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Coupling of RG A1 and displaced amacrine cells was preserved in Cx36-, Cx45-, and Cx40-de fi cient mice. ( A ) RG A1 cell injected in
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Mammalian retinae express multiple connexins that mediate the metabolic and electrical coupling of various cell types. In retinal neurons, only connexin 36, connexin 45, connexin 50, and connexin 57 have been described so far. Here, we present an analysis of a novel retinal connexin, connexin 30.2 (Cx30.2), and its regulation in the mouse retina. T...
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... we tested whether other known connexins are involved in this gap junction. In the mouse retina, three neuronal connexins (Cx36, Cx45, and Cx57) have been described so far. Cx57 is exclusively expressed in horizontal cells of the mouse retina ( Hombach et al., 2004;Shelley et al., 2006). Therefore, we did not test for Cx57 but for Cx36 and Cx45 (Fig. ...
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... only been described in one type of amacrine cell: the AII amacrine cell Mills et al., 2001). To test whether Cx36 is the connexin involved in the heterologous coupling between RG A1 and displaced amacrine cells, we injected RG A1 cells in Cx36 À/À mice ( Güldenagel et al., 2001). However, RG A1 cells were still coupled to displaced amacrine cells (Fig. 5A and 5D). There was no significant difference between the coupling in WT (4.7 6 2.3 cells; n 5 15 from nine retinae) and Cx36- deficient (4.5 6 1.5 cells; n 5 4 from three retinae, P . 0.85) mice. These results are consistent with previous findings ( Schubert et al., 2005a;Pan et al., 2010) and suggest that Cx36 does not participate at the ...
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... ( Maxeiner et al., 2005; Pérez de Sevilla Müller et al., 2007;Dedek et al., 2009). To examine whether Cx45 forms heterotypic channels with Cx30.2, RG A1 cells were tracer injected in Cx45-deficient mice ). We found that RG A1 cells were still coupled to displaced amacrine cells in Cx45-deficient mice (5.3 6 2.8 cells, n 5 3 from three retinae; Fig. 5B and 5D). Coupling in WT and Cx45-deficient mice was not significantly different (P . 0.66), indicating that these heterotypic gap junctions do not involve ...
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... with Cx30.2 ( Kreuzberg et al., 2005). To test whether this is also the case for the coupling between displaced amacrine cells and RG A1 cells, we analyzed this coupling in Cx40-deficient mice ( Kirchhoff et al., 1998). We found that RG A1 cells were still coupled to displaced amacrine cells in Cx40-deficient mice (n 5 3 from three retinae; Fig. 5C and 5D). The extent of coupling was similar in WT and Cx40-deficient retinae (P . 0.24), indicating that Cx40 is not involved in the gap junction between displaced amacrine and RG A1 cells. Instead, our data suggest that either the reporter gene expression in the coupled displaced cells is very low or an unidentified gap junction protein ...
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Purpose:
RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the...
Citations
... It has been shown for instance that Cx36 is expressed by α-GCs and that Cx45 comprises GJs of ON-OFF direction-selective RGCs, serving spike synchronization and encoding of the direction of movement, respectively [39][40][41][42]. In addition to Cx36 and Cx45, only one other subunit, Cx30.2, has been shown to be expressed by A1 type of RGCs [43]. Besides the retinal distribution pattern of the Cx protein subunits, the expression levels of corresponding Cx36 mRNA transcripts have been studied as well, and their distribution patterns correlated with those of the subunit proteins [44][45][46]. ...
... These latter connections include rod hemichannels [27,[31][32][33][34][35][36], GJs that connect α-GCs into an array [39,40,42] or the ACs that form GJs with Cx30.2-expressing A1 type RGCs [43]. ...
... Besides the above two prevalent Cx subunits, Cx30.2 is also expressed in some RGC connections [43], whereas Cx50 and Cx57 subunits form horizontal cell GJs [29,61,62]. The developmental changes of these latter Cx subunits have not been established yet; although, our unpublished observations indicate that the Cx57 mRNA transcript level is monotonously increasing during postnatal development P0-P15 and displays a plateau in the adult tissue (unpublished observations, Figure 3c). ...
