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

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

All experimental procedures were conducted according to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, set out by the National Health and Medical Research Council in Australia. Ethics clearance was obtained from the University of Melbourne, Science Faculty, Animal Ethics Committee (approval number 0911322.1).
1. Pre-implantation of Chronic VEP Electrodes
Note: If concurrent ERG and VEP signals are to be collected animals must be surgically implanted with VEP electrode...

<ol>
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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 ...

Research

JoVE Journal

Neuroscience

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 ...

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 ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

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

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Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

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

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 ...

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 ...

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

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 ...

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 ...

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 ...