Quantifying the size of gap junctions in the retina by fluorescence microscopy

Abstract

Purpose : Gap junction (GJ) contains a cluster of connexin channels which electrically couple neighboring neurons. Quantitative detection of the channels at GJ clusters is important but challenging. Previous electron microscopy (EM) studies visualized GJ of 50 – 2,500 channels in different structural types (string, crystalline plaque, etc.) among nervous tissues, but EM techniques cannot detect GJs in large volumes of tissue. In the retina, connexin36 (Cx36) channels form GJs between rods and cones, as well as between some types of amacrine and/or bipolar cells. We hypothesized that the number of Cx36 channels is related to the fluorescence intensity (FI) signal at GJ clusters, and it may help us to evaluate the effects of mutations on Cx36 channels.

Methods : Linearity between the amount of fluorescent dye and the detected FI signal is necessary for quantification. Linearity in our system was confirmed by standard beads of various diameters measured by confocal microscope (Zeiss LSM800). Mouse retinal sections were collected and labeled with Cx36 antibodies and a Cy3-conjugated secondary; then a subset (3 – 12 μm) of 50-μm thick section was imaged and analyzed. The integrated 3D FI of each Cx36 cluster was measured using Imaris. Comparisons were made between the outer plexiform layer (OPL), the inner plexiform layer sublamina a and b (IPLa & b), as well as between wild type and Cx36 mutant mice.

Results : FI of Cx36 clusters was 4 and 8 times stronger in the IPLa & b, respectively than in the OPL. Cx36 clusters in the IPLb are known to form large crystalline arrays containing 200 – 300 channels, suggesting that rod/cone GJs contain about 30-40 channels, comparable with previous EM studies. Rod/cone-specific Cx36 knockout mice showed no fluorescence in the OPL as expected, but a 30% reduction of Cx36 cluster size in the IPL. Mice expressing constitutively open Cx36 in the retina showed a comparable level of cluster size in the OPL, but produced more than 4-times bigger clusters in the IPL compared with the wild-type.

Conclusions : Quantification of the GJ size in the retina using FI of Cx36 clusters was established. The low FI of OPL GJs is consistent with the presence of GJ strings between rods and cones containing 30-40 channels. The string-like morphology was also confirmed by super resolution STED microscopy. Mutant analyses indicate that signaling via rod/cone GJ plays an important role in homeostasis of GJ in the IPL.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.