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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#246;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 ~0.7mm 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&#160;</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&#160;</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 watt and one 1 watt 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&#160;</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&#160;</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&#160;</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&#38;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&#160;</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 &#38; Watson&#39;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&#160;</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&#160;</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&#160;</td>
			<td>Eppendorf Pty Ltd</td>
			<td>0030 073.169</td>
			<td>Eppendorf epTIPS 100 - 5000 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>

Watch this Scientific Journal Video about Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats at JoVE.com

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

12.2K 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

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Extracting Visual Evoked Potentials from EEG Data Recorded During fMRI-guided Transcranial Magnetic Stimulation

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological effects. Specifically, by creating a transient interference with the normal activity of a target region and measuring changes in an electrophysiological signal, we can establish a causal link between the stimulated brain area or network and the electrophysiological signal that we record. If target brain areas are functionally defined with prior fMRI scan, TMS could be used to link the fMRI activations with evoked potentials recorded. However, conducting such experiments presents significant technical challenges given the high amplitude artifacts introduced into the EEG signal by the magnetic pulse, and the difficulty to successfully target areas that were functionally defined by fMRI. Here we describe a methodology for combining these three common tools: TMS, EEG, and fMRI. We explain how to guide the stimulator&#39;s coil to the desired target area using anatomical or functional MRI data, how to record EEG during concurrent TMS, how to design an ERP study suitable for EEG-TMS combination and how to extract reliable ERP from the recorded data. We will provide representative results from a previously published study, in which fMRI-guided TMS was used concurrently with EEG to show that the face-selective N1 and the body-selective N1 component of the ERP are associated with distinct neural networks in extrastriate cortex. This method allows us to combine the high spatial resolution of fMRI with the high temporal resolution of TMS and EEG and therefore obtain a comprehensive understanding of the neural basis of various cognitive processes.

This paper describes a method for collecting and analyzing electroencephalography (EEG) data during concurrent transcranial magnetic stimulation (TMS) guided by activations revealed with functional magnetic resonance imaging (fMRI). A method for TMS artifact removal and extraction of event related potentials is described as well as considerations in paradigm design and experimental setup.

Transcranial Magnetic Stimulation (TMS) is an effective method for establishing a causal link between a cortical area and cognitive/neurophysiological ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

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 ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

Stimulus-specific Cortical Visual Evoked Potential Morphological Patterns

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using 128-channel high-density electroencephalography (EEG). The specific aim of the described stimuli and analyses is to examine whether it is feasible to replicate previously reported CVEP morphological patterns elicited by an apparent motion stimulus, designed to simultaneously stimulate both ventral and dorsal central visual networks, using object and motion stimuli designed to separately stimulate ventral and dorsal visual cortical networks.&#160; Four visual paradigms are presented: 1. Randomized visual objects with consistent temporal presentation. 2. Randomized visual objects with inconsistent temporal presentation (or jitter).&#160; 3. Visual motion via a radial field of coherent central dot motion without jitter.&#160; 4. Visual motion via a radial field of coherent central dot motion with jitter.&#160; These four paradigms are presented in a pseudo-randomized order for each participant.&#160; Jitter is introduced in order to view how possible anticipatory-related effects may affect the morphology of the object-onset and motion-onset CVEP response.&#160; EEG data analyses are described in detail, including steps of data exportation from and importation to signal processing platforms, bad channel identification&#160;and removal, artifact rejection, averaging, and categorization of average CVEP morphological pattern type based upon latency ranges of component peaks. Representative data show that the methodological approach is indeed sensitive in eliciting differential object-onset and motion-onset CVEP morphological patterns and may, therefore, be useful in addressing the larger research aim. Given the high temporal resolution of EEG and the possible application of high-density EEG in source localization analyses, this protocol is ideal for the investigation of distinct CVEP morphological patterns and the underlying neural mechanisms which generate these differential responses.&#160; &#160; &#160;&#160;

In this paper, we present a protocol to investigate differential cortical visual evoked potential morphological patterns through stimulation of ventral and dorsal networks using high-density EEG. Visual object and motion stimulus paradigms, with and without temporal jitter, are described. Visual evoked potential morphological analyses are also outlined.&#160;&#160;

This paper presents a methodology for the recording and analysis of cortical visual evoked potentials (CVEPs) in response to various visual stimuli using ...