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 ...

<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>

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 次观看.  Washington University School of Medicine. 我们描述一个transretinal视网膜电图（ERG）录音获得完整的小鼠视网膜的杆状和锥状光反应相对简单的方法。这种做法从感光细胞的突触传递块孤立的光反应，并记录他们使用场电极置于整个平板式的孤立视网膜。