Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina

doi:10.1016/j.neuroscience.2007.06.003

Neuroscience

Volume 148, Issue 2, 24 August 2007, Pages 473-488

https://doi.org/10.1016/j.neuroscience.2007.06.003 Get rights and content

Abstract

Horizontal cells form gap junctions with each other in mammalian retina, and lacZ reporter analyses have recently indicated that these cells express the Cx57 gene, which codes for the corresponding gap junctional protein. Using anti-connexin57 antibodies, we detected connexin57 protein in immunoblots of mouse retina, and found punctate immunolabeling of this connexin co-distributed with calbindin-positive horizontal cells in the retinal outer plexiform layer. Double immunofluorescence labeling was conducted to determine the spatial relationships of connexin36, connexin57, the gap junction-associated protein zonula occludens-1 and the photoreceptor ribbon synapse-associated protein bassoon in the outer plexiform layer. Connexin36 was substantially co-localized with zonula occludens-1 in the outer plexiform layer, and both of these proteins were frequently located in close spatial proximity to bassoon-positive ribbon synapses. Connexin57 was often found adjacent to, but not overlapping with, connexin36-positive and zonula occludens-1-positive puncta, and was also located adjacent to bassoon-positive ribbon synapses at rod spherules, and intermingled with such synapses at cone pedicles. These results suggest zonula occludens-1 interaction with connexin36 but not with Cx57 in the outer plexiform layer, and an absence of connexin57/connexin36 heterotypic gap junctional coupling in mouse retina. Further, an arrangement of synaptic contacts within rod spherules is suggested whereby gap junctions between horizontal cell terminals containing connexin57 occur in very close proximity to ribbon synapses formed by rod photoreceptors, as well as in close proximity to Cx36-containing gap junctions between rods and cones.

Section snippets

Antibodies and animals

Primary antibodies used in this study are listed in Table 1, with indications of source and dilutions employed. Monoclonal mouse anti-calbindin C-9848 antibody was obtained from Sigma (Sigma Aldrich Canada, Oakville, ON, Canada), and monoclonal anti-bassoon VAM-PS00 antibody was purchased from Stressgen Biotechnologies Corporation (Victoria, BC, Canada). Affinity-purified anti-Cx57, anti-Cx36 and anti-ZO-1 antibodies were obtained from Invitrogen/Zymed (Camarillo, CA, USA). Anti-Cx57 antibodies

Characterization of anti-Cx57 antibodies

Immunofluorescence detection of Cx57 was examined in HeLa cells transiently transfected with a Cx57-eGFP fusion protein. No immunosignals corresponding to gap junctional plaques in the plasma membrane of transfected cells were found. Instead, fluorescence for eGFP in isolated cells was distributed around the periphery of cell nuclei (Fig. 1A1, B1). All cells containing eGFP fluorescence were immunolabeled for Cx57 with affinity-purified Ab40-5000MID (Fig. 1A2) and Ab40-4800CT (Fig. 1B2). As

Discussion

We demonstrate the expression of Cx57 protein in mouse retina, and establish its localization within the OPL. Our results are consistent with those of Hombach et al. (2004), who replaced a portion of the Cx57 gene coding region with the LacZ reporter in mice and observed β-galactosidase activity in cell bodies of retinal horizontal cells. However, as with many other connexins that fail to be detected in the somata of neural cells in which they are expressed (Nagy and Rash 2000, Nagy et al 2004

Acknowledgments

This work was supported by grants from the Canadian Institutes of Health Research to J.I.N. and by grants from the German Research Association to K.W. (Wi 270/22-5,6). We thank B. McLean and N. Nolette for excellent technical assistance. We thank Dr. D. Paul (Harvard University) for provision of Cx36 knockout mice, and C. Olson for help in maintaining and genotyping these mice. We also thank Dr. S. Schein (University of California, Los Angeles) for invaluable discussions on the 3-D organization

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Functionally, Cx36 is thought to contribute to regulation of cone-rod interactions and rod synaptic transmission based on post-mortem examination (Asteriti et al., 2014; Güldenagel et al., 2001). Cx36 is expressed at the outer plexiform layer in a laminar and non-interacting arrangement with cx57 (Ciolofan et al., 2007; Deans and Paul, 2001; O'Brien et al., 2012a,b). Here, new data are presented of dark adapted cx36−/− mice (a kind gift from Dr. Marla Feller) demonstrating focal regions of subnormal uptake, consistent with a role for Cx36 in the inner and outer segments, and in the inner plexiform and ganglion cells layers (Fig. 9).
Rod cell oxidative stress is a major pathogenic factor in retinal disease, such as diabetic retinopathy (DR) and retinitis pigmentosa (RP). Personalized, non-destructive, and targeted treatment for these diseases remains elusive since current imaging methods cannot analytically measure treatment efficacy against rod cell compartment-specific oxidative stress in vivo. Over the last decade, novel MRI-based approaches that address this technology gap have been developed. This review summarizes progress in the development of MRI since 2006 that enables earlier evaluation of the impact of disease on rod cell compartment-specific function and the efficacy of anti-oxidant treatment than is currently possible with other methods. Most of the new assays of rod cell compartment-specific function are based on endogenous contrast mechanisms, and this is expected to facilitate their translation into patients with DR and RP, and other oxidative stress-based retinal diseases.
Gap junctional coupling in the vertebrate retina: Variations on one theme?
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Gap junctions connect cells in the bodies of all multicellular organisms, forming either homologous or heterologous (i.e. established between identical or different cell types, respectively) cell-to-cell contacts by utilizing identical (homotypic) or different (heterotypic) connexin protein subunits. Gap junctions in the nervous system serve electrical signaling between neurons, thus they are also called electrical synapses. Such electrical synapses are particularly abundant in the vertebrate retina where they are specialized to form links between neurons as well as glial cells. In this article, we summarize recent findings on retinal cell-to-cell coupling in different vertebrates and identify general features in the light of the evergrowing body of data. In particular, we describe and discuss tracer coupling patterns, connexin proteins, junctional conductances and modulatory processes. This multispecies comparison serves to point out that most features are remarkably conserved across the vertebrate classes, including (i) the cell types connected via electrical synapses; (ii) the connexin makeup and the conductance of each cell-to-cell contact; (iii) the probable function of each gap junction in retinal circuitry; (iv) the fact that gap junctions underlie both electrical and/or tracer coupling between glial cells. These pan-vertebrate features thus demonstrate that retinal gap junctions have changed little during the over 500 million years of vertebrate evolution. Therefore, the fundamental architecture of electrically coupled retinal circuits seems as old as the retina itself, indicating that gap junctions deeply incorporated in retinal wiring from the very beginning of the eye formation of vertebrates. In addition to hard wiring provided by fast synaptic transmitter-releasing neurons and soft wiring contributed by peptidergic, aminergic and purinergic systems, electrical coupling may serve as the ‘skeleton’ of lateral processing, enabling important functions such as signal averaging and synchronization.
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For some of these connexins antibodies are available such that the localization could be determined. All connexins studied from this group (Cx57, Cx55.5, Cx52.6, Cx52.9) appear to be localized in horizontal cells (Dermietzel et al., 2000; Zoidl et al., 2004; Ciolofan et al., 2007; Shields et al., 2007; Janssen-Bienhold et al., 2009; Klaassen et al., 2011). An antibody against Cx57 also stains horizontal cells in rabbit retina Puller et al. (2009).
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.
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Variety of horizontal cell gap junctions in the rabbit retina
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In the rabbit retina, there are two types of horizontal cell (HC). The axonless A-type HCs form a coupled network via connexin 50 (Cx50) gap junctions in the outer plexiform layer (OPL). The axon-bearing B-type HCs form two independently coupled networks; the dendritic network via gap junctions consisted of unknown Cx and the axon terminal network via Cx57. The present study was conducted to examine the localization and morphological features of Cx50 and Cx57 gap junctions in rabbit HCs at cellular and subcellular levels. The results showed that each gap junction composed of Cx50 or Cx57 showed distinct features. The larger Cx50 gap junctions were located more proximally than the smaller Cx50 gap junctions. Both Cx50 plaques formed symmetrical homotypic gap junctions, but some small ones had an asymmetrical appearance, suggesting the presence of heterotypic gap junctions or hemichannels. In contrast, Cx57 gap junctions were found in the more distal part of the OPL but never on the axon terminal endings entering the rod spherules, and they were exclusively homotypic. Interestingly, about half of the Cx57 gap junctions appeared to be invaginated. These distinct features of Cx50 and Cx57 gap junctions show the variety of HC gap junctions and may provide insights into the function of different types of HCs.

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Cognitive neuroscienceSpatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina

Abstract

Section snippets

Antibodies and animals

Characterization of anti-Cx57 antibodies

Discussion

Acknowledgments

Neuron

Exp Eye Res

Neuroscience

J Biol Chem

Int Rev Cytol

Brain Res Brain Res Rev

Brain Res Rev

Brain Res Rev

Brain Res Rev

J Biol Chem

Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina

Eur J Neurosci

Connexin 36 in photoreceptor cells: studies on transgenic rod-less and cone-less mouse retinas

Mol Vis

Mouse horizontal cells do not express connexin26 or connexin36

Cell Commun Adhes

Localization of heterotypic gap junctions composed of connexin45 and connexin36 in the rod pathway of the mouse retina

Eur J Neurosci

Ultrastructure and cytochemistry of the synaptic region

Science

Organization of the primate retina: electron microscopy

Proc R Soc Lond B Biol Sci

Expression of neuronal connexin36 in AII amacrine cells of the mammalian retina

J Neurosci

Expression of connexin36 in cone pedicles and OFF-cone bipolar cells of the mouse retina

J Neurosci

A reporter allele for investigating connexin 26 gene expression in the mouse brain

Eur J Neurosci

Gap junctions and connexin-interacting proteins

Cardiovasc Res

Expression patterns of connexin genes in mouse retina

J Comp Neurol

Visual transmission deficits in mice with targeted disruption of the gap junction gene connexin36

J Neurosci

An alternative pathway for rod signals in the rodent retina: rod photoreceptors, cone bipolar cells, and the localization of glutamate receptors

Proc Natl Acad Sci U S A

Electrical synapses in retinal ON cone bipolar cells: subtype-specific expression of connexins

Proc Natl Acad Sci U S A

Immunocytochemical analysis of the mouse retina

J Comp Neurol

The synaptic architecture of AMPA receptors at the cone pedicle of the primate retina

J Neurosci

Endogenous dopaminergic regulation of horizontal cell coupling in the mammalian retina

J Comp Neurol

PDZ proteins and proximal ion transport

Curr Opin Nephrol Hypertens

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex

Science

Functional expression of connexin57 in horizontal cells of the mouse retina

Eur J Neurosci

Electrical coupling between red and green cones in primate retina

Nat Neurosci

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Cell Res

Identification and localization of connexin26 within the photoreceptor-horizontal cell synaptic complex

Vis Neurosci

Hemichannel-mediated inhibition in the outer retina

Science

Organization of the outer plexiform layer of the primate retina: electron microscopy of Golgi-impregnated cells

Philos Trans R Soc Lond B Biol Sci

Cognitive neuroscience
Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina