, Tohyama M. A simple method for the separation of retinal sublayers from the entire retina with special reference to application for cell culture. J Neurosci Methods 10: 229–235, 1984.
402 403 404
Walston ST, Chow RH, Weiland JD. Patch clamp recordings of retinal bipolar cells in response to extracellular electrical stimulation in wholemount mouse retina. Proc Annu Int Conf IEEE Eng Med Biol Soc EMBS 2015-Novem: 3363–3366, 2015.
405 406
Yamashita M, Wässle H. Responses of rod bipolar cells isolated from the rat retina to the glutamate agonist 2-amino-4-phosphonobutyric acid (APB). J Neurosci 11: 2372–82, 1991.
407 408
Zenisek D, Matthews G. Calcium action potentials in retinal bipolar neurons. Vis Neurosci 15: 69–75, 1998.
22
409
Tables
410
(none)
411
23
412
Equations
413
(none)
24
Figure 1. Flow diagram for the photoreceptor peel technique.
Figure 2. Photoreceptor peeling allows visualization of bipolar cells.
EGFP
Live Assay
Dead Assay
Infrared
A
B
C
D
E
F
G
H
Figure 3. Live-dead assay of the retina after performing the photoreceptor peel technique.
A
ON-type Bipolar Cells Ethidium Homodimer-1
ONL INL IPL RGC Layer
50µm
Figure 4. Vertical retina slice view of the photoreceptor peel technique.
B
Figure 5. I-V curves recorded from ON-type bipolar cells in normal retina.
Figure 6. Bipolar cell response to current injections in current clamp in normal retina.
1
Title Page
2
Title:
3
Method to Remove Photoreceptors from Wholemount Retina in vitro
4
Authors:
5
Steven T. Walston1, Yao-Chuan Chang1, James D. Weiland1,2,3, Robert H. Chow1,3,4
6 7 8
STW performed experiments, analyzed data, and wrote the manuscript. YCC performed experiments, JDW and RHC guided experimental design and analysis, and edited the manuscript.
9
Affiliations:
10 11 12 13 14 15 16 17
1
18
Running Head:
19 20
“Method to Remove Photoreceptors from Wholemount Retina”
21
Correspondence:
22 23 24 25 26 27
Robert. H. Chow, University of Southern California, 1501 San Pablo St., #323, Los Angeles, CA 90033 (e-mail: [email protected]).
Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089 2 USC Roski Eye Institute, University of Southern California, Los Angeles, CA 90033 3 Institute for Biomedical Therapeutics, University of Southern California, Los Angeles, CA 90033 4 Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90033
James D. Weiland, University of Michigan, 2800 Plymouth Road, Ann Arbor, MI, 48105 ([email protected])
1 Copyright © 2017 by the American Physiological Society.
28
Abstract
29
Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct
30
in a wholemount retina preparation because surrounding neurons block the path of the patch
31
pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at
32
the cost of severing lateral connection between neurons. We have developed a technique to
33
remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing
34
access for patch clamp recording. Repeated application and removal of filter paper to the
35
photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells
36
and, in degenerate retina, hypertrophied Müller cell endfeet. Live-dead assays applied to neurons
37
remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings
38
from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated
39
cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner
40
retinal neurons in a wholemount retina preparation for investigation of signal processing. A
41
disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The
42
technique may be useful for investigations of extracellular electrical stimulation, photoreceptor
43
DNA analysis, and non-pharmacological removal of light input.
2
44
News and Noteworthy
45
This study reports a method for removing photoreceptors from rodent wholemount retina while
46
preserving the architecture of the inner retina. The method enables easier access to the inner
47
retina for studies of neural processing, such as by patch clamp recording.
