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Electrical coupling, mediated by gap junctions, contributes to signal averaging, synchronization, and noise reduction in neuronal circuits. In addition, gap junctions may also provide alternative neuronal pathways. However, because they are small and especially difficult to image, gap junctions are often ignored in large-scale 3D reconstructions. Here, we reconstruct gap junctions between photoreceptors in the mouse retina using serial blockface-scanning electron microscopy, focused ion beam-scanning electron microscopy, and confocal microscopy for the gap junction protein Cx36. An exuberant spray of fine telodendria extends from each cone pedicle (including blue cones) to contact 40-50 nearby rod spherules at sites of Cx36 labeling, with approximately 50 Cx36 clusters per cone pedicle and 2-3 per rod spherule. We were unable to detect rod/rod or cone/cone coupling. Thus, rod/cone coupling accounts for nearly all gap junctions between photoreceptors. We estimate a mean of 86 Cx36 channels per rod/cone pair, which may provide a maximum conductance of ~1200 pS, if all gap junction channels were open. This is comparable to the maximum conductance previously measured between rod/cone pairs in the presence of a dopamine antagonist to activate Cx36, suggesting that the open probability of gap junction channels can approach 100% under certain conditions.Neurons can talk to each other in two ways: they can send chemical messengers across specialized junctions between two cells, or they can directly pass electrical signals to one another. This latter process is made possible by gap junctions, a system of channel-like structures which connect neighbouring cells and let ions move between them. In most neurons, gap junction channels are made from a specialized protein called connexin 36. Gap junctions are small, difficult to observe, and therefore often ignored by researchers studying neural circuits. In response, Ishibashi et al. focused on nerve cells in the mouse retina, in particular the cones (which detect color during the day) and the rods (which are essential for night vision). Gap junctions between rods and cones allow them to communicate; for example, they enable rod signals to directly activate cones. This provides an alternative route for rod signaling known as the ‘secondary rod pathway’, which seems to be open at night and switches to closed around dawn. Both rods and cones only produce connexin 36, so Ishibashi et al. labeled these proteins with fluorescent tags to pinpoint gap junctions. This showed that each cone makes around 50 gap junctions with nearby rods; however, gap junctions were not detected between cells of the same type. In addition, 3D reconstruction helped to establish the length of each gap junction. Further experiments showed that a typical rod was connected to a cone by about 80 connexin 36 channels. Finally, calculations revealed that the gap junction channels would all need to open to account for the level of electrical activity required for the secondary rod pathway. This suggests that gap junctions may be much more active and important than previously thought. The work by Ishibashi et al. provides a new understanding of the number, size and activity of gap junctions in the retina, potentially paving the way to prevent diseases where light-sensing cells degenerate and cause blindness.

There are many examples of neuronal coupling via gap junctions in the retina. Of these, perhaps the best known is the extensive coupling between horizontal cells. In the rabbit retina, there are two types of horizontal cells, A-type and B-type, both of which are independently coupled. Connexin 50 (Cx50) cDNA, encoding a 440 aa protein, was successfully isolated from rabbit retina RNA. Cx50 was also obtained from isolated A-type horizontal cells (A-type HCs) by single-cell RT-PCR. A-type HCs were visualized by intracellular dye injection or with an antibody against calbindin. Confocal analysis revealed all Cx50 labeling occurred on the A-type HC matrix, typically at dendritic intersections. The Cx50 plaques varied in size, from punctate signals in which fine dendrites cofasciculated, to giant plaques, >50 μm2, in which large dendrites crossed. The numerous Cx50 plaques between A-type HCs may adequately account for the remarkable coupling observed in this network. We could not detect Cx50 staining on the tips of horizontal cell dendrites within the cone pedicle invagination. This distribution does not support a role for Cx50 in hemichannel-mediated feedback. In addition, the absence of Cx50 in B-type HCs suggests the presence of a different connexin for this cell type. In summary, these results suggest that gap junctions in the A-type horizontal cell matrix are composed from Cx50. Multiple neuronal connexins are expressed in the mammalian retina and different cell types express specific connexins.

Mammalian retinal circuits are broadly divided into rod and cone pathways, responsible for dark- and light-adapted vision, respectively. The classic rod pathway employs a single type of rod bipolar cell, which synapses with AII amacrine cells. AII amacrine cells then pass the signal to ON and OFF cone bipolar cells, respectively. Alternatively, rod signals may enter cones via gap junctions between rods and cones, and then pass from cones to cone bipolar cells. Thus, this second rod pathway does not utilize rod bipolar cells. Finally, in rodents, a third rod pathway, involving direct connections between rods and certain OFF cone bipolar cells, has been suggested. In this study, 56 OFF cone bipolar cells in the rabbit retina were dye-injected with Lucifer Yellow and their photoreceptor connections were examined by confocal microscopy in wholemount. The locations of rod and cone terminals were marked with antibodies to mGluR6 or synaptic proteins. Most OFF cone bipolar dendrites terminated at cone pedicles but some made potential contacts with rod spherules. The synaptic nature of these sites was confirmed by the presence of GluR2 receptors. All three OFF bipolar cell types had dendrites that terminated at rod spherules. However, ∼80% of Ba2 and Ba3, but only 26% of Ba1 OFF cone bipolar cells made rod contacts. This variability suggests differential rod input to certain retinal pathways. In summary, we report anatomical evidence for direct connections between rods and OFF cone bipolar cells in a nonrodent mammal. Our data suggest that this alternative rod pathway may be a common feature of the mammalian retina. J. Comp. Neurol. 474:1–12, 2004. © 2004 Wiley-Liss, Inc.

Gap junctions are abundant in the mammalian retina and many neuronal types form neural networks. Several different neuronal connexins have now been identified in the mammalian retina. Cx36 supports coupling in the AII amacrine cell network and is essential for processing rod signals. Cx36 is probably also responsible for photoreceptor coupling. Horizontal cells appear to be extensively coupled by either Cx50 or Cx57. These results indicate that multiple neuronal connexins are expressed in the mammalian retina and that different cell types express different connexins.