Test of the paired-flash electroretinographic method in mice lacking b-waves
ABSTRACT Previous studies of rod photoreceptors in vivo have employed a paired-flash electroretinographic (ERG) technique to determine rod response properties. To test whether absence versus presence of the ERG b-wave affects the photoreceptor response derived by the paired-flash method, we examined paired-flash-derived responses obtained from nob mice, a mutant strain with a defect in signal transduction between photoreceptors and ON bipolar cells that causes a lack of the b-wave. Normal littermates of the nob mice served as controls. The normalized amplitude-intensity relation of the derived response determined in nob mice at the near-peak time of 86 ms was similar to that determined for the controls. The full time course of the derived rod response was obtained for test flash strengths ranging from 0.11 to 17.38 scotopic cd s m(-2) (sc cd s m(-2)). Time-course data obtained from nob and control mice exhibited significant but generally modest differences. With saturating test flash strengths, half-recovery times for the derived response of nob versus control mice differed by approximately 60 ms or less about the combined (nob and control) average respective values. Time course data also were obtained before versus after intravitreal injection of L-2-amino-4-phosphonobutyrate (APB) (which blocks transmission from photoreceptors to depolarizing bipolar cells) and of cis 2,3-piperidine dicarboxylic acid (PDA) (which blocks transmission to OFF bipolar cells, and to horizontal, amacrine and ganglion cells). Neither APB nor PDA substantially affected derived responses obtained from nob or control mice. The results provide quantitative information on the effect of b-wave removal on the paired-flash-derived response in mouse. They argue against a substantial skewing effect of the b-wave on the paired-flash-derived response obtained in normal mice and are consistent with the notion that, to good approximation, this derived response represents the isolated flash response of the photoreceptors in both nob and normal mice.
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ABSTRACT: Reliable signal transduction via G-protein-coupled receptors requires proper receptor inactivation. For example, signals originating from single rhodopsin molecules vary little from one to the next, requiring reproducible inactivation of rhodopsin by phosphorylation and arrestin binding. We determined how reduced concentrations of rhodopsin kinase (GRK1) and/or arrestin1 influenced the kinetics and variability of the single-photon responses of mouse rod photoreceptors. These experiments revealed that arrestin, in addition to its role in quenching the activity of rhodopsin, can tune the kinetics of rhodopsin phosphorylation by competing with GRK1. This competition influenced the variability of the active lifetime of rhodopsin. Biasing the competition in favor of GRK1 revealed that rhodopsin remained active through much of the single-photon response under the conditions of our experiments. This long-lasting rhodopsin activity can explain the characteristic time course of single-photon response variability. Indeed, explaining the late time-to-peak of the variance required an active lifetime of rhodopsin approximately twice that of the G-protein transducin. Competition between arrestins and kinases may be a general means of influencing signals mediated by G-protein-coupled receptors, particularly when activation of a few receptors produces signals of functional importance.The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 09/2009; 29(38):11867-79. DOI:10.1523/JNEUROSCI.0819-09.2009 · 6.75 Impact Factor
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ABSTRACT: The purpose of this study was to determine the contributions of postreceptoral neurons to the light-adapted ERG of the Nob mouse, a model for complete-type congenital stationary night blindness (CSNB1) that lacks a b-wave from depolarizing bipolar cells. Ganzfeld ERGs were recorded from anesthetized adult control mice, control mice injected intravitreally with L-2-amino-4-phosphonobutyric acid (Control APB mice) to remove On pathway activity, and Nob mice. ERGs also were recorded after PDA (cis-2,3-piperidine-dicarboxylic acid, 3-5mM) was injected to block transmission to hyperpolarizing (Off) bipolar and horizontal cells, and all third-order neurons. Stimuli were brief (<4ms, 0.4-2.5log sc td s) and long (200ms, 2.5-4.6log sc td) LED flashes (lambda(max)=513nm, on a rod suppressing background (2.6log sc td). Sinusoidal modulation of the LEDs (mean, 2.6log sc td; contrast, 100%; 3-36Hz) was used to study flicker ERGs. Brief-flash ERGs of Nob mice presented as long-lasting negative waves with a positive-going intrusion that started about 50ms after the flash and peaked around 120ms. Control APB mice had similar responses, and in both cases, PDA removed the positive-going intrusion. For long flashes, PDA removed a small, slow "d-wave" after light offset. With sinusoidal stimulation, the fundamental (F1) amplitude of control mice ERG peaked at 8Hz ( approximately 70microV). For Nob mice the peak was approximately 20microV at 6Hz before PDA and approximately 10muV at 3Hz or lower after PDA. F1 responses were present up to 21Hz in control and Nob eyes and 15Hz in Nob eyes after PDA. Between 3 and 6Hz, F1 phase was 170-210 degrees more delayed in Nob than control mice; phase was hardly altered by PDA. With vector analysis, a substantial postreceptoral input to the Nob flicker ERG was revealed. In control mice, the second harmonic (F2) response showed peaks of approximately 10mocrpV at 3Hz and 13Hz. Nob mice showed almost no F2. In summary, in this study it was found that in Nob mice, postreceptoral neurons from the Off pathway make a positive-going contribution to the light-adapted flash ERG, and contribute substantially to sinusoidal flicker ERG.Experimental Eye Research 07/2008; 86(6):914-28. DOI:10.1016/j.exer.2008.03.008 · 3.02 Impact Factor
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ABSTRACT: We characterize the dark-adapted photoresponses from mouse cones intact in the isolated retina, their virtually natural environment, by isolating pharmacologically the photoreceptor light responses from the electroretinogram (ERG). Due to the different photoresponse kinetics and sensitivity of rods and cones, the cone responses were readily attained by using a rod-saturating preflash. The stimulus wavelength (544 nm) was chosen to selectively stimulate the green sensitive ("M"-)pigment. Obtained responses were monophasic, showing fast kinetics (mean t(p)=51 ms) and low sensitivity (fractional single-photon response ca. 0.23%). The amplification coefficient of cones (4.6 s(-2)) was very close to that of rods (5.6 s(-2)), while the dominant time constant of recovery was clearly smaller for cones (33 ms) than for rods (160 ms).Vision Research 02/2008; 48(2):264-72. DOI:10.1016/j.visres.2007.11.005 · 2.38 Impact Factor