Contribution of GABAa, GABAc and glycine receptors to rat dark-adapted oscillatory potentials in the time and frequency domain

Abstract

Retinal oscillatory potentials (OPs) consist of a series of relatively high-frequency rhythmic wavelets, superimposed onto the ascending phase of the b-wave of the electroretinogram (ERG). However, the origin of OPs is uncertain and methods of measurement of OPs are diverse. In this study, we first isolated OPs from the rat ERG and fitted them with Gabor functions and found that the envelope of the OP contained information about maximum amplitude and time-to-peak to enable satisfactory quantification of the later OPs. And the OP/b-wave ratio should be evaluated to exclude an effect of the b-wave on the OPs. Next, we recorded OPs after intravitreal injection of 2-amino-4-phosphonobutyric acid (APB), tetrodotoxin (TTX), γ-aminobutyric acid (GABA), strychnine (STR), SR95531 (SR), isoguvacine (ISO), (1,2,5,6-tetrahydropyridin-4-yl) methylphosphinic acid (TPMPA) and GABA+TPMPA. We showed that GABA and APB only removed the later OPs, when compared to control eyes. TTX delayed the peak time, and STR, SR and ISO reduced the amplitude of OPs. TPMPA delayed the peak time but increased the ratio of OPs to b-wave. Furthermore, administration of combined GABA and TPMPA caused the later OPs to increase in amplitude with time, compared with those after delivery of GABA alone. Finally, we observed that GABAc and glycine receptors contributed to a low-frequency component of the OPs, while GABAa contributed to both components. These results suggest that the early components of the OPs are mainly generated by the photoreceptors, whilst the later components are mainly regulated by GABAa, GABAc and glycine receptors.

Keywords: GABA receptors; frequency domain; glycine receptors; oscillatory potentials.

Figure 1. Measurement of OPs in the time domain

(A) A Gabor function is a Gaussian function, g(x), multiplied by a sinusoidal carrier function, f(x). (B) Flowchart showing OPs processing. The envelope of the raw OPs was calculated and then fitted with a Gaussian function to get the three parameters of g(x). The sine wave carrier was then modeled by dividing out the Gaussian envelope and fitting the resulting signal with a double-sine function to get the parameters of f(x). (C) Left: Dark-adapted OPs showing the four distinct OPs components (OP1 to OP4). The amplitude and time-to-peak of the largest OPs component (OP4) could be employed as a surrogate for the amplitude and time-to-peak of the overall OPs. Middle: Simple linear regression between parameter a and the amplitude of OP4. Right: Simple linear regression between parameter m and the time-to-peak of OP4. (D) OP1 and OP2 diminished when subtracting the PIII component from the raw waveforms of ERG.

Figure 2. OPs attenuation after intravitreal injection of APB

(A) Left panels: Representative ERG traces evoked by six different light intensities, from −4.5 log(cd·s·m^-2) at the top to 1 log(cd·s·m^-2) at the bottom. Right panels: OP waveforms isolated from the ERG waveforms by bandpass filtering (60–300 Hz). Red line, APB-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for APB-treated and PBS-treated eyes (n=6 each). (D) Amplitude of OP1 and OP2 in B (n=6 each). **p<0.01. Line and bar charts show mean ± SEM.

Figure 3. OPs attenuation after intravitreal injection of STR

(A) Left panels: Representative light-evoked ERG traces for different light intensities. Right panels: OP waveforms isolated by bandpass filtering. Red line, STR-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for STR-treated and PBS-treated eyes (n=5 each). (D) Amplitude of OP1 and OP2 in B (n=5 each). *p<0.05, **p<0.01. Line and bar charts show mean ± SEM.

Figure 4. The effect of TTX on dark-adapted OPs

(A) Left panels: Representative light-evoked ERG traces for different light intensities. Right panels: OP waveforms isolated by bandpass filtering. Red line, TTX-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for TTX-treated and PBS-treated eyes (n=5 each). (D) Ratio of OPs amplitude to b-wave amplitude at different light intensities. (E) Time-to-peak of OPs evoked by the highest five light intensity in B (n=5 each). *p<0.05, **p<0.01. Line and bar charts show mean ± SEM.

Figure 5. OPs attenuation after intravitreal injection of GABA

(A) Left panels: Representative light-evoked ERG traces for different light intensities. Right panels: OP waveforms isolated by bandpass filtering. Red line, GABA-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for GABA-treated and PBS-treated eyes (n=5 each). (D) Average amplitude of OP1 and OP2 in B (n=5 each). **p<0.01. Line and bar charts show mean ± SEM.

Figure 6. The effect of SR and ISO on dark-adapted OPs

(A) Left panels: Representative light-evoked ERG traces for different light intensities. Right panels: OP waveforms isolated by bandpass filtering. Red line, SR-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for SR-treated and PBS-treated eyes (n=8 each). (D) OPs/b-wave amplitude ratio at different light intensities (n=8 each). (E-H) As per A–D, but for ISO-treated eyes (red) vs. PBS-treated control eyes (blue). *, p<0.05; **, p<0.01. Line charts show mean ± SEM.

Figure 7. The effect of TPMPA on dark-adapted OPs

(A) Left panels: Representative light-evoked ERG traces for different light intensities. Middle panels: OP waveforms isolated by bandpass filtering. Right panels: Normalized ERG traces. Red line, TPMPA-treated; blue line, PBS-treated. (B) Magnification of those traces outlined in A. (C) Stimulus-response curves, showing OPs amplitude for TPMPA-treated and PBS-treated eyes (n=5 each). (D) Stimulus-response curves, showing normalized OP amplitude for TPMPA-treated and PBS-treated eyes (n=5 each). (E) OPs/b-wave amplitude ratio at different light intensities (n=5 each). (F) Time-to-peak of OPs evoked by the highest five light intensities (n=5). **p<0.01. Line charts show mean ± SEM.

Figure 8. The effect of TPMPA+GABA on the dark-adapted OPs

(A) Representative OP waveforms, isolated from light-evoked ERG traces, evoked by six different light intensities, and at three different times post-injection. Red line, TPMPA+GABA; blue line, PBS+GABA. (B) Magnification of traces outlined in A. (C) OPs amplitude for TPMPA+GABA-treated and PBS+GABA-treated eyes (n=5) at three different time points after drug delivery. **p<0.01. Line chart shows mean ± SEM.

Figure 9. GABAa, GABAc and glycine receptors made differing contributions to OPs: frequency domain analysis

(A) Flowchart of OPs analysis in the frequency domain. (B) Representative frequency-amplitude spectra of the OPs at six different light intensities after administration of different drugs. (C) Mean (± SEM) relative values of the parameters of a double-Gaussian model fitted to the data at the highest light intensity (1 log(cd·s·m^-2)). (n=5, 8, 5). *p<0.05, **p<0.01.