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 electrode...

<ol>
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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. 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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...

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 10...

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 ...

The overall goal of this procedure is to simultaneously measure the electroretinogam and visual evoked potential in a rat. This method can help answer key questions in the field of fissure neuroscience. Such as whether the effect of aging or diseases affect certain components in the visual pathway.The main advantage of this technique is that it allows comprehensive assessment of the retinal function and its upstream affect on the visual evoked cortical function. To begin this procedure, pre-fashion the custom made ERG inactive electrodes by cutting a 70 millimeter length of silver wire and forming a loop of eight millimeters in diameter to encircle the rat eye. Prepare a uniform circle and adjust the size of the loop by shaping it on a one millimeter pipe head tip.This is important because a ring that is too small may inadvertently raise inarticular pressure, where as an oversized ring can increase the variability of signals. Next, pre-fashion the custom made VEP inactive electrode by cutting a 70 millimeter length of silver. And forming an ellipse of eight millimeters in diameter to hook on to the rat incisors.Then, pre-fashion the custom made ERG active electrodes by cutting a 30 millimeter length of silver wire and form a small loop of about one to two millimeters in diameter which is used to gently contact the rat cornea with minimal corneal abrasions. Afterward, securely attach the electrodes to the electrode leads by entwining the silver with the exposed inner wire. Insulate excess exposed metal with masking tape to reduce photo-voltaic artifacts.For the ERG active electrodes, more layers of masking tape can be applied to achieve a thicker and more rigid wrapping so that the electrode is easier to be held with a micro-manipulator. On each ERG inactive electrode, stick a small piece of hook and loop fastener to the masking tape to enable stable attachment to the rodent neck strap. Then, for each VEP active electrode, attach an alligator clip to the inner wire of the electrode lead.Immediately before recordings, electro plate the exposed surfaces of the silver wires with chloride to improve signal conduction. To do so, connect the cathode to the negative terminal of the battery and immerse the other end into the saline. Subsequently, immerse the silver tip on the anode wire into normal saline and connect the other end of this electrode wire, to the positive terminal of a nine volt battery.Disconnect them after 20 seconds and the silver tip of the electrode wire should be coated evenly in white. Repeat the chloriding procedure for all the silver ERG and VEP electrodes. In this procedure, disinfect the shaved area of an anesthetized animal with 10%povidone iodine three times.Make a sagittal incision along the mid-line on the head with a scalpel and excise a 20 millimeter diameter circle of dermal tissue to expose the cranial bone. Next, remove the underlying periosteum by scraping and drying with gauze, to expose the coronal and sagittal cranial sutures. Drill two holes through the skull on both hemispheres.Screw in stainless steel screws into the two premade holes up to a depth of one millimeter to allow firm anchorage. Then, prepare the surgical area for dental amalgam by drying the cranial bone with gauze and retracting the skin with two sutures. Subsequently, spread the dental amalgam over the exposed skull to secure the screw electrodes in place and ensure about 1.5 millimeters of the screws are exposed for recording.After that, remove the retraction sutures. For scotopic recordings, all the procedures are performed in a dark room. For illustration purposes, electrode positioning is conducted under normal room lighting here.The animal was dark adapted fore 12 hours and anesthetized with ketamine and xylazine. After pupil dilatation and topical anesthesia, hook the inactive VEP electrode around the bottom incisors. Then, place the animal on the ERG platform in front of the Ganzfeld bowl situated in the Faraday cage.Secure the animal to the platform with a strip of hook and loop fastener placed firmly but not tightly around the nape. Next, position the ERG inactive electrodes by encircling the sclaral ring non invasively around the eyes equator. Approximately three meters behind the limbus.Stabilize this by attaching the electrode to the hook and loop fastener strip around the nape. Repeat the procedure for contralateral eye. Now, fasten the VEP active electrodes by attaching alligator clips to the stainless steel screws, pre-implanted on the skull.Prior to the ERG active electrode placement, place a small drop of 1%carboxymethylcellulose sodium on the electrode to improve signal quality. Position the ERG active electrodes to lightly touch the central corneal surface using a micro manipulator attached to the custom built stereotaxic arm. Ensure the carboxymethylcellulose sodium only contacts the cornea and not the sclara, by wiping excess fluid away.Then insert two to five millimeters of the stainless steel ground needle electrode, subcutaneously into the tail. Slide the platform closer to the Ganzfeld bowl and ensure the animals eyes align with the opening of the bowl to enable even illumination of both retinas. Subsequently, close the Faraday cage to reduce extraneous noise.Using the above protocol, a stack of ERG wave forms can be recorded in response to a range of stimuli with the dimmest flash shown at the bottom and the brightest at the top. At very dim light levels, scotopic threshold responses can be recorded. With increasing stimulus brightness, the wave forms become dominated by the B wave and then the negative going A wave emerge at the brightest stimuli.The rod photoreceptor function can be assaid by using a P3 to model the A wave. This figure shows that P2 amplitude grows with increasing stimulus energy. Rod bipolar cell function can be assaid by modeling the intensity response theories of the rod P2 with a naka rushtin function.Retinal ganglion cell function is assaid at dim luminous energies and quantified by the pSTR peak amplitude and timing. Cone bipolar cell function is elicited with a paired flash paradime and quantified by cone P2 peak amplitude and timing. Amplitude analysis of the VEP is taken as peak the troph and troph to peak amplitudes which gives the timing of these responses.The amplitude of the VEP wave forms increasing with increasing stimulus energy. When it's mastered, this technique can be done within about 30 to 45 minutes excluding the time for preparing the electrode and placing the electrode and adakadaptation. While attempting this procedure, it is particularly important to pay attention to how accurately you place the electrodes and allow enough time for adakadaptation.Following this procedure, other procedures such as disease modification or therapies can be performed to answer specific questions. After watching this video, you should have a good understanding of how to prepare and place the electrodes as well as how to record and analyze the ERG and the VEP wave forms.

simultaneous-recording-electroretinography-visual-evoked-potentials

Research

JoVE Journal

Neuroscience

12.1K visualizações.  University of Melbourne. The overall goal of this procedure is to simultaneously measure the electroretinogam and visual evoked potential in a rat. This method can help answer key questions in the field of fissure neuroscience. Such as whether the effect of aging or diseases affect certain components in the visual pathway.The main advantage of this technique is that it allows comprehensive assessment of the retinal function and its upstream affect on the visual evoked cortical function. To begin this procedure...