Post-receptoral contributions to the rat scotopic electroretinogram a-wave
- First Online:
- Cite this article as:
- Dang, T.M., Tsai, T.I., Vingrys, A.J. et al. Doc Ophthalmol (2011) 122: 149. doi:10.1007/s10633-011-9269-y
- 137 Views
The electroretinogram is a widely used objective measure of visual function. The best characterised feature of the full-field dark-adapted flash ERG, is the earliest corneal negativity, the a-wave, which primarily reflects photoreceptoral responses. However, recent studies in humans and primates show that there are post-receptoral contributions to the a-wave. It is not clear if such contributions exist in the rat a-wave. We consider this issue in the rat a-wave, using intravitreal application of pharmacological agents that isolate post-receptoral ON-pathways and OFF-pathways. In anaesthetised adult long Evans rats, we show that the ON-pathway (2-amino-4-phosphonobutyric acid, APB sensitive) makes negligible contribution to the a-wave. In contrast, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) or PDA (cis-piperidine-2,3-dicarboxylic acid) sensitive mechanisms modify the a-wave in two ways. First, for bright luminous energies, OFF-pathway inhibition (CNQX or PDA) results in a 22% reduction to the early phase of the leading edge of the a-wave up to 14 ms. Second, OFF-pathway inhibition removed a corneal negativity that resides between the a-wave trough and the b-wave onset.
The a-wave of the full-field dark-adapted flash electroretinogram is thought to arise largely from photoreceptor activity. However, studies in humans, non-human primates [1, 2] and cats  show that the dark-adapted a-wave also receives contributions from post-receptoral responses. It is not clear if similar post-receptoral contributions exist in the rodent scotopic a-wave.
In past work, the post-receptoral contribution to the dark-adapted a-wave has been characterised using brief flashes [1, 2]. However, the balance of activity induced in ON-pathway and OFF-pathway cells is known to depend on flash duration, so it is appropriate to probe with both brief and long (longer than time to a-wave peak) flashes as has been employed in previous studies of scotopic ERG responses . The waveforms are measured with and without drugs that selectively inhibit glutamatergic neurotransmission via group III metabotropic glutamate receptors (2-amino-4-phosphonobutyric acid, APB) and AMPA/KA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainite sensitive) ionotropic glutamate receptors (6-cyano-7-nitroquinoxaline-2,3-dione, CNQX, cis-piperidine-2,3-dicarboxylic acid, PDA). APB removes contributions from the ON-pathway, whereas CNQX and PDA will remove contributions from the OFF-pathway, as well as AMPA/KA sensitive responses arising from the outer retina (horizontal cells), and inner retina (amacrines and ganglion cells).
All animal experimental procedures were conducted with approval from our institutional Animal Experimentation Ethics Committee (A04001) and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Subjects were adult long Evans rats between 10 and 12 weeks of age (180–270 g, Monash Animal Services, Clayton, VIC, Aust). The rats were maintained at 22°C in a 30–70 lux environment with a 12-h light/dark cycle (on at 8 am). Water and rat chow were provided ad libitum.
Prior to experimentation, animals were dark adapted overnight (>12 h). All animal preparation was performed in the dark using a dim red light, which has very little output below 580 nm (λmax~650 nm). Anaesthesia was induced with intramuscular injections of 60 mg/kg ketamine (ketamil 100 mg/ml, Troy laboratories, Smithfield, Aust) and 5 mg/kg xylazine (xylazil 100 mg/ml, Troy laboratories). Supplemental injections of half the original dose were given every hour after the initial injection. Corneal anaesthesia was obtained by instillation of one drop of proxymetacaine (Opthetic 0.5%, Allergan, Irvine, CA). Mydriasis was induced with one drop of 0.5% tropicamide (Mydriacyl, Alcon Laboratories, Frenchs Forest, NSW, Aust). Following anaesthesia, core temperature was maintained by covering the animal with an insulating blanket and placing the rat over a circulating-water heat-pad (MGW pump Lauda, Lauda-Königshoffen, Ger). The animal and heating pad were mounted, and stabilised on a custom-built platform with the rat’s neck and abdomen lightly secured to the stage with Velcro to minimise movement during recordings.
Single full-field ERGs were recorded using a custom-built Ganzfeld sphere (Photometric Solutions International, Huntingdale, VIC, Aust) using methods common to our laboratory . Recordings were taken simultaneously from both eyes using chlorided silver electrodes. The recording electrode was placed at the centre of the cornea. The reference was a ring electrode placed around the equator of the eye. The ground was a stainless steel needle (F-E2-30, Grass Telefactor, West Warwick, RI) inserted subcutaneously into the tail. Electrode contact was improved by applying a small amount of 1.0% carboxymethylcellulose sodium (Celluvisc, Allergan, Irvine, CA, USA). A readaptation period of 10 min was allowed following electrode placement prior to data collection. The signals were amplified and band-pass filtered (0.3 Hz–1 kHz) over a 2,560 (640 ms) epoch with a 4 kHz sampling rate. A 50-Hz notch filter was applied post hoc to eliminate line noise.
Stimuli were either relatively short (1–4 ms) or long (256 ms) white flashes (5 Watt LEDs, Luxeon, Philips Lumileds Lighting Co., CA, USA) delivered via a Ganzfeld integrating sphere. Stimulus energy was calibrated using an IL-1700 Research Radiometer (International light, Newbury Port, MA) with a scotopic filter in place (Z CIE, International Light). Values for short flashes are specified by their luminous energies (scotopic cd.s.m−2), whereas those for long flashes are specified as luminances (scotopic cd.m−2). Modification of Lyubarsky et al.’s  formulæ to account for the different ocular dimensions in rats means that 1 scot cd.s.m−2 produces 828 Φ/rod . ERG responses were obtained for luminous energies ranging from −1.15 to 1.55 log cd.s.m−2 for short flashes and for luminances between 0.76 and 3.11 log cd.m−2 (0.76, 0.97, 1.27, 1.68, 2.18, 2.53, 2.82 and 3.11) for 256 ms flashes. For flash durations from 1 to 4 ms, time 0 is specified from the middle of the stimulus. The interstimulus interval was varied between 45 and 240 s depending on luminous energy, to allow for complete recovery of a-wave amplitude between successive flashes.
As described previously , pharmacological agents were delivered into the vitreal chamber via a 30-gauge needle attached with fine polyethelyne tubing (inner diameter = 0.38 mm) to a Hamilton syringe (SGE syringes, Ringwood, Aust). The needle was introduced through the pars plana, approximately 1 mm posterior to the superior limbus and at an angle of 45 degrees to avoid the lens. Animals were excluded from analysis if opacification of the lens occurred (<10%). Aliquots of 2–5 μl were used with the upper limit chosen to minimise intraocular pressure changes . Contributions from the ON-pathway are removed using APB (400 μM, n = 4 for long flashes and n = 5 for short flashes), whereas the OFF-pathway is removed using either CNQX (250 μM, n = 4 for long flashes) or PDA (5 mM, n = 6 for short flashes). All agents were obtained from Sigma–Aldrich (Sigma–Aldrich Pty. Ltd., Castle Hill, NSW, Aust). APB and PDA were diluted using distilled H2O, whereas CNQX was mixed in the minimal volume of dimethyl sulphoxide (DMSO, <0.1%) that gave full dilution. This concentration of DMSO has been shown to have no effect on the rat ERG . These concentrations represent the estimated vitreal concentrations assuming complete dilution and a rat vitreous volume of 50 μl .
Control data represent a group of control eyes (n = 22 for long flashes, n = 11 sham injected for short flashes). Following drug injection, single ERG waveforms (−0.74 log cd s.m−2) were collected at 5-min intervals, to track drug onset for 40 min, ensuring stabilisation before the ERG protocol began.
A hyperbolic function was used to fit the amplitude–energy curves for various criterion times following stimulus onset . The function is given by a maximal amplitude (Vmax, μV) and semi-saturation constant (K, log cd.m2).
Statistical analysis was performed using Prism® software (v4.0, GraphPad Software Inc., 2003, CA, USA). Two-way ANOVA was conducted to consider interactions between the main effects of treatment (control vs APB, CNQX or PDA) and luminous energy or luminance nested within treatment. A Bonferroni post hoc test was applied in the presence of significant interactions. Throughout this study, group data are reported as means ± SEM.
Contributions from APB sensitive elements
Figure 1 shows representative control and APB-treated waveforms in response to short (Panels A and B) and long duration flashes (256 ms, Panels C and D) of varying luminous energy and luminances, respectively. Figure 1 shows that APB (thick black) completely abolishes the positive b-wave, leaving a corneal negative waveform. The leading edge was unaffected by APB (Fig. 1A and 1B) for short or long duration flashes (Fig. 1D).
Treatment effects for the different criterion times (Ax) for controls and eyes treated with APB (short and long flashes), CNQX or PDA (short flash, n = 6)
Criterion time (s)
Contributions from CNQX and PDA sensitive elements
The possibility that a scaling process can explain the a-wave reduction caused by PDA and CNQX was evaluated by normalising treated waveforms to those of controls. The scaling factor needed to achieve this alignment for a fixed amplitude at 12 ms was 22 ± 13% for PDA-treated eyes with short flashes and 23 ± 14% for CNQX-treated eyes with long flashes.
