DP. An asymmetric outer retinal response to drifting sawtooth gratings.

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1 J Neurophysiol 1: 39 3, 1. First published February 17, 1; doi:.1/jn..1. An asymmetric outer retinal response to drifting sawtooth gratings Nina Riddell, 1, * Laila Hugrass, * Jude Jayasuriya, Sheila G. Crewther, 1 and David P. Crewther 1 School of Psychology and Public Health, La Trobe University, Melbourne, Victoria, Australia; and Centre for Human Psychopharmacology, Swinburne University of Technology, Melbourne, Victoria, Australia Submitted 1 January 1; accepted in final form 17 February 1 Riddell N, Hugrass L, Jayasuriya J, Crewther SG, Crewther DP. An asymmetric outer retinal response to drifting sawtooth gratings. J Neurophysiol 1: 39 3, 1. First published February 17, 1; doi:.1/jn..1. Electroretinogram (ERG) studies have demonstrated that the retinal response to temporally modulated fast-on and fast-off sawtooth flicker is asymmetric. The response to spatiotemporal sawtooth stimuli has not yet been investigated. Perceptually, such drifting gratings or diamond plaids shaded in a sawtooth pattern appear brighter when movement produces fast-off relative to fast-on luminance profiles. The neural origins of this illusion remain unclear (although a retinal basis has been suggested). Thus we presented toad eyecups with sequential epochs of sawtooth, sine-wave, and square-wave gratings drifting horizontally across the retina at temporal frequencies of. Hz. All ERGs revealed a sustained direct-current (DC) transtissue potential during drift and a peak at drift offset. The amplitudes of both phenomena increased with temporal frequency. Consistent with the human perceptual experience of sawtooth gratings, the sustained DC potential effect was greater for fast-off cf. fast-on sawtooth. Modeling suggested that the dependence of temporal luminance contrast on stimulus device frame rate contributed to the temporal frequency effects but could not explain the divergence in response amplitudes for the two sawtooth profiles. The difference between fast-on and fast-off sawtooth profiles also remained following pharmacological suppression of postreceptoral activity with tetrodotoxin (TTX), -amino--phosphonobutric acid (APB), and,3 cis-piperidine dicarboxylic acid (PDA). Our results indicate that the DC potential difference originates from asymmetries in the photoreceptoral response to fast-on and fast-off sawtooth profiles, thus pointing to an outer retinal origin for the motion-induced drifting sawtooth brightness illusion. sawtooth; electroretinogram; flicker; motion; spatiotemporal; brightness illusion ELECTROPHYSIOLOGICAL STUDIES of retinal ON-OFF processing have demonstrated that the response to temporally modulated sawtooth flicker is asymmetric (Alexander et al. 1; Barnes et al. ; Dryja et al. ; Khan et al. ; Kremers 13; Kremers et al. 1993; Pangeni and Kremers 13; Pangeni et al. 1; Rodrigues et al. ; Vukmanic et al. 1). It is unknown whether such asymmetries also occur for more complex spatiotemporal sawtooth stimuli (i.e., moving sawtooth grating or diamond patterns). Indeed, relatively little is known about electroretinographic responses to moving stimuli in any species. The motion electroretinograms (ERGs) recorded across studies to date have identified some similar waveforms, most notably a positivity following motion onset that increases * N. Riddell and L. Hugrass contributed equally to this work. Address for reprint requests and other correspondence: D. P. Crewther, Centre for Human Psychopharmacology, Advanced Technologies Centre, Swinburne Univ. of Technology, Burwood Rd., Hawthorn, VIC 31, Australia ( dcrewther@swin.edu.au). in amplitude with temporal frequency (Bach and Hoffmann ; Dodt and Kuba 199; Korth 197; Korth et al. ). These studies, however, have primarily used short motion durations, square-wave stimuli, and alternating current (AC)- coupled recording systems (that do not allow study of sustained effects). Moreover, although the motion ERG has been compared to the pattern offset-onset response (Korth et al. ), no attempts have been made to dissect pharmacologically the level at which the response is generated. In addition to extending the ON-OFF pathway and motion processing literature, ERG recordings of the retinal response to drifting sawtooth gratings may clarify the cellular origins of the perceptual effects that these stimuli elicit in humans. Such moving patterns generating fast-off profiles appear brighter than those moving in the opposite direction generating fast-on profiles (despite no change in mean luminance). This illusion was explored by Cavanagh and Anstis (19) using shaded grating patterns drifting leftward or rightward on the horizontal axis and has also been demonstrated for drifting shaded diamonds (Watanabe et al. 199), drifting shaded boxes (Ashida and Scott-Samuel 1), and spatially uniform sawtooth flicker (Cavanagh and Anstis 19; Wu et al. 199). An