Name: simultaneous recording of electroretinography and visual evoked potentials in anesthetized rats
Uploaded: 2016-07-01T14:00:00.000+00:00
Duration: 10 min 30 s
Description: Watch this Scientific Journal Video about Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats at JoVE.com

Funding for this project was provided by the National Health and Medical Research Council (NHMRC) 1046203 (BVB, AJV) and Melbourne Neuroscience Institute Fellowship (CTN).

All experimental procedures were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, set out by the National Health and Medical Research Council in Australia. Ethics clearance was obtained from the University of Melbourne, Science Faculty, Animal Ethics Committee (approval number 0911322.1).
1. Pre-implantation of Chronic VEP Electrodes
Note: If concurrent ERG and VEP signals are to be collected animals must be surgically implanted with VEP electrodes at least 1 week prior to signal collection.
<ol>
	<li>Sterilize the surgical bench prior to experimentation by cleaning with chlorhexidine (0.5% in 70% ethanol). Autoclave all surgical equipment before use. Cover the animal with a sterilized surgical drape. Ensure all experimenters wear surgical masks, gowns and sterilized gloves.</li>
	<li>Induce anesthesia with 3 - 3.5% isoflurane with O2 at a flow rate of 3 L/min. Maintain anesthesia at 1.5% and 2 L/min throughout the surgery. Ensure sufficient depth of anesthesia by the absence of paw pinch reflex.</li>
	<li>Apply 1% carboxymethylcellulose sodium on the cornea to prevent drying of the eyes.</li>
	<li>Shave a 30 mm x 30 mm area over the forehead, posterior to the eyes and anterior to the ears.</li>
	<li>Place animal on a heat pad (37 &#730;C) to maintain body temperature and stabilize animals&#39; head with a stereotaxic frame.</li>
	<li>Disinfect the shaved area with 10% povidone-iodine three times. Avoid the use of alcohol-based antiseptics for area near the eye, being consistent with the Standard of Practice set out by the Association of Surgical Technologists.</li>
	<li>Make a median-sagittal incision on the head with a scalpel and from this excise a ~ 20 mm diameter circle of dermal tissue to expose the cranial bone.</li>
	<li>Remove underlying periosteum by scraping and drying with gauze to expose the coronal and sagittal cranial sutures.</li>
	<li>Using a dental burr attached to a drill, trepan two holes (0.7 mm diameter, depth ~ 1 mm) through the skull on both hemispheres at the stereotaxic co-ordinates: 7 mm caudal to bregma, 3 mm lateral to midline.</li>
	<li>Screw in stainless steel screws (diameter 0.7 mm, length 3 mm, sterilized with chlorhexidine) into the two pre-made holes up to a depth of ~ 1 mm (2 mm of screw exposed) to allow firm anchorage. This contacts the dura without damaging the underlying cortical tissue.</li>
	<li>Prepare surgical area for dental amalgam by drying the cranial bone with gauze, and retracting loose skin with two 3 - 0 sutures at ~ 4 and 8 o&#39;clock.</li>
	<li>Spread dental amalgam over the exposed skull to secure the screw electrodes (stainless steel screws described in step 1.10) in place. Ensure ~ 1.5 mm of the screws remain exposed for recording.</li>
	<li>Remove the retraction sutures.</li>
	<li>Inject 0.5% carprofen subcutaneously (5 mg/kg) for analgesia and saline (sodium chloride 0.9%, 1.5 ml) subcutaneously for fluid replacement.</li>
	<li>Allow animal to recover in separate cages. Do not leave animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Do not return animal to the company of other animals until it has fully recovered from surgery (minimum 5 days).</li>
	<li>Continue to administer 0.5% carprofen subcutaneously for analgesia (5 mg/kg) once a day for 4 days.</li>
	<li>Record ERG and VEP 1 week following surgery.</li>
</ol>
2. ERG and VEP Recording&#160;
<ol>
	<li>Data collection preparation
	<ol>
		<li>Use computer software to simultaneously trigger stimulus and acquire data2 according to the settings recommended below.
		<ol>
			<li>Amplify the signals3 (ERG: &#215;1,000, VEP: x10,000) with the gain internally set by an isolated pre-amplifier and amplifier, and with both eyes matched for impedance.</li>
			<li>Set sampling rate for the ERG to 4 kHz over a 650 msec recording window (2,560 points), To do this, click the tab for &#34;time base&#34; in the data acquisition software (for name and version of software see Table for Materials), select &#34;2,560&#34; for Samples, and &#34;500 ms&#34; for time which will return a 650 msec recording window.
			<ol>
				<li>Use the same method to set the sampling rate for the VEP to 10 kHz over a 250 msec epoch. Allow a 10 msec pre-stimulus baseline for both ERG and VEP recordings. To do this, click the &#34;Setup&#34; tab; select &#34;Stimulator&#34; to bring up a new dialogue window; in that window select &#34;pulse&#34; from the drop down list for &#34;Mode&#34;; and set the value for &#34;delay&#34; to &#34;10 ms&#34;.</li>
			</ol>
			</li>
			<li>Set ERG band-pass filtering to 0.3 - 1,000 Hz (- 3 dB). This is done by clicking &#34;Bio Amplifier&#34; in the data acquisition software. Then set the value for &#34;High Pass&#34; to &#34;0.3Hz&#34;, and the value for &#34;low pass&#34; to &#34;1 kHz&#34;.</li>
			<li>Using the abovementioned method in 2.1.1.3, set VEP band-pass settings to 0.1 - 100 Hz (- 3 dB) as recommended by the International Society for Clinical Electrophysiology of Vision (ISCEV) for human VEP recordings6.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Electrode preparation
	<ol>
		<li>Custom-make the ERG active/inactive and VEP active/inactive electrodes by attaching silver wire or an alligator clip to an electrode lead, respectively2. Commercially obtain ground electrode.</li>
		<li>For the 4 custom-made electrodes, cut the male end from the electrode lead extension. Remove 1 cm of the outer polytetrafluoroethylene insulation coating with a scalpel blade ensuring the inner wire is not damaged.</li>
		<li>Pre-fashion the ERG inactive electrodes by cutting a 70 mm length of silver wire (0.3 mm thickness) and forming a loop ~ 8 mm in diameter to encircle the rat eye. Prepare a uniform circle by shaping the loop on a 1 ml pipette tip.</li>
		<li>Pre-fashion VEP inactive electrodes by cutting a 70 mm length of silver and forming an ellipse ~ 8 mm in length-wise diameter to hook onto rat incisors.</li>
		<li>Pre-fashion the ERG active electrodes by cutting a 30 mm length of silver wire and forming a small loop to gently contact the rat cornea (~ 1-2 mm in diameter)</li>
		<li>Securely attach electrodes (2 ERG active, 2 ERG inactive, 1 VEP inactive) to the electrode lead by entwining the silver with the exposed inner wire.</li>
		<li>Insulate excess exposed metal with masking tape to reduce photovoltaic artifacts.</li>
		<li>On the ERG inactive electrodes stick a small piece of hook-and-loop fastener (~ 5 mm &#215; 20 mm) to the masking tape to enable stable attachment to the rodent neck strap.</li>
		<li>Attach alligator clip to the inner wire of the electrode leads to make the VEP active electrodes.</li>
		<li>Prior to recordings, electroplate the exposed surfaces of the silver wires (i.e., the inactive ring and active tip) with chloride using a 9 V DC source for 20 sec to improve signal conduction.
		<ol>
			<li>To do this, immerse the silver tip of the ERG electrode wire (acting as the anode of a primary cell) into normal saline; connect the other end of this electrode wire to the positive terminal of a 9 V battery.</li>
			<li>Connect another wire (the cathode) to the negative terminal of the battery, and immerse the other end into saline as well. Disconnect after 20 sec and observe the sliver tip of the ERG electrode wire to be coated evenly in white color. 
			Note: Prepare new ERG electrodes for each experimental session (~ up to 8 hr) to ensure patency of the chloride coating.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Animal preparation
	<ol>
		<li>Dark-adapt the animals overnight (&#8805; 8 hr) prior to recordings in a light tight room. Ensure maximal dark adaptation by turning off room lights, closing all doors and blinds. Minimize light leakage by placing light-proof materials around junctions of doors/windows and placing computer screens outside thick black curtains.</li>
		<li>Conduct animal preparation in a dark room with the aid of dim red light-emitting diode (LED; 17.4 cd.m-2, &#955;max = 600 nm) to sustain rod sensitivity.</li>
		<li>Anesthetize rat by injecting ketamine/xylazine (60 : 5 mg/kg) intramuscularly. Confirm sufficient depth of anesthesia by the absence of a paw pinch reflex.</li>
		<li>To maintain sedation, administer a further dose of anesthesia (50% of initial dose) after 50 min if necessary.</li>
		<li>For additional topical anesthesia apply one drop of 0.5% proxymetacaine to each eye, and blink off excess fluid.</li>
		<li>For pupil dilation apply one drop of 0.5% tropicamide to each eye, then dry off excess fluid.</li>
	</ol>
	</li>
	<li>ERG and VEP electrode positioning
	<ol>
		<li>Place animal on the ERG platform in front of the Ganzfeld bowl situated in the Faraday cage. Avoid using an electrical heating pad, as it can introduce electrical noise into the electrophysiological recordings. Note: The platform is attached to a circulated heated water platform to maintain body temperature.</li>
		<li>Secure animal to platform with a strip of hook-and-loop fastener placed firmly but not tightly around the nape.</li>
		<li>Hook the inactive VEP electrode around bottom incisors of anesthetized rat.</li>
		<li>Position the ERG inactive electrodes by encircling the scleral ring non-invasively around the eye&#39;s equator. Stabilize this by attaching electrodes to the hook-and-loop fastener strip around the nape. Repeat for the contralateral eye.</li>
		<li>Fasten VEP active electrodes by attaching alligator clips to stainless-steel screws pre-implanted on the skull.</li>
		<li>Place a small drop of 1% carboxymethylcellulose sodium on cornea prior to placement of the ERG active electrode to improve signal quality. Note: The viscose fluid also helps maintain corneal hydration throughout experimentation to minimize the formation of desiccation-type cataract in rodents7.</li>
		<li>Place a small drop of 1% carboxymethylcellulose sodium on the lower incisors to improve contact of the VEP inactive electrode and thus signal quality.</li>
		<li>Position the ERG active electrodes to lightly touch the central corneal surface using a micromanipulator attached to a custom-built stereotaxic arm.</li>
		<li>Insert 2 - 5 mm of the ground needle electrode (stainless steel) subcutaneously into the tail.</li>
		<li>If necessary dry any excess fluid from the inferior eyelid prior to recording to improve signal quality.</li>
		<li>Slide platform closer to the Ganzfeld bowl ensuring the animal&#39;s eyes align with the opening of the bowl to enable even illumination of both retinas (see step 2.4.1).</li>
		<li>Close the Faraday cage to reduce extraneous noise.</li>
	</ol>
	</li>
	<li>Data collection
	<ol>
		<li>Use a dim test-flash (- 0.52 log cd.s.m-2) to assess whether electrode placement is satisfactory2. Under control conditions this would result in an ERG amplitude of ~ 800 &#956;V and an inter-eye variability of no greater than 10%. If required reposition electrodes.</li>
		<li>Following the test-flash allow animals to dark-adapt for 10 min in complete darkness prior to recording.</li>
		<li>Present flashes of light stimuli using a Ganzfeld bowl whilst collecting ERG and VEP signals simultaneously over a ~ 500 msec time-window. Progress from dimmer to brighter light levels in order to maintain sufficient dark-adaptation for particular waveforms.</li>
		<li>Collect signals over a range of luminous energies to elicit STR, b-wave and a/b-wave waveforms of the ERG. Average more signals at the dimmer light levels (20 repeats) and less at the brighter luminous energies (1 repeat). Gradually lengthen the inter-stimulus interval from 1 to 180 sec from dimmest to the brightest light level. See Table 1 for an example protocol.</li>
		<li>To isolate the ERG rod and cone responses, utilize a paired-flash paradigm8. Initiate four flashes at 1.52 log cd.s.m-2 with a 500 msec inter-stimulus interval2 in-between. Digitally subtract the cone waveform (3rd or 4th flash) from the mixed waveform (1st flash) to derive the putative rod response.</li>
		<li>To record VEP signals, average 20 repeats at the brighter luminous energies (i.e., - 0.52 to 1.52 log cd.s.m-2, 5 sec inter-stimulus interval). Note that the first flash in this sequence returns the conventional dark-adapted ERG response.</li>
		<li>Allow 1 - 3 min for re-adaptation after (20) VEP sweeps before the next brighter ERG step, depending on the luminous energy.</li>
		<li>After completion of data collection, euthanize the anesthetized animal with intracardiac injection of pentobarbital sodium (325 mg/ml, 3 ml).</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Waveform</td>
			<td>Stimulus light energy (log cd.s.m-2)</td>
			<td>Number of repeats</td>
			<td>interstimulus interval (sec)</td>
		</tr>
		<tr>
			<td>STR</td>
			<td>-6.24</td>
			<td>20</td>
			<td>2</td>
		</tr>
		<tr>
			<td>STR</td>
			<td>-5.93</td>
			<td>20</td>
			<td>2</td>
		</tr>
		<tr>
			<td>STR</td>
			<td>-5.6</td>
			<td>20</td>
			<td>2</td>
		</tr>
		<tr>
			<td>STR</td>
			<td>-5.33</td>
			<td>20</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-4.99</td>
			<td>10</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-4.55</td>
			<td>10</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-4.06</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-3.51</td>
			<td>5</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-3.03</td>
			<td>1</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-2.6</td>
			<td>1</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Rod b-wave</td>
			<td>-1.98</td>
			<td>1</td>
			<td>15</td>
		</tr>
		<tr>
			<td>Mixed a-/b-wave</td>
			<td>-1.38</td>
			<td>1</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Mixed a-/b-wave</td>
			<td>-0.94</td>
			<td>1</td>
			<td>30</td>
		</tr>
		<tr>
			<td rowspan="2">Flash 1: Mixed a-/b-wave Average of 20: VEP</td>
			<td rowspan="2">-0.52</td>
			<td rowspan="2">20</td>
			<td>5</td>
		</tr>
		<tr>
			<td>(90 sec before next)</td>
		</tr>
		<tr>
			<td rowspan="2">Flash 1: Mixed a-/b-wave Average of 20: VEP</td>
			<td rowspan="2">0.04</td>
			<td rowspan="2">20</td>
			<td>5</td>
		</tr>
		<tr>
			<td>(120 sec before next)</td>
		</tr>
		<tr>
			<td rowspan="2">Flash 1: Mixed a-/b-wave Average of 20: VEP</td>
			<td rowspan="2">0.58</td>
			<td rowspan="2">20</td>
			<td>5</td>
		</tr>
		<tr>
			<td>(180 sec before next)</td>
		</tr>
		<tr>
			<td rowspan="2">Flash 1: Mixed a-/b-wave Average of 20: VEP</td>
			<td rowspan="2">1.2</td>
			<td rowspan="2">20</td>
			<td>5</td>
		</tr>
		<tr>
			<td>(180 sec before next)</td>
		</tr>
		<tr>
			<td rowspan="2">Flash 1: Mixed a-/b-wave Average of 20: VEP</td>
			<td rowspan="2">1.52</td>
			<td rowspan="2">20</td>
			<td>5</td>
		</tr>
		<tr>
			<td>(180 sec before next)</td>
		</tr>
		<tr>
			<td>Cone a-/b-wave&#160;</td>
			<td>1.52</td>
			<td>4</td>
			<td>0.5</td>
		</tr>
	</tbody>
</table>
Table 1. ERG and VEP Recording Protocol Using a Range of Stimulus Energy. Stimulus presentations progress from dim (top) to bright (bottom) flashes, with sufficient inter-stimulus interval to ensure dark adaption. At the end of protocol, repetition of four flashes with short interval is presented to elicit the cone mediated response.
3. Analysis of ERG Waveforms&#160;
Note: ERG and VEP analysis has been described in detail previously.3,9,10 The following sections provide a brief overview.
<ol>
	<li>Export signals in digital voltage-time format to a spreadsheet software for data analysis.</li>
	<li>Rod photoreceptoral function
	<ol>
		<li>Model the leading edge of the a-wave PIII with a delayed Gaussian (Equation 1)11. 
		PIII (i,t) = RmPIII&#8729; [1 - exp(-i&#8729; S (t - td)2)] for t &#62; td (Equation 1)</li>
		<li>Optimize the fit over an ensemble of two brightest luminous energies12,13 (i.e., 1.22 and 1.52 log cd.s.m-2).</li>
		<li>Model up to 90% of the a-wave amplitude to avoid post-receptoral intrusions14. 
		Note: The model returns the saturated amplitude (RmPIII, &#181;V), sensitivity (S, m2.cd-1.s-3) and delay (td, msec) of the photoreceptoral response.</li>
	</ol>
	</li>
	<li>Rod bipolar cell function
	<ol>
		<li>Digitally subtract the PIII model (see above) from the mixed waveforms to return the mixed PII with overlying oscillatory potentials.</li>
		<li>To extract the rod PII from the mixed PII, digitally subtract the cone response (3rd or 4th flash at 1.52 log cd.s.m-2) from the mixed PII (1st flash at 1.52 log cd.s.m-2).</li>
		<li>Then, apply a low-pass filter to the waveform (46.9 Hz, -3 dB, Blackman window) to remove oscillatory potentials. The remaining waveform is the rod PII response10.</li>
		<li>Extract the rod PII peak amplitude and plot it against all stimulus intensities (below -2 log cd.s.m-2 and rod-isolated PII at 1.52 log cd.s.m-2 )10.</li>
		<li>Model these data using a hyperbolic function (Equation 2), which provides a measure of inner retinal cell integrity. 
		V (i) = Vmax (in/(in + kn)) (Equation 2) 
		Note: This equation returns maximal PII response (Vmax, &#181;V), 1/sensitivity (k,log cd.s.m-2) and the slope of the function (n)15.</li>
	</ol>
	</li>
	<li>Cone bipolar cell function 
	Note: As the cone response is taken at a single intensity (1.52 log cd.s.m-2) the amplitude and timing are retuned at this light level.
	<ol>
		<li>Extract maximal cone PII response2,16.</li>
		<li>Extract implicit time to which this maximal response corresponds2,16.</li>
	</ol>
	</li>
	<li>Ganglion cell function
	<ol>
		<li>As the STR is a small signal, apply a low pass filter with 50 Hz notch to waveform to eliminate high frequency and line noise (46.9 Hz, -3 dB, Blackman window).</li>
		<li>Extract maximal pSTR response3,17.</li>
		<li>Extract implicit time to which this maximal response corresponds3,17.</li>
	</ol>
	</li>
</ol>
4. Analysis of VEP Waveforms
<ol>
	<li>Extract maximum and minimum components of the VEP (P1, N1 and P2). For detail see references3,6.</li>
	<li>Express amplitudes as trough-to-peak amplitudes from their preceding peak or trough (P1N1 and N1P2)3,6.</li>
	<li>Extract implicit time (it) to which this maximal response corresponds (P1it, N1it, P2it)3,6.</li>
</ol>

