Supported by Career Development Award from Research to Prevent Blindness, NIH grants EY19312 and EY19543 (VJK), as well as by unrestricted grant from Research to Prevent Blindness and EY02687 (Department of Ophthalmology and Visual Sciences at Washington University).

1. Making Electrodes
<ol>
	<li>Prepare glass electrodes. Weigh 120 mg agar and mix it in 10 mL distilled water (final agar concentration 1.2%). Melt the agar solution in hot water bath. Fill glass capillaries (we use Word Presision Instruments TW100-4 capillaries with the following dimensions: length = 100 mm, OD/ID = 1/0.75 mm, and internal volume = 44 &mu;L) with the agar solution using plastic syringe. Solidify the agar at room temperature for 10 min. Thus, up to ~200 capillaries can be filled (the rest of the agar mixture can be stored at -4 &deg;C and re-used several times).</li>
	<li>Cut the capillaries into halves using a diamond knife.</li>
	<li>Soak the resulting glass electrodes in mouse electrode solution for at least 24 hours at 4 &deg;C. Store the electrodes at 4 &deg;C and use them over the period of 3-4 months.</li>
</ol>
2. Setting up the Experiment
<ol>
	<li>Dark-adapt a mouse overnight. Use wild type (for instance, C57Bl/6) mice for rod-driven transretinal recordings and T&alpha;-/- mice lacking rod signaling8 for cone transretinal recordings.</li>
	<li>Prepare 1 L perfusion solution, 100 mL electrode solution, and 20 mL incubation solution (see below) and filter the perfusion solution trough a Millipore 0.45 &mu;m or 0.22 &mu;m filter (optional).</li>
	<li>Calibrate the light source (505-nm LED or halogen bulb optical stimulator) using a photometer placed at the plane of the retina. In the case of LED light source, use an optical diffuser to achieve light uniformity.</li>
	<li>Prepare the perfusion chamber for experiment (chamber constructions may vary). Our chamber is made of 3 mm Plexiglas, with a bottom made from the lid of a small plastic Petri dish, and has an active perfusion volume of ~400 &mu;L (L = 25 mm, W = 8 mm, H &asymp; 2 mm). Fill the lower electrode space of the chamber with electrode solution containing 2 mM L-glutamate and 10 mM BaCl2. Using plastic tubing and a glass capillary, make a connection between the solution in lower electrode space (made of plastic tubing connector attached to the chamber bottom) and the electrode holder. Make sure there are no bubbles in the electrode, the connecting tubing, or the electrode holder (purge the solution using plastic syringe if needed).</li>
	<li>Mount the recording chamber on the stage of the microscope (optional: the microscope can be mounted on an anti-vibration table and shielded within a Faraday cage to minimize mechanical and electromagnetic noise). Connect the lower electrode holder to the positive headstage pole of the differential amplifier (e.g. Warner Instruments DP-311 amplifier). Center and align the chamber lower electrode opening with the stimulus light spot. Connect the perfusion line to the recording chamber and turn the perfusion on.</li>
	<li>Bubble the perfusion solution with 95% O2/5% CO2 while incubating it in 40 &deg;C water bath. CO2 adjusts the pH of the solution to 7.2. The solution flows from the bottle to the recording chamber by gravity passing through a ceramic resistor reheating it to 36-37 &deg;C. To reduce heat loss, the heater should be located as close to the recording chamber as possible, ideally on the stage of the microscope.</li>
	<li>With a flow regulator adjust the flow rate to about 1 mL/min.</li>
	<li>Connect the upper electrode to the second (negative) pole of the headstage using a second electrode holder. Bind the thermocouple probe to the upper electrode using an O-ring. Make sure that the electrode and thermocouple tips are close together so that the temperature reading is taken as close to the center of the retina as possible. Immerse the tip of the upper electrode in the perfusion solution and place it in the center of the chamber. Allow 10-15 min for baseline stabilization.</li>
	<li>Adjust the DC voltage for the heater so that the temperature of the solution is 36-37 &deg;C.</li>
</ol>
3. Isolating the Mouse Retina
<ol>
	<li>Under dim red light, euthanize the dark-adapted mouse with CO2 and cervical dislocation. Optional: make a reference mark on each mouse eyeball by cauterizing the most dorsal point of the sclera with a cautery pen or heated dissecting pin. All subsequent procedures are performed under infrared light using infrared image converters fit to a dissecting microscope.</li>
	<li>Dissect out the eyes with curved scissors by applying gentle pressure and stretching the skin on the sides of each eye. Hemisect both eyeballs under infrared illumination using microscissors or a razor blade.</li>
	<li>Remove the cornea and lens. Remove as much of the vitreous as possible.</li>
	<li>Peel the first retina from the pigment epithelium layer and, if necessary, thoroughly clean it with forceps to remove remaining granules of pigment epithelium. We are typically using whole retina for both rod and cone transretinal recordings. However, if you wish to specifically record from the dorsal part of mouse retina that has more M/L-cones9, before peeling the retina remove the ventral part of the eyecup with a razor blade or microscissors using the cautery mark as a reference.</li>
	<li>Place the second hemisected eyeball in a small Petri dish with incubation solution and incubate the preparation in a light-tight box saturated with pure oxygen until use. Typically, under these conditions the second mouse eyecup can be stored for several hours at room temperature and used for recordings.</li>
	<li>Using a glass or plastic pipette transfer the isolated retina to a drop of perfusion solution (ca. 300 &mu;l) on the lid of a Petri dish. Add 5-6 small drops of electrode solution containing BaCl2 to pre-incubate the retina with BaCl2. Place a square piece (ca. 5x5 mm) of back side corner-greased (we use Dow corning 111 valve lubricant and sealant) Millipore filter paper (0.45 &mu;m HARG type) with pre-made hole in its center (2-2.5 mm in diameter) to the same solution drop. Using forceps position the retina on top of the filter paper (photoreceptor side up) and press its edges on the periphery to flat-mount it. The retina should be centered over the opening in the filter paper.</li>
	<li>Using forceps transfer the filter paper with the retina to the perfusion chamber and place it above the stimulus spot-aligned lower electrode space. Slightly press the greased edges of the filter paper to bond it with the chamber.</li>
	<li>Place the upper electrode over the retina center (slightly touch the photoreceptor layer). Centering the upper electrode on the retina increases the signal amplitude (up to ~20-40% as compared to placing it closer to the retina edge). Although such electrode location distorts slightly the effective light stimulus intensity reaching a small sector of the retina (~12-15% of its total area), it is still the optimal way of placing the electrode. The preparation is now ready for transretinal recordings.</li>
</ol>
4. Transretinal Recordings
<ol>
	<li>Depending on your mouse line, record rod or M/L-cone-driven test flash responses from dim to bright intensity of calibrated 505 nm light provided from LED light source or optical stimulator. In the case of our LED light source, the flash intensity is controlled by a combination of computerized control of LED input voltage, switchable resistors, and a set of neutral density filters (we use E-Colour #211 0.9 ND film filters from Rosco Laboratories) placed between the LED and the retina. The duration of the LED test flash (20 ms) is controlled by a computer. If using a halogen bulb optical stimulator, test flash intensity and wavelength can be controlled by a set of neutral density filters and narrow band interference filters, respectively. Test flash duration can be controlled by computer-driven shutters.</li>
	<li>Optional: for maintaining efficient suppression of the glial component of photoresponse with BaCl2 throughout the day, change the lower electrode solution occasionally (e.g. prior to testing each retina) by purging it using a plastic syringe filled with fresh solution.</li>
</ol>
5. Representative Results
Solutions
<ol>
	<li>Mouse retina perfusion solution: 112.5 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 10 mM HEPES (pH 7.2), 20 mM NaHCO3, 3 mM Na succinate, 0.5 mM Na glutamate, 0.02 mM EDTA, and 10 mM glucose. In addition, the solution is supplemented with 2 mM L-glutamate and 10 &mu;M DL-AP-4 to block higher order components of the photoresponse10,11, and with 0.1% MEM vitamins and MEM amino acids solutions (Sigma) to improve retina viability.</li>
	<li>Mouse electrode solution: 140 mM NaCl, 3.6 mM KCl, 2.4 mM MgCl2, 1.2 mM CaCl2, 3 mM HEPES (pH 7.4, NaOH). The solution in the lower electrode space also contains 2 mM L-glutamate to block the synaptic transmission10 and, in addition, 10 mM BaCl2 to suppress the glial component of the photoresponse3,12.</li>
	<li>Retina incubation solution: dissolve 272 mg L-15 medium (Sigma) and 20 mg BSA in 20 mL deionized water. Final L-15 and BSA concentrations are 13.6 mg/mL and 1 mg/mL, respectively. Adjust pH to 7.4 with 1 M HCl.</li>
</ol>
<img alt="Figure 1." src="/files/ftp_upload/3424/3424fig1.jpg" /> 
Figure 1. Schematic diagram showing the isolation of mouse retina for transretinal ERG recordings.
<img alt="Figure 2." src="/files/ftp_upload/3424/3424fig2.jpg" /> 
Figure 2. Photograph of the perfusion chamber and recording electrodes with their holders. Red arrows indicate the direction of perfusion flow.
<img alt="Figure 3." src="/files/ftp_upload/3424/3424fig3.jpg" /> 
Figure 3. Schematic diagram of the experimental setup for transretinal ERG recordings. Red arrows indicate the direction of perfusion flow.
<img alt="Figure 4." src="/files/ftp_upload/3424/3424fig4.jpg" /> 
Figure 4. Representative results: recorded family of mouse rod-driven transretinal responses. Photoresponses were low-pass filtered at 30 Hz (8-pole Bessel), digitized at 1 kHz and stored on a computer for further analysis. Shown traces represent averages of 5-6 responses at dim light intensities and 2-3 responses at saturating light intensities.
<img alt="Figure 5." src="/files/ftp_upload/3424/3424fig5.jpg" /> 
Figure 5. Representative results: recorded family of mouse cone-driven transretinal responses. Photoresponses were low-pass filtered at 30 Hz (8-pole Bessel), digitized at 1 kHz and stored on a computer for further analysis. Shown traces are averages of 5-10 responses at dim light intensities and 3-5 responses at saturating light intensities.

