AII amacrine cells are essential interneurons of the primary rod pathway and transmit rod-driven signals to ON cone bipolar cells to enable scotopic vision. Gap junctions made of connexin36 (Cx36) mediate electrical coupling among AII cells and between AII cells and ON cone bipolar cells. These gap junctions underlie a remarkable degree of plasticity and are modulated by different signaling cascades. In particular, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized as an important regulator of Cx36, capable of potentiating electrical coupling in AII cells. However, it is unclear which CaMKII isoform mediates this effect. To obtain a more detailed understanding of the isoform composition of CaMKII at retinal gap junctions, we analyzed the retinal distribution of all four CaMKII isoforms using confocal microscopy. These experiments revealed a differential distribution of CaMKII isoforms: CaMKII-α was strongly expressed in starburst amacrine cells, which are known to lack electrical coupling. CaMKII-β was abundant in OFF bipolar cells, which form electrical synapses in the outer and the inner retina. CaMKII-γ was diffusely distributed across the entire retina and could not be assigned to a specific cell type. CaMKII-δ labeling was evident in bipolar and AII amacrine cells, which contain the majority of Cx36-immunoreactive puncta in the inner retina. We double-labeled retinas for Cx36 and the four CaMKII isoforms and revealed that the composition of the CaMKII enzyme differs between gap junctions in the outer and the inner retina: in the outer retina, only CaMKII-β colocalized with Cx36-containing gap junctions, whereas in the inner retina, CaMKII-β and -δ colocalized with Cx36. This finding suggests that gap junctions in the inner and the outer retina may be regulated differently although they both contain the same connexin. Taken together, our study identifies CaMKII-β and -δ as Cx36-specific regulators in the mouse retina with CaMKII-δ regulating the primary rod pathway.

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.

In the mammalian retina, amacrine cells represent the most diverse cell class and are involved in the spatio-temporal processing of visual signals in the inner plexiform layer. They are connected to bipolar, other amacrine and ganglion cells, forming complex networks via electrical and chemical synapses. The small-field A8 amacrine cell was shown to receive non-selective glutamatergic input from OFF and ON cone bipolar cells at its bistratified dendrites in sublamina 1 and 4 of the inner plexiform layer. Interestingly, it was also shown to form electrical synapses with ON cone bipolar cells, thus resembling the rod pathway-specific AII amacrine cell. In contrast to the AII cell, however, the electrical synapses of A8 cells are poorly understood. Therefore, we made use of the Ier5-GFP mouse line, in which A8 cells are labeled by GFP, to study the gap junction composition and frequency in A8 cells. We found that A8 cells form <20 gap junctions per cell and these gap junctions consist of connexin36. Connexin36 is present at both OFF and ON dendrites of A8 cells, preferentially connecting A8 cells to type 1 OFF and type 6 and 7 ON bipolar cells and presumably other amacrine cells. Additionally, we show that the OFF dendrites of A8 cells co-stratify with the processes of dopaminergic amacrine cells from which they may receive GABAergic input via GABAA receptor subunit α3. As we found A8 cells to express dopamine receptor D1 (but not D2), we also tested whether A8 cell coupling is modulated by D1 receptor agonists and antagonists as was shown for the coupling of AII cells. However, this was not the case. In summary, our data suggests that A8 coupling is differently regulated than AII cells and may even be independent of ambient light levels and serve signal facilitation rather than providing a separate neuronal pathway.

Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14-3-3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

In the outer plexiform layer (OPL) of the mammalian retina, cone photoreceptors (cones) provide input to more than a dozen types of cone bipolar cells (CBCs). In the mouse, this transmission is modulated by a single horizontal cell (HC) type. HCs perform global signaling within their laterally coupled network but also provide local, cone-specific feedback. However, it is unknown how HCs provide local feedback to cones at the same time as global forward signaling to CBCs and where the underlying synapses are located. To assess how HCs simultaneously perform different modes of signaling, we reconstructed the dendritic trees of five HCs as well as cone axon terminals and CBC dendrites in a serial block-face electron microscopy volume and analyzed their connectivity. In addition to the fine HC dendritic tips invaginating cone axon terminals, we also identified "bulbs," short segments of increased dendritic diameter on the primary dendrites of HCs. These bulbs are in an OPL stratum well below the cone axon terminal base and make contacts with other HCs and CBCs. Our results from immunolabeling, electron microscopy, and glutamate imaging suggest that HC bulbs represent GABAergic synapses that do not receive any direct photoreceptor input. Together, our data suggest the existence of two synaptic strata in the mouse OPL, spatially separating cone-specific feedback and feedforward signaling to CBCs. A biophysical model of a HC dendritic branch and voltage imaging support the hypothesis that this spatial arrangement of synaptic contacts allows for simultaneous local feedback and global feedforward signaling by HCs.