Abstract
Purpose: :
The electroretinogram (ERG) has become a common tool for assessing retinal physiology of both normal and genetically–altered zebrafish. While positive flashes are useful for examining the characteristics of the b–wave component, assessment of the d–wave component with positive flashes can be problematic. Here, we examined the response of the b– and d–wave components to both positive and negative flashes. In addition, pharmacologic agents were employed to determine the neural contributions to the b– and d–wave components.
Methods: :
ERG responses to positive and negative square–wave flashes of various contrasts were recorded from adult zebrafish (Danio rerio). Two white light emitting diodes formed the background (5 µW/cm2) and stimulus (125 µW/cm2) channels. Once responses were obtained over a range of positive and negative contrasts, subjects were intravitreally injected with DL–2–amino–4–phosphonobutyric acid (APB; 100 µM), 6–cyano–7–nitroquinoxaline–2,3–dione (CNQX; 200 µM), cis–2,3–piperidinedicarboxylic acid (PDA; 200 µM), and N–methyl–D–aspartate (NMDA; 100 µM). The sequence of administration was either APB or CNQX followed by either CNQX or APB depending upon the first drug. The remaining drug sequence was PDA (following both APB and CNQX) and NMDA.
Results: :
Under normal conditions, ERG responses to positive flashes consisted of an a– and b–wave to stimulus onset and a d–wave to stimulus termination (decrement). ERG responses to negative flashes (decrement) consisted of an initial d–wave to stimulus onset (decrement) and an a– and b–wave to stimulus termination (increment). Separate injections of APB and CNQX altered the b– and d–wave components of the ERG but in different fashions. APB reduced the amplitudes of both the b– and d–waves, while CNQX suppressed the d–wave but enhanced the b–wave; this was independent of stimulus polarity. Addition of PDA to an APB + CNQX cocktail suppressed both b– and d–waves; addition of NMDA to the final cocktail eliminated the b– and d–waves and appeared to leave a slight hyperpolarization to stimulus increments and a slight depolarization to stimulus decrements.
Conclusions: :
These results illustrate the utility of using both positive and negative flashes to separate and evaluate the neural contributions to the zebrafish ERG, and are consistent with previously proposed "push–pull" models of on– and off–bipolar cell contributions to the ERG b– and d–waves.
Keywords: electroretinography: non-clinical • retina: proximal (bipolar, amacrine, and ganglion cells) • drug toxicity/drug effects