<ol>
	<li>Yau, K. W., Lamb, T. D., Baylor, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+fluctuations+in+membrane+current+of+single+toad+rod+outer+segments.">Light-induced fluctuations in membrane current of single toad rod outer segments.</a> Nature. 269, 78-80 (1977).</li><li>Nikonov, S. S., Kholodenko, R., Lem, J., Pugh, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+features+of+the+S-+and+M-cone+photoreceptors+of+wild-type+mice+from+single-cell+recordings.">Physiological features of the S- and M-cone photoreceptors of wild-type mice from single-cell recordings.</a> J. Gen. Physiol. 127, 359-374 (2006).</li><li>Nymark, S., Heikkinen, H., Haldin, C., Donner, K., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+responses+and+light+adaptation+in+rat+retinal+rods+at+different+temperatures.">Light responses and light adaptation in rat retinal rods at different temperatures.</a> J. Physiol. 567, 923-938 (2005).</li><li>Heikkinen, H., Nymark, S., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cone+photoresponses+obtained+with+electroretinogram+from+the+isolated+retina.">Mouse cone photoresponses obtained with electroretinogram from the isolated retina.</a> Vision Res. 48, 264-272 (2008).</li><li>Frank, R. N., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhodopsin+photoproducts:+effects+on+electroretinogram+sensitivity+in+isolated+perfused+rat+retina.">Rhodopsin photoproducts: effects on electroretinogram sensitivity in isolated perfused rat retina.</a> Science. 161, 487-489 (1968).</li><li>Wang, J. S., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+alternative+pathway+mediates+the+mouse+and+human+cone+visual+cycle.">An alternative pathway mediates the mouse and human cone visual cycle.</a> Curr. Biol. 19, 1665-1669 (2009).</li><li>Kolesnikov, A. V., Tang, P. H., Parker, R. O., Crouch, R. K., Kefalov, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+cone+visual+cycle+promotes+rapid+M/L-cone+pigment+regeneration+independently+of+the+interphotoreceptor+retinoid-binding+protein.">The mammalian cone visual cycle promotes rapid M/L-cone pigment regeneration independently of the interphotoreceptor retinoid-binding protein.</a> J. Neurosci. 31, (2011).</li><li>Calvert, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototransduction+in+transgenic+mice+after+targeted+deletion+of+the+rod+transducin+alpha+-subunit.">Phototransduction in transgenic mice after targeted deletion of the rod transducin alpha -subunit.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 13913-13918 (2000).</li><li>Applebury, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+cone+photoreceptor:+a+single+cone+type+expresses+both+S+and+M+opsins+with+retinal+spatial+patterning.">The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning.</a> Neuron. 27, 513-523 (2000).</li><li>Sillman, A. J., Ito, H., Tomita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mass+receptor+potential+of+the+isolated+frog+retina.+I.+General+properties+of+the+response.">Studies on the mass receptor potential of the isolated frog retina. I. General properties of the response.</a> Vision Res. 9, 1435-1442 (1969).</li><li>Vinberg, F., Koskelainen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+sets+the+physiological+value+of+the+dominant+time+constant+of+saturated+mouse+rod+photoresponse+recovery.">Calcium sets the physiological value of the dominant time constant of saturated mouse rod photoresponse recovery.</a> PLoS One. 5, e13025-e13025 (2010).</li><li>Bolnick, D. A., Walter, A. E., Sillman, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barium+suppresses+slow+PIII+in+perfused+bullfrog+retina.">Barium suppresses slow PIII in perfused bullfrog retina.</a> Vision Res. 19, 1117-1119 (1979).</li></ol>