example of the illusion is provided in Supplemental Video S1 (available in the data supplement online at the Journal of Neurophysiology Web site). Psychophysically, the strength of the drifting sawtooth grating illusion has been shown to increase with temporal frequency up to 3.7 Hz, the highest speed tested to date (Cavanagh and Anstis 19). Related brightness perception measures for full-field sawtooth flicker (Wu et al. 199) and moving sawtooth boxes (Ashida and Scott-Samuel 1) suggest that the drifting sawtooth illusion may also occur at moderate temporal frequencies (i.e., up to 1 Hz). Although the neural basis of this perceptual effect remains unclear, Cavanagh and Anstis (19) psychophysical experiments led them to suggest that the illusion may result from saturation of transient responses to the fast phase of the sawtooth (although they did not specify the level of the visual system where they expected this to occur). In the present study, we used direct-current (DC) ERG and pharmacological dissection to explore the retinal response to drifting sawtooth gratings. In doing so, we aimed to extend the temporal sawtooth flicker literature into the spatiotemporal domain and to elucidate the neural origins of Cavanagh and Anstis (19) drifting sawtooth illusion. Toad eyecups, for which robust retinal responses have been well-studied (Gallemore et al. 1997), were chosen as the model system as comparatively long recording times were required to facilitate pharmacological dissection of functional responses using a within-subjects design. In accordance with the temporal saw /1 Copyright 1 the American Physiological Society 39

2 3 DRIFTING SAWTOOTH GRATINGS tooth flicker literature and the effects of spatiotemporal sawtooth on brightness perception in humans (Cavanagh and Anstis 19), we expected that the DC ERG response amplitudes for fast-off and fast-on sawtooth profiles would be significantly different and sensitive to the temporal frequency of stimulation. METHODS Subjects Forty-six male cane toads (Rhinella marina) were obtained from a local supplier (Peter Douch, Mareeba, Queensland, Australia) and housed under a 1:1-h light-dark cycle before experimentation. Toads were anesthetized in a bath containing.% MS- (Tricaine methanesulfonate; ph was adjusted to neutral with NaHCO 3 ) before pithing and tissue preparation. All procedures were conducted in strict accordance with the approved Swinburne University of Technology Animal Ethics Committee (AEC) protocols and adhere to the National Health and Medical Research Council (NHMRC) Code for the Care and Use of Animals for Scientific Purposes (13). Setup Toads were removed from housing during the light cycle and remained under normal room lighting to facilitate light adaptation during the 3-min anesthesia and dissection protocol. Eyes were enucleated immediately following anesthesia, and the cornea and lens were removed. The remaining eyecup was supported by a custommade wax mold in a bath of amphibian Ringer solution at room temperature ( C). The Ringer solution, containing (in mm). NaCl, KCl, 7. NaHCO 3, 1 MgCl, 1. CaCl, and glucose (Miller and Steinberg 1977), was oxygenated with 9% O -% CO, resulting in a ph of 7.. Transtissue potential was recorded using Ag/AgCl electrodes with agar bridges (3% agar in 1 M KCl) on a Warner Dual Channel Epithelial Voltage Clamp (EC-A). Analog voltage output ( voltage amplification) was digitized at 1 khz, notch-filtered ( Hz), and low-pass filtered ( Hz) using a PowerLab data acquisition system (ADInstruments) with LabChart software. Trigger outputs from the video stimulator marking the onset of stimulation blocks were recorded together with the electrophysiological recordings. Stimuli were presented using a DATAPixx video stimulator (VPixx Technologies) and a custom-made projection system consisting of a backlit liquid-crystal display (LCD) projector screen ( cm, frame rate Hz, native resolution 3 ) and Minolta camera lens (f/1., FL mm). The LCD screen, which was removed from an HDMI Portable Mini LED Projector (UC model), was driven by video graphics array (VGA) output from the DATAPixx and backlit with a DC light source. The screen was positioned at the top of a -cm length of polyvinyl chloride (PVC) tube that kept it at the hyperfocal distance from the lens. Lens focus was adjusted before start of each experiment such that when the eyecup was placed in the bath, the plane of focus fell at the level of the central retina. Experimental Protocols Data were collected across three experiments. The main experiment used a within-subjects design to measure the DC ERG response to drifting sawtooth, square-wave, and sine-wave gratings before and after drug delivery. Two additional experiments were conducted to test whether the response to drifting sawtooth diamonds was similar to that for drifting sawtooth gratings and to measure toad ERGs for Fig. 1. Methods summary for drifting grating and diamond and temporal sawtooth flicker experiments.

3 DRIFTING SAWTOOTH GRATINGS 31 temporally varied sawtooth flicker. The protocols for these experiments are outlined in Fig. 1. Drifting grating experiment. Sawtooth, sine-wave, and squarewave gratings (cropped examples in Fig. of RESULTS) with an equal number of dark and light bars were created using VPixx (v.3.3). When projected onto the eyecup, the gratings covered a - -mm patch (subtending..7 at the lens) with a spatial frequency of 1.3 cycles per degree and a mean luminance of cd/m. The stimulus was surrounded by a rectangular black mask with a luminance of.1 cd/m. During a presentation sequence, stimuli drifted leftward for s, remained stationary for 3 s, drifted rightward for s, and then remained stationary for 3 s before the sequence was repeated. This horizontal drift was lined up with the long direction of the rectangular patch. Leftward and rightward drift of sawtooth gratings produced local fast-off and fast-on temporal luminance profiles, respectively, with no change in net luminance of the stimulus. Preliminary experiments confirmed that flipping the luminance profile of the sawtooth stimulus sequence also reversed the amplitude of the ERG response (when recording sequentially from the same eyecups). This reversed stimulus was not included in subsequent experiments due to time constraints (recovery time was required following the flip). Sine- and square-wave stimuli produced a symmetric profile during leftward and rightward drift. Preliminary experiments confirmed that flipping the luminance profile had no effect on ERG responses to these symmetric stimuli. The presentation sequence described above was used to test the effects of grating profile and temporal frequency on ERG response amplitudes in eyecups (see Fig. 1. for schematic flowchart of experimental protocol). Preliminary data collection indicated that experiments should be limited to h per eyecup to ensure stable and healthy recordings. To keep within this time frame, sawtooth, sinewave, and square-wave grating sequences were presented 7 times to each eyecup. Each presentation sequence was repeated across drift frequencies (.,, 7.,, and Hz) with order counterbalanced between eyecups. This series of presentations formed a run lasting min. Spatially uniform temporal square-wave flash ERGs were recorded at the beginning and end of each run to assess the ongoing integrity of the eyecup preparation. Each eyecup was presented with three stimulus runs. Run 1 was recorded with Ringer superfusate for all eyecups (n ). Subsequently, eyecups were divided into three groups, and drugs were added to the bath to suppress cellular activity as follows. Postreceptoral ON pathway activity was suppressed by specific inhibition of the metabotropic glutamate receptor of ON-bipolar cells (mglur) using 1 mm -amino--phosphonobutric acid (APB) in Ringer (Jardon et al. 199; Slaughter and Miller 191). Postreceptoral OFF pathway activity was suppressed by inhibition of ionotropic glutamate receptors found on hyperpolarizing second-order (OFF-bipolar and horizontal) cells using 1 mm,3 cis-piperidine dicarboxylic acid (PDA) in Ringer (Jardon et al. 199). Spiking amacrine and ganglion cell activity was A Drift Offset B Stationary Amplitude (µv) C Amplitude (µv) fast-off fast-on 1 3 Square-wave D fast-off fast-on 1 3 Sine-wave Fig.. Mean DC ERG responses ( SE) to directionally moving sawtooth gratings (A), sawtooth diamonds (B), square-wave gratings (C), and sine-wave gratings (D) drifting across the retina at Hz. Each stimulus (cropped examples shown in insets) drifted leftward and rightward (resulting in fast-on and fast-off temporal profiles for the sawtooth stimuli). Shading indicates drift offset and the beginning of the 3-s stationary period. Note that although the DC potential shift and drift offset peak were not strong at Hz for sine-wave and fast-on gratings, these waveforms become larger at higher temporal frequencies (see Fig. 3) Time (s) Time (s)

4 3 DRIFTING SAWTOOTH GRATINGS suppressed using or M tetrodotoxin (TTX) in Ringer (Awatramani et al. 1). Drug dosage choices were based on the published amphibian studies referenced above. See Fig. 1 for eyecup numbers and the sequence of pharmacological suppression used in each condition. Drifting diamond experiment. The sawtooth perceptual illusion in humans occurs for both drifting gratings and drifting diamond plaids (Cavanagh and Anstis 19; Watanabe et al. 199). To test whether the DC ERG response to sawtooth gratings and diamond plaids is also similar, ERGs were recorded from 11 additional eyecups presented with shaded diamonds drifting at Hz (see Fig. 1 for an outline of procedures and Fig. for a cropped example of the diamond plaid stimulus). These recordings were conducted with Ringer superfusate only. Temporal sawtooth flicker experiment. Although the DC ERG response to drifting sawtooth gratings has not previously been examined in any species, many studies have investigated the retinal response to temporal sawtooth flashes (as noted in the Introduction and DISCUSSION). To provide a basis for comparison with these studies, ERG responses to 1-Hz spatially uniform fast-on and fast-off sawtooth flicker were recorded from an additional eyecups. As with the drifting grating experiment, these data were collected across multiple runs to allow pharmacological dissection (see Fig. 1 for details). Data Analysis Electrophysiological data were analyzed with LabVIEW (v.11.1; National Instruments). The preprocessing stage involved removing high-amplitude spiking interference and applying a -Hz digital low-pass filter. Low-frequency drift was observed across experimental runs [presumably resulting from changes in electrode junction potentials (Barry and Diamond 197), slight variations in bath temperature, and declining tissue integrity over time]. To control for this slope in A Mean amplitude (µv) for 1-s epoch during drift Drift Offset 1 3 fast-off fast-on sine-wave square-wave Fig. 3. Effects of grating profile and temporal frequency (.,, 7.,, and Hz) on the sustained potential increase during drift and the peak amplitude at drift offset. A: mean ERG amplitudes ( SE) for the 1 s epoch following drift onset for all grating profiles across temporal frequencies (epoch time frame indicated relative to an example response at Hz in inset figure). B: mean troughpeak amplitude ( SE) for the drift offset peak (example offset peak indicated in -Hz inset figure). B Mean trough to peak amplitude (µv). 7. Drift Offset Temporal frequency (Hz)

5 DRIFTING SAWTOOTH GRATINGS 33 the signal that was not due to experimental conditions, the fourthorder polynomial regression fit line (chosen for goodness of fit) was subtracted from the entire -min trace. Starting from the onset of drift, -s epochs were extracted for each leftward and rightward drift sequence ( s drift, 3 s stationary). Data for each condition were averaged relative to a baseline taken from the -ms stationary presentation preceding each drift onset trigger. Averaged epochs for each eyecup were downsampled to Hz. As there were no significant differences in electrophysiological responses from leftward/ rightward motions of symmetric sine- and square-wave gratings, these data were averaged for each eyecup. RESULTS Drifting Gratings and Diamonds ERG responses to -Hz drift are illustrated in Fig.. In general, these grand mean ERGs revealed a sustained positive shift in transtissue DC potential during drift and a peak at drift offset. For both gratings and diamonds, the DC potential shift was larger for the fast-off sawtooth (perceptual brightening) than for the fast-on sawtooth (perceptual darkening). Oscillations at the fundamental frequency of local flicker were apparent in the responses to gratings but not diamond plaids. Although both of the projected patterns were designed to be equated for light and dark regions, there can be no guarantee that all sections of the retina were uniformly responsive. The effects of a small imbalance in effective light and dark regions would be greater in gratings cf. diamonds due to their simpler spatiotemporal luminance pattern and to the fact that the orientation of the grating pattern was parallel to the edge of the stimulus. Two parameters were extracted for further analyses: the DC potential shift was defined as the average potential from 1 to s postdrift onset and the drift offset peak as the trough-to-peak amplitude of the response at the cessation of stimulus motion. The responses displayed in Fig. 3 illustrate mean DC shift (Fig. 3A) and drift offset peak potentials (Fig. 3B) for the grating profiles at each temporal frequency. As shown in Fig. 3A, the difference in DC potential shift between the two sawtooth modulations increased with temporal frequency up to Hz. Over all temporal frequencies, the square-wave DC amplitude was consistently greater than that of the sine-wave profile and for frequencies Hz was roughly parallel (Fig. 3A). However, the sawtooth profiles showed lesser slope with temporal frequency compared with either square- or sine-wave stimulation. A (sawtooth profile) (frequency) within-groups ANOVA confirmed that across temporal frequency, the sustained DC potential during drift was greater for the fast-off than fast-on sawtooth [F(1, 17) 3.9, P.1, partial.7]. The sawtooth profile by frequency interaction shows that the effect of temporal frequency on the DC potential was significantly different for the fast-off and fast-on profiles [F(,) 1.3, P.1, partial.]. Although we were specifically interested in the DC potential shift during drift (corresponding to the time frame when illusory brightness has been demonstrated in humans), we observed a similar pattern of results for the drift offset peak (Fig. 3B). As with the DC potential, both the main effect of temporal frequency [F(1, 17).11, P.1, partial.73] and the sawtooth profile by frequency interaction [F(, ) 1.9, P.1, partial.] were significant. It should be noted that the frame-based display used here affected the stimulus in a temporally dependent manner. As the temporal frequency increases, there are progressively fewer luminance steps covered across one cycle of a moving grating. Thus at the -Hz frame rate used here and drift frequencies of 7.,, and Hz, a single photoreceptor will receive one cycle of the stimulus in eight, four, and three steps of luminance, respectively. For the sawtooth stimuli, this has the unusual effect that the proportion of increments and decrements occurring on each frame across the retinal photoreceptors becomes more equal as the drift frequency increases (i.e., as the number of frames per cycle of the pattern decreases). The effects of temporal frequency of stimulation on the different stimulus waveforms were modeled using LabVIEW software. Sample waves (square, sine, and sawtooth) were defined over 1 sample points for a single cycle. Whereas the linear estimates of brightening and darkening are for all waves (corresponding to the sum of deviations away from the mean over a full cycle), a nonlinear estimate based on the root mean square (RMS) of the changes for each frame of stimulation showed generally increasing values with increasing temporal frequency over the range of Hz (Fig. ). There is a strong qualitative similarity between the empirical data (Fig. 3) and the model (Fig. ), particularly with respect to the lower slope with temporal frequency for sawtooth compared with square or sine wave stimulation. The square-wave showed a greater RMS effect than either the sine or the sawtooth waves, whereas the RMS effects of the sine and sawtooth waves cross over at Hz (at approximately the same point as the sine and the average of the fast-on and fast-off sawtooth waves in the empirical data). The outstanding difference, not predicted in some form by the model, is the difference between the two sawtooth profiles. Although there is a clear empirical difference, the model prediction is identical for the fast-on RMS temporal contrast per point (Hz) Temporal frequency (Hz) Sawtooth Sine-wave Square-wave Fig.. Modeling the effects of alteration in temporal contrast on the rootmean-square (RMS) temporal contrast per point. For square, sine, and sawtooth waveforms, each defined on 1 points per cycle, the effects of increasing temporal frequency of drift were modeled by estimating the RMS difference per stimulus frame over a spatial cycle of each wave. The sawtooth polarities (fast-on and fast-off) produced identical results (shown as sawtooth).

6 3 DRIFTING SAWTOOTH GRATINGS A fast-off Amplitude (µv) 3 TTX µm Stationary TTX µm Ringer APB 1mM APB+PDA Ringer PDA 1mM PDA+APB Ringer Fig.. Effects of pharmacological suppression on mean DC ERG responses ( SE) to directionally moving fast-off (A) and fast-on (B) sawtooth gratings drifting at Hz. C shows the difference wave (fast-off minus fast-on) for each drug condition. Each set of 3 graphs (fast-off, fast-on, and difference) explores responses collected from the same eyecups before and after drug delivery. For TTX eyecups, superfusate changes were as follows: run 1 Ringer, run M TTX, and run 3 M TTX. For APB eyecups: run 1 Ringer, run 1 mm APB, and run 3 1mMAPB 1mM PDA. For PDA eyecups: run 1 Ringer, run 1 mm PDA, and run 3 1mMAPB 1 mm PDA. B fast-on Amplitude (µv) 3 C Difference (fast-off - fast-on) Amplitude (µv) Time (s) 1 3 Time (s) 1 3 Time (s) and fast-off sawtooth RMS effects as a function of temporal frequency. The cellular basis of differences in response to the two sawtooth grating polarities was further investigated by addition of TTX, APB, and PDA to the bath superfusate. Flash ERGs recorded at the beginning and end of each stimulus block confirmed the expected elimination of the b-wave/on-bipolar response by APB and a substantial but not complete suppression of the OFF-bipolar response with PDA (Hare and Ton ; Stockton and Slaughter 199). Figure illustrates the effects of pharmacological manipulation on the response to -Hz fast-on and fast-off sawtooth gratings. As shown in this figure, TTX and PDA did not affect the DC shift during drift or peak at drift offset for either sawtooth polarity (P. for all paired-samples t-tests). Application of APB, however, increased the DC shift [fast-on: t().7, P.1; fast-off: t() 7., P.1] and completely abolished the peak at drift offset for both fast-off [t().3, P.1] and fast-on [t().1, P.] gratings [this effect is evident in APB only (red trace) and APB PDA (purple trace) responses]. Importantly, as illustrated in Fig. C, the DC shift amplitudes for fast-off and fast-on gratings remained significantly different following APB delivery [t().1, P.]. This pattern of responses was consistent across all of the temporal frequencies tested. Figure illustrates the mean DC shift (Fig. A) and drift offset peak (Fig. B) potentials for fast-off and fast-on sawtooth grating profiles drifting at. Hz. Separate (sawtooth profile) by (drug delivery) by (drift frequency) repeated-measures ANOVAs were performed to test the effects of TTX, PDA, and APB on the DC potential shift and the peak after drift offset. Neither TTX nor PDA had a significant main effect on the DC potential shift or peak at drift offset (P.). Furthermore, there were no significant interactions between drug delivery and sawtooth polarity on the DC shift or peak at drift offset for either PDA

7 DRIFTING SAWTOOTH GRATINGS 3 A DC Potential during drift TTX APB PDA fast-off fast-on Difference (foff-fon) 3 3 B Drift offset peak fast-off fast-on Difference (foff-fon) Mean drift offset trough to peak amplitude (µv) Mean amplitude (µv) for 1-s epoch during drift Pre-drug TTX µm TTX µm Pre-drug APB 1mM APB+PDA 1mM Temporal frequency (Hz) Temporal frequency (Hz) or TTX (P.). Application of APB enhanced the DC shift during drift [main effect: F(1, ) 33., P., partial.7] and abolished the peak at drift offset for both waveforms across temporal frequency [main effect: F(1, ) 3., P., partial.]. The significant drug by sawtooth profile interaction indicates that APB has a greater Pre-drug PDA 1mM TTX APB PDA PDA+APB 1mM Fig.. Effects of pharmacological suppression on the DC potential increase during drift (A) and the peak amplitude at drift offset (B) for sawtooth gratings across temporal frequency. Mean ERG amplitudes ( SE) for the 1- to -s epoch following onset of leftward drift (fast-off) and rightward drift (fast-on) across temporal frequencies (.,, 7.,, and ) are shown in A. Mean trough-peak amplitude ( SE) for the drift offset peak are shown in B. The final line of graphs in both A and B shows the difference (fast-off minus fast-on) for each drug condition. Each set of graphs (fast-off, fast-on, and difference in A and B) explores responses collected from the same set of eyecups before and after drug delivery. For TTX eyecups, superfusate changes were as follows: run 1 Ringer, run M TTX, and run 3 M TTX. For APB eyecups: run 1 Ringer, run 1 mm APB, and run 3 1mMAPB 1mM PDA. For PDA eyecups: run 1 Ringer, run 1 mm PDA, and run 3 1mMAPB 1mM PDA. For an example of epoch time frames relative to original waveforms, see Fig. 3. effect on the DC potential shift for fast-on than fast-off waveforms [F(1, ) 7.1, P.1, partial.9]. Similar drug effects occurred for square-wave and sine-wave gratings (data not shown). It should also be noted that there was some variation in baseline ERG response amplitudes across the three drug groups (i.e., mean Ringer traces in TTX,

8 3 DRIFTING SAWTOOTH GRATINGS A fast-off Ringer TTX APB PDA 1 1 Amplitude (µv) Amplitude (µv) 1 B fast-on Ringer TTX APB PDA APB+PDA APB, and PDA graphs of Figs. ). These small variations in the quality of response obtained from each set of eyecups are not expected to affect the results given that all statistical analyses used a within-subjects design. Temporal Sawtooth Flicker Figure 7 illustrates ERG responses to 1-Hz temporal sawtooth flicker before and after addition of TTX, APB, and PDA to the bath. Although TTX can alter the timing and magnitude of the square-wave flash ERG b-wave (Dong and Hare ; Hare and Ton ), it caused no obvious changes to the sawtooth flicker waveforms recorded here. By comparison, APB abolished the ON response and enhanced the OFF response to both fast-on and fast-off sawtooth. PDA effects were weaker, primarily resulting in a suppression of the OFF response to both sawtooth profiles. DISCUSSION This study demonstrated that the DC ERG response to fast-on and fast-off drifting sawtooth gratings is asymmetric. At temporal frequencies Hz, all grating and diamond profiles elicited a sustained positive DC potential shift during drift and a peak at drift offset. These waveforms were also evident at. and Hz for all stimuli except sine-wave and fast-on gratings, which showed weak responses at low temporal frequencies. Consistent with the temporal sawtooth flicker literature (Alexander et al. 1; Barnes et al. ; Dryja et al. ; Khan et al. ; Kremers 13; Kremers et al. 1993; Pangeni and Kremers 13; Pangeni et al. 1; Rodrigues et al. ; Vukmanic et al. 1) and brightness perception measures in humans (Cavanagh and Anstis 19; Watanabe et al. 199), the DC potential shift for spatiotemporal fast-off sawtooth was larger than that for fast-on sawtooth across all of the conditions tested (shaded gratings drifting at. Hz and shaded diamonds drifting at Hz) APB+PDA 1 1 Fig. 7. Mean DC ERG responses to 1-Hz spatially uniform fast-on and fast-off sawtooth flicker in Ringer and following pharmacological suppression with TTX, APB, PDA, or APB PDA. As with the drifting grating data, these full-field flicker responses were recorded over successive runs. Run 1 was recorded with Ringer. Run was recorded following pharmacological suppression with M TTX, 1 mm APB, or 1 mm PDA. For eyecups with APB or PDA in run, a 3rd run was recorded following suppression of all postreceptoral activity with 1 mm APB 1 mm PDA. Modeling suggested that differences in root-mean-square (RMS) temporal luminance contrast resulting from the stimulus device frame rate may have contributed to ERG response patterns for drifting stimuli, particularly when considering the effects of temporal frequency. This modeling shows that as temporal frequency increases, so does the RMS temporal contrast and in a way that depends on the particular grating, sine versus square versus sawtooth. These effects are quite general for all frame-based stimuli involving moving spatiotemporal patterns, including those used in previous studies of the drifting sawtooth illusion (Ashida and Scott-Samuel 1; Cavanagh and Anstis 19). Importantly, RMS temporal luminance contrast was identical for the two directions of sawtooth drift at any given temporal frequency. Thus the DC ERG responses recorded for these stimuli reflect a physiological asymmetry in the encoding of fast-on and fast-off spatiotemporal sawtooth by toad retina. Pharmacological dissection of ERG responses to drifting sawtooth gratings suggested an ON-bipolar source for the drift offset peak. The DC potential during drift appears to have more complex origins. The amplitude of this potential was not altered by TTX, which inhibits sodium-based action potentials of spiking amacrine and ganglion cells (Narahashi et al. 19). It was also unaltered by PDA, an antagonist of ionotropic glutamate receptors on OFF-bipolar and horizontal cells and kainite receptors in the inner retina. In contrast, mglur agonist APB increased the DC shift, indicating that the ON-bipolars make an antagonistic contribution to this waveform. This increase in the DC potential following APB occurred for both fast-on and fast-off profiles. Thus, if we compare fast-on and fast-off responses following APB or APB PDA delivery, they remain significantly different. This suggests that, although the ON-bipolars contribute to the DC potential, the asymmetry originates in the outer retina.

9 DRIFTING SAWTOOTH GRATINGS 37 To our knowledge, this is the first demonstration of a larger outer retinal response to fast-off versus fast-on spatiotemporal sawtooth. This finding builds on an extensive crossspecies literature demonstrating that the retinal response to temporal fast-off and fast-on sawtooth is asymmetric (Alexander et al. 1; Barnes et al. ; Dryja et al. ; Khan et al. ; Kremers 13; Kremers et al. 1993; Pangeni and Kremers 13; Pangeni et al. 1; Rodrigues et al. ; Vukmanic et al. 1). Within this temporal domain, the toad ERG response appears comparable to that of humans and monkeys, where the fast phase of fast-on flicker generally elicits slightly larger ON-bipolar responses relative to the combined photoreceptor and OFF-bipolar response elicited by fast-off flicker (Alexander et al. 1; Barnes et al. ; Khan et al. ; Kremers 13; Pangeni and Kremers 13; Vukmanic et al. 1). Moreover, like in monkey (Khan et al. ), our pharmacological dissection of the toad temporal sawtooth ERG (Fig. 7) revealed push-pull interactions between ON- and OFF-bipolar cell contributions to both fast-on and fast-off waveforms. In this respect, our pharmacological results for temporal and spatiotemporal sawtooth profiles are somewhat similar, as APB increased the DC potential shift in response to drifting fast-on and fast-off gratings (Figs. and A) and substantially increased the amplitude of OFF responses to both fast-on and fast-off temporal flicker (Fig. 7). It should be noted that this study is also the first, to our knowledge, to dissect the DC motion ERG pharmacologically. The only previous studies to investigate ERG responses to sustained motion (i.e., ms) were conducted using squarewave stimuli in humans (Dodt and Kuba 199; Korth et al. ). These studies identified an initial fast positive waveform [not seen in toad, where temporal resolution is slow (Donner et al. 199)] followed by a sustained potential with similarities to the DC shift identified in the present study. As noted above, our pharmacological findings suggest that this sustained waveform primarily reflects photoreceptoral and ONbipolar activity. Also similar to the present study, Korth et al. () identified a peak following motion offset in human motion ERGs that appeared related to the pattern onset response. Our pharmacological results suggest that this peak could be generated by the ON-bipolars rather than cells in the proximal retina. In summary, this study aimed to characterize the retinal response to spatiotemporal sawtooth stimuli and to clarify the neural basis of the perceptual effects that such stimuli elicit in humans. We demonstrated pharmacologically that the response to drifting fast-off and fast-on sawtooth gratings is asymmetric at the photoreceptoral level in toad. Further comparison of our findings with published AC motion ERG and sawtooth flicker studies demonstrated that the toad response to both temporal and spatiotemporal stimuli shares many similarities with that of humans and monkeys. Together, these findings point to an outer retinal origin for the perceptual brightness illusion generated by drifting sawtooth stimuli. GRANTS This research was supported by Australia Research Council Grant DP137 to D. P. Crewther and S. G. Crewther. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS N.R., L.H., S.G.C., and D.P.C. conception and design of research; N.R., L.H., and J.J. performed experiments; N.R., L.H., and D.P.C. analyzed data; N.R., L.H., J.J., S.G.C., and D.P.C. interpreted results of experiments; N.R., L.H., and D.P.C. prepared figures; N.R. and L.H. drafted manuscript; N.R., L.H., J.J., S.G.C., and D.P.C. edited and revised manuscript; N.R., L.H., J.J., S.G.C., and D.P.C. approved final version of manuscript. REFERENCES Alexander KR, Fishman GA, Barnes CS, Grover S. On-response deficit in the electroretinogram of the cone system in X-linked retinoschisis. Invest Ophthalmol Vis Sci : 3 9, 1. Ashida H, Scott-Samuel NE. Motion influences the perception of background lightness. Iperception : 1 9, 1. Awatramani G, Wang J, Slaughter MM. Amacrine and ganglion cell contributions to the electroretinogram in amphibian retina. Vis Neurosci 1: 17, 1. Bach M, Hoffmann MB. Visual motion detection in man is governed by non-retinal mechanisms. Vision Res : 379 3,. Barnes CS, Alexander KR, Fishman GA. A distinctive form of congenital stationary night blindness with cone ON-pathway dysfunction. Ophthalmology 9: 7 3,. Barry PH, Diamond JM. Junction potentials, electrode standard potentials, and other problems in interpreting electrical properties of membranes. J Membr Biol 3: 93 1, 197. Cavanagh P, Anstis SM. Brightness shift in drifting ramp gratings isolates a transient mechanism. Vision Res : 99 9, 19. Dodt E, Kuba M. Simultaneously recorded retinal and cerebral potentials to windmill stimulation. Doc Ophthalmol 9: 7 9, 199. Dong CJ, Hare WA. Contribution to the kinetics and amplitude of the electroretinogram b-wave by third-order retinal neurons in the rabbit retina. Vision Res : 79 9,. Donner K, Copenhagen DR, Reuter T. Weber and noise adaptation in the retina of the toad Bufo marinus. J Gen Physiol 9: , 199. Dryja TP, McGee TL, Berson EL, Fishman GA, Sandberg MA, Alexander KR, Derlacki DJ, Rajagopalan AS. Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM gene encoding mglur. Proc Natl Acad Sci USA : 9,. Gallemore RP, Hughes BA, Miller SS. Retinal pigment epithelial transport mechanisms and their contributions to the electroretinogram. Prog Retin Eye Res 1: 9, Hare WA, Ton H. Effects of APB, PDA, and TTX on ERG responses recorded using both multifocal and conventional methods in monkey. Doc Ophthalmol : 19,. Jardon B, Yücel H, Bonaventure N. Glutamatergic separation of ON and OFF retinal channels: possible modulation by glycine and acetylcholine. Eur J Pharmacol 1:, 199. Khan NW, Kondo M, Hiriyanna KT, Jamison JA, Bush RA, Sieving PA. Primate retinal signaling pathways: suppressing ON-pathway activity in monkey with glutamate analogues mimics human CSNB1-NYX genetic night blindness. J Neurophysiol 93: 1 9,. Korth M. Electric responses of the human retina to moving stimuli. Graefes Arch Clin Exp Ophthalmol : 9 9, 197. Korth M, Rix R, Sembritzki O. The sequential processing of visual motion in the human electroretinogram and visual evoked potential. Vis Neurosci 17: 31,. Kremers J. Asymmetries in the contributions of On- and Off-mechanisms to the ERG signal. Psychol Neurosci : , 13. Kremers J, Lee BB, Pokorny J, Smith VC. Responses of macaque ganglion cells and human observers to compound periodic waveforms. Vision Res 33: , Miller SS, Steinberg RH. Passive ionic properties of frog retinal pigment epithelium. J Membr Biol 3: , Narahashi T, Moore JW, Scott WR. Tetrodotoxin blockage of sodium conductance increase in lobster giant axons. J Gen Physiol 7: 9 97, 19. Pangeni G, Kremers J. Human photopic ON- and OFF-ERG responses elicited by square wave and sawtooth stimuli. Psychol Neurosci :, 13.

10 3 DRIFTING SAWTOOTH GRATINGS Pangeni G, Lämmer R, Tornow RP, Horn FK, Kremers J. On- and off-response ERGs elicited by sawtooth stimuli in normal subjects and glaucoma patients. Doc Ophthalmol 1: 37, 1. Rodrigues AR, da Silva Filho M, Silveira LC, Kremers J. Spatial distributions of on- and off-responses determined with the multifocal ERG. Doc Ophthalmol 1:,. Slaughter MM, Miller RF. -Amino--phosphonobutyric acid: a new pharmacological tool for retina research. Science 11: 1, 191. Stockton RA, Slaughter MM. B-wave of the electroretinogram. A reflection of ON bipolar cell activity. J Gen Physiol 93: 1 1, 199. Vukmanic E, Godwin K, Shi P, Hughes A, DeMarco P Jr. Full-field electroretinogram response to increment and decrement stimuli. Doc Ophthalmol 19: 9, 1. Watanabe I, Cavanagh P, Anstis S, Shrira I. Shaded diamonds give an illusion of brightness. Invest Ophthalmol Vis Sci 3: S, 199. Wu S, Burns SA, Reeves A, Elsner AE. Flicker brightness enhancement and visual nonlinearity. Vision Res 3: 73 3, 199.