Gap junctions (GJs) are not static bridges; instead, GJs as well as the molecular building block connexin (Cx) proteins undergo major expression changes in the degenerating retinal tissue. Various progressive diseases, including retinitis pigmentosa, glaucoma, age-related retinal degeneration, etc., affect neurons of the retina and thus their neuronal connections endure irreversible changes as well. Although Cx expression changes might be the hallmarks of tissue deterioration, GJs are not static bridges and as such they undergo adaptive changes even in healthy tissue to respond to the ever-changing environment. It is, therefore, imperative to determine these latter adaptive changes in GJ functionality as well as in their morphology and Cx makeup to identify and distinguish them from alterations following tissue deterioration. In this review, we summarize GJ alterations that take place in healthy retinal tissue and occur on three different time scales: throughout the entire lifespan, during daily changes and as a result of quick changes of light adaptation.
... The cells of the retina express several connexin isoforms [6]. Retinal pigment epithelial cells express Cx43, cone and rod photoreceptors express Cx36 [11,12], horizontal cells express Cx50 [13] or Cx57 [14], bipolar and amacrine cells express Cx36 [12] or Cx45 [15,16], and retinal ganglion cells express Cx30.2 [17], Cx36 [18], or Cx45 [19]. ...
Electrical coupling between retinal neurons contributes to the functional complexity of visual circuits. "Cut-loading" methods allow simultaneous assessment of cell-coupling between multiple retinal cell-types, but existing analysis methods impede direct comparison with gold standard direct dye injection techniques. In the current study, we both improved an existing method and developed two new approaches to address observed limitations. Each method of analysis was applied to cut-loaded dark-adapted Guinea pig retinae (n = 29) to assess coupling strength in the axonless horizontal cell type ('a-type', aHCs). Method 1 was an improved version of the standard protocol and described the distance of dye-diffusion (space constant). Method 2 adjusted for the geometric path of dye-transfer through cut-loaded cells and extracted the rate of dye-transfer across gap-junctions in terms of the coupling coefficient (kj). Method 3 measured the diffusion coefficient (De) perpendicular to the cut-axis. Dye transfer was measured after one of five diffusion times (1-20 mins), or with a coupling inhibitor, meclofenamic acid (MFA) (50-500μM after 20 mins diffusion). The standard protocol fits an exponential decay function to the fluorescence profile of a specified retina layer but includes non-specific background fluorescence. This was improved by measuring the fluorescence of individual cell soma and excluding from the fit non-horizontal cells located at the cut-edge (p
... Paradoxically, in uninjured retinas, Pten deletion at the onset of neurogenesis in retinal progenitor cells or in a conditional Pten loss-of-function allele, results in reduced number of RGCs and rod photoreceptors, and disrupts amacrine cell dendritic arborizations (Cantrup et al., 2012;Jo et al., 2012;Sakagami et al., 2012;Tachibana et al., 2016). In cell cultures, silencing Pten with non-targeting small-interfering RNA (NT-siRNA) decreases gap junctional intercellular communication between glia cells (González-Sánchez et al., 2016), perhaps due to Pten's effect on protein kinases (Preiss et al., 2007), which mediate phosphorylation of connexins (Xia and Mills, 2004;Urschel et al., 2006;Pérez de Sevilla Müller et al., 2010a). ...
... Immunohistochemical labeling was performed using our published protocols (Pérez de Sevilla Müller et al., 2007, 2010a,b, 2013. Whole-mounted retinas were fixed for 15 min by immersion in 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.4) at RT. ...
... Intracellular injections of Lucifer Yellow and Neurobiotin were performed as described earlier (Pérez de Sevilla Müller et al., 2007, 2010aVuong et al., 2015). Borosilicate glass electrodes (#60200; A-M Systems; Sequim, WA, USA) were pulled and filled at their tips with 0.5% Lucifer Yellow (Sigma-Aldrich) and 4% N-(2-aminoethyl)-biotinamide hydrochloride (Neurobiotin; Vector Laboratories, Burlingame, CA, USA), and back-filled with 0.1 M Tris buffer, pH 7.4. ...
Manipulation of the phosphatase and tensin homolog (PTEN) pathway has been suggested as a therapeutic approach to treat or prevent vision loss due to retinal disease. In this study, we investigated the effects of deleting one copy of Pten in a well-characterized class of retinal ganglion cells called α-ganglion cells in the mouse retina. In Pten+/– retinas, α-ganglion cells did not exhibit major changes in their dendritic structure, although most cells developed a few, unusual loop-forming dendrites. By contrast, α-ganglion cells exhibited a significant decrease in heterologous and homologous gap junction mediated cell coupling with other retinal ganglion and amacrine cells. Additionally, the majority of OFF α-ganglion cells (12/18 cells) formed novel coupling to displaced amacrine cells. The number of connexin36 puncta, the predominant connexin that mediates gap junction communication at electrical synapses, was decreased by at least 50% on OFF α-ganglion cells. Reduced and incorrect gap junction connectivity of α-ganglion cells will affect their functional properties and alter visual image processing in the retina. The anomalous connectivity of retinal ganglion cells would potentially limit future therapeutic approaches involving manipulation of the Pten pathway for treating ganglion cell degeneration in diseases like glaucoma, traumatic brain injury, Parkinson’s, and Alzheimer’s diseases.