Authors have nothing to disclose.

The method of rod- and cone-driven transretinal ERG recordings described above is becoming a powerful tool for investigating the function of mouse photoreceptors in both wild type and genetically modified animals. In addition to the easy characterization of basic photoresponse properties, this simple technique provides great response stability during long-lasting experiments performed on close-to-intact retina preparations. Both dark-adapted rod maximal response amplitude and photosensitivity in wild type mice are stable...

<table border="1">
<tbody>
 <tr>
 <td>Material Name</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>DL-AP-4</td>
 <td>&nbsp;</td>
 <td>TOCRIS bioscience</td>
 <td>0101</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>All other reagents</td>
 <td>&nbsp;</td>
 <td>Sigma-Aldrich</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass capillaries</td>
 <td>&nbsp;</td>
 <td>World Precision Instruments</td>
 <td>TW100-4</td>
 <td>For making electrodes</td>
 </tr>
 <tr>
 <td>Filter paper</td>
 <td>HARG</td>
 <td>Millipore</td>
 <td>HABG01300</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Photometer</td>
 <td>&nbsp;</td>
 <td>UDT Instruments</td>
 <td>S350</td>
 <td>For light calibration</td>
 </tr>
 <tr>
 <td>Radiometric silicon 			sensor</td>
 <td>&nbsp;</td>
 <td>UDT Instruments</td>
 <td>221</td>
 <td>For light calibration</td>
 </tr>
 <tr>
 <td>Anti-vibration table</td>
 <td>&nbsp;</td>
 <td>Technical Manufacturing Corporation</td>
 <td>TMC 78-239-02R, TMC63-26012-01</td>
 <td>To minimize mechanical noise</td>
 </tr>
 <tr>
 <td>Air compressor Panther P 15TC</td>
 <td>&nbsp;</td>
 <td>Werther International</td>
 <td>P 15TC</td>
 <td>Connected to anti-vibration table</td>
 </tr>
 <tr>
 <td>Stereomicroscope </td>
 <td>&nbsp;</td>
 <td>LEICA</td>
 <td>MZ9.5</td>
 <td>For mouse eye dissection</td>
 </tr>
 <tr>
 <td>Infrared image converters</td>
 <td>&nbsp;</td>
 <td>B.E.Meyers</td>
 <td>ProwlerTM</td>
 <td>Bound to stereomicroscope</td>
 </tr>
 <tr>
 <td>Differential 			amplifier 
DP-311</td>
 <td>&nbsp;</td>
 <td>Warner Instruments</td>
 <td>DP-311</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Axon Digidata 1440A Digitizer</td>
 <td>&nbsp;</td>
 <td>Molecular Devices</td>
 <td>1440A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dual Channel 8-pole Filter</td>
 <td>&nbsp;</td>
 <td>KROHN-HITE Corporation</td>
 <td>3382</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ceramic resistor</td>
 <td>&nbsp;</td>
 <td>TE Connectivity</td>
 <td>CGS SBCHE618RJ</td>
 <td>For reheating the perfusion solution</td>
 </tr>
 <tr>
 <td>Thermocouple</td>
 <td>T</td>
 <td>Physitemp Instruments</td>
 <td>IT-18</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Temperature monitor</td>
 <td>&nbsp;</td>
 <td>Omega</td>
 <td>DPi32</td>
 <td>Connected to thermocouple</td>
 </tr>
 <tr>
 <td>LED 505 nm</td>
 <td>&nbsp;</td>
 <td>TT Electronics/ Optek Technology</td>
 <td>Digi-key P/N 365-1185-ND</td>
 <td>To apply test flashes/bleaching light </td>
 </tr>
 <tr>
 <td>Cautery pen</td>
 <td>&nbsp;</td>
 <td>Bovie</td>
 <td>AA25</td>
 <td>For marking the dorsal part of the mouse 			eyeball</td>
 </tr>
 <tr>
 <td>pCLAMP 10 Electrophysiology 			Data Acquisition and Analysis Software</td>
 <td>&nbsp;</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

transretinal erg recordings from mouse retina: rod and cone photoresponses

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light whereas cones operate continuously under rapidly changing bright light conditions. Absorption of light by rod- and cone-specific visual pigments in the outer segments of photoreceptors triggers a phototransduction cascade that eventually leads to closure of cyclic nucleotide-gated channels on the plasma membrane and cell hyperpolarization. This light-induced change in membrane current and potential can be registered as a photoresponse, by either classical suction electrode recording technique1,2 or by transretinal electroretinogram recordings (ERG) from isolated retinas with pharmacologically blocked postsynaptic response components3-5. The latter method allows drug-accessible long-lasting recordings from mouse photoreceptors and is particularly useful for obtaining stable photoresponses from the scarce and fragile mouse cones. In the case of cones, such experiments can be performed both in dark-adapted conditions and following intense illumination that bleaches essentially all visual pigment, to monitor the process of cone photosensitivity recovery during dark adaptation6,7. In this video, we will show how to perform rod- and M/L-cone-driven transretinal recordings from dark-adapted mouse retina. Rod recordings will be carried out using retina of wild type (C57Bl/6) mice. For simplicity, cone recordings will be obtained from genetically modified rod transducin &alpha;-subunit knockout (T&alpha;-/-) mice which lack rod signaling8.

Watch this Scientific Journal Video about Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses at JoVE.com

Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

We describe a relatively simple method of transretinal electroretinogram (ERG) recordings for obtaining rod and cone photoresponses from intact mouse retina. This approach takes advantage of the block of synaptic transmission from photoreceptors to isolate their light responses and record them using field electrodes placed across the isolated flat-mounted retina.

