Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina.

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

Extracellular ATP hydrolysis inhibits synaptic transmission by increasing ph buffering in the synaptic cleft.

Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca²âº channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.

Ephaptic communication in the vertebrate retina.

In the vertebrate retina, cones project to the horizontal cells (HCs) and bipolar cells (BCs). The communication between cones and HCs uses both chemical and ephaptic mechanisms. Cones release glutamate in a Ca(2+)-dependent manner, while HCs feed back to cones via an ephaptic mechanism. Hyperpolarization of HCs leads to an increased current through connexin hemichannels located on the tips of HC dendrites invaginating the cone synaptic terminals. Due to the high resistance of the extracellular synaptic space, this current makes the synaptic cleft slightly negative. The result is that the Ca(2+)-channels in the cone presynaptic membrane experience a slightly depolarized membrane potential and therefore more glutamate is released. This ephaptic mechanism forms a very fast and noise free negative feedback pathway. These characteristics are crucial, since the retina has to perform well in demanding conditions such as low light levels. In this mini-review we will discuss the critical components of such an ephaptic mechanism. Furthermore, we will address the question whether such communication appears in other systems as well and indicate some fundamental features to look for when attempting to identify an ephaptic mechanism.

Action spectra of zebrafish cone photoreceptors.

Zebrafish is becoming an increasingly popular model in the field of visual neuroscience. Although the absorption spectra of its cone photopigments have been described, the cone action spectra were still unknown. In this study we report the action spectra of the four types of zebrafish cone photoreceptors, determined by measuring voltage responses upon light stimulation using whole cell patch clamp recordings. A generic template of photopigment absorption spectra was fit to the resulting action spectra in order to establish the maximum absorption wavelength, the A2-based photopigment contribution and the size of the ß-wave of each cone-type. Although in general there is close correspondence between zebrafish cone action- and absorbance spectra, our data suggest that in the case of MWS- and LWS-cones there is appreciable contribution of A2-based photopigments and that the ß-wave for these cones is smaller than expected based on the absorption spectra.

Connexin hemichannel mediated ephaptic inhibition in the retina.

Connexins are the building blocks of gap-junctions; sign conserving electrical synapses. Recently it has been shown that connexins can also function as hemichannels and can mediate a sign inverting inhibitory synaptic signal from horizontal cells to cones via an ephaptic mechanism. In this review we will discuss the critical requirements for such an ephaptic interaction and relate these to the available experimental evidence. The highly conserved morphological structure of the cone synapse together with a number of specific connexin proteins and proteoglycans present in the synaptic complex of the cones creates a synaptic environment that allows ephaptic interactions. The connexins involved are members of a special group of connexins, encoded by the GJA9 and GJA10 genes. Surprisingly, in contrast to many other vertebrates, mouse and other rodents seem to lack a GJA9 encoded connexin. The specific combination of substances that block feedback and the highly specific modification of feedback in a zebrafish lacking Cx55.5 hemichannels all point to an ephaptic feedback mechanism from horizontal cells to cones. This article is part of a Special Issue entitled Electrical Synapses.

Synaptic transmission from horizontal cells to cones is impaired by loss of connexin hemichannels.

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.