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.
2. ERG and VEP Recording
Waveform | Stimulus light energy (log cd.s.m-2) | Number of repeats | interstimulus interval (sec) |
STR | -6.24 | 20 | 2 |
STR | -5.93 | 20 | 2 |
STR | -5.6 | 20 | 2 |
STR | -5.33 | 20 | 2 |
Rod b-wave | -4.99 | 10 | 2 |
Rod b-wave | -4.55 | 10 | 2 |
Rod b-wave | -4.06 | 5 | 5 |
Rod b-wave | -3.51 | 5 | 5 |
Rod b-wave | -3.03 | 1 | 15 |
Rod b-wave | -2.6 | 1 | 15 |
Rod b-wave | -1.98 | 1 | 15 |
Mixed a-/b-wave | -1.38 | 1 | 30 |
Mixed a-/b-wave | -0.94 | 1 | 30 |
Flash 1: Mixed a-/b-wave Average of 20: VEP | -0.52 | 20 | 5 |
(90 sec before next) | |||
Flash 1: Mixed a-/b-wave Average of 20: VEP | 0.04 | 20 | 5 |
(120 sec before next) | |||
Flash 1: Mixed a-/b-wave Average of 20: VEP | 0.58 | 20 | 5 |
(180 sec before next) | |||
Flash 1: Mixed a-/b-wave Average of 20: VEP | 1.2 | 20 | 5 |
(180 sec before next) | |||
Flash 1: Mixed a-/b-wave Average of 20: VEP | 1.52 | 20 | 5 |
(180 sec before next) | |||
Cone a-/b-wave | 1.52 | 4 | 0.5 |
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
Note: ERG and VEP analysis has been described in detail previously.3,9,10 The following sections provide a brief overview.
4. Analysis of VEP Waveforms
The ERG a-wave (> -1.38 log cd.s.m-2), b-waves (> – 4.99 log cd.s.m-2) STRs (< – 4.99 log cd.s.m-2) and the VEPs (> – 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).
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. Please click here to view a larger version of this figure.
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, ○) are fit as an ensemble with a PIII (grey lines, Equation 1) to 90% of the minimum which returns RmPIII (saturated amplitude, µV) S (sensitivity, m2.cd-1.s-3) and td (timing delay, msec) parameters. (B) Rod bipolar cell function (mean ± SEM) can be assayed by modelling the intensity response series of the rod PII (unfilled circles ○) with a Naka-Rushton function (grey line). This returns Vmax (saturated amplitude, µV), 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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ± SEM) increases with increasing stimulus energy. Please click here to view a larger version of this figure.
Alligator clip | generic brand | HM3022 | Stainless steel 26 mm clip for connecting VEP screw electrodes to cables |
Bioamplifier | ADInstruments | ML 135 | For amplifying ERG and VEP signals |
Carboxymethylcellulose sodium 1.0% | Allergan | CAS 0009000-11-7 | Viscous fluid for improving signal quality of the active ERG electrode |
Carprofen 0.5% | Pfizer Animal Health Group | CAS 53716-49-7 | Proprietary name: Rimadyl injectable (50 mg/mL). For post-surgery analgesia, diluted to 0.5% (5 mg/mL) in normal saline |
Chlorhexadine 0.5% | Orion Laboratories | 27411, 80085 | For disinfecting surgical instruments |
Circulating water bath | Lauda-Königshoffen | MGW Lauda | For maintaining body temperature of the anesthetized animal during surgery and electrophysiological recordings |
Dental amalgam | DeguDent GmbH | 64020024 | For encasing the electrode-skull assembly to make it more robust |
Dental burr | Storz Instruments, Bausch and Lomb | #E0824A | A miniature drill head of ~0.7mm diameter for making a small hole in the skull over each hemisphere to implant VEP screws |
Drill | Bosch | Dremel 300 series | An automatic drill for trepanning |
Electrode lead | Grass Telefactor | F-E2-30 | Platinum cables for connecting silver wire electrodes to the amplifier |
Faraday Cage | custom-made | Ensures light proof to maintain dark adaptation. Encloses the Ganzfeld setup to improve signal to noise ratio | |