... Out of over 20 mammalian Cx subunits, only a handful are abundant in the neuronal retina, including Cx 30, Cx30.2, Cx36, Cx43, Cx45, Cx50, and Cx57 (Güldenagel et al., 2001;Massey et al., 2003;Hombach et al., 2004;Maxeiner et al., 2005;Müller et al., 2010;Kántor et al., 2018). Based on research with KO models, we can determine the exact roles the different Cx subunit types play in retinal mechanisms. ...
Vision altering diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration, myopia, retinal vascular disease, traumatic brain injuries and others cripple many lives and are projected to continue to cause anguish in the foreseeable future. Gap junctions serve as an emerging target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells, thereby playing a crucial role in pathophysiology. Since they are permeable for macromolecules, able to cross the cellular barriers, they show duality in illness as a cause and as a therapeutic target. In this review, we take recent advancements in gap junction neuromodulation (pharmacological blockade, gene therapy, electrical and light stimulation) into account, to show the gap junction’s role in neuronal cell death and the possible routes of rescuing neuronal and glial cells in the retina succeeding illness or injury.
... 21 saw normal coupling in their "G 1 " ganglion cells, which correspond to the ON α-like "RG A1 ", "cluster 11", and "M10" types. [62][63][64] Reinforcing the latter finding, Müller et al. 65 showed that RG A1 -amacrine coupling was unaffected in Cx36-deficient mice but abolished in Cx30.2-deficient mice. However, a more recent study proposed that RG A1 corresponds to M2 ipRGCs rather than ON α and that M2amacrine coupling uses Cx30.2 exclusively, 60 which would contradict our result, although in our opinion, RG A1 cells' somas are too large for them to be M2 ipRGCs. ...
Purpose:
Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with amacrine cells, (2) the neuromodulator contents of ipRGC-coupled amacrine cells, and (3) whether connexin36 (Cx36) contributes to ipRGC-amacrine coupling.
Methods:
Gap junction-permeable Neurobiotin tracer was injected into green fluorescent protein (GFP)-labeled ipRGCs in Opn4Cre/+; Z/EG mice to stain coupled amacrine cells, and immunohistochemistry was performed to reveal the neuromodulator contents of the Neurobiotin-stained amacrine cells. We also created Opn4Cre/+; Cx36flox/flox; Z/EG mice to knock out Cx36 in GFP-labeled ipRGCs and looked for changes in the number of ipRGC-coupled amacrine cells.
Results:
Seventy-three percent of ipRGCs, including all six types (M1-M6), were tracer-coupled with amacrine somas 5.7 to 16.5 µm in diameter but not with ganglion cells. Ninety-two percent of the ipRGC-coupled somas were in the ganglion cell layer and the rest in the inner nuclear layer. Some ipRGC-coupled amacrine cells were found to accumulate serotonin or to contain nitric oxide synthase or neuropeptide Y. Knocking out Cx36 in M2 and M4 dramatically reduced the number of coupled somas.
Conclusions:
Heterologous gap junction coupling with amacrine cells is widespread across mouse ipRGC types. ipRGC-coupled amacrine cells probably comprise multiple morphologic types and use multiple neuromodulators, suggesting that gap junctional ipRGC-to-amacrine signaling likely exerts diverse modulatory effects on retinal physiology. ipRGC-amacrine coupling is mediated partly, but not solely, by Cx36.
... including Cx 30, Cx30.2, Cx36, Cx43, Cx45, Cx50, and Cx57 (Güldenagel et al., 2001;Massey et al., 2003;Hombach et al., 2004;Maxeiner et al., 2005;Müller et al., 2010;Kántor et al., 2018 ). The constitutive Cx45 KO mice die before birth due to the importance of this subunit during development and heart muscle formation, thus the role of Cx45 in retinal functioning have been studied in conditional Cx45 ...
