Paired Recording to Study Electrical Coupling Between Photoreceptors in Mouse Retina

Abstract

Gap junction-mediated electrical coupling between retinal photoreceptors is an important determinant of photoreceptor function. Yet, quantitative measurements of the junctional conductance between coupled photoreceptors are required to fully assess the effects of coupling on visual performance. Such measurements have been obtained in salamander and other lower vertebrate retinas but are difficult to acquire in mammalian retinas, in part because of the much smaller size of photoreceptors in mammals. Here, we describe in detail a dual whole-cell patch-clamp technique we recently developed to measure the junctional conductance between photoreceptor pairs in the mouse retina. With this method, electrical coupling strength between mouse photoreceptors can be estimated with high accuracy and its impact on retinal processing of visual information further evaluated.

Keywords: Cx36; Dual patch clamp; Electrical coupling; Gap junction; Mouse; Photoreceptor.

Fig. 1

Identification of photoreceptor pairs in the living mouse retinal slice. (a) Overview of the perfusion chamber with retinal sections in place. Note the large size of the chamber, which allows for laminar flow of the perfusion solution and ensures stability during recordings. (b)-(d) Views of a mouse retinal slice fixed in paraformaldehyde, reacted with an antibody against cone arrestin (green), and visualized under a confocal microscope. A magnified view of the outer portion (white square in [b]) is shown in (c)-(d). DAPI staining (blue) of the nuclei is shown in (b)-(c) and omitted in (d) for clarity. Note the position of the cone somas in the outermost part of the ONL (white arrows) and that of the cone pedicles at the bottom of the ONL and in the OPL (white arrowheads). (e) DIC image of the outer portion of a mouse retinal slice and of the recording electrodes placed on adjacent rod somas under infrared illumination in the experimental setup. Dashed lines delimit the area that includes only rod somas. This is the area targeted for recording from rods, in rod/rod or rod/cone paired recordings. To record from cones, we target the cone terminals or pedicles that are visible as contrasted structures in DIC mode (white circles). OS, Outer segments; IS, inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bars: (a), 2 cm, (b), 50 μm, (c)-(e), 25 μm. Figures (c)-(e) modified from Figure 1 of [9], with permission.

Fig. 2

Simultaneous patch-clamp recording of photoreceptor pairs in the living mouse retinal slice illustrated for a pair of rods (a,a’,a”,a”’), a pair of cones (b,b’,b”,b”’), and a rod/cone pair (c,c’,c”,c”’). (a)-(c) Schematic representation of the experimental setup. (a’)-(c’) Visualization of the photoreceptors simultaneously patch-clamped and filled with Lucifer Yellow through the 2 recording pipettes. (a”)-(c”) Simultaneous voltage-clamp recording from pairs of neighboring photoreceptors. Shown are junctional currents (I2) during a series of steps in transjunctional voltage (V1). (a”’)-(c”’) Plot of I2 as a function of V1. The junctional conductance (Gj) is estimated from the slope of the linear regression curve that fits the experimental data. Scale bars: (a’)-(c’), 5 μm. Figure (a’) is from Figure 1 of [9], with authorization.