The synaptic ribbon is a unique presynaptic structure with an intricate morphology in photoreceptors. Because of the resolution limit in conventional fluorescence microscopy, investigating ribbon protein locations has been challenging, especially in the early development stages of model animals. Here, we used stimulated emission depletion microscopy, a super-resolution imaging technique, to look at retina sections in 4 days post-fertilization (dpf) zebrafish. We observed that in photoreceptor cells, RIBEYE and RIM2 are expressed along the synaptic ribbon, with RIM2 consistently located inside of the horseshoe-shaped synaptic ribbon structure with RIBEYE located on the outside. The L-type calcium channel subunit, CACNA1F, exhibited small spot-like staining beneath the RIM2 and RIBEYE structures. Using morpholino antisense oligonucleotides to knock down RIBEYE expression, we observed fewer and shorter ribbons in the photoreceptor outer plexiform layers of 4 dpf fish retina as well as a reduction in RIM2 expression. The clustering of CACNA1F in these blind fish was no longer observed, but instead showed a diffuse expression in the photoreceptor terminal.

The development of synaptic ribbons in rod and cone photoreceptor terminals of the cat retina was studied using quantitative electron microscopy. At the region of the area centralis, synaptic ribbon profiles are initially recognized at PCD (postconception day) 59. Synaptic ribbon density increases rapidly, reaching a peak of 0.55 ribbons/μm3 at PCD 68 (postnatal day 3) and maintains approximately that value for an additional 8 d. Following PCD 76, ribbon density begins to decrease, to 0.37 ribbons/μm3 at PCD 82 and 0.25 ribbons/μm3 at PCD 102. Although ribbon density drops by approximately 50% during this 39-d period, the outer plexiform layer (OPL) volume at the area centralis increases by about 20%. Ribbon density continues to decrease gradually over a protracted period to reach a final adult value of 0.11–0.14 ribbons/μm3. During the period of high ribbon density, rod spherules with two, or even three ribbon profiles, were routinely observed. In contrast, in the adult, spherules with more than one ribbon profile are only rarely encountered. During development, the length of synaptic ribbon profiles increases from a mean of 0.22 μm at PCD 62 to the 0.47 μm mean length found in the adult.

Connexin26 (Cx26) is a member of the family of integral membrane proteins that normally form intercellular gap junctional channels. We have used Western blotting, immunofluorescence, immunoelectron microscopy, and single-cell reverse-transcriptase polymerase chain reaction amplification (RT-PCR) to analyze the expression and cellular localization of Cx26 in the carp retina. In the outer plexiform layer, strong clustered Cx26 immunolabeling was concentrated at and restricted to the terminal dendrites of horizontal cells. Single-cell RT-PCR confirmed the expression of Cx26 in carp retinal horizontal cells. 248-bp fragments amplified from cDNAs of four different horizontal cells were cloned and each nucleotide sequence encodes a protein fragment (AA 104-185) with highly significant homology to rat and mouse Cx26. Immunoelectron microscopy revealed that only the invaginating dendrites of horizontal cells in intimate lateral association with the presynaptic ribbon complex were labeled. No labeling was found at the photoreceptor membrane and there was no septalaminar structure, indicative of gap junctions, between photoreceptors and horizontal cells. The focal location of Cx26 at the membrane of the dendritic tips of horizontal cells and the lack of gap junctional morphology suggests that Cx26 might form hemichannels.