There are two distinct classes of image-forming photoreceptors in the vertebrate retina: rods and cones. Rods are able to detect single photons of light ...

transretinal-erg-recordings-from-mouse-retina-rod-cone

Research

JoVE Journal

Neuroscience

17.6K Aufrufe.  Washington University School of Medicine. Wir beschreiben eine relativ einfache Methode der transretinal Elektroretinogramm (ERG) Aufnahmen für den Erhalt von Stäbchen und Zapfen Photomorphosen aus intakten Maus Netzhaut. Dieser Ansatz nutzt die Block der synaptischen Übertragung von Photorezeptoren ihre Lichtantworten isolieren und speichert sie über das Feld über den Elektroden isoliert flachen montiert Netzhaut zu liegen.

Serie: Transretinal ERG Recordings from Mouse Retina: Rod and Cone Photoresponses

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

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 ...

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 ...

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 ...

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 ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Visualizing Axonal Growth Cone Collapse and Early Amyloid &amp;#946; Effects in Cultured Mouse Neurons

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Cone-Enriched Cultures from the Retina of Chicken Embryos to Study Rod to Cone Cellular Interactions

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form of inherited retinal degeneration, retinitis pigmentosa, lose night vision because of mutation driven loss of rod photoreceptors, a phenomenon followed by a progressive loss of function and death of cones leading to blindness. Geneticists have identified many genes with mutations causing this disease, but the first mutations identified questioned the mechanisms of secondary cone degeneration and how a dominant mutation in the rhodopsin gene encoding for the visual pigment expressed exclusively in rods can trigger cone degeneration.
This result of transplantations in a genetic model of the disease led to the concept of cell interactions between rods and cones and of non-cell autonomous degeneration of cones in all genetic forms of retinitis pigmentosa.
Cones comprise 5% of all photoreceptors in humans and only 3% in the mouse, so their study is difficult in these species, but cones outnumber rods in bird species. We have adapted 96-well plates to culture retinal precursors from the retina of chicken embryos at stage 29 of their development. In these primary cultures, cones represent 80% of the cells after in vitro differentiation. The cells degenerate over a period of one week in the absence of serum. Here, we describe the methods and its standardization.
This cone-enriched culture system was used to identify the epithelium-derived cone viability factor (EdCVF) by high content screening of a rat retinal pigmented epithelium normalized cDNA library. Recombinant EdCVF prevents the degeneration of the cones.

We describe a method to obtain primary cultures of cone photoreceptors from the retina of chicken embryos and its use for high content screening.

Human daytime vision relies on the function of cone photoreceptors at the center of the retina, the fovea. Patients suffering from the most prevalent form ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Mouse Retina Isolation and Flattening

After fixing the enucleated eyeball of the euthanized mouse, transfer it to a glass slide and place it under a dissecting microscope. Using forceps, clamp the margin of the cornea to prevent eyeball movement. With the dissecting scissors, cut around the cornea at a depth of approximately one millimeter through the cornea scleral limbus, and then remove the lens and vitreous body.Holding the optic disc at the center, cut the retina radially along the four quadrants, reaching approximately 2/3 of the retina's radius. Clamp one of the scleral edges using toothed forceps. Hold the sclera edge with untoothed forceps and move from the toothed forceps toward the sclera base.Carefully peel the retina. Wet the retina with 200 microliters of PBS using a pipette. Remove excess PBS using absorbent paper, and flatten the retina with a small bristle brush if needed.Slowly add cold methanol drop-by-drop onto the retina's inner surface until it covers completely. Once the retina turns white and develops increased tenacity, gently flatten the retina and remove impurities, such as residual pigment membranes and the vitreous body, with a small bristle brush. After inverting the retina, treat it with methanol as previously demonstrated, and transfer it into a well of 24 well plate, Soak it in methanol and store it at 20 degrees Celsius for one to two hours for immediate immunostaining.Then rinse the retina with PBS after removing methanol from the plate. The retinol ganglion cells of the retinas before and after methanol treatment were visualized using confocal microscopy, which indicated that the 12 months of methanol preservation did not affect the RGC immunostaining.