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

Klaassen LJ, Sun Z, Steijaert MN, Bolte P, Fahrenfort I, Sjoerdsma T, Klooster J, Claassen Y, Shields CR, Ten Eikelder HM, Janssen-Bienhold U, Zoidl G, McMahon DG, Kamermans M - PLoS Biol. (2011)

Bottom Line: A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized.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.

View Article: PubMed Central - PubMed

Affiliation: Research Unit Retinal Signal Processing, The Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.

ABSTRACT
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.

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Localization of cellular markers in retinas from wild-type and mutant zebrafish.The organization of the outer retina was evaluated by the distribution of a number of cellular markers for double cones (A, FRet43) in red, horizontal cells (B, GluR2) in green, OFF-bipolar cells (C, GluR4) in red, and interplexiform cells (E, TH) in red. The blue stain is the nuclear marker DAPI. No difference in the distribution of these markers could be observed. In both preparations, GluR2 forms horseshoe-like structures in the OPL (B, D). Previous studies have shown that these structures are horizontal cell dendrites invaginating the cone terminal [57],[58]. GluR4 labeling is present as small puncta at the level of the OPL (C, D). These puncta were identified as the tips of the dendrites of OFF bipolar cells [57],[58]. (E) TH labels interplexiform cells and the synapses these cells make onto horizontal cells. The immunoreactivity pattern is similar in wild-type and mutant retinas. These results indicate that all major neuron types in the OPL have developed normally. (A to E): Scale bar = 10 µm. (F) Ultrastructure of the cone synaptic terminal in wild-type (left) and mutant (right) retinas. No differences were found in the ultrastructural organization of the cone synaptic terminal. In both animal models, the characteristic shape of the synaptic triad was easily identified. R, synaptic ribbon; Sp, spinules; HC, horizontal cell; * bipolar cell. Scale bar = 0.5 µm.
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pbio-1001107-g001: Localization of cellular markers in retinas from wild-type and mutant zebrafish.The organization of the outer retina was evaluated by the distribution of a number of cellular markers for double cones (A, FRet43) in red, horizontal cells (B, GluR2) in green, OFF-bipolar cells (C, GluR4) in red, and interplexiform cells (E, TH) in red. The blue stain is the nuclear marker DAPI. No difference in the distribution of these markers could be observed. In both preparations, GluR2 forms horseshoe-like structures in the OPL (B, D). Previous studies have shown that these structures are horizontal cell dendrites invaginating the cone terminal [57],[58]. GluR4 labeling is present as small puncta at the level of the OPL (C, D). These puncta were identified as the tips of the dendrites of OFF bipolar cells [57],[58]. (E) TH labels interplexiform cells and the synapses these cells make onto horizontal cells. The immunoreactivity pattern is similar in wild-type and mutant retinas. These results indicate that all major neuron types in the OPL have developed normally. (A to E): Scale bar = 10 µm. (F) Ultrastructure of the cone synaptic terminal in wild-type (left) and mutant (right) retinas. No differences were found in the ultrastructural organization of the cone synaptic terminal. In both animal models, the characteristic shape of the synaptic triad was easily identified. R, synaptic ribbon; Sp, spinules; HC, horizontal cell; * bipolar cell. Scale bar = 0.5 µm.

Mentions: Connexins are proteins with four transmembrane domains, two highly conserved extracellular loops, one intracellular loop, and a long C-terminal end. To eliminate Cx55.5 function, we generated a zebrafish with a stop codon in the first extracellular loop of Cx55.5(C54X) using the TILLING technique (see Methods). The site of this mutation prevents the formation of a functional protein. The mutants develop normally. Body length (wild-type: 22±3 mm, n = 10; mutant: 22±1 mm, n = 10; p>0.05) and eye size (wild-type: 2.1±0.2 mm, n = 40; mutant: 2.1±0.2 mm, n = 41; p>0.05) did not differ significantly. Also the cell density in the outer nuclear layer (ONL) (p = 0.73), the inner nuclear layer (INL) (p = 0.80), and the ganglion cell layer (GCL) (p = 0.09) were equal. Finally, the localization of cell-specific markers was studied. The immunoreactivity of FRet43 (double cones), GluR2 (horizontal cell dendrites), GluR4 (OFF-bipolar cell dendrites), and TH (interplexiform cells) did not differ between wild-type and mutant and the ultrastructure of the cone synaptic terminal was normal (Figure 1).


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

Klaassen LJ, Sun Z, Steijaert MN, Bolte P, Fahrenfort I, Sjoerdsma T, Klooster J, Claassen Y, Shields CR, Ten Eikelder HM, Janssen-Bienhold U, Zoidl G, McMahon DG, Kamermans M - PLoS Biol. (2011)

Localization of cellular markers in retinas from wild-type and mutant zebrafish.The organization of the outer retina was evaluated by the distribution of a number of cellular markers for double cones (A, FRet43) in red, horizontal cells (B, GluR2) in green, OFF-bipolar cells (C, GluR4) in red, and interplexiform cells (E, TH) in red. The blue stain is the nuclear marker DAPI. No difference in the distribution of these markers could be observed. In both preparations, GluR2 forms horseshoe-like structures in the OPL (B, D). Previous studies have shown that these structures are horizontal cell dendrites invaginating the cone terminal [57],[58]. GluR4 labeling is present as small puncta at the level of the OPL (C, D). These puncta were identified as the tips of the dendrites of OFF bipolar cells [57],[58]. (E) TH labels interplexiform cells and the synapses these cells make onto horizontal cells. The immunoreactivity pattern is similar in wild-type and mutant retinas. These results indicate that all major neuron types in the OPL have developed normally. (A to E): Scale bar = 10 µm. (F) Ultrastructure of the cone synaptic terminal in wild-type (left) and mutant (right) retinas. No differences were found in the ultrastructural organization of the cone synaptic terminal. In both animal models, the characteristic shape of the synaptic triad was easily identified. R, synaptic ribbon; Sp, spinules; HC, horizontal cell; * bipolar cell. Scale bar = 0.5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139627&req=5

pbio-1001107-g001: Localization of cellular markers in retinas from wild-type and mutant zebrafish.The organization of the outer retina was evaluated by the distribution of a number of cellular markers for double cones (A, FRet43) in red, horizontal cells (B, GluR2) in green, OFF-bipolar cells (C, GluR4) in red, and interplexiform cells (E, TH) in red. The blue stain is the nuclear marker DAPI. No difference in the distribution of these markers could be observed. In both preparations, GluR2 forms horseshoe-like structures in the OPL (B, D). Previous studies have shown that these structures are horizontal cell dendrites invaginating the cone terminal [57],[58]. GluR4 labeling is present as small puncta at the level of the OPL (C, D). These puncta were identified as the tips of the dendrites of OFF bipolar cells [57],[58]. (E) TH labels interplexiform cells and the synapses these cells make onto horizontal cells. The immunoreactivity pattern is similar in wild-type and mutant retinas. These results indicate that all major neuron types in the OPL have developed normally. (A to E): Scale bar = 10 µm. (F) Ultrastructure of the cone synaptic terminal in wild-type (left) and mutant (right) retinas. No differences were found in the ultrastructural organization of the cone synaptic terminal. In both animal models, the characteristic shape of the synaptic triad was easily identified. R, synaptic ribbon; Sp, spinules; HC, horizontal cell; * bipolar cell. Scale bar = 0.5 µm.
Mentions: Connexins are proteins with four transmembrane domains, two highly conserved extracellular loops, one intracellular loop, and a long C-terminal end. To eliminate Cx55.5 function, we generated a zebrafish with a stop codon in the first extracellular loop of Cx55.5(C54X) using the TILLING technique (see Methods). The site of this mutation prevents the formation of a functional protein. The mutants develop normally. Body length (wild-type: 22±3 mm, n = 10; mutant: 22±1 mm, n = 10; p>0.05) and eye size (wild-type: 2.1±0.2 mm, n = 40; mutant: 2.1±0.2 mm, n = 41; p>0.05) did not differ significantly. Also the cell density in the outer nuclear layer (ONL) (p = 0.73), the inner nuclear layer (INL) (p = 0.80), and the ganglion cell layer (GCL) (p = 0.09) were equal. Finally, the localization of cell-specific markers was studied. The immunoreactivity of FRet43 (double cones), GluR2 (horizontal cell dendrites), GluR4 (OFF-bipolar cell dendrites), and TH (interplexiform cells) did not differ between wild-type and mutant and the ultrastructure of the cone synaptic terminal was normal (Figure 1).

Bottom Line: A reduction of feedback was also found when horizontal cells were pharmacologically hyperpolarized but was absent when they were pharmacologically depolarized.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.

View Article: PubMed Central - PubMed

Affiliation: Research Unit Retinal Signal Processing, The Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus