Use of extended protocols with nonstandard stimuli to characterize rod and cone contributions to the canine electroretinogram

Abstract

Purpose

In this study, we assessed several extended electroretinographic protocols using nonstandard stimuli. Our aim was to separate and quantify the contributions of different populations of retinal cells to the overall response, both to assess normal function and characterize dogs with inherited retinal disease.

Methods

We investigated three different protocols for measuring the full-field flash electroretinogram—(1) chromatic dark-adapted red and blue flashes, (2) increasing luminance blue-background, (3) flicker with fixed frequency and increasing luminance, and flicker with increasing frequency at a fixed luminance—to assess rod and cone contributions to electroretinograms recorded in phenotypically normal control dogs and dogs lacking rod function.

Results

Temporal separation of the rod- and cone-driven responses is possible in the fully dark-adapted eye using dim red flashes. A- and b-wave amplitudes decrease at different rates with increasing background luminance in control dogs. Flicker responses elicited with extended flicker protocols are well fit with mathematical models in control dogs. Dogs lacking rod function demonstrated larger amplitude dark-adapted compared to light-adapted flicker responses.

Conclusions

Using extended protocols of the full-field electroretinogram provides additional characterization of the health and function of different populations of cells in the normal retina and enables quantifiable comparison between phenotypically normal dogs and those with retinal disease.

Introduction

Electroretinography is an invaluable tool in ophthalmology. The electroretinogram (ERG) has been used in dogs for decades for clinical assessment and research into retinal function and dysfunction. However, assessment of canine ERGs is often restricted to responses elicited by a limited number of stimuli [1, 2]. While these short protocols provide a general overview of retinal function and aid in the diagnosis of retinal disease, they are somewhat limited in the information that they can provide. For example, receptor response threshold is not obtained by the standard protocols. However, additional extended protocols provided for flash ERG by the International Society for Clinical Electrophysiology of Vision (ISCEV) include the dark-adapted red flash, a strong flash to saturate the rod a-wave, a dark- and light-adapted luminance/response series, light-adapted ON–OFF with long duration flashes, photopic negative response (PhNR), and S-cone response [3–9].

Standard ERG protocols typically feature white light flashes (which consists of wavelengths comprising the visible spectrum) presented in the dark to a dark-adapted eye or to a light-adapted eye superimposed on a constant white background light [1, 2]. Chromatic flashes utilize stimuli of specific wavelengths in order to preferentially stimulate different photoreceptors. These protocols typically feature long wavelength red flashes and may also include short wavelength blue flashes [3, 10–12]. In humans, dark-adapted red flashes elicit a cornea-positive cone-driven response, known as the x-wave that precedes the rod-driven b-wave. Chromatic flashes have also been utilized in investigation of disease processes such as glaucoma [13–16]. Several studies in dogs have demonstrated separation of dark-adapted rod- and cone-driven responses [17–22]. Dogs have two type types of cones—M/L-cones, which are mainly stimulated by longer wavelength light (peak sensitivity at 555 nm) and S-cones, which are mainly stimulated by shorter wavelength light (peak sensitivity at 430–435 nm). Additionally, rods have a peak sensitivity at 508 nm [23]. Chromatic stimuli have also been used in analysis of retinal dysfunction, such as evaluation of drug-induced retinal toxicity [24]. However, chromatic stimuli are used less frequently in current canine ERG studies [2].

Photoreceptors are also differentially affected by the strength of background light. Cones signal through ON and OFF pathways to depolarizing and hyperpolarizing bipolar cells, and the relative and opposing contributions of these pathways exhibit what has been described as a ‘push–pull’ mechanism that that leads to the resulting shape and amplitude of the b-wave response [25–27]. The primary rod pathway is through rod bipolar cells; however, studies in mice and primates indicate that rods also signal through gap junctions with cones as well as via direct synapses with cone OFF-bipolar cells, with the relative contribution of each pathway influenced by ambient light levels [28–32]. In primates and rodents, both the degree and length of light adaptation have been shown to differentially affect responses originating from different classes of bipolar cells and photoreceptors [25, 33–36]. However, the electroretinographic responses to different background lighting conditions have not been quantified or modeled in the dog.

ERG protocols that vary in flash frequency and stimulus strength have been used to separate rod- and cone-driven signaling pathways. Differences have been found between flicker stimuli presented in dark- and light-adapted conditions, as well as changes in the shape and amplitude of responses to flicker stimuli of increasing stimulus strength [37–39]. Moreover, a study in mice demonstrated differential responses of second-order neurons to flicker stimuli, with ON-bipolar cells driving responses to lower-frequency flicker stimuli and OFF-bipolar cells driving responses to higher-frequency flicker stimuli [40]. Although both ERG and behavioral studies have examined the critical flicker fusion frequency in dogs, there are limited reported investigations of responses to flicker stimuli at different frequency or stimulus strength [19, 20, 41].