48
3
49
Keywords
50
retina wholemount, photoreceptor removal, patch clamp, bipolar cell
4
51
Glossary
52
(none)
5
53 54
Main Text
55
Introduction
56
Patch clamp recordings of rodent retinal bipolar cell somas from in vitro wholemount retina
57
preparations are difficult to obtain because bipolar cells are located between several layers of
58
retinal neurons. In the normal mouse retina, a pipette approaching from the photoreceptor side
59
would have to pass through 23 µm-long (approximate), densely packed outer segments, 10-14
60
layers of 4-7 µm-diameter photoreceptors somas, and the outer plexiform layer before reaching
61
bipolar cell somas (Carter-Dawson and LaVail 1979; Barhoum et al. 2008). From the ganglion
62
cell side, the pipette would have to traverse the inner limiting membrane, the ganglion cell layer
63
and a 60-µm (approximate) thick inner plexiform layer (IPL) (Fisher 1979). Each approach is
64
technically difficult because debris may impair visualization of the neurons, and the pipette tip
65
may become soiled, preventing the giga-seal required for electrical recording. Assuming that a
66
successful giga-seal is obtained on a neuron in the inner nuclear layer (INL), there is a possibility
67
that the neuron will be a horizontal cell, amacrine cell or Müller cell instead of a bipolar cell,
68
further reducing the efficiency of the intended experiment.
69
Vertical slice or retinal dissociation techniques are commonly used for patch clamp recording
70
from inner retinal neurons. The vertical slice preparation provides ready access to retinal neurons
71
and is useful for interrogating the vertical phototransduction pathway through the retina
72
(Edwards et al. 1989; Boos et al. 1993; Van Hook and Thoreson 2013). However, the lateral
73
processes of horizontal and amacrine cells are severed. Dissociated cell preparations are prepared
74
by enzymatic digestion followed by mechanical trituration of the whole retina to prepare a
75
primary culture (Sarthy and Lam 1979; Yamashita and Wässle 1991; Grozdanov et al. 2010).
76
This technique facilitates single cell recordings from any neuron, but the neuron is completely 6
77
disconnected from the retinal network and exhibits altered ion channel functionality due to
78
enzymatic digestion (Klumpp et al. 1995).
79
Although the slice and dissociated cell preparations have demonstrated great utility in retinal
80
research, some studies would benefit from bipolar cell recordings conducted in wholemount
81
retinas. Retinal prosthesis studies examining the response of INL neurons to electrical
82
stimulation will benefit from preparation conditions that better mimic the operational conditions
83
of the prosthesis. Prior studies of bipolar cell responses have relied on the vertical slice
84
preparation for bipolar cell access, though each study notes that the slice preparation may not be
85
the ideal model because there is significant current shunting around the retina, and the laterally
86
projecting neurites have been severed (Margalit and Thoreson 2006; Margalit et al. 2011;
87
Cameron et al. 2013).
88
To overcome the difficulty of obtaining patch clamp recordings from INL neurons in
89
wholemount retina, we have developed a method to mechanically remove photoreceptors from
90
the retina by repeated application and peeling away of filter paper adhering to tissue on the
91
photoreceptor side of an isolated retina. The photoreceptor peel technique described exposes the
92
INL for single cell recording while maintaining cell viability. A subset of these results has been
93
previously reported (Walston et al. 2015).
94
Methods
95
Transgenic
96
EGFP)GI206Gsat (PD > 300) mice with a C57BL/6J background were used (Gong et al. 2003;
97
Huang et al. 2003). These transgenic mice express enhanced green fluorescent protein (EGFP) in
98
ON-type bipolar cells. Normal mice were gifts from A. Sampath (University of Southern
99
California, Los Angeles). Normal mice were crossed with rd10 mice to produce rd10 mice
normal
Tg(Gng13-EGFP)GI206Gsat
(PD
>
60)
and
rd10
Tg(Gng13-
7
100
containing EGFP. Mice were anaesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg)
101
followed by cervical dislocation. Eyes were enucleated and placed on moist filter paper. A small
102
slit was made across the cornea with a razor blade. The eye was then placed into 30 °C Ames’
103
media (#A1420, Sigma Aldrich, St. Louis, MO) buffered with sodium bicarbonate and penicillin-
104
streptomycin (#P4333, Sigma-Aldrich) (280mOsm, pH 7.3). The cornea was excised above the
105
ora serrata followed by removal of the lens. The vitreous was removed with forceps and four
106
radial relief cuts were made around the eyecup. The retina was severed from the optic nerve and
107
removed from the eyecup. A quarter of the retina was isolated such that it contained the optic
108
disc. All procedures were approved by the Institutional Animal Care and Use Committee of the
109
University of Southern California.
