In the vertebrate retina, horizontal cells generate the inhibitory surround of bipolar cells, an essential step in contrast enhancement. For the last decades, the mechanism involved in this inhibitory synaptic pathway has been a major controversy in retinal research. One hypothesis suggests that connexin hemichannels mediate this negative feedback signal; another suggests that feedback is mediated by protons. Mutant zebrafish were generated that lack connexin 55.5 hemichannels in horizontal cells. Whole cell voltage clamp recordings were made from isolated horizontal cells and cones in flat mount retinas. Light-induced feedback from horizontal cells to cones was reduced in mutants. A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized. Hemichannel currents in isolated horizontal cells showed a similar behavior. The hyperpolarization-induced hemichannel current was strongly reduced in the mutants while the depolarization-induced hemichannel current was not. Intracellular recordings were made from horizontal cells. Consistent with impaired feedback in the mutant, spectral opponent responses in horizontal cells were diminished in these animals. A behavioral assay revealed a lower contrast-sensitivity, illustrating the role of the horizontal cell to cone feedback pathway in contrast enhancement. Model simulations showed that the observed modifications of feedback can be accounted for by an ephaptic mechanism. A model for feedback, in which the number of connexin hemichannels is reduced to about 40%, fully predicts the specific asymmetric modification of feedback. To our knowledge, this is the first successful genetic interference in the feedback pathway from horizontal cells to cones. It provides direct evidence for an unconventional role of connexin hemichannels in the inhibitory synapse between horizontal cells and cones. This is an important step in resolving a long-standing debate about the unusual form of (ephaptic) synaptic transmission between horizontal cells and cones in the vertebrate retina.

Connexins in retinal horizontal cells (HC) function in the processing of visual information. For example, gap junction-forming connexins may contribute to the spatial integration of visual stimuli. Additionally, connexin hemichannels have been hypothesized to participate in the feedback pathway from HCs to cones. To verify the identities of the zebrafish HC connexins, we performed promoter expression and immunohistochemical studies of connexin 52.6 (Cx52.6) and Cx55.5. Zebrafish embryos were microinjected with Cx52.6 or Cx55.5 promoter sequences and a green fluorescent protein reporter construct. Light and electron microscopic (EM) analysis showed green fluorescent protein expression exclusively in retinal HCs. Immunohistochemistry confirmed that HCs express Cx52.6 and Cx55.5 proteins. Light microscopy revealed Cx52.6 and Cx55.5 in the retinal inner nuclear and outer plexiform layers. Double labeling for Cx55.5 or Cx52.6 and cell-specific markers (tyrosine hydroxylase, protein kinase C-alpha, or GluR2) demonstrated that these connexins do not localize to interplexiform or ON bipolar cells, but most likely are present in HCs. Preembedding immuno-EM confirmed the HC-specific expression of Cx52.6 and Cx55.5 and illustrated the presence of these two connexins in gap junctions between HCs. The EM data also revealed robust labeling for Cx55.5 in hemichannels on HC dendrites in photoreceptor synaptic terminals. Voltage-clamp experiments in cultured cells demonstrated that Cx55.5-containing hemichannels can open at physiological membrane potentials. These results offer the first in vivo demonstration of the HC-specific activities of the Cx52.6 and Cx55.5 promoters. Furthermore, these data provide the first proof at the protein level for retinal HC-specific connexins in the zebrafish.