Gauze swabs | Multigate Medical Products Pty Ltd | 57-100B | For drying the surgical incision and exposed skull surface during surgery |
Ganzfeld integrating sphere | Photometric Solutions International | Custom designed light stimulator: 36 mm diameter, 13 cm aperture size | |
Velcro | VELCRO Australia Pty Ltd | VELCRO Brand Reusable Wrap | Hook-and-loop fastener to secure the electrodes and the animal on the recording platform |
Isoflurane 99.9% | Abbott Australasia Pty Ltd | CAS 26675-46-7 | Proprietary Name: Isoflo(TM) Inhalation anaaesthetic. Pharmaceutical-grade inhalation anesthetic mixed with oxygen gas for VEP electrode implant surgery |
Ketamine | Troy Laboratories | Ilium Ketamil | Proprietary name: Ketamil Injection, Brand: Ilium. Pharmaceutical-grade anesthetic for electrophysiological recording |
Luxeon LEDs | Phillips Lighting Co. | For light stimulation twenty 5 watt and one 1 watt LEDs. | |
Micromanipulator | Harvard Apparatus | BS4 50-2625 | Holds the ERG active electrode during recordings |
Needle electrode | Grass Telefactor | F-E2-30 | Subcutaneously inserted in the tail to serve as the ground electrode for both the ERG and VEP |
Phenylephrine 2.5% minims | Bausch and Lomb | CAS 61-76-7 | Instilled with Tropicamide to achieve maximal dilation for ERG recording |
Povidone iodine 10% | Sanofi-Aventis | CAS 25655-41-8 | Proprietory name: Betadine, Antiseptic to prepare the shaved skin for surgery 10%, 500 mL |
Powerlab data acquisition system | ADInstruments | ML 785 | Controls the LEDs |
Proxymetacaine 0.5% | Alcon Laboratories | CAS 5875-06-9 | For corneal anaesthesia during ERG recordings |
Saline solution | Gelflex | Non-injectable, for electroplating silver wire electrodes | |
Scope Software | ADInstruments | version 3.7.6 | Simultaneously triggers the stimulus via the Powerlab system and collects data |
Silver (fine round wire) | A&E metal | 0.3 mm | Used to make active and inactive ERG electrodes, and the inactive VEP electrode |
Stainless streel screws | MicroFasterners | 0.7 mm shaft diameter, 3 mm in length to be implanted over the primary visual cortex and serve as the active VEP electrodes | |
Stereotaxic frame | David Kopf | Model 900 | A small animal stereotaxic instrument for locating the primary visual cortices according to Paxinos & Watson's 2007 rat brain atlas coordinates |
Surgical blade | Swann-Morton Ltd. | 0206 | For incising the area of skin overlaying the primary visual cortex to implant the VEP electrodes |
Suture | Shanghai Pudong Jinhuan Medical Products Co.,Ltd | 3-0 silk braided suture non-absorbable, for skin retraction during VEP electrode implantation surgery | |
Tobramycine eye ointment 0.3% | Alcon Laboratories | CAS 32986-56-4 | Proprietary name: Tobrex. Prophylactic antibiotic ointment applied around the skin wound after surgery |
Tropicamide 0.5% | Alcon Laboratories | CAS 1508-75-4 | Proprietary name: 0.5% Mydriacyl eye drop, Instilled to achieve mydriasis for ERG recording |
Xylazine | Troy Laboratories | Ilium Xylazil-100 | Pharmaceutical-grade anesthetic for electrophysiological recording |
Pipette tip | Eppendorf Pty Ltd | 0030 073.169 | Eppendorf epTIPS 100 – 5000 mL, for custom-made electrodes |
Microsoft Office Excel | Microsoft | version 2010 | spreadsheet software for data analysis |
Lethabarb Euthanazia Injection | Virbac (Australia) Pty Ltd | LETHA450 | 325 mg/mL pentobarbital sodium for rapid euthanazia |
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 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 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.