F. Pan

Screening of gap junction antagonists on dye coupling in the rabbit retina

Many cell types in the retina are coupled via gap junctions and so there is a pressing need for a potent and reversible gap junction antagonist. We screened a series of potential gap junction antagonists by evaluating their effects on dye coupling in the network of A-type horizontal cells. We evaluated the following compounds: meclofenamic acid (MFA), mefloquine, 2-aminoethyldiphenyl borate (2-APB), 18-alpha-glycyrrhetinic acid, 18-beta-glycyrrhetinic acid (18-beta-GA), retinoic acid, flufenamic acid, niflumic acid, and carbenoxolone. The efficacy of each drug was determined by measuring the diffusion coefficient for Neurobiotin (Mills & Massey, 1998). MFA, 18-beta-GA, 2-APB and mefloquine were the most effective antagonists, completely eliminating A-type horizontal cell coupling at a concentration of 200 muM. Niflumic acid, flufenamic acid, and carbenoxolone were less potent. Additionally, carbenoxolone was difficult to wash out and also may be harmful, as the retina became opaque and swollen. MFA, 18-beta-GA, 2-APB and mefloquine also blocked coupling in B-type horizontal cells and AII amacrine cells. Because these cell types express different connexins, this suggests that the antagonists were relatively non-selective across several different types of gap junction. It should be emphasized that MFA was water-soluble and its effects on dye coupling were easily reversible. In contrast, the other gap junction antagonists, except carbenoxolone, required DMSO to make stock solutions and were difficult to wash out of the preparation at the doses required to block coupling in A-type HCs. The combination of potency, water solubility and reversibility suggest that MFA may be a useful compound to manipulate gap junction coupling.

Coupling between A-Type Horizontal Cells Is Mediated by Connexin 50 Gap Junctions in the Rabbit Retina

There are many examples of neuronal coupling via gap junctions in the retina. Of these, perhaps the best known is the extensive coupling between horizontal cells. In the rabbit retina, there are two types of horizontal cells, A-type and B-type, both of which are independently coupled. Connexin 50 (Cx50) cDNA, encoding a 440 aa protein, was successfully isolated from rabbit retina RNA. Cx50 was also obtained from isolated A-type horizontal cells (A-type HCs) by single-cell RT-PCR. A-type HCs were visualized by intracellular dye injection or with an antibody against calbindin. Confocal analysis revealed all Cx50 labeling occurred on the A-type HC matrix, typically at dendritic intersections. The Cx50 plaques varied in size, from punctate signals in which fine dendrites cofasciculated, to giant plaques, >50 μm2, in which large dendrites crossed. The numerous Cx50 plaques between A-type HCs may adequately account for the remarkable coupling observed in this network. We could not detect Cx50 staining on the tips of horizontal cell dendrites within the cone pedicle invagination. This distribution does not support a role for Cx50 in hemichannel-mediated feedback. In addition, the absence of Cx50 in B-type HCs suggests the presence of a different connexin for this cell type. In summary, these results suggest that gap junctions in the A-type horizontal cell matrix are composed from Cx50. Multiple neuronal connexins are expressed in the mammalian retina and different cell types express specific connexins.

Rod and cone input to horizontal cells in the rabbit retina

In the rabbit retina, there are two types of horizontal cell (HC). The A‐type HC is axonless and extensively coupled. The B‐type HC is axon bearing; the somatic dendrites are radially symmetric and form a second coupled network, while the axon branches expansively to form a complex terminal structure. The B‐type axon terminals (ATs) are independently coupled to form a third network in the outer plexiform layer. We have modified our dye‐injection methods to obtain detailed fills of the three different horizontal cell networks for analysis via confocal microscopy. We have confirmed that A‐type HCs and the somatic dendrites of B‐type HCs receive input exclusively from cones, whereas the B‐type ATs receive input only from rods. A single B‐type AT may receive input from as many as 1,000 rods, but, surprisingly, our data reveal only one end terminal per rod spherule. The somatic dendrites of A‐ and B‐type HCs form clusters at each cone pedicle coincident with GluR2/3 and GluR4 glutamate receptor subunits. The B‐type ATs have GluR2/3‐ or GluR4‐labeled glutamate receptors in two locations: small puncta on the end terminals within the rod spherule invagination and large clusters on the terminal stalks, approximately 1.5 μm from the rod synaptic ribbon. We conclude that AMPA receptors of the same or similar composition mediate photoreceptor input to all types of HCs. J. Comp. Neurol. 500:815–831, 2007.

Connexin 57 Is Expressed by the Axon Terminal Network of B-type Horizontal Cells in the Rabbit Retina

In the rabbit retina there are two types of horizontal cell (HC). A‐type HCs (AHC) are axonless and extensively coupled via connexin (Cx)50 gap junctions. The B‐type HC (BHC) is axon‐bearing; the somatic dendrites form a second network coupled by gap junctions while the axon terminals (ATs) form a third independent network in the outer plexiform layer (OPL). The mouse retina has only one type of HC, which is morphologically similar to the B‐type HC of the rabbit. Previous work suggested that mouse HCs express Cx57 (Hombach et al. [2004] Eur J Neurosci 19:2633–2640). Therefore, we cloned rabbit Cx57 and raised an antibody to determine the distribution of Cx57 gap junctions among rabbit HCs. Dye injection methods were used to obtain detailed fills for all three HC networks for analysis by confocal microscopy. We found that Cx57 was associated with the B‐type AT plexus. Cx57 plaques were anticorrelated with the B‐type somatic dendrites and the A‐type HC network. Furthermore, there was no colocalization between Cx50 and Cx57. We conclude that in the rabbit retina, Cx57 is only found on BHC‐AT processes. Thus, in species where there are two types of HC, different connexins are expressed. The absence of Cx57 labeling in the somatic dendrites of B‐type HCs suggests the possibility of an additional unidentified HC connexin in the rabbit. J. Comp. Neurol. 520:2256–2274, 2012.

Variety of horizontal cell gap junctions in the rabbit retina

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.