<ol>
	<li>Nguyen, C. T. O., Vingrys, A. J., Bui, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+omega-3+fatty+acids+and+ganglion+cell+function.">Dietary omega-3 fatty acids and ganglion cell function.</a> Invest Ophthalmol Vis Sci. 49, 3586-3594 (2008).</li><li>Weymouth, A. E., Vingrys, 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=Rodent+electroretinography:+methods+for+extraction+and+interpretation+of+rod+and+cone+responses.">Rodent electroretinography: methods for extraction and interpretation of rod and cone responses.</a> Prog Retin Eye Res. 27, 1-44 (2008).</li><li>Tsai, T. I., Bui, B. V., Vingrys, 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=Effect+of+acute+intraocular+pressure+challenge+on+rat+retinal+and+cortical+function.">Effect of acute intraocular pressure challenge on rat retinal and cortical function.</a> Invest Ophthalmol Vis Sci. 55, 1067-1077 (2014).</li><li>Cowey, A., Franzini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinal+origin+of+uncrossed+optic+nerve+fibres+in+rats+and+their+role+in+visual+discrimination.">The retinal origin of uncrossed optic nerve fibres in rats and their role in visual discrimination.</a> Exp Brain Res. 35, 443-455 (1979).</li><li>Weinstein, G. W., Odom, J. V., Cavender, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visually+evoked+potentials+and+electroretinography+in+neurologic+evaluation.">Visually evoked potentials and electroretinography in neurologic evaluation.</a> Neurol Clin. 9, 225-242 (1991).</li><li>Odom, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+evoked+potentials+standard+(2004).">Visual evoked potentials standard (2004).</a> Doc Ophthalmol. 108, 115-123 (2004).</li><li>Ridder, W. H., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+cataract+development+in+anesthetized+mice.">Causes of cataract development in anesthetized mice.</a> Exp Eye Res. 75, 365-370 (2002).</li><li>Nixon, P. J., Bui, B. V., Armitage, J. A., Vingrys, 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=The+contribution+of+cone+responses+to+rat+electroretinograms.">The contribution of cone responses to rat electroretinograms.</a> Clin Experiment Ophthalmol. 29, 193-196 (2001).</li><li>Bui, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+electroretinogram+to+understand+how+intraocular+pressure+elevation+affects+the+rat+retina.">Using the electroretinogram to understand how intraocular pressure elevation affects the rat retina.</a> J Ophthalmol. 2013, 262467 (2013).</li><li>Nguyen, C. T., Vingrys, A. J., Bui, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+omega-3+fatty+acids+and+ganglion+cell+function.">Dietary omega-3 fatty acids and ganglion cell function.</a> Invest Ophthalmol Vis Sci. 49, 3586-3594 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+measure+of+the+electrical+activity+of+human+rod+photoreceptors+using+electroretinography.">A quantitative measure of the electrical activity of human rod photoreceptors using electroretinography.</a> Vis Neurosci. 5, 379-387 (1990).</li><li>Birch, D. G., Hood, D. C., Locke, K. G., Hoffman, D. R., Tzekov, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+electroretinogram+measures+of+phototransduction+in+cone+and+rod+photoreceptors+-+Normal+aging,+progression+with+disease,+and+test-retest+variability.">Quantitative electroretinogram measures of phototransduction in cone and rod photoreceptors - Normal aging, progression with disease, and test-retest variability.</a> Arch Ophthalmol. 120, 1045-1051 (2002).</li><li>Bui, B. V., Vingrys, 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=Development+of+receptoral+responses+in+pigmented+and+albino+guinea-pigs+(Cavia+porcellus).">Development of receptoral responses in pigmented and albino guinea-pigs (Cavia porcellus).</a> Doc Ophthalmol. 99, 151-170 (1999).</li><li>Robson, J. G., Saszik, S. M., Ahmed, J., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+and+cone+contributions+to+the+a-wave+of+the+electroretinogram+of+the+macaque.">Rod and cone contributions to the a-wave of the electroretinogram of the macaque.</a> J Physiol. 547, 509-530 (2003).</li><li>Severns, M. L., Johnson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+care+and+fitting+of+Naka-Rushton+functions+to+electroretinographic+intensity-response+data.">The care and fitting of Naka-Rushton functions to electroretinographic intensity-response data.</a> Doc Ophthalmol. 85, 135-150 (1993).</li><li>Bui, B. V., Fortune, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+electroretinogram+amplitude+growth+during+light+adaptation+in+pigmented+rats.">Origin of electroretinogram amplitude growth during light adaptation in pigmented rats.</a> Vis Neurosci. 23, 155-167 (2006).</li><li>Bui, B. V., Fortune, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+contributions+to+the+rat+full-field+electroretinogram.">Ganglion cell contributions to the rat full-field electroretinogram.</a> J Physiol. 555, 153-173 (2004).</li><li>Tremblay, F., Laroche, R. G., Debecker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Electroretinographic+Diagnosis+of+the+Incomplete+Form+of+Congenital+Stationary+Night+Blindness.">The Electroretinographic Diagnosis of the Incomplete Form of Congenital Stationary Night Blindness.</a> Vision Res. 35, 2383-2393 (1995).</li><li>Bayer, A. U., Keller, O. N., Ferrari, F., Maag, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+glaucoma+with+neurodegenerative+diseases+with+apoptotic+cell+death:+Alzheimer's+disease+and+Parkinson's+disease.">Association of glaucoma with neurodegenerative diseases with apoptotic cell death: Alzheimer's disease and Parkinson's disease.</a> Am J Ophthalmol. 133, 135-137 (2002).</li><li>Wostyn, P., Audenaert, K., De Deyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+abnormal+high+trans-lamina+cribrosa+pressure+difference:+A+missing+link+between+Alzheimer's+disease+and+glaucoma.">An abnormal high trans-lamina cribrosa pressure difference: A missing link between Alzheimer's disease and glaucoma.</a> Clinical Neurology and Neurosurgery. 110, 753-754 (2008).</li><li>Yucel, Y. H., Zhang, Q. A., Weinreb, R. N., Kaufman, P. L., Gupta, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+retinal+ganglion+cell+loss+on+magno-,+parvo-,+koniocellular+pathways+in+the+lateral+geniculate+nucleus+and+visual+cortex+in+glaucoma.">Effects of retinal ganglion cell loss on magno-, parvo-, koniocellular pathways in the lateral geniculate nucleus and visual cortex in glaucoma.</a> Prog Retin Eye Res. 22, 465-481 (2003).</li><li>Gupta, N., Yucel, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+changes+can+we+expect+in+the+brain+of+glaucoma+patients.">What changes can we expect in the brain of glaucoma patients.</a> Survey of Ophthalmology. 52, 122-126 (2007).</li><li>Kong, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+aging+and+diet+restriction+on+retinal+function+during+and+after+acute+intraocular+pressure+injury.">Impact of aging and diet restriction on retinal function during and after acute intraocular pressure injury.</a> Neurobiol Aging. 33, 1115-1125 (2012).</li><li>Bui, B. V., Sinclair, A. J., Vingrys, 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=Electroretinograms+of+albino+and+pigmented+guinea-pigs+(Cavia+porcellus).">Electroretinograms of albino and pigmented guinea-pigs (Cavia porcellus).</a> Aust N Z J Ophthalmol. 26, 98-100 (1998).</li><li>Jobling, A. I., Wan, R., Gentle, A., Bui, B. V., McBrien, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+and+choroidal+TGF-beta+in+the+tree+shrew+model+of+myopia:+isoform+expression,+activation+and+effects+on+function.">Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function.</a> Exp Eye Res. 88, 458-466 (2009).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+dark-adapted+electroretinogram.">Dissecting the dark-adapted electroretinogram.</a> Doc Ophthalmol. 95, 187-215 (1998).</li><li>Robson, J. G., Frishman, L. 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+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Prog Retin Eye Res. 39, 1-22 (2014).</li><li>Hudnell, H. K., Boyes, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparability+of+rat+and+human+visual-evoked+potentials.">The comparability of rat and human visual-evoked potentials.</a> Neurosci Biobehav Rev. 15, 159-164 (1991).</li><li>Charng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conscious+wireless+electroretinogram+and+visual+evoked+potentials+in+rats.">Conscious wireless electroretinogram and visual evoked potentials in rats.</a> PLoS One. 8, e74172 (2013).</li><li>Hetzler, B. E., Berger, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ketamine-Induced+Modification+of+Photic+Evoked-Potentials+in+the+Superior+Colliculus+of+Hooded+Rats.">Ketamine-Induced Modification of Photic Evoked-Potentials in the Superior Colliculus of Hooded Rats.</a> Neuropharmacology. 23, 473-476 (1984).</li></ol>

The authors have no disclosures relevant to this work.

The ERG and VEP are objective measures of visual function from the retina and cortex, respectively. The advantage of simultaneous recording is that a more comprehensive view of the entire visual pathway is afforded. Specifically, the complementary information from their concurrent assessment could provide a clearer delineation of the site of injury in the visual pathway (e.g., for disorders with overlapping ERG yet distinct VEP manifestations18, when optic neuropathy may co-exist with primary cerebral atrophy19,20, or when VEP loss may be confounded by manifestation of injuries at several locations in visual pathway21,22). By measuring the ERG and VEP concurrently, an index of the gain between retinal and cortical response can also be derived. This may provide a useful tool to detect subtle pathological changes. The current protocol allows for ERG and VEP measurement in commonly used laboratory rats but can readily be adapted to other mammalian species23-25. ERG and VEP waveforms from rodents provide a reasonable preclinical surrogate for responses observed in human eyes26-28.
By designing a specific stimulus protocol, both ERG and VEP response can be obtained during a single recording session. Table 1 shows a progression in light levels with appropriate consideration of the recovery time between consecutive flashes. This protocol provides a balance between the need to maximize signal-to-noise characteristics and to limit recording time within the anesthetic window provided by a single dose of ketamine:xylazine. Therefore, this technique may be useful for an objective quantitative measure of visual function for research into basic physiology and disease.
A comprehensive assessment of the visual system can be achieved by concurrently assessing bilateral retinal responses and visually evoked cortical responses. However, each technique can also be conducted in isolation and monocularly instead of binocularly to simplify the procedure. The current protocol describes scotopic ERG and VEP signals chosen to isolate the rod-pathway given that rats have a rod-dominated retina. If light adapted responses are of greater interest to the study, it is also possible to conduct photopic ERG and VEP signals by pre-adaption to a background light.
One major limitation of this technique is the need to conduct the procedure under anaesthetized conditions to enable stable electrode placement. Nevertheless this approach provides robust signal-to-noise characteristics enabling detection of subtle treatment changes.
Due to the small amplitude of the STR and its sensitivity to light adaptation, several steps need to be closely observed to ensure successful recording of this response. Firstly, sufficient dark adaptation needs to be implemented, which includes overnight dark adaptation (&#8805; 8 hr), electrode placement under dim red lighting (17.4 cd.m-2, &#955;max = 600 nm), and re-dark adaptation following a dim test-flash (10 min for - 0.52 log cd.s.m-2). Furthermore, the signal-to-noise characteristics of the STR can be improved by averaging over multiple signals (i.e., 20 signals) collected with short inter-stimulus intervals (i.e., 2 sec). One of the advantages of this comprehensive assessment of both eyes and cortices is to enable comparison to the contralateral recording3. As such, particular care should be taken in electrode making (i.e., same sized and shaped electrodes), to ensure minimal inter-eye and inter-cortical variability.
Given the extensive usage of both ERG and VEP techniques to provide in vivo measures of the visual pathway and its disease-related processes, it would be useful to collate other pathway-specific protocols (e.g., ON/OFF or cone sub-type specific), and perform simultaneous ERG/VEP recordings with different stimulus modalities (e.g., flicker, pattern, sawtooth) to extend the application of this technique in clinical diagnoses. Another logical step of this application in the future would also be to record the ERG and VEP concurrently from conscious29, freely-moving animals to avoid anesthetic influences on neural physiology30.

Measurement of the electroretinogram (ERG) and visual evoked potential (VEP) provide useful quantitative assessments of the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. This manuscript describes a protocol for the recording and analysis of ERG and VEP responses in a commonly used laboratory model, the rat.
The ERG provides an index of the functional integrity of a number of key retinal cell classes by quantifying the retina&#39;s gross electrical response to a flash of light. A coordinated series of ionic fluxes initiated by light onset and offset, produce detectable changes in voltage that can be measured using surface electrodes placed outside the eye. The resultant waveform represents the combination of a series of well-defined components, differing in amplitude, timing and frequency. A substantial body of research has shown that these components are relatively well conserved across many vertebrate retinae and that the components can be separated from each other. By judiciously selecting the stimulus (flash stimulus, background, interstimulus interval) conditions and choosing specific features of the composite waveform to analyze one can be confident of returning a measure of a specific group of retinal cells1,2. These characteristics underlie the utility and hence the widespread applications of the ERG as a non-invasive measure of retina function. This manuscript focuses on the methodology for measuring the ERG and analyzing its features to return information about some of the major cell classes in the retina, namely photoreceptors (the PIII component), bipolar cells (the PII component) and retinal ganglion cells (the positive scotopic threshold response or pSTR).
The VEP provides an assay of the cortical response to light; first originating from the retina and thereafter communicated serially via the optic nerve, optic tract, thalamus (lateral geniculate nucleus, LGN) and optic radiation to area V1 of the cortex3. In rodents, the majority (90 - 95%) of optic nerve fibers from each eye decussate4 and innervate the contralateral mid-brain. Unlike the ERG, it is as yet not possible to attribute different components of the VEP to specific cell classes,5 thus changes anywhere along the visual pathway could affect the VEP waveform. Nevertheless, the VEP is a useful non-invasive measure of visual performance and visual pathway integrity. The VEP, when used in conjunction with the ERG, can provide a more complete assessment of the visual system (i.e., retina/visual pathway).
ERG and VEP recordings can be conducted in isolation or in combination, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres in anesthetized rats. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

<table><tbody><tr><td>Alligator clip</td><td>generic brand</td><td>HM3022</td><td>Stainless steel 26 mm clip for connecting VEP screw electrodes to cables</td></tr><tr><td>Bioamplifier</td><td>ADInstruments</td><td>ML 135</td><td>For amplifying ERG and VEP signals</td></tr><tr><td>Carboxymethylcellulose sodium 1.0%</td><td>Allergan</td><td>CAS 0009000-11-7</td><td>Viscous fluid for improving signal quality of the active ERG electrode</td></tr><tr><td>Carprofen 0.5%</td><td>Pfizer Animal Health Group</td><td>CAS 53716-49-7</td><td>Proprietary name: Rimadyl injectable (50 mg/ml). For post-surgery analgesia, diluted to 0.5% (5 mg/ml) in normal saline</td></tr><tr><td>Chlorhexadine 0.5%</td><td>Orion Laboratories</td><td>27411, 80085</td><td>For disinfecting surgical instruments</td></tr><tr><td>Circulating water bath</td><td>Lauda-K&amp;ouml;nigshoffen</td><td>MGW Lauda</td><td>For maintaining body temperature of the anesthetized animal during surgery and electrophysiological recordings</td></tr><tr><td>Dental amalgam</td><td>DeguDent GmbH</td><td>64020024</td><td>For encasing the electrode-skull assembly to make it more robust</td></tr><tr><td>Dental burr</td><td>Storz Instruments, Bausch and Lomb</td><td>#E0824A</td><td>A miniature drill head of ~ 0.7 mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws</td></tr><tr><td>Drill</td><td>Bosch</td><td>Dremel 300 series</td><td>An automatic drill for trepanning</td></tr><tr><td>Electrode lead</td><td>Grass Telefactor&amp;nbsp;</td><td>F-E2-30</td><td>Platinum cables for connecting silver wire electrodes to the amplifier</td></tr><tr><td>Faraday Cage</td><td>custom-made</td><td></td><td>Ensures light proof to maintain dark adaptation. Encloses the Ganzfeld setup to improve signal to noise ratio</td></tr><tr><td>Gauze swabs</td><td>Multigate Medical Products Pty Ltd</td><td>57-100B</td><td>For drying the surgical incision and exposed skull surface during surgery</td></tr><tr><td>Ganzfeld integrating sphere</td><td>Photometric Solutions International</td><td></td><td>Custom designed light stimulator: 36 mm diameter, 13 cm aperture size</td></tr><tr><td>Velcro</td><td>VELCRO Australia Pty Ltd</td><td>VELCRO Brand Reusable Wrap</td><td>Hook-and-loop fastener to secure the electrodes and the animal on the recording platform</td></tr><tr><td>Isoflurane 99.9%</td><td>Abbott Australasia Pty Ltd</td><td>CAS 26675-46-7</td><td>Proprietary Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery</td></tr><tr><td>Ketamine&amp;nbsp;</td><td>Troy Laboratories</td><td>Ilium Ketamil</td><td>Proprietary name: Ketamil Injection, Brand: Ilium. Pharmaceutical-grade anesthetic for electrophysiological recording</td></tr><tr><td>Luxeon LEDs</td><td>Phillips Lighting Co.</td><td></td><td>For light stimulation twenty 5 W and one 1 W LEDs.</td></tr><tr><td>Micromanipulator</td><td>Harvard Apparatus</td><td>BS4 50-2625</td><td>Holds the ERG active electrode during recordings</td></tr><tr><td>Needle electrode</td><td>Grass Telefactor&amp;nbsp;</td><td>F-E2-30</td><td>Subcutaneously inserted in the tail to serve as the ground electrode for both the ERG and VEP</td></tr><tr><td>Phenylephrine 2.5% minims&amp;nbsp;</td><td>Bausch and Lomb</td><td>CAS 61-76-7</td><td>Instilled with Tropicamide to achieve maximal dilation for ERG recording</td></tr><tr><td>Povidone iodine 10%</td><td>Sanofi-Aventis</td><td>CAS 25655-41-8</td><td>Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 ml</td></tr><tr><td>Powerlab data acquisition system</td><td>ADInstruments</td><td>ML 785</td><td>Controls the LEDs</td></tr><tr><td>Proxymetacaine 0.5%</td><td>Alcon Laboratories&amp;nbsp;</td><td>CAS 5875-06-9</td><td>For corneal anaesthesia during ERG recordings</td></tr><tr><td>Saline solution</td><td>Gelflex</td><td></td><td>Non-injectable, for electroplating silver wire electrodes</td></tr><tr><td>Scope Software</td><td>ADInstruments</td><td>version 3.7.6</td><td>Simultaneously triggers the stimulus via the Powerlab system and collects data</td></tr><tr><td>Silver (fine round wire)</td><td>A&amp;amp;E metal</td><td>0.3 mm</td><td>Used to make active and inactive ERG electrodes, and the inactive VEP electrode</td></tr><tr><td>Stainless streel screws&amp;nbsp;</td><td>MicroFasterners</td><td></td><td>0.7 mm shaft diameter, 3 mm in length to be implanted over the primary visual cortex and serve as the active VEP electrodes</td></tr><tr><td>Stereotaxic frame</td><td>David Kopf</td><td>Model 900</td><td>A small animal stereotaxic instrument for locating the primary visual cortices according to Paxinos &amp;amp; Watson&#039;s 2007 rat brain atlas coordinates</td></tr><tr><td>Surgical blade</td><td>Swann-Morton Ltd.</td><td>0206</td><td>For incising the area of skin overlaying the primary visual cortex to implant the VEP electrodes</td></tr><tr><td>Suture</td><td>Shanghai Pudong Jinhuan Medical Products Co.,Ltd</td><td></td><td>3-0 silk braided suture non-absorbable, for skin retraction during VEP electrode implantation surgery</td></tr><tr><td>Tobramycine eye ointment 0.3%</td><td>Alcon Laboratories&amp;nbsp;</td><td>CAS 32986-56-4</td><td>Proprietary name: Tobrex. Prophylactic antibiotic ointment applied around the skin wound after surgery</td></tr><tr><td>Tropicamide 0.5%</td><td>Alcon Laboratories&amp;nbsp;</td><td>CAS 1508-75-4</td><td>Proprietary name: 0.5% Mydriacyl eye drop, Instilled to achieve mydriasis for ERG recording</td></tr><tr><td>Xylazine</td><td>Troy Laboratories</td><td>Ilium Xylazil-100</td><td>Pharmaceutical-grade anesthetic for electrophysiological recording</td></tr><tr><td>Pipette tip</td><td>Eppendorf Pty Ltd</td><td>0030 073.169</td><td>Eppendorf epTIPS 100 - 5,000 ml, for custom-made electrodes</td></tr><tr><td>Microsoft Office Excel</td><td>Microsoft</td><td>version 2010</td><td>spreadsheet software for data analysis</td></tr><tr><td>Lethabarb Euthanazia Injection</td><td>Virbac (Australia) Pty Ltd</td><td>LETHA450</td><td>325 mg/ml pentobarbital sodium for rapid euthanazia</td></tr></tbody></table>

