Method to remove photoreceptors from whole mount retina in vitro

Abstract

Patch clamp recordings of neurons in the inner nuclear layer of the retina are difficult to conduct in a whole mount retina preparation because surrounding neurons block the path of the patch pipette. Vertical slice preparations or dissociated retinal cells provide access to bipolar cells at the cost of severing the lateral connection between neurons. We have developed a technique to remove photoreceptors from the rodent retina that exposes inner nuclear layer neurons, allowing access for patch clamp recording. Repeated application to and removal of filter paper from the photoreceptor side of an isolated retina effectively and efficiently removes photoreceptor cells and, in degenerate retina, hypertrophied Müller cell end feet. Live-dead assays applied to neurons remaining after photoreceptor removal demonstrated mostly viable cells. Patch clamp recordings from bipolar cells reveal responses similar to those recorded in traditional slice and dissociated cell preparations. An advantage of the photoreceptor peel technique is that it exposes inner retinal neurons in a whole mount retina preparation for investigation of signal processing. A disadvantage is that photoreceptor removal alters input to remaining retinal neurons. The technique may be useful for investigations of extracellular electrical stimulation, photoreceptor DNA analysis, and nonpharmacological removal of light input. NEW & NOTEWORTHY This study reports a method for removing photoreceptors from rodent whole mount retina while preserving the architecture of the inner retina. The method enables easier access to the inner retina for studies of neural processing, such as by patch clamp recording.