Vision altering diseases, like Glaucoma, Diabetic Retinopathy (DR), Age-Related Macular Degeneration (AMD), Myopia, Retinal Vascular Disease (RVD), Traumatic brain injuries (TBI), and others cripple many lives and are projected to continue to cause anguish in the foreseeable future. Gap junctions serve as an emergent target for neuromodulation and possible regeneration as they directly connect healthy and/or diseased cells, thereby playing a crucial role in pathophysiology. Since they are permeable for macromolecules, able to cross the cellular barriers, they show a duality in illness as a cause and as a therapeutic target. In this review we take recent advancements in GJ neuromodulation (pharmacological blockade, gene therapy, electrical and light stimulation) into account, to show the GJ's role neuronal cell death and the possible routes of reviving neuronal and glial cells in the retina succeeding illness or injury.
Research highlights: This article shows recent advancements in GJ neuromodulation (pharmacological blockade, gene therapy, electrical and light stimulation), to show the GJ's role neuronal cell death and the possible routes of reviving neuronal and glial cells in the retina succeeding illness or injury.
... This was confirmed in studies of genetically modified mice lacking the connexin subunit proteins Cx36 or Cx45 (Schubert et al., 2005a,b;Pan et al., 2010;Völgyi et al., 2013;Roy et al., 2017). Cx36 is the major connexin subunit involved in RGC coupling (Pan et al., 2010) in addition to Cx45 and possibly Cx30.2 (Schubert et al., 2005a;Müller et al., 2010), and the guinea pig retina shows a distribution of Cx36 common to mammals (Kovács-Ö ller et al., 2017). Therefore, it is likely that heterotypic Figure 1L (n ϭ 14). ...
Electrical coupling has been reported to occur only between homotypic retinal ganglion cells, in line with the concept of parallel processing in the early visual system. Here, however, we show reciprocal correlated firing between heterotypic ganglion cells in multielectrode array recordings during light stimulation in retinas of adult guinea pigs of either sex. Heterotypic coupling was further confirmed via tracer spread after intracellular injections of single cells with neurobiotin. Both electrically coupled cell types were sustained ON center ganglion cells but showed distinct light response properties and receptive field sizes. We identified one of the involved cell types as sustained ON α-ganglion cells. The presence of electrical coupling between heterotypic ganglion cells introduces a network motif in which the signals of distinct ganglion cell types are partially mixed at the output stage of the retina.
SIGNIFICANCE STATEMENT The visual information is split into parallel pathways, before it is sent to the brain via the output neurons of the retina, the ganglion cells. Ganglion cells can form electrical synapses between dendrites of neighboring cells in support of lateral information exchange. To date, ganglion-to-ganglion cell coupling is thought to occur only between cells of the same type. Here, however, we show that electrical coupling between different types of ganglion cells exists in the mammalian retina. We provide functional and anatomical evidence that two different types of ganglion cells share information via electrical coupling. This new network motif extends the impact of the heavily studied coding benefits of homotypic coupling to heterotypic coupling across parallel neuronal pathways.
... GJs connect neurons throughout the central nervous system in all examined mammals, where they allow the passage of signals between neurons (Furshpan and Potter, 1957;Watanabe, 1958). The mammalian retina has been a popular model of GJ studies as a number of Cx subunits are expressed by various retinal neurons, including Cx30.2, Cx36, Cx45, Cx50, and Cx57 (Güldenagel et al., 2001;Lee et al., 2003;Massey et al., 2003;Hombach et al., 2004;Maxeiner et al., 2005;Müller et al., 2010;Han and Massey, 2005). It has been shown, that similar to other mammalian species the human retina expresses Cx36 and Cx45 both in the IPL and OPL (Söhl et al., 2010) and the expression patterns resemble those of other mammalian species, including the mouse, rabbit, and rat Mills et al., 2001;Deans et al., 2002;Kihara et al., 2006Kihara et al., , 2010O'Brien et al., 2012;Kovács-Öller et al., 2014Kovács-Öller et al., , 2017Kántor et al., 2016aKántor et al., , 2017. ...