simultaneous recording of electroretinography and visual evoked potentials in anesthetized rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

The ERG a-wave (&#62; -1.38 log cd.s.m-2), b-waves (&#62; - 4.99 log cd.s.m-2) STRs (&#60; - 4.99 log cd.s.m-2) and the VEPs (&#62; - 0.52 log cd.s.m-2) were recorded simultaneously (Figure 1 and 3). At very dim flashes, a positive STR (pSTR) is seen at approximately 110 msec after the flash, and a negative STR (nSTR) at approximately 220 msec (Figures 1 and 2). An ERG with a large b-wave, peaks between 50 to 100 msec after the onset of a moderate flash which can be analyzed for its PII response (Figures 1 and 2). At this stimulus energy, the negative a-wave before the peak is negligible. At brighter luminous energies the negative deflection a-wave becomes more prominent which can be quantified with the PIII response (Figure 2). The scotopic VEP waveform shows a negative response (P1N1; 15 - 70 msec window) followed by a positive deflection (N1P2; 30 - 100 msec) (Figures 3 and 4).
<img alt="Figure 1" src="/files/ftp_upload/54158/54158fig1.jpg" /> 
Figure 1. Group Average ERG Waveforms. The ERG alters with increasing stimulus intensity. Numbers to the left of waveform indicate the luminous exposure used to elicit the waveform. Note the different amplitude and time scales for each panel. At dimmer luminous energies the positive and negative components of the scotopic threshold response can be elicited (pSTR, nSTR). As stimulus energies get brighter, the a and b-wave response can be assayed, and a paired-flash paradigm allows the cone response to be measured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54158/54158fig2.jpg" /> 
Figure 2. ERG analysis. (A) Rod photoreceptor function can be assayed by using a PIII to model the a-wave. A-waves at 1.22 and 1.52 log cd.s.m-2 (unfilled circles, &#9675;) are fit as an ensemble with a PIII (grey lines, Equation 1) to 90% of the minimum which returns RmPIII (saturated amplitude, &#181;V) S (sensitivity, m2.cd-1.s-3) and td (timing delay, msec) parameters. (B) Rod bipolar cell function (mean &#177; SEM) can be assayed by modelling the intensity response series of the rod PII (unfilled circles &#9675;) with a Naka-Rushton function (grey line). This returns Vmax (saturated amplitude, &#181;V), k (1/sensitivity, log cd s m-2) and n (slope). (C) Retinal ganglion cell function is assayed at dim luminous energies and quantified by pSTR peak amplitude (pSTRamp) and timing (pSTRit). (D) Cone bipolar cell function is elicited with a paired-flash paradigm quantified by cone PII peak amplitude (cone PIIamp) and timing (cone PIIit). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54158/54158fig3.jpg" /> 
Figure 3. Group Average VEP Waveforms. The shape of the VEP waveform alters with increasing stimulus energy. Numbers to the left of the waveform indicate the luminous exposure used to elicit the waveform. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54158/54158fig4.jpg" /> 
Figure 4. VEP Analysis and Intensity Response Function. (A) Amplitude analysis of the VEP is taken as peak to trough (P1N1) and trough to peak (N1P2) amplitudes. The implicit times (it) of these responses is also returned (P1it, N1it, P2it). (B) The VEP P1N1 amplitude (mean &#177; SEM) increases with increasing stimulus energy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54158/54158fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

simultaneous-recording-electroretinography-visual-evoked-potentials

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.3K Views.  University of Melbourne. This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats. 

