June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Confocal and STED microscopy reveals single strings of rod/cone gap junctions
Author Affiliations & Notes
  • Munenori Ishibashi
    University of Houston College of Optometry, Houston, Texas, United States
  • Joyce Keung
    The University of Texas Health Science Center at Houston John P and Katherine G McGovern Medical School, Houston, Texas, United States
  • Stephen C Massey
    The University of Texas Health Science Center at Houston John P and Katherine G McGovern Medical School, Houston, Texas, United States
  • Christophe Ribelayga
    University of Houston College of Optometry, Houston, Texas, United States
  • Footnotes
    Commercial Relationships   Munenori Ishibashi None; Joyce Keung None; Stephen Massey None; Christophe Ribelayga None
  • Footnotes
    Support  NIMH Grant RF1MH127343
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 1930. doi:
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    • Get Citation

      Munenori Ishibashi, Joyce Keung, Stephen C Massey, Christophe Ribelayga; Confocal and STED microscopy reveals single strings of rod/cone gap junctions. Invest. Ophthalmol. Vis. Sci. 2023;64(8):1930.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Gap junctions (GJ) are aggregates of channels which connect neighboring neurons and support electrical coupling, but they are small and difficult to observe. Previous electron microscopy (EM) studies revealed a large variability in GJ size and structure in various nervous tissues, but EM techniques are time-consuming for detecting GJs in large volumes of tissue. In the retina, GJ channels made from connexin36 (Cx36) have been found between many cell types, including between rods and cones, and between certain types of amacrine and/or bipolar cells. We hypothesized that the immunofluorescence intensity (FI) signal of Cx36 clusters is directly correlated with GJ size or number of channels, and that super-resolution microscopy may help us resolve GJ nanostructure.

Methods : Using standard beads of various diameters we confirmed the linearity of the FI signal in our confocal microscope system (Zeiss LSM800). Mouse retinal sections were collected and reacted with an antibody against Cx36. A subset (3 – 12 μm) of the 50-μm thick sections was imaged and analyzed to quantify FI. The integrated FI of each Cx36 cluster was measured using Imaris. Background noise intensity was subtracted, and the signal was calibrated against standard beads. In addition, Stimulated Emission and Depletion (STED) microscopy was applied to visualize the structure of GJs under the diffraction limit of 250 nm.

Results : FI of Cx36 clusters was 8 times dimmer on average in the outer plexiform layer (OPL) than in sublamina b of the inner plexiform layer (IPL-b) where Cx36 GJs are known to form large crystalline arrays containing 200 – 300 channels. This suggests that rod/cone GJs contain about 30-40 channels. STED microscopy revealed that rod/cone GJs were elongated structures, such as strings, which is consistent with previous observations made in primate retina (Raviola and Gilula, 1973).

Conclusions : We have established and validated a method to determine the size and nanostructure of GJs in the retina. This approach may provide insights on the conductance and function of GJs in specific retinal circuits and can be readily applied to other brain regions and across species.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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