Connexin36 (Cx36) subunits form gap junctions (GJ) between neurons throughout the central nervous system. Such GJs of the mammalian retina serve the transmission, averaging and correlation of signals prior to conveying visual information to the brain. Retinal GJs have been exhaustively studied in various animal species, however, there is still a perplexing paucity of information regarding the presence and function of human retinal GJs. Particularly little is known about GJ formation of human retinal ganglion cells (hRGCs) due to the limited number of suitable experimental approaches. Compared to the neuronal coupling studies in animal models, where GJ permeable tracer injection is the gold standard method, the post-mortem nature of scarcely available human retinal samples leaves immunohistochemistry as a sole approach to obtain information on hRGC GJs. In this study Lucifer Yellow (LY) dye injections and Cx36 immunohistochemistry were performed in fixed short-post-mortem samples to stain hRGCs with complete dendritic arbors and locate dendritic Cx36 GJs. Subsequent neuronal reconstructions and morphometric analyses revealed that Cx36 plaques had a clear tendency to form clusters and particularly favored terminal dendritic segments.
... Cx45 is found in some cone bipolar cells, where it is involved in heterologous electrical synapses made with AII amacrine cells (Maxeiner et al. 2005;Li et al. 2008), and in some RGCs (Schubert et al. 2005b). Cx30.2 expression has been found in one or more types of RGC and some amacrine cells (Muller et al. 2010). Cx30.2, like Cx36, has very low voltage sensitivity and an extremely small 9 pS single channel conductance (Kreuzberg et al. 2005). ...
... A number of other modulators control electrical coupling in retinal neurons through protein kinase signaling pathways. Electrical synapses containing Cx30.2, present in several types of RGCs and amacrine cells, are modulated by protein kinase C activity (Muller et al. 2010). Connexins on the cone bipolar cell side of AII amacrine-ON cone bipolar cell electrical synapses (Mills & Massey 1995;Xia & Mills 2004) and in horizontal cells (Pottek et al. 1997) are uncoupled by nitric oxide through a signaling pathway involving cGMP and presumably protein kinase G activity. ...
Electrical synaptic transmission via gap junctions underlies direct and rapid neuronal communication in the central nervous system. The diversity of functional roles played by electrical synapses is perhaps best exemplified in the vertebrate retina in which gap junctions are expressed by each of the five major neuronal types. These junctions are highly plastic; they are dynamically regulated by ambient illumination and circadian rhythms acting through light-activated neuromodulators. The networks formed by electrically coupled neurons provide plastic, reconfigurable circuits positioned to play key and diverse roles in the transmission and processing of visual information at every retinal level. Recent work indicates gap junctions also play a role in the progressive cell death and aberrant activity seen in various pathological conditions of the retina. Gap junctions thus form potential targets for novel neuroprotective therapies in the treatment of neurodegenerative retinal diseases such as glaucoma and ischemic retinopathies. Expected final online publication date for the Annual Review of Vision Science Volume 4 is September 15, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... The technique is based on the fact that gap junctions are not only permeable to small ions but also to small molecules which can then be detected in the gap junction- coupled cells. By prelabeling cells with markers for DNA [4,6-diamino-2-phenylindole (DAPI), acridine orange] [9,10] or with other fluorescent dyes (EGFP, fluorescein) in genetically modified mouse lines [10,11] the cell of interest can be pre- cisely targeted (Fig. 1). Normally, a gap junction-permeable (neurobiotin) and impermeable tracer (e.g., Alexa 488) are injected together (Fig. 2): the impermeable tracer serves to test for correct impalement whereas the permeable tracer can be visualized after the injection, e.g., using a fluorophore-coupled streptavidin complex. ...
... We also successfully used a fluorogenic substrate for β-galactosidase (encoded by lacZ DNA) to visualize lacZ- expressing cells prior to injection [11]. ...
... 8. To study the regulation of gap junctions, injections can be combined with pharmacology [19]. The retina is then incu- bated in, for example, protein kinase inhibitors prior to injection [11]. ...
In the mammalian retina, gap junctions, made of connexin proteins, are found in all neuronal cell types and are important for the transmission of rod photoreceptor signals, spike synchronization, noise reduction, and signal averaging. There are several methods available to assess gap junctional coupling in the retina: simultaneous electrical recordings from two adjacent cells, cut-loading, and intracellular injection of gap junction-permeable tracers. Here, we focus on the latter as it allows precise targeting of the cell of interest and is suitable to assess tracer coupling in a wide variety of retinal cell types, e.g., horizontal cells, amacrine cells, and ganglion cells. Tracer coupling experiments are usually performed in the intact retina and can provide information on the extent of coupling, the identity of synaptic partners, and (when combined with immunohistochemistry or pharmacology) the underlying connexin or the regulation of gap junctions.
