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. 2019 Jul;224(6):2183-2197.
doi: 10.1007/s00429-019-01876-y. Epub 2019 Jun 6.

Connexin-36 distribution and layer-specific topography in the cat retina

Ildikó Telkes  1   2   3 , Péter Kóbor  1   2   3 , József Orbán  2   4 , Tamás Kovács-Öller  2   5   6   3 , Béla Völgyi  2   5   6   3 , Péter Buzás  7   8   9
Affiliations

Connexin-36 distribution and layer-specific topography in the cat retina

Ildikó Telkes et al. Brain Struct Funct. 2019 Jul.

Abstract

Connexin-36 (Cx36) is the major constituent of mammalian retinal gap junctions positioned in key signal pathways. Here, we examined the laminar and large-scale topographical distribution of Cx36 punctate immunolabels in the retina of the cat, a classical model of the mammalian visual system. Calretinin-immunoreactive (CaR-IR) cell populations served to outline the nuclear and plexiform layers and to stain specific neuronal populations. CaR-IR cells included horizontal cells in the outer retina, numerous amacrine cells, and scattered cells in the ganglion cell layer. Cx36-IR plaques were found among horizontal cell dendrites albeit without systematic colocalization of the two labels. Diffuse Cx36 immunoreactivity was found in the cytoplasm of AII amacrine cells, but no colocalization of Cx36 plaques was observed with either the perikarya or the long varicose dendrites of the CaR-IR non-AII amacrine cells. Cx36 puncta were seen throughout the entire inner plexiform layer showing their highest density in the ON sublamina. The densities of AII amacrine cell bodies and Cx36 plaques in the ON sublamina were strongly correlated across a wide range of eccentricities suggesting their anatomical association. However, the high number of plaques per AII cell suggests that a considerable fraction of Cx36 gap junctions in the ON sublamina is formed by other cell types than AII amacrine cells drawing attention to extensive but less studied electrically coupled networks.

Keywords: AII amacrine cell; Calretinin; Eccentricity; Gap junction.

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Conflict of interest statement

The authors have no conflicts of interest.

Figures

Fig. 1
Fig. 1
Immunohistochemical labeling of calretinin (magenta) and connexin-36 (green) in the outer layers of the cat retina. a Cone photoreceptor outer segments revealed by the anti-CaR antibody in the flat-mounted retina. Eccentricity, 13.9 mm. b Side view reconstruction of the same confocal stack as in a showing all retinal layers. c Composite image of both markers with focus on the outer plexiform layer. Connexin-36 plaques were not systematically associated with horizontal cells dendrites. Eccentricity, 4.4 mm. d Same frame as A showing Cx36-positive plaques and occasional elongated structures (arrows). e Cell bodies and proximal dendrites of CaR-positive horizontal cells. Same field of view as A but focus is slightly deeper on the top of the inner nuclear layer. Asterisks indicate type-A horizontal cells with thicker but more weakly labeled proximal dendrites. Scale bar, 20 μm
Fig. 2
Fig. 2
Calretinin-immunoreactive amacrine cells and connexin-36 immunoreactivity of the inner nuclear layer. Eccentricity, 4.4 mm. a Composite image of calretinin (magenta) and connexin-36 (green) double label. Arrowheads indicate strongly CaR-positive amacrine cells. AII cells show weaker CaR-labeling and diffuse cytoplasmic Cx36 expression. b Close-up of the strongly CaR-positive amacrine cell (indicated by the open arrowhead in panels a and c) surrounded by several AII amacrine cells. Large Cx36 plaques are often seen associated with double labeled AII cells (arrows). c, d Show CaR and Cx36 immunolabeling, respectively, for the same region as in a. Note the lack of Cx36 label at the locations of strongly CaR-positive cell bodies (arrowheads in d). Scale bar, 20 μm
Fig. 3
Fig. 3
Classification of amacrine cells in the inner plexiform layer using cluster analysis based on CaR and Cx36 staining intensity. Scatter plot shows the labeling intensity of a sample of cell profiles (n = 182) on the imaging channels for CaR (abscissa) and Cx36 (ordinate) in units of standard deviations around the mean. Filled and open markers indicate the two clusters defined by k-means clustering, respectively. Marker shape indicates the two cell types established by visual inspection. Marginal histograms show the distributions of staining intensities
Fig. 4
Fig. 4
Horizontal view of the inner plexiform layer double labeled for calretinin and connexin-36. a–c Focus on stratum 2 of the IPL where calretinin label had the highest density. d–f Focus on stratum 4 of the IPL where connexin-36 label had the highest density. a, d Composite image. b, e CaR only. c, f, Cx36 only. Scale bar, 20 μm. Eccentricity, 2.8 mm. g Fluorescence intensity profiles along the Z-axis of the confocal stack shown in a–f for CaR (magenta) and Cx36 (green). The ordinate shows depth within the IPL with the five strata indicated. Fluorescence intensity is scaled between 0 and 1 within the IPL. The differential staining of the IPL sublaminae can also be seen in Fig. 1b
Fig. 5
Fig. 5
a Strongly CaR-positive (magenta) regular amacrine cells were the main source of the dense meshwork of varicose dendrites in the OFF sublamina of the IPL (also see Fig. 4a, b). b In the ganglion cell layer, CaR was expressed by small cell bodies (arrowheads) and larger, weakly labeled ganglion cells (asterisks). CaR-immunoreactive optic fibers (arrow) and connexin-36 plaques (green) from the inner plexiform layer are also seen. Eccentricity a 10.6 mm; b 11.3 mm. Scale bar, 20 μm
Fig. 6
Fig. 6
Pairwise comparisons of the densities of the three CaR-IR amacrine cell types innervating the inner plexiform layer, Cx36 puncta, and eccentricity. Each data point (diamonds) corresponds to one region of interest. Pearson’s correlation coefficients (r) and their significance levels are shown for each pair of measures; p < 0.05 was regarded as significant. Alignment of data points in some panels is due to the small (integer) number of strongly labeled amacrine cells in a region of interest. Curves in a, g, and h show best-fit models of the data. a Exponential decay (R2 = 0.60). h Dashed line, linear model (R2 = 0.58); solid line, square root model (R2 = 0.62). Curves in g were obtained by substituting the respective models in h into the equation of the curve in a to estimate the eccentricity dependence of Cx36 density (R2 = 0.49 and R2 = 0.50, respectively)
Fig. 7
Fig. 7
a The density ratio of Cx36 puncta and AII amacrine cells as a function of eccentricity for each region of interest (diamonds). Curves are derived as the ratio of the respective linear (dashed line) or square root models (solid line) of Fig. 6g and a. Note that an estimate of gap junctions per AII cell must take into account the type of connections that these gap junctions are involved in (see text). b Estimates of the numbers of gap junctions per AII amacrine cell as a function of eccentricity. Curves are obtained from the linear (dashed lines) or square root models (solid lines) assuming either only homocellular (Q = 2) or only heterocellular (Q = 1) gap junctions
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