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.

Keywords: Blue-background; Chromatic; Dogs; ERG; Flicker.

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

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)

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