Video: Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

Implantation and Recording of Wireless Electroretinogram and Visual Evoked Potential in Conscious Rats

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both laboratory and clinical settings. Currently, preclinical ERG and VEP measurements are performed with anesthesia to ensure stable electrode placements. However, the very presence of anesthesia has been shown to contaminate normal physiological responses. To overcome these anesthesia confounds, we develop a novel platform to assay ERG and VEP in conscious rats. Electrodes are surgically implanted sub-conjunctivally on the eye to assay the ERG and epidurally over the visual cortex to measure the VEP. A range of amplitude and sensitivity/timing parameters are assayed for both the ERG and VEP at increasing luminous energies. The ERG and VEP signals are shown to be stable and repeatable for at least 4 weeks post surgical implantation. This ability to record ERG and VEP signals without anesthesia confounds in the preclinical setting should provide superior translation to clinical data.

We show surgical implantation and Recording procedures to measure visual electrophysiological signals from the eye (electroretinogram) and brain (visual evoked potential) in conscious rats, which is more analogous to the human condition where recordings are conducted without anesthesia confounds.

The full-field electroretinogram (ERG) and visual evoked potential (VEP) are useful tools to assess retinal and visual pathway integrity in both ...

Behavior

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Electroretinogram Recording for Infants and Children under Anesthesia to Achieve Optimal Dark Adaptation and International Standards

Electroretinogram (ERG) is the only clinical objective test available to assess retinal function. Full-field ERG (ffERG) measures the panretinal rod and cone photoreceptor function as well as inner retinal function and is an important measure in the diagnosis and management of inherited retinal diseases as well as inflammatory, toxic, and nutritional retinopathies. Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid and reliable dark-adapted (scotopic) and light-adapted (photopic) ffERG responses. Performing ffERG in infants and children is challenging and often requires general anesthesia in the operating room. However, maintaining retinal dark adaptation in the operating room is becoming increasingly difficult given the numerous light sources from anesthesiology monitoring systems and other equipment. A practical and widely applicable method for ffERG testing is described in the operating room that optimizes retinal dark adaptation. The method reduces operating room time by dark-adapting the patient before general anesthesiology is instituted. The operating room is modified for dark adaptation and any remaining light source in the darkened operating room is minimized with the use of a modified portable foldable darkroom that encloses the patient&#8217;s head and the ERG examiner during ffERG scotopic recordings. The simple method adheres to ffERG international standards and provides valid reliable scotopic and photopic ffERG recordings that are critical to assess objective retinal function in this young age group where subjective assessment of visual function such as visual acuity and visual fields are not possible. Furthermore, the ffERG is the gold standard clinical test in detecting early onset inherited retinal diseases including Leber congenital amaurosis where approved gene therapy has become available. In sedated conditions, very low amplitude ffERG signals can be detected due to minimal orbicularis muscle activity interference, which is particularly relevant in patients after gene therapy to detect improved amplitude responses.

Adhering to international standards and maintaining retinal dark adaptation are critical to acquire valid full-field electroretinogram responses in the diagnosis and management of inherited retinal diseases. A practical protocol using a portable darkroom is provided to obtain full-field&#160;electroretinogram for infants and children under sedation or general anesthesia in the operating room setting.

Electroretinogram (ERG) is the only clinical objective test available to assess retinal function. Full-field ERG (ffERG) measures the panretinal rod and ...

Medicine

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Preparing a Rat Model to Assess Visual Pathway Integrity Using an Electrophysiology Setup

Source: Nguyen, C. T. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54158">Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats.</a>&#160;J. Vis. Exp. (2016)
This video demonstrates the preparation of a rat model to study retinal health and visual pathway integrity. Visual evoked potential and electroretinography reference and active electrodes are connected to the rat, followed by aligning the platform with the rat in front of the Ganzfeld bowl for even retinal illumination and closing the Faraday cage to avoid external interference.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Direct-Coupled Electroretinogram Recording of the Retinal Pigment Epithelium in a Mouse

Source: Miyagishima, K. J., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61491">Direct-Coupled Electroretinogram (DC-ERG) for Recording the Light-Evoked Electrical Responses of the Mouse Retinal Pigment Epithelium.</a> J. Vis. Exp. (2020).
This video demonstrates the recording of a direct-coupled electroretinogram (DC-ERG) on a dark-adapted, anesthetized mouse retinal pigment epithelium (RPE). After applying dilating drops, the animal is placed in a recording setup, and the capillary electrode filled with a salt buffer is placed on the cornea. After establishing the baseline, DC-ERG is recorded.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Mouse ...

Imaging and Optical Techniques

Recording of Visual Evoked Potentials via the Skull Electrodes in a Rat Model

Source: You, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/52934">Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination.</a> J. Vis. Exp. (2015).
This video demonstrates the process of measuring visual evoked potentials or VEPs in a dark-adapted, anesthetized rat with pre-implanted skull electrodes, which record the electrical signals from the brain&#39;s visual cortex in response to light stimuli.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. VEP ...

Concurrent Electroencephalographic and Local Field Potential Recordings in an Anesthetized Rat

Source: Kang, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56447">Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent.</a> J. Vis. Exp. (2017).
This video demonstrates the procedure for concurrent recordings of co-localized electroencephalography (EEG) and local field potentials (LFPs) in an anesthetized rat. The process involves preparing the rat and attaching stimulating electrodes to its whisker pads. An EEG spider electrode is placed on the rat&#39;s skull for surface EEG measurements. At the same time, a multi-channel microelectrode is inserted into a specific brain region through a burr hole in the skull to record neural activity from a localized brain area.

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

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