110
Photoreceptor Peel Technique
111
To remove photoreceptor outer segments, inner segments, and somas, the photoreceptor peel
112
technique was performed as depicted in Figure 1. The quarter of retina was placed
113
photoreceptor-side down on a piece of VWR 415 Grade Qualitative Filter Paper (#28320, VWR
114
International, Radnor, PA) bathed in oxygenated solution. The size of the filter paper piece was
115
5x5 mm (approximately 3 times larger than the quarter of retina). The coarse filter paper is made
116
of pure cellulose with 25-µm particle retention. The filter paper-containing retina was removed
117
from the solution and placed filter paper-side down on a paper towel. Drops of solution were
118
applied atop the retina with a transfer pipette. The solution was allowed to move through the
119
retina and filter paper. Care was taken to keep the retina hydrated at all times. Additionally, the
120
retina was outside of the oxygenated solution for no more than 10 seconds at any time. The retina
121
was placed back into the medium and gently removed from the filter paper using forceps to lift
122
up the retina by grasping at the optic disc. When necessary, the sides of the retina were also lifted
8
123
with forceps. This process was repeated approximately 10-15 times to remove the majority of
124
photoreceptor outer segments, inner segments and somas. The photoreceptor peel technique was
125
performed only 1-2 times on the remaining outer nuclear layer of the rd10 retina, leaving out
126
Step 5 and medium application in Step 4 shown in Figure 1. Only a quarter of the retina was used
127
because it was easier to lay flat and because grasping at the optic disc (at the corner of the
128
quarter) reduced retinal tears. The VWR 415 filter paper was used because its surface roughness
129
was sufficiently coarse to remove layers of neurons yet fine enough to avoid tearing the retina in
130
its entirety during the peeling process, but filter paper properties were not investigated
131
systematically.
132
Fluorescence imaging was used to determine the thoroughness of the photoreceptor peel process.
133
The retina was mounted ganglion cell-side down on a glass-bottom petri dish, held in place with
134
a PTFE membrane (JVWP01300, Millipore) weighted by a titanium ring, and viewed with an
135
upright microscope. Photoreceptor removal was judged adequate by the clarity of EGFP
136
fluorescence arising from bipolar cells and by presence of three or fewer layers of photoreceptor
137
somas. The retina was removed from the microscope stage and the peeling process was resumed
138
if necessary.
139
Live-Dead Assay
140
We assessed retinal health 45 minutes after performing the photoreceptor peel technique using a
141
live-dead assay. The free-floating retina was incubated in Ames’ media supplemented with 4 µM
142
calcein-blue AM (#C1429, Invitrogen, Grand Island, NY) and 1 µM ethidium homodimer-1
143
(#E1169, Invitrogen, Grand Island, NY) for the live and dead assay, respectively. The solution
144
was continuously oxygenated and maintained at 30 °C for 25 minutes. After incubation, the
145
retina was mounted ganglion cell side-down on glass-bottom petri dish and perfused in Ames’ 9
146
media for 30 minutes while being gently held down with the weighted membrane. Fluorescence
147
imaging was performed at multiple regions across the retina under 60x magnification. Images
148
were manually post-processed in ImageJ with the Cell Counter plugin to quantify the number of
149
cells labeled with EGFP, calcein-blue, and ethidium homodimer-1, and to determine
150
fluorescence colocalization.
151
Imaging of Vertical Slices
152
Retinas with and without photoreceptors removed were prepared for imaging in vertical slices.
