Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Co-IP of Cx36 with Cx45 from mouse retina and HeLa cells transfected with Cx36 and Cx45. A, Immunoblot showing detection of Cx36 after IP of Cx45 from mouse retina (lane 2) and from HeLa cells co-transfected with Cx36/Cx45 (lane 1), and absence of Cx36 detection after IP of Cx45 from HeLa cells co-transfected with Cx45 and c-terminus-truncated Cx36 (lane 3) or after IP with omission of anti-Cx45 (lane 4). B, Immunoblot showing detection of Cx45 after IP of Cx36 from mouse retina (lane 2) and from HeLa cells co-transfected with Cx36/Cx45 (lane 1), and absence of Cx45 detection after IP of Cx36 from HeLa cells co-transfected with Cx45 and c-terminus-truncated Cx36 (lane 3) or after IP with omission of anti-Cx36 (lane 4).

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Co-Immunoprecipitation Assay, Transfection, Western Blot

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Antibodies used for western blotting and immunohistochemistry

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Western Blot

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Numbers of gap junctions labeled for Cx36, Cx45, or Cx36+Cx45 in FRIL single-replicas of mouse and rat retinas that were double-labeled for Cx36 plus Cx45.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Labeling

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Numbers of gap junctions in single and double replicas, per cent of Cx45+Cx36 double-labeled gap junctions, and lower and upper confidence intervals (CI) at 95% confidence levels for Cx45-containing gap junctions expressing both connexins.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Expressing

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Images and the interpretation of labeling in matched double-replicas. A-C, Scale diagram showing gap junction before and after freeze fracturing and retrieval of complementary replicas. A, The fracture plane (dashed line) separates connexons at their points of contact in the extracellular space, leaving all connexons of the lower cell for subsequent immunogold labeling. B-C, Creation of double-replicas having complementarity of E- and P-fracture faces. Each matching replica contains connexons from only one cell, but the E-face of one cell and the P-face of the other cell. D,D′, Images from dissecting microscope showing matched double-replicas containing three strips of retina after SDS washing and immunogold labeling. E, Low-magnification image of one side of a complementary replica, with locations of two of the mapped gap junctions indicated by arrows. F,F′,G,G′, High magnification stereoscopic views of complementary replicas of two gap junctions, with 10-nm gold labeling Cx36 and 5-nm gold labeling Cx45. Cx36 labeling is present beneath three quadrants of both gap junctions (E-face pits in top replica and complementary P-face particles in bottom replica). 5-nm Cx45 labels are present in the lower right quadrant. Initial pre-coating with 2-3 nm of carbon partially obscured E-face connexon pits. Arrow (in F) indicates a gold bead beyond the 28-nm radius of immunogold labeling. Arrowhead (in G′) indicates a 5-nm gold label for Cx45, with possible corresponding label in the complementary image (G, arrow). Scale bars, 0.1 μm unless otherwise indicated.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Labeling, Microscopy

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: FRIL localization of Cx45 vs. Cx36 in mouse (A-D) and rat (E) IPL. A, Neuronal gap junctions in adjacent cells, one of which (1; 210 connexons) is labeled for both Cx45 (one 18-nm gold and two 6-nm gold beads, arrowheads) and Cx36 (ca. 450 connexons; eight 12-nm gold beads) and the second (2) is labeled for Cx36 only (10 12-nm gold beads). #1 overall LE = 1:19 and #2 LE = 1:45. B, Medium-size plaque gap junction (139 connexons) labeled for Cx45 (three 6-nm gold beads, arrowheads, and one 18-nm gold bead) and Cx36 (five 12-nm gold beads); overall LE = 1:15. C, Medium-size reticular gap junction (106 connexons) labeled for Cx45 (six 6-nm, arrowheads) and for Cx36 (three 12-nm gold beads). Two gold beads (thin cross-bars) within the apparent “radius of uncertainty” for connexon labeling represent “false-positive” labeling (i.e., “noise”) because stereoscopic analysis (not shown) revealed that both were >1 μm above the replica on an unstable wisp of undissolved Lexan plastic. Overall LE = 1:12. D, Small gap junction (68 connexons) in rat IPL labeled for Cx45 only (one 18-nm and three 6‐nm gold beads; zero 12-nm gold beads are present for Cx36). Cx45 LE = 1:17. This gap junction may be too small to allow positive analysis regarding Cx36. A and C from same replica. E, Large plaque gap junction (797 connexons) with small area of P-face and larger area of E-face; labeled for both Cx45 (three 12-nm gold beads, arrows; and one 30-nm gold bead) and for Cx36 (17 6-nm gold beads; arrowheads and 41 18-nm gold beads; labeled for ca. twice as long as all other FRIL images shown; see MATERIALS AND METHODS for details). Overall LE = 1:13. Scale bars, 0.1 μm.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Labeling

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Immunolabeling of Cx45 and Cx36 in adult mouse retina. A, B, Vertical retinal sections labeled by immunofluorescence, with the right portion of each field shown by counterstaining with fluorescent NeuroTrace. Punctate labeling with polyclonal anti-Cx45 is seen in the inner half of the IPL (A, arrow), and more sparsely in the outer half of the IPL (A). Labeling of Cx36 is very dense in the inner half of the IPL (B, large arrowheads), less dense in the outer half of the IPL (B, small arrowheads) and moderate in the OPL (B, arrows). C-E, Higher magnifications showing punctate labeling for Cx45 at the outer margin of the INL (C, arrows), and along blood vessels in the retina (D, arrows), and in cerebral cortex (E, arrow). ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars, 50 μm.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Immunolabeling, Labeling, Immunofluorescence

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Double-immunofluorescence images showing co-localization of Cx45 with Cx36 in vertical sections of the IPL of adult mouse retina. Images in A-D show the entire vertical span of IPL in single confocal scans. A, The same field (A1-A3) labeled with monoclonal anti-Cx45 and polyclonal anti-Cx36, showing Cx45-positive puncta labeled for Cx36 in retina after perfusion of mice with 20 ml of tissue fixative. B, The same set of confocal double immunofluorescence images as in A, showing minimal Cx45/Cx36 co-localization in the IPL after horizontal flipping of the image showing labeling for Cx36. C, The same field (C1-C3) of IPL double-labeled for Cx36 with monoclonal Ab37-4600 and polyclonal Ab36-4600, showing nearly total overlap of immunopositive puncta. D, The same field (D1-D3) in a tangential section through the IPL double-labeled for Cx45 with monoclonal MAB3101 and polyclonal Ab40-7000, showing nearly total overlap of immunopositive puncta. Scale bars, 10 μm.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Immunofluorescence, Labeling

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Quantitative immunofluorescence analysis of Cx45 co-localization relationships with Cx36, ZO-1, ZO-2 and ZONAB in the IPL of adult mouse retina

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Immunofluorescence

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Eight generalized connexin/connexon configurations proposed to occur within neuronal gap junctions. A, B, Homomeric homotypic Cx36-to-Cx36 channels (A) and Cx45-to-Cx45 channels (B). C, Cx36 in the upper cell and Cx45 in the lower cell, with Cx36 linking to Cx45 (“homomeric heterotypic”). D, Cx43 in the upper cell and Cx45 in the lower cell, with Cx43 linking to Cx45 (“homomeric heterotypic”). E, Both Cx36 and Cx45 in both cells, with Cx36 linked only to Cx36 and Cx45 linked only to Cx45 (“homomeric bi-homotypic). F, Both Cx36 and Cx45 in both cells, with both connexins on one side linked to both connexins on the other side (“homomeric bi-heterotypic). G, Cx36 only in the upper cells linking to connexons containing a mixture of Cx36/Cx45 in the lower cell (homomeric to heteromeric channels). H, Each connexon contains both Cx36 and Cx45 (heteromeric connexons), linking to heteromeric connexons in the other cell (heteromeric to heteromeric channels).

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques:

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Double immunofluorescence labeling of connexins in cultures containing mixtures of HeLa cells expressing Cx45, Cx43 or Cx36. A, Co-culture of cells stably expressing Cx45 with those expressing Cx43, showing co-localization of Cx45/Cx43 at abutments between Cx45-HeLa and Cx43-HeLa cells (A1-A3, arrows, yellow puncta in A3). B, Co-culture of cells expressing Cx45 with those expressing Cx36, showing Cx45-positive puncta at cell appositions among colonies of Cx45-HeLa cells (B1, lower right half of field, and arrows) and Cx36-positive puncta at appositions among Cx36-HeLa cells (B2, upper left half of field and arrowheads). There is a lack of Cx45/Cx36 co-localization at abutments between Cx45-HeLa and Cx36 HeLa cells (B3, double-headed arrows). C, HeLa cells stably expressing Cx36 and transiently transfected with Cx45, showing two apposed cells (arrows) expressing both connexins, and Cx45/Cx36 co-localization at points of cell-cell contact (arrowhead). D, Two HeLa cells stably expressing Cx36 and transiently transfected with Cx45, showing Cx45 localized intracellularly (D1, double-headed arrows), Cx36/Cx45 co-localization at points of cell-cell contact between cells expressing both connexins (arrowhead), and absence of Cx45 at points where double-expressing cells make contacts with cells expressing only Cx36 (arrow). Scale bars, 10 μm.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Immunofluorescence, Labeling, Expressing, Co-Culture Assay, Stable Transfection, Transfection

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Cx45/Cx36 double-labeled gap junction adjacent to a ribbon of E-face particles / P-face pits (rectangles) that are found postsynaptic to ribbon synapses, which in IPL, are characteristic of rod and cone bipolar cells. Outer oval delineates the area with increased Cx45 labeling (12 and 11 5-nm gold beads, A vs. B), and the inner oval delineates the domain of Cx36 labeling (11 and nine 10-nm gold beads, A vs. B). P, P-face; E, E-face. Scale bars, 0.1 μm

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Labeling

Journal:

Article Title: Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bi-homotypic gap junctions, with scaffolding contributed by zonula occludens-1

doi: 10.1523/JNEUROSCI.2137-08.2008

Figure Lengend Snippet: Laser scanning confocal double immunofluorescence showing relationships of Cx45 with ZO-1, ZO-2 and ZONAB in the IPL of adult mouse retina, and Cx36, Cx45 and ZO-1 in the IPL of adult wild-type and Cx36 ko mice. Images show the IPL from inner (bottom) to outer (top) edge, and represent z-stacks of five confocal scans in A-D, and single scans in E-I. Co-localization of green and red labeling is seen as yellow in image overlays. A, The same field (A1-A3) showing a high proportion of Cx45-positive puncta labeled for ZO-1. B, The same set of laser scanning confocal double immunofluorescence images as in A, showing minimal Cx45/ZO-1 co-localization in the IPL after horizontal flipping of the image showing labeling for ZO-1. C, Double immunofluorescence overlay showing lack of Cx45 (green) co-localization with ZO-2 (red). D, Double immunofluorescence overlay showing very few Cx45-positive puncta (green) labeled for ZONAB (red). E, Double immunofluorescence overlay showing the persistence of Cx45/ZO-1 co-localization seen as yellow puncta (arrows) in the IPL of Cx36 ko retina. F-I, Confocal scans showing Cx36-puncta (F) and Cx45-puncta (G) in the IPL of wild-type retina, and an absence of labeling for Cx36 (H) and a reduction of labeling for Cx45 (I) in IPL of Cx36 ko retina. Scale bars, 10 μm.

Article Snippet: Statistical analysis of Cx45 relationships with Cx36 in retinal neurons and gap junctions To test various hypotheses regarding expression and localization of Cx36 and Cx45 in the retina, we used a web-based online statistical analysis system to calculate confidence intervals ( http://www.measuringusability.com/wald.htm ) (see Hypothesis 3, below).

Techniques: Immunofluorescence, Labeling