In order to better visualise this feature, treated waveforms were subtracted from controls (Fig. 3B) following scaling. For the brightest stimulus, the departure between control and APB-treated waveforms begins after 18 ms. This departure for CNQX treatment in long duration flashes begins at 14 ms. Also, plotted on Fig. 3B is the difference for PDA treatment assessed using short flashes. This shows that CNQX and PDA modify the descending limb of the a-wave in similar ways.
We show that the pigmented rat scotopic a-wave like non-human primates [1, 2] and cats  also contains post-receptoral contributions. Green and Kapousta–Bruneau  using trans-retinal microelectrode recordings in the isolated albino rat retina report that the scotopic a-wave is entirely due to photoreceptor-derived currents. The lack of post-receptoral involvement in these rat a-waves is surprising, and may represent differences in the albino strain of rats [14, 15] and/or the isolated retinal approach employed by Green and Kapousta–Bruneau .
Rat dark-adapted a-wave receives no ON-pathway contributions
The rat scotopic a-wave, contains little contribution from the ON-pathway. This is consistent with previous literature showing that it is unchanged following APB application in rats . APB is an agonist to group III metabotropic receptors , and since no change was observed with APB, it is unlikely that any of the Group III mGluRs make any contribution to the rat dark-adapted a-wave. Interestingly, Jamison et al.  find a 15–30% scotopic a-wave increase after APB application in non-human primates. This discrepancy may reflect species differences.
Rat dark-adapted a-wave is scaled by an ionotropic sensitive mechanism
To explore the contributions of the hyperpolarising bipolar cells and horizontal cells to the rat dark-adapted a-wave, CNQX and PDA were used to inhibit AMPA/KA receptors. These drugs had two effects on the scotopic a-wave. The first was an amplitude reduction. Indeed, the treated a-waves could be scaled to match control waveforms along their entire leading edge (Fig. 3). This ‘scaling’ of approximately 22% might reflect a decreased receptor output or the loss of an ionotropic sensitive corneal negative response that shares a similar time course to the ERG a-wave.
Jamison et al.  have reported that PDA caused an amplitude increase ‘scaling’ without affecting phototransduction sensitivity in primates. Similarly, modelling our CNQX data using the Hood and Birch  formulation of the delayed Gaussian model , produces significantly smaller phototransduction amplitudes (control −467 ± 23 vs CNQX, −346 ± 38 μV, 26% loss, unpaired t-test, P < 0.01), with a similar sensitivity (control, 2.56 ± 0.04 vs. CNQX 2.46 ± 0.06 log m2.cd−1.s−3, unpaired t-test, P = 0.32).
Alternatively, AMPA/KA sensitive elements in the outer retina may also be involved. Shiells and Falk  show in dogfish retina that the ERG a-wave is reduced in the absence of horizontal cell responses, and that horizontal cells produced a negativity with a similar time course to the photoreceptors. Hanitzsch and colleagues , simultaneously recorded intracellular potentials from horizontal cells and ERG responses, and showed that the negative horizontal cell response overlaps in time and has a similar intensity response function to the PIII in rabbits. Despite this evidence, it is not clear how currents from the lateral oriented horizontal cell can manifest in the a-wave.
Rat a-wave trough modulated by an ionotropic sensitive mechanism
The second effect of ionotropic receptor blockade is to remove a contribution that modulates the a-wave leading edge after 14 ms for the brightest stimulus energies employed here (Fig. 3). This is consistent with findings following blockade of AMPA/KA receptors in primate scotopic a-waves . The timing of the hyperpolarising bipolar cell intrusion in rat a-waves (>14 ms at 3.11 log cd.m−2) is similar to that reported for primate rod responses . Although the exact origins are unclear, it is likely to arise from inner retinal elements . Robson and Frishman  have shown that NMDA, results in a reduction of the cat dark-adapted a-waves, recorded using vitreal electrodes. Moreover, these authors find that such contributions are removed by a dim light background suggesting that the elements responsible arise from the rod pathway. These outcomes they interpret as contributions from NMDA sensitive elements in inner retinal rod pathway. Consistent with this idea, responses collected using a short interstimulus interval (500 ms) are unaffected by PDA treatment (data not shown). Thus, it is likely that the differences noted here arise from the rod pathway.
The findings from this experiment show that the rat dark-adapted a-wave is unaffected by application of APB. CNQX and PDA however, result in changes that appear to arise from two different mechanisms. First, a scaling of the rat a-wave along the entire leading edge, and second a modification of the a-wave trough.
NHMRC CJ Martin Postdoctoral Fellowship (BVB), NHMRC project grant 400127 (BVB).