153
After dissections, retinas were fixed in 4% paraformaldehyde for 2 hours. They were incubated
154
in 0.01M phosphate buffered saline 3 times for 5 minutes on a shaker. The retinas were mounted
155
flat in blocks of 4% agarose. Each agarose block was prepared on a vibratome and sectioned at
156
50-µm increments to obtain vertical retina slices. Retinal slices were mounted on microscope
157
slides, covered with an ethanol-based mounting solution supplemented with 1 µM ethidium
158
homodimer-1, covered with glass cover slips, and imaged.
159
Patch Clamp Electrophysiology
160
After performing the photoreceptor peel technique, the retina was mounted ganglion cell-side
161
down on a glass-bottom petri dish. The retina was held in place with a PTFE membrane
162
containing manually perforated 1 mm-diameter access holes and weighted by a titanium
163
horseshoe. The retina was perfused with buffered Ames’ media at 30 °C at a flow rate of 4-
164
6mL/min. A silver-silver/chloride pellet (#64-1305, Warner Instruments, Hamden, CT) reference
165
electrode was placed in solution. Borosilicate glass (outer diameter=1.2 mm, inner diameter=1.0
166
mm, Sutter Instruments, Novato, CA, #B120-69-10) was pulled to obtain resistances of 7-14MΩ
167
(when pipette was filled with internal solution described below) using a laser pipette puller
168
(Sutter Instruments, Novato, CA, #P-2000). Pipettes were filled with internal solution containing
10
169
the following components (mM): 120 K-Gluconate, 10 NaCl, 3 ATP-Mg, 0.3 GTP-Na, 10
170
HEPES, 0.5 EGTA, 10 Phosphocreatine Disodium Hydrate, adjusted to pH=7.3 with 1M KOH
171
(Sigma, Louis, MO) and an osmolality of 270 mOsm. K-gluconate was substituted for 140 mM
172
Cs-gluconate, and 20 mM tetraethylammonium (TEA) was added to the external solution for
173
tests blocking potassium channels. The junction potential of 14 mV and 15.2 mV was corrected
174
in control and blocker solutions, respectively (Neher 1992). An EPC9 amplifier (HEKA,
175
Bellmore, New York) under control of PatchMaster software was used for data acquisition. Input
176
resistance from recorded neurons averaged 2.78±1.31 GΩ, and membrane capacitance averaged
177
4.59±1.55 pF (mean±std).
178
Whole-cell voltage-clamp recordings were collected from ON-type bipolar cell somas. Cells
179
were held at -80 mV and stepped in increments of 10 mV from -80 mV to 60 mV. Each step was
180
held for 100ms. Current-voltage (I-V) curves were obtained from the sustained current in
181
response to each voltage step. In current clamp, the holding current was 0 pA, current injections
182
of -10 pA to 20 pA in steps of 2 pA were delivered through the pipette for 6 or 8 seconds.
183
Membrane potentials were estimated from the median membrane potential and oscillation
184
amplitudes were estimated from the standard deviation of the membrane potential during the
185
current step.
186 187
Results
188
Photoreceptor Removal
189
The photoreceptor peel technique effectively removes photoreceptor outer segments, inner
190
segments, and somas from normal retina. EGFP fluorescence identifies ON-type bipolar cells in
191
the INL, but the fluorescence is scattered by photoreceptors when imaging from the subretinal
11
192
side (Figure 2A). After removing photoreceptors, the contours of bipolar cells are readily
193
visualized (Figure 2B). The majority of photoreceptor outer segments were removed from the
194
retina after the first few peels, however 10-15 cycles of the process were necessary to provide
195
adequate bipolar cell visualization and access. Remaining photoreceptor soma layers ranged
196
from 0-3 depending on the location. In the rd10 retina, the objective of the photoreceptor peel
197
process is to loosen the hypertrophied glial seal covering the INL. Only 1-2 gentle peels were
198
necessary. In rd10 retina, the fluorescence from the ON-type bipolar cells is visible without the
199
photoreceptor peel technique, but we found that successful “giga-seal” recordings were easier to
200
obtain, due to less soiling of the patch pipette tip from the glial cell contact.
201
After mechanically removing photoreceptors in three retina preparations, we performed a live-
202
dead assay to assess the health of bipolar cells. Calcein blue-AM and ethidium homodimer-1
203
served as the live and dead assay, respectively. Figure 3 illustrates representative images of how
204
the assays labeled the retina. The ON-type bipolar cells are labeled with EGFP (Figure 3A).
205
Figure 3D illustrates the cell contours in the INL. The live assay stains cells in the INL including
206
the ON-type bipolar cells (Figure 3B). The labeling of the live assay appears to be limited to the
207
outer surface of the retina which restricts our evaluation to the most exposed neurons. From 34
208
image regions, we identified 886 ON-type bipolar cells, 691 (77.9%) of which were co-labeled
209
by the live assay. In the same image regions, 151 cells were labeled by the dead assay in total.
210
Though these cells were labeled by the dead assay, none were colocalized with the EGFP-labeled
211
bipolar cells (Figure 3C). This result demonstrates that the majority of labeled ON-type bipolar
212
cells have intact cell membranes, suggesting healthy cells. The same live-dead assay procedure
213
was applied to two rd10 retina preparations after applying one or two peels with the filter paper.
214
From 23 image regions, we identified 868 ON-type bipolar cells (Figure 3E), 679 (78.2%) of
12
215
which were labeled with the live assay (Figure 3F). The contours of cell somas are illustrated in
216
the infrared image (Figure 3H). 103 cells were labeled with the dead assay (Figure 3G), but none
217
were colocalized with the EGFP-labeled bipolar cells. 0.3% of cells labeled by the live assay
218
were also labeled by the dead assay. The live-dead assay was also performed on 4 rd10 retinas
219
without applying the peel technique in order to assess potential effects of the peel technique. The
220
staining performance of the live assay was heterogeneous and less robust, which may be due to
221
the glial seal. In 11 image areas with discernible labeling, 236 (73%) of EGFP labeled bipolar
222
cells were colocalized with the live assay. None were colocalized with the dead assay.
223
Vibratome sectioning of the retina reveals that the retina is still morphologically intact. The
224
normal retina without photoreceptor peeling contains 10-14 layers of photoreceptors somas
225
above the INL (Figure 4A). ON-type bipolar cells are identified in the INL by EGFP
226
fluorescence. Neurons not labeled with EGFP in the INL are OFF-type bipolar cells, horizontal
227
cells, amacrine cells, and Müller cells. ON-type bipolar cell terminals are also observed in the
228
lower strata of the IPL. Ganglion cells and displaced amacrine cells in the ganglion cell layer are
229
also labeled by ethidium homodimer-1. Figure 4B depicts the retina after the photoreceptor peel
230
technique has been performed. The majority of photoreceptor somas in the outer nuclear layer
231
have been removed. The number of somas removed is largely determined by the number of peel
232
cycles. The remaining inner retina appears intact, with a similar structural morphology to the
233
control retina.
234
Electrophysiological Measurements
235
Whole-cell patch clamp recordings were performed on ON-type bipolar cells in wholemount
236
mouse retina after photoreceptor removal. The transmembrane current was recorded in response
237
to voltage steps from a holding potential of -80 mV (Figure 5A). Membrane voltage was stepped 13
238
from -80 mV to +60 mV in steps of 10 mV. Outward currents are observed above approximately
239
-40 mV and increase above -10 mV. Steady-state current responses are plotted as a function of
240
step potential in Figure 5B. Recordings with 140 mM Cs-gluconate internal and 20 mM TEA in
241
the external solution significantly reduced the outward currents indicating that they are largely
242
dependent on potassium channels (Figure 5B). Though reduced, the potassium current at steady-
243
state increases approximately linearly with membrane potential for step potentials greater than
244
approximately -10 mV. The addition of potassium channel blockers also revealed small inward
245
currents when voltage was stepped to -30 and -40 mV.
246
In current clamp, we injected current into ON-type bipolar cells (N=30) in the normal retina and
247
measured the voltage response. Injections ranged from -10 pA to 20 pA in steps of 2 pA.
248
Negative current steps resulted in hyperpolarization of the membrane potential and reduction in
249
the amplitude of membrane oscillations (Figure 6A). Positive current steps elevated the
250
membrane potential, which resulted in fast depolarizing transients. Figure 6B shows the relation
251
between resting membrane potential and the membrane potential at which oscillations were
252
maximal for 30 bipolar cells. The average membrane potential at which the maximum oscillation
253
amplitude occurred was -40.6±4.7 mV (mean±std). The average resting membrane potential of
254
these cells was -58.8±7.6 mV.
255
Discussion
256
Mechanically peeling photoreceptors from wholemount retina using filter paper is an effective
257
technique to expose neurons in the INL. The removal of retina layers is relatively uniform across
258
the treated area (Figure 4), the retina is kept in viable physiological condition, and the progress
259
of removing photoreceptors can be monitored. Live-dead assays indicate that majority of
260
remaining bipolar cells are still viable after the procedure (Figure 3). In our investigation, we 14
261
used a transgenic mouse line expressing EGFP in ON-type bipolar cells to provide a priori
262
discrimination of these neurons. This strategy can be adapted with other transgenic lines to allow
263
visualization of other retinal cells. Adjusting the number of times the filter paper is applied and
264
removed provides some control over the number of photoreceptor somas removed from the
265
retina. This feature is beneficial as it allows one to efficiently adjust which layer(s) of neurons
266
will be removed and exposed. For example, in this study we determined that only a couple peels
267
were necessary to carefully remove the glial seal covering the INL of the rd10 degenerate retina
268
in contrast to the 10-15 required to remove the majority of photoreceptor outer segments, inner
269
segments, and somas in the normal retina. This investigation did not test the ability of filter paper
270
to remove the inner limiting membrane.
271
Patch clamp electrophysiology with this preparation provided baseline bipolar cell measurements
272
similar to those obtained in traditional slice and dissociated cell preparations (Klumpp et al.
273
1995; Zenisek and Matthews 1998; Frech and Backus 2004). I-V curves reveal strong outward
274
rectifying potassium currents, initiating at membrane potentials more positive than -40 mV.
275
Though complete blockade was not observed, the K+ channel blockers cesium and TEA
276
significantly reduced this outward current and revealed small inward currents that are consistent
277
with previous reports of calcium channels in bipolar cell terminals (De La Villa 1998). Further,
278
application of current steps into bipolar cells revealed that the bipolar cell membrane potentials
279
transition into and out of oscillatory behavior peaking at approximately -40 mV. The magnitude
280
of the responses showed dependence on membrane potential and appears to be in agreement with
281
previously reported bipolar cell calcium spikes (Zenisek and Matthews 1998; Protti et al. 2000;
282
Palmer 2006; Baden et al. 2011). The correspondence between the various techniques is
283
encouraging because each preparation approach is different. However, the advantage of this
15
284
preparation is that it exposes INL neurons in a wholemount retina, which may preserve certain
285
aspects of signal processing.
286
Adjusting the number of peel steps makes the photoreceptor removal technique adaptable to
287
different types of experiments. Proteomics or DNA analyses can be performed on either
288
individual retinal layers that are removed or on the retinal tissue remaining after performing the
289
peel technique, and, because no chemicals or enzymes are used during the processing,
290
contamination by chemicals and enzymes is minimal. Individual bipolar cell subclass signaling
291
pathways can be examined without contamination from broad photoreceptor input to the entire
292
retina. Activity could be elicited, for example, by targeting optogenetic channels to single bipolar
293
cell subtypes and then measuring the response characteristics of postsynaptic ganglion cells or
294
amacrine cells. A few peel steps could be used to remove light input into photoreceptors without
295
the use of pharmacological blockers. The technique is also useful for recording INL responses to
296
electrical stimulation from microelectrode arrays. A large portion of the retina wholemount
297
would remain intact and cover the stimulating electrode. This provides a better model of a human
298
implant because electric current paths will be directed through the retina rather than shunting
299
around the tissue as in the retinal slice or dissociated cell preparations.
300
Removing retinal layers in rodent wholemount retina has also been reported using other
301
methodologies (Shiosaka et al. 1984; Hayashida et al. 2004; Enoki et al. 2006; Feigenspan and
302
Babai 2015). One method uses a mounting medium to hold the retina before slicing through the
303
retina horizontally with a fine blade (Enoki et al. 2006; Feigenspan and Babai 2015). Drawbacks
304
to this preparation are that it is difficult to align the retina relative to the blade, which is
305
necessary for precise removal of a specific layer. Care must also be taken to prevent exposing the
306
retina to high temperatures when covering it in mounting medium and to maintain a healthy 16
307
retinal environment throughout the entire process. Manual horizontal slicing with a blade has
308
also been described atop filter paper without mounting media (Hayashida et al. 2004). Another
309
method uses enzymatic trypsin digestion and two sets of filter paper attached on both retinal
310
surfaces to remove retinal layers (Shiosaka et al. 1984). Cell exposure to digestion enzymes
311
loosens the extracellular matrix holding neurons in place, resulting in as few as 5 applications of
312
filter paper to remove the entire ONL and INL from the retina in comparison to the 10-15
313
required to remove the ONL without enzymatic digestion in our preparation. However, enzymes
314
must be used with caution because their effect on ion channels may fundamentally change neural
315
activity (Klumpp et al. 1995; Holt et al. 2001).
316
No chemicals or digestion enzymes are used during our photoreceptor peel technique. It does not
317
require the retina to be submerged in a mounting medium. Nor does it require the alignment of a
318
fine blade to cut the retina. The limitation of the photoreceptor peel technique is that it severs the
319
connection between photoreceptors and INL neurons. As such, it is not possible to use this
320
technique and record light-activated responses from the remaining retina. Additionally, the
321
removal of photoreceptors will alter the input to bipolar cells and horizontal cells, which could
322
affect baseline retinal activity. In our study, we did not directly assess the integrity of lateral
323
connections in the INL. Maintenance of such connections is a potential benefit of this approach
324
compared to retinal slice, but must be proven through further experimentation.
325
In conclusion, we have developed and assessed a method for improving the access to bipolar
326
cells of both normal and degenerate mammalian wholemount retina for patch clamp recording.
327
Photoreceptor and glial membranes are removed by repeated application and peeling of
328
commercially available filter paper to the retina. Cell viability and electrophysiological measures
329
suggest
that
the
bipolar
cells
are
viable
after
this
procedure. 17
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Appendix
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(none)
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Footnotes
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Acknowledgements
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(none)
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Grants
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This research has been supported by the National Eye Institute (Grants RO1 EY022931 and
338
EY022931-S1), National Science Foundation (Grant CBET 1343193), the USC Institute for
339
Biomedical Therapeutics, and a Departmental grant from Research to Prevent Blindness.
340
Disclosures
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The authors do not have any disclosures with regard to this study.
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Tables
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24
Figure 1. Flow diagram for the photoreceptor peel technique.
Figure 2. Photoreceptor peeling allows visualization of bipolar cells.
EGFP
Live Assay
Dead Assay
Infrared
A
B
C
D
E
F
G
H
Figure 3. Live-dead assay of the retina after performing the photoreceptor peel technique.
A
ON-type Bipolar Cells Ethidium Homodimer-1
ONL INL IPL RGC Layer
50µm
Figure 4. Vertical retina slice view of the photoreceptor peel technique.
B
Figure 5. I-V curves recorded from ON-type bipolar cells in normal retina.
Figure 6. Bipolar cell response to current injections in current clamp in normal retina.
