Impediments to eye transplantation: ocular viability following optic-nerve transection or enucleation.

Maintenance of ocular viability is one of the major impediments to successful whole-eye transplantation. This review provides a comprehensive understanding of the current literature to help guide future studies in order to overcome this hurdle. A systematic multistage review of published literature was performed. Three specific questions were addressed: (1) Is recovery of visual function following eye transplantation greater in cold-blooded vertebrates when compared with mammals? (2) Is outer retina function following enucleation and reperfusion improved compared with enucleation alone? (3) Following optic-nerve transection, is there a correlation between retinal ganglion cell (RGC) survival and either time after transection or proximity of the transection to the globe? In a majority of the studies performed in the literature, recovery of visual function can occur after whole-eye transplantation in cold-blooded vertebrates. Following enucleation (and reperfusion), outer retinal function is maintained from 4 to 9 h. RGC survival following optic-nerve transection is inversely related to both the time since transection and the proximity of transection to the globe. Lastly, neurotrophins can increase RGC survival following optic-nerve transection. This review of the literature suggests that the use of a donