An eyecup preparation for the rat and mouse

Abstract

The eyecup preparation has traditionally been used to study retinal physiology in lower vertebrates and in some mammals. The procedures for preparing eyecups of the rat and mouse have not been described, however. We now describe methods for preparing and maintaining viable eyecups for these two species. Eyecups were everted over a plastic dome and held in place between the two halves of a superfusion chamber. Fluid exchange in the chamber was rapid, with near total exchange occurring in 9 s. Eyecup viability was tested by monitoring light-evoked retinal responses as the preparation aged. In both rat and mouse, the amplitude of the electroretinogram (ERG) b-wave decreased slowly, declining to $\text{1}\text{2}$ maximal amplitude in ≈70 min. Light-evoked spike activity of neurons in the ganglion cell layer remained stable for ≈3 h and attenuated responses were recorded for an additional 1–2 h. Eyecups were able to dark adapt. Retinal sensitivity, tested by monitoring b-wave amplitude, recovered following exposure to an adapting light.

Introduction

The eyecup preparation has been used to great advantage to study retinal physiology in lower vertebrates, including the frog, turtle, and skate (Dowling, 1987). Electrical recording from retinal cells and rapid exchange of superfusion solutions, which are difficult to achieve in the intact eye, are easily obtained in the eyecup. In mammals, the eyecup of the rabbit has also been used successfully (Bloomfield and Miller, 1986, Miller et al., 1986). It has proven more difficult, however, to prepare viable eyecups from the rat and mouse and there are few published studies that have employed eyecups of these species to study retinal physiology.

The techniques employed to prepare rabbit eyecups cannot be used successfully for the rat and mouse. The retinas of these smaller eyes tend to detach as the eyecup is prepared. In addition, the retinas of the rat and mouse are vascularized and thicker than the retina of the rabbit, making them more difficult to oxygenate.

The isolated retina (Meister et al., 1991, Meister et al., 1994, Soucy et al., 1998, Winkler et al., 1999) and retinal slice (Boos et al., 1993, Hartveit, 1996, Euler et al., 1996) preparations have been used successfully in recent years to study retinal physiology in a number of vascularized mammalian species, including the mouse, rat, cat, and ferret. The retina is separated from the retinal pigment epithelium in these preparations, preventing recovery from photopigment bleach following exposure to bright stimuli. The retinas remain responsive to light for many hours as long as exposure to intense light is avoided. These isolated retina preparations cannot be used, however, to study adaptation to bright stimuli or to monitor ion concentrations using fluorescent indicator dyes, which require bright excitation illumination. Under bright illumination, regeneration of photopigments and maintained responsiveness to photopic stimuli only occurs when the retina remains in direct contact with the pigment epithelium, as it does in the eyecup.

In recent years, with the development of transgenic and knockout strains of mouse and rat, it has become increasingly important to develop a successful eyecup preparation for these species. Genomic techniques offer unique advantages for studying retinal physiology (Goto et al., 1995, Shaaban et al., 1998), advantages that cannot be fully exploited without a viable eyecup preparation.

This paper describes a method for preparing eyecups that utilizes a new design for a superfusion and recording chamber. The chamber enables everted eyecups of the rat and mouse to be prepared quickly and reliably. Measurements of retinal responses indicate that eyecups maintained in the chamber remain viable for many hours.