A major aim of these studies is the analysis of rod and cone contributions to the canine ERG using extended protocols tested in both phenotypically normal dogs and identified canine models of specific retinal dysfunction. Animals with gene mutations are often utilized to investigate rod and cone contributions; in this study we used a retinitis pigmentosa dog model with non-functional rods and an achromatopsia model with non-functional cones. For a model with non-functional rods, we used dogs homozygous for a PDE6A null mutation [42]. This allowed us to assess cone-only responses with the presence of little or no active rod interaction. In young PDE6A^−/− dogs, cone b-waves are of normal amplitude but of slightly delayed peak time, while cone a-waves are slightly reduced. They do progressively lose remaining cone function over the first 12 months of age [43]. We additionally tested an abbreviated protocol on a dog with compound heterozygous mutations in the CNGB3 gene (a CNGB3*^del dog with a D262N missense mutation and genomic deletion), who have almost no cone responses by 12 weeks of age and serve as a model of rod-driven function [44].

In designing this current study, we considered multiple approaches to characterize rod and cone pathways in the dog. We were particularly interested in methods that have enabled separation of these pathways with different states of retinal adaptation. We ultimately tested three different types of protocols. We assessed temporal separation of dark-adapted rod and cone responses using red and blue chromatic flashes, an older technique that has been infrequently used in recent canine ERG studies. Additionally, we considered the effect of light adaptation on the individual contributions of rod and cone pathways using a luminance/response protocol with progressively increasing background luminance. Finally, we examined the temporal differences in rod- and cone-driven responses using extended dark- and light-adapted flicker protocols which varied in either flash frequency or stimulus strength. Not only do these protocols have potential utility in routine ERG recordings, but it may be possible to develop mathematical models for these protocols, similar to the Naka–Rushton b-wave fitting, to quantify normal response ranges as well as parameterize pathological ERG recordings [45–47]. Although some of these models have been researched and utilized in humans and many other species, less is known about their capacity to characterize retinal function in normal dogs as well as dog models of retinal disease.

Methods

Animals

Purpose bred PDE6A^−/−, CNGB3^*/del, and phenotypically normal control dogs maintained in a colony at Michigan State University were investigated in this study. They were housed under 12 h:12 h light/dark cycles. The PDE6A mutation arose in the Cardigan Welsh Corgi breed but has been bred onto a laboratory beagle background. The CNGB3 mutations arose in the Alaskan Malamute (genomic deletion) and German Shorthaired Pointer (missense mutation) breeds but has been bred onto a laboratory beagle background.

With exceptions noted below, subjects included 10 phenotypically normal control dogs (4 male and 6 female) and 5 PDE6A^−/− dogs (2 male and 3 female). Control subjects were all 2 months of age (dogs have a measurable ERG by 3 weeks of age, which grows in amplitude to reach adult ERG amplitudes by 8 weeks of age [48]), while PDE6A^−/− dogs ranged from 1 to 2 months of age. (Younger dogs were used for this study to preclude significant cone loss in PDE6A^−/− subjects.)

Anesthesia

General anesthesia was induced by isoflurane mask for puppies and by IV propofol for older animals (4–6 mg/kg, PropoFlo, Abbott Laboratories, North Chicago, IL, USA). The animals were intubated and subsequently maintained under anesthesia with isoflurane (IsoFlo, Abbott Laboratories, North Chicago, IL, USA) [between 2 and 3.5% in a 1–2 L/min oxygen flow via a rebreathing circle system for dogs over 10 kg and via a Bain system for dogs under 10 kg].

Electroretinography (ERG)

General procedures for ERGs were described previously [49]. Differences in apparatuses and protocols are noted below. Briefly, prior to anesthesia dogs were dark-adapted for 1 h and pupils dilated with tropicamide (Tropicamide Ophthalmic Solution UPS 1%, Falcon Pharmaceuticals Ltd., Fort Worth, TX, USA). A monopolar gold-ringed electrode contact lens (ERG-Jet electrode, Fabrinal Eye Care, La Chaux-De-Fonds, Switzerland) was used and for reference, and grounding platinum needle skin electrodes (Grass Technologies, Warwick, RI, USA) were placed 5 mm lateral to the lateral canthus and over the occiput, respectively. ERGs were recorded using an Espion E² Electrophysiology system with ColorDome Ganzfeld (Diagnosys LLC, Lowell, MA, USA).

Chromatic ERG

Flashes were presented at 1 Hz (< 4 mS flash duration) for a dark-adapted eye. Red LED flash stimuli, with a wavelength centered at 630 nm and half-bandwidth of 20 nm, ranged from −1.3 to 0.4 log cd s/m² (0.05–2.5 cd s/m²). Blue LED flash stimuli, with a wavelength centered at 445 nm and half-bandwidth of 20 nm, ranged from −4.3 to −1.3 log cd s/m² (0.00005–0.05 cd s/m²).

For the purpose of scotopic matching of red and blue flashes in control dogs, we performed Naka–Rushton fitting of b-wave amplitudes. Amplitudes were measured from the trough of the preceding a-wave to the peak of the following b-wave, accounting for oscillatory potential (OP) intrusion along the leading edge of the b-wave. Model parameters were calculated by fitting the Naka–Rushton function to the first limb of the b-wave luminance/response plot. The Naka–Rushton function and parameters are as follows:

$$R/V_m=L^n/\left(L^n+K^n\right)$$

V_m represents the maximum response amplitude of the first limb of the b-wave luminance/response plot, k is a semi-saturation constant considered a measure of retinal sensitivity, and n is dependent on of the slope of the plot at the position of k, which may reflect retinal homogeneity [47, 50, 51].

We also tested a short scotopic matching protocol on one adult (4 years of age) CNGB3^*/del dog. (Additional dogs were not available for inclusion in statistical analyses.)

Blue-background ERG

Each flash was presented at one second intervals (< 4 mS flash duration) on a dark background. White-flash stimuli ranged from −2 to 0.4 log cd s/m² (0.01–2.5 cd s/m²) and were presented on no background, as well as blue-backgrounds of 0.01, 0.1, 1.0, and 10 cd/m², with a wavelength centered at 445 nm and half-bandwidth of 20 nm, for gradual suppression of rod responses. Dogs were dark-adapted for 1 h prior to recording and were adapted for 5 min to each background luminance.

Flicker ERG

Two different protocols, adapted from previous work by Seeliger et al. [52], were tested—one with a stimulus of 3.2 cd s/m² white light with flicker stimuli of increasing frequency from 0.5 to 30 Hz and the other with a frequency of 6 Hz and stimuli luminance ranging from −4.9 to 1.5 log cd s/m² (1 × 10⁻⁵ to 32.0 cd s/m²; both protocols had < 4 mS flash duration). The flicker stimuli were presented in both dark-adapted (in the dark) and light-adapted (30 cd/m² white background) conditions. Dogs were dark-adapted for 1 h prior to the dark-adapted flicker and light-adapted for 10 min on a 30 cd/m² white background prior to light-adapted flicker.

For the extended flicker protocols, subjects included 11 phenotypically normal control dogs (4 male and 7 female) and 3 PDE6A^−/− dogs (3 female). Subjects ranged in age from 3 to 7 months. (All PDE6A^−/− dogs were tested at 3 months of age.)

ERG response averages

All stimulus conditions were repeated to generate an averaged response detectable against background noise. The number of responses recorded varied depending on stimulus and background characteristics, with additional sweeps performed in light-adapted conditions (which have more underlying background noise) as well as for lower amplitude responses (such as in PDE6A^−/− dogs).

Statistical analysis

All statistical tests were performed in Python with the Statsmodels package [53]. ANCOVA testing was used to compare statistical significance between the means of different groups when modeled with linear regression:

$$y_{ij}=\mu +{\tau }_i+B\left(x_{ij}-\overline{x}\right)+{\epsilon }_{ij}$$

The variables i and j are the jth observation of the ith categorical group, the dependent variable y is modeled as a function of independent variable x, μ and $\overline{x}$ are mean parameters derived from the data, and the fitted variables are the effect parameter τ, slope parameter B, and error term ε.

After assessment of linearity of regression, homogeneity of error variances, independence and normality of error terms, and homogeneity of regression slopes, mean group differences were assessed using the F-test [54].

Results

Rod and cone contributions to the chromatic ERG in the dark-adapted eye in control dogs and dogs lacking rod function (Figs. 1, 2, 3 and 4)

Fig. 1.

Representative chromatic dark-adapted ERG tracings. Red and blue flash dark-adapted ERG montage of a control dog (a) and PDE6A^−/− dog (b). Red stimuli had flash strengths ranging from −1.3 to 0.4 log cd s/m² (0.05–2.5 cd s/m²). Blue stimuli had flash strengths ranging from −4.3 to −1.3 log cd s/m² (0.00005–0.05 cd s/m²). Note the scale difference between A and B

Fig. 2.

Dark-adapted ERGs as a result of matched red and blue flashes in six control dogs. A single red flash with strength −1.0 log cd s/m² (red tracing) and blue flash with strength −3.2 log cd s/m² (blue tracing) were used for comparison. Although the appearance differed between dogs, a small positive deflection was noted preceding the rod-driven b-wave in all dogs. This likely represents the dark-adapted cone-driven response to red-flash stimulus known as the x-wave. Tracings recorded from six different dogs are shown to demonstrate variability of responses, with magnification of the responses preceding the rod-driven b-wave shown in the inserts

Fig. 3.

Comparing red and blue flash ERG responses in dark-adapted control dogs. These figures denote the peak times (a) and amplitudes (b) of the rod-driven b-wave in control dogs in response to red and blue flashes (red and blue tracings, respectively). The stimulus strength used for the blue flashes is shown above the graph, and those for the red flashes are below the graph. The comparisons between the two flashes were based on the difference of the Naka–Rushton K parameter. Error bars denote standard deviation

Fig. 4.

Comparing red-flash ERG responses in dark-adapted control and PDE6A^−/− dogs. Comparison of responses to a −1.0 (a) and 0.4 (b) log cd s/m² red-flash stimulus of a control dog (black) with a PDE6A^−/− dog (red). Error bars denote standard deviation. With dimmer flashes, the slope, amplitude, and latency of the small deflection that appears before the leading edge of the rod b-wave in the control dog matches those of ascending limb of the b-wave in the PDE6A^−/− dog. With stronger flashes, the leading edge of the positive deflection preceding the b-wave in the control dog (the x-wave) was superimposed on the growing a-wave, Magnification of the cone-driven responses is shown in the insert. Average latency (time from flash onset to start of the x-wave, shown in c) and slope (d) of the x-wave (control dogs, shown in black) and b-wave (PDE6A^−/− dogs, shown in red) vs. stimulus strength with red-flash stimulus. Note that in PDE6A^−/− dogs there was a slight delay in response times compared to controls. Comparison of average peak time (e) and amplitude (f) of the leading edge of the cone-driven b-wave in control (black) and PDE6A^−/− dogs (green), and the full cone-driven b-wave in PDE6A^−/− dogs (blue). In PDE6A^−/− dogs, stronger flash resulted in temporal separation of an initial peak and subsequent larger peak amplitude and peak time were comparable between the initial peak in both dogs

Red and blue flash dark-adapted ERG series were performed in control dogs and young PDE6A^−/− dogs (Fig. 1). Naka–Rushton fitting (supplemental Fig. 1) was performed to match stimulus strengths based on the constant K in control dogs in determination of the ½ saturation parameter K, with model parameters as follows:

$$\mathrm{Blue}:V_{\text{m}}=162.68\pm 30.26\mu \text{V},K=0.0012\pm 3.6\mathrm{cd}.\text{s}/{\text{m}}^2,n=1.019\pm 0.232$$

$$\mathrm{Red}:V_{\text{m}}=151.25\pm 24.36\mu \text{V},K=0.257\pm 0.047\mathrm{cd}.\text{s}/{\text{m}}^2,n=1.064\pm 0.103$$

Comparison of model parameters indicates that the semi-saturation parameter V_m was similar across both tested flash colors, as was the slope parameter n. However, the greatest differences were revealed in comparison of the K parameter, reflecting the differences in spectral sensitivity with different flash colors (see Supplemental Fig. 1). Note that the difference in the K parameter likely reflects the calibration of the flashes provided by the unit in photopic cd s/m² for the human eye rather than in scotopic units for canine eye.

In control dogs specifically, this scotopic matching revealed a small positive deflection preceding the larger rod-driven b-wave in response to the red stimulus and not the blue stimulus. Overlaying dark-adapted responses to red and blue flashes (with −1.0 and −3.3 log cd s/m² flash strengths, respectively—see Fig. 2 showing overlays in 6 control dogs) suggests that this difference may reflect the cone contribution to the dark-adapted ERG (the x-wave). Although this positive deflection was present in all ERGs recorded in control dogs, there was great variability in both the amplitude and appearance of the deflection (Fig. 2). The x-wave response is small, which is not unexpected given the relatively low-amplitude responses seen in the white-flash light-adapted ERG series.

Using ANCOVA to compare linear regressions of the matched red and blue flash parameters in control dogs, there was a small but statistically significant difference between the peak time of the b-waves, with the red having a shorter peak time (Fig. 3a blue and red tracings; ANCOVA F-test: τ = 5.67 ± 1.994; F = 2.84, p-value = 0.016). The luminance/response plots for dark-adapted red and blue flash were similar in shape and are shown in Fig. 3b with scales adjusted to overlay the plots and thus compensate for the use of the calibration of the unit in photopic units.

Using the results of scotopic matching of red and blue flashes in control dogs, we devised a short protocol consisting of a single red and blue flash with −1.0 and −3.3 log photopic cd s/m² stimulus strengths, respectively. Overlaying these responses in a PDE6A^−/− dog, a model of cone-driven function, demonstrated a small response to the red-flash stimulus and no response to the blue flash (Supplemental Fig. 2A). In the CNGB3^*/del dog, a model of rod-driven function with absence of cone function, the red-flash b-wave had a substantially lower amplitude than that of the blue flash and had no discernible x-wave response (Supplemental Fig. 2B).

Comparison of control to PDE6A^−/− dog responses in the red-flash ERG further supports the presence of a small x-wave in control dogs. Overlaying responses between control and PDE6A^−/− dogs shows that the leading edge of the cone-mediated b-wave in the PDE6A^−/− dogs closely aligns with the positive deflection preceding the rod-mediated b-wave seen in control dogs (Fig. 4). This similarity was particularly noticeable in response to dimmer red-flash stimuli, such as −1.0 log cd s/m², when there was greater separation between the cone-driven x-wave and rod-driven b-wave (Fig. 4a). Note that both the a- and b-waves of the PDE6A^−/− dogs are somewhat slower than those of controls. This is consistent with the timing differences seen when comparing both dark- and light-adapted ERGs recorded from young PDE6A^−/− dogs with those from light-adapted young control dogs [unpublished findings].

Using ANCOVA to compare linear regressions of the matched red and blue flash parameters in control dogs, there was a small but statistically significant difference between the peak time of the b-waves, with the red having a shorter peak time (Fig. 3a). The peak time of the a-wave was not assessed due to intrusion of the cone-driven response. We performed further analysis of the latency (time from flash to onset of waveform), peak time, slope, and amplitude of the rod- and cone-driven recordings in the control dogs and the responses (cone-driven) in the PDE6A^−/− dogs (Fig. 4c–f). In the PDE6A^−/− dogs, weak flash stimuli elicited a cone-driven component with a single peak, but with stronger flashes additional peaks were visualized (red tracings, Fig. 4a–b). In control dogs, the cone-driven components became overwhelmed by the growth of the rod-driven a-wave and earlier onset of the rod-driven b-wave with increasing luminance (Fig. 4b; black tracing shows the x-wave superimposed on a small a-wave). For accurate analysis, we restricted direct comparisons to the leading slope of the cone-driven x-wave. Using ANCOVA to compare the slope, amplitude, and peak time of this first limb between control and PDE6A^−/− dogs demonstrated no significant differences in the means of tested parameters (Fig. 4c–f). A- and b-wave amplitudes change at different rates depending on background luminance (Fig. 5)

Fig. 5.

Representative ERG tracings on a blue-background light. a Blue-background ERG montage of a control dog. White stimulus flash strengths ranged from −2 to 0.4 log cd s/m² (0.01–2.5 cd s/m²). Background luminances from left to right were 0 cd/m² (dark-adapted—no background), 0.01, 0.1, 1.0, and 10 cd/m² blue-backgrounds. b Blue-background ERG montage of a PDE6A^−/− dog. Note the smaller scale of response amplitude. A-wave amplitudes were similar for all background luminances, while b-wave amplitudes had a small decrease with the strongest background. A magnified view of the 10 cd/m² background condition is shown for control (c) and PDE6A^−/− (d) dogs (both calibrated to 15 μV amplitude)

The stimuli in this protocol were chosen, so in the dark-adapted eye the weakest flashes result in rod responses and with increasing flash strength mixed rod and cone responses are obtained. This protocol was performed in both control and PDE6A^−/− dogs (Fig. 5).

A major area of investigation with this protocol was the relative changes in a- and b-wave amplitudes with increases in background luminance. In control dogs between 0 and 0.1 cd/m² luminance, the b-wave amplitudes declined significantly more than the a-wave amplitudes—this led to a negative-type ERG appearance in the mesopic conditions (see Fig. 5a). This was further demonstrated in comparison of the b/a ratio, which declined substantially across the dimmest three luminances. Although the a-wave amplitudes decreased significantly between the 0.1 and 1 cd/m² backgrounds, there appeared to a continued small rod contribution to the response resulting in an underlying negativity. Additionally with stronger flashes (above −0.5 log cd s/m²) on the 1 cd/m² background, the b/a ratio declined similarly to the weaker backgrounds, which may reflect remaining rod contributions. The b/a ratio was flat in the control group at 10 cd/m², and the response is likely driven predominately by cones. Overall, these results supported previous work in a number of species that the postreceptoral response saturated at significantly dimmer background luminances compared to rod photoreceptors.

Another area of investigation with this protocol was the relative changes in cone-driven responses to increasing background luminance. These changes were studied by examining the responses in PDE6A^−/− dogs which have no rod function. Although analysis of these responses could not compare direct interactions between rods and cones, it did enable characterization of changes in response amplitude of isolated cone photoreceptors and postreceptoral pathways. In these dogs, a- and b-wave amplitudes were remarkably consistent across all background luminances, albeit with a slight decrease in b-wave amplitude for stronger flashes on the strongest background. As such, the b/a ratio in these dogs remained largely similar in contrast to control dogs who exhibited substantial decreases in gain from a-wave to b-wave increasing from 0 to 1 cd/m² background luminance. However, with the 10 cd/m² background, responses between the two groups were comparable (Fig. 5c–d).

The constant luminance and increasing frequency flicker ERG demonstrate the transition from rod to cone-driven responses. Measured amplitudes of both flicker protocols were well-approximated with nonlinear equations. (Figs. 6, 7, 8 and 9).

Fig. 6.

Representative 3.2 cd s/m² flicker ERG tracings. Flash stimulus was held constant at 3.2 cd s/m², and flash frequency varied from 0.5 to 30 Hz. The first column (column 1) shows responses recorded from a control dog, and the second column (column 2) shows responses recorded from a PDE6A^−/− dog. The first row (row A) shows responses starting with a dark-adapted eye, and the second row (row B) shows the light-adapted responses. The inset in A1 shows the responses from 7 to 30 Hz, to the same scale as the other three panels

Fig. 7.

3.2 cd s/m² flicker ERG measurements and models. Comparison of dark-adapted (black) and photopic (red) flicker amplitudes vs. flash frequency in the control (a) and PDE6A (b) dog. The inset in A shows the responses to frequencies between 5 and 30 Hz in the control dog. Above 7 Hz, the difference in amplitude was similar in both control and PDE6A^−/− dogs. Error bars denote standard deviation. c The decline in amplitude of the dark-adapted flicker amplitude with increasing flash frequency in the control dog was well-modeled by a negative exponential function. The function and parameters are given in the inset

Fig. 8.

Representative 6 Hz flicker ERG tracings. Flash frequency was held constant at 6 Hz. The first column (column 1) shows responses recorded from a control dog, and the second column (column 2) shows responses recorded from a PDE6A^−/− dog. The first row (row A) shows dark-adapted responses with stimulus strength varied from −4.9 to 1.5 log cd s/m², and the second row (row B) shows photopic responses with stimulus strength varied from −2.5 to 1.5 log cd s/m²

Fig. 9.

6 Hz flicker ERG measurements and models. Error bars denote standard deviation. a Comparison of dark-adapted (black) and light-adapted (red) flicker amplitudes vs. stimulus strength in the control dog. b Comparison of dark-adapted (black) and light-adapted (red) flicker amplitudes vs. stimulus strength in the PDE6A^−/− dog. c The increase and subsequent decline of the dark-adapted flicker amplitude with increasing stimulus strength in the control dog was well-modeled with a piecewise Michaelis–Menten equation. Parameters are given in the inset. d The increase in light-adapted flicker amplitude with increasing stimulus strength in the control dog was well-modeled with a Michaelis–Menten equation. Parameters are given in the inset

We recorded ERGs in responses to a constant stimulus strength (3.2 cd s/m²), delivered at increasing frequency in both control and PDE6A^−/− dogs (Fig. 6). The dark-adapted response in the control dogs (that have both rod and cone function) had high amplitudes at low frequencies. Response amplitudes decreased with progressively higher frequency, but with only a very small decrease in amplitude above 7 Hz. The reduction in decreasing amplitude with increasing frequency suggests that rods were no longer able to recover between flashes or had become light-adapted (Figs. 6A1, 7a, black tracing). However, even after the noted flattening of the curve after 7 Hz the dark-adapted amplitudes were slightly higher than those in the light-adapted series (compare Fig. 6A1 and 6B1—noting the scale difference—and see Fig. 7a inset). The response amplitude in the light-adapted flicker series in normal dogs was similar across the range of frequencies tested (Figs. 6B1, 7a red tracing). In contrast to the normal dogs, the response amplitude of PDE6A^−/− dogs (cone-only responses) in the dark-adapted series was similar across the range of flicker stimuli used (Figs. 6A2, 7b). Similar to the light-adapted normal dogs, the amplitude in the light-adapted flicker series of PDE6A^−/− dogs was similar across all tested frequencies. However, the light-adapted series amplitudes were slightly lower than the dark-adapted series (compare Figs. 6A2 and B2, and 7b). The small but persistent difference in amplitudes between dark- and light-adapted cone responses in the PDE6A^−/− dogs as well as the control dogs above 7 Hz may reflect a reduction of the dark current in cones due to the 30 cd/m² background luminance in light-adapted conditions (Fig. 7a–b). We also noted a difference in the shape of the light-adapted series waveforms between controls and PDE6A^−/− dogs, with the latter showing broader peaks and absent post-peak negativity (Fig. 6B1–2).

The decline in amplitude of the control dogs’ dark-adapted ERG (Fig. 7c) is well approximated by a negative exponential function of the form $a*e^{^}bx+c$. The a parameter scales the response to its maximal value and reflects the b-wave amplitude with 0.5 Hz flashes, and b is a function of the rate of change and reflects the decline in amplitude with progressively higher-frequency flashes. The c is parameter is likely driven by dark-adapted cones and shifts the model upward by the constant amplitude seen in the light-adapted ERG as well as the difference between dark- and light-adapted cones (Fig. 7a). For this model, parameters were:

$$a=189.3\mu \text{V};b=-0.537{\mathrm{Hz}}^{-1};c=32.9\mu \text{V}$$

We additionally recorded ERGs in control and PDE6A^−/− dogs using a constant flash frequency (6 Hz) and increasing stimulus strength (Fig. 8). In control dogs, both the initial increase and subsequent decrease in flicker amplitude with increasing luminance (Fig. 8A1) can be modeled by semi-saturation kinetics. Both of these findings are interesting, as they suggest that the Naka–Rushton equation traditionally fit to the first limb of the single flash dark-adapted ERG b-wave luminance/response series is also suitable for the rod component of the luminance/response plot dark-adapted b-wave from a slow flicker series. Furthermore, this suggests that the initial increase in rod-mediated amplitude and, with progressive light adaptation, decrease in the dark-adapted 6 Hz ERG series follow semi-saturation kinetics. The PDE6A^−/− dog 6 Hz luminance/response series indicates that cone responses are similar in both dark- and light-adapted conditions (Fig. 9b), with the light-adapted responses tending to have a lower amplitude as found with the responses of increased flicker frequency with a set luminance described above.

At 6 Hz frequency, the peak dark-adapted amplitude occurred between −2 and −1 log cd s/m² flash stimulus (Fig. 9a). The increase in amplitude was approximated with model parameters V_m = 99.13 μV, K = 0.0043 cd s/m², and n = 0.888, while the decrease in amplitude was approximated with model parameters V_m = 72.21 μV, K = 0.271 cd s/m², and n = −0.693 (with an additional additive vertical shift of 26 μV, reflecting the amplitude of cone-driven responses with higher stimulus strength) (Fig. 9c). Furthermore, the light-adapted amplitude increase could be modeled with parameters V_m = 21.21 μV, K = 1.592 cd s/m², and n = 1.138 (Fig. 9d). These results suggest that the decline in dark-adapted amplitudes (driven by rod saturation with progressively stronger flashes) is somewhat offset by an increase in the cone-driven response.

Discussion

In this study, we tested three different ERG techniques to further assess canine retinal function. We showed that scotopic match of dark-adapted ERG responses to red and blue stimuli over a range of stimulus strengths could be fit with a Naka–Rushton equation to obtain the relative sensitivity of the rods to the two stimuli. Using this method and comparing responses in control and PDE6A^−/−) dogs that lack rod responses, we demonstrated temporal separation of rod and cone contributions to the dark-adapted ERG b-waves. We also showed that blue-background ERGs can be used to quantify the differential effects of background light on the response and saturation of rod photoreceptors and rod bipolar cells. We used extended flicker protocols to demonstrate that cone response amplitudes are similar with flash frequency under 30 Hz, whereas rod responses predominate up to about 7 Hz. Finally, we fit models to the amplitude of flicker responses in control dogs; the response to constant luminance and increasing frequency dark-adapted ERG can be modeled with a negative exponential function, whereas the constant frequency and increasing luminance dark-adapted and light-adapted ERG follow Michaelis–Menten-type saturation kinetics.

The selective use of red and blue flashes reveals the dark-adapted cone-driven component known as the x-wave. The x-wave is elicited by red-flash stimuli and has been demonstrated in humans as well as other species such as rats and monkeys. It is used to characterize the dark-adapted ERG, with normal x-waves and absent b-waves in diseases such as Oguchi disease, and normal b-waves and absent x-waves in diseases such as protanopia and complete achromatopsia [14, 55]. The x-wave has also been described in the past in ERG studies of dogs and used to characterize dogs with either rod or cone dystrophy. However, these studies either measured the x-wave early during dark-adaptation [19, 20, 22] or did not demonstrate complete temporal separation of rod- and cone-driven components in the fully dark-adapted dog [21].

We demonstrated that identification and isolation of the dark-adapted cone-driven x-wave is possible in dogs using sufficiently dim red stimuli. We found significantly earlier peak times in the b-waves with red stimuli compared to blue, which may reflect cone-driven contributions to the dim red-flash response. Additionally, we found that using a single red flash with strength −1.0 log cd s/m² and blue flash with strength −3.2 log cd s/m² is sufficient to elicit the x-wave and temporally separate dark-adapted rod and cone responses in dogs and could be easily incorporated into any dark-adapted ERG protocol for assessment of dark-adapted cone function (Supplemental Fig. 2).

Psychophysical studies of human vision show that humans have similar ability to discriminate across multiple magnitudes of background illumination. The remarkable adaptability of the visual response depends on a gain control mechanism, which prevents saturation of postreceptoral responses to rod-mediated signaling [56–59]. In dogs, we show that the photoreceptor-driven a-wave remains remarkably consistent across dim background lighting conditions (between 0 and 0.1 cd/m²), while the bipolar cell-driven b-wave experiences a significant decrease in amplitude with the introduction of even dim background luminance (see Fig. 6). These findings agree with those in humans and provide additional insight to the change in response characteristics of photoreceptors and second-order neurons when exposed to progressively brighter background light [33, 34, 36].

In the PDE6A^−/− dogs, the absence of rod function enabled characterization of cone-driven responses to increasing background luminance. In these dogs, a-wave responses, although very small in amplitude, were remarkably consistent irrespective of background light. This indicates that cone photoreceptors adapt and respond similarly across a wide range of lighting conditions, consistent with findings in other species such as humans and mice [59–62]. Furthermore, there was no significant difference in the a-wave amplitudes in these dogs at any background luminance, and a slight decrease in b-wave amplitudes only with the brightest background luminance. These findings suggest that cone ON- and OFF-bipolar cells may experience similar changes in response amplitude as rod bipolar cells, albeit with a higher threshold for changes to be seen. However, cone responses exhibit a ‘photopic hill’ effect whereby cone-driven b-wave amplitudes decrease with increasing stimulus strength, potentially reflecting a relative shift in signaling from the ON pathway to the OFF pathway (i.e., the ‘push–pull’ mechanism) or temporal separation of the ON and OFF responses, and this may contribute to these findings [25–27].

The use of flicker stimuli provides a better characterization of how rod- and cone-driven responses change as a function of altered temporal stimulation. As the interstimulus interval shortens with increasing flash frequency, it reaches a stage that the slower responding rods cannot recover between flashes (and also likely lose their dark-adapted state). In our study, we found that rod responses in dogs predominate with stimuli up to about 7 Hz (Fig. 7), in agreement with findings in other species such as mice [40]. Additionally, we found that cone-driven responses are similar across a wide range of frequencies, with comparable amplitudes in the light-adapted ERG between 0.5 and 30 Hz stimuli. We did note a difference in the shape of the waveforms in the light-adapted ERG (Fig. 6B1–2). With lower-frequency flashes, control dogs had a large cornea-negative deflection similar in appearance to the photopic negative response (PhNR), which is thought to derive from ganglion cells with possible amacrine cell contributions [63, 64]. This negative component declined with higher-frequency flicker, perhaps reflecting reduced ganglion cell recovery or masking of the response (due to shorter interstimulus interval). This negative response was not seen in PDE6A^−/− dogs, which may be attributable to differences seen in the single flash light-adapted ERG (including a wider b-wave and attenuated a-wave).

Additionally, we provided baseline mathematical models of the amplitude changes seen in two flicker protocols in phenotypically normal dogs. In PDE6A^−/− dogs, the difference in amplitudes in the light-adapted compared to the dark-adapted ERG was similar at all frequencies (see Fig. 7b), while the difference in amplitudes was similar with higher-flicker frequency (above 7 Hz) in control dogs (see Fig. 7a). This may reflect a difference in basal activity of cone photoreceptors and bipolar cells, as well as differences in inner retinal contributions to the overall recorded waveform, but without a notable impact on the cone recovery [61, 65, 66].

In this study, we designed protocols to separate and better characterize rod and cone contributions to the canine ERG. We measured responses to these protocols in both phenotypically normal control dogs and PDE6A^−/− dogs as a model of cone-only function. Using these protocols, we preferentially stimulated isolated photoreceptor responses and provided further insight into the separate contributions of rods and cones in the dark- and light-adapted ERG. Additionally, we showed how rod and cone responses vary with stimulus frequency as well as background luminance. Finally, we showed how the inclusion of a single red and blue flash in the dark-adapted ERG could be of use in ophthalmological clinics to test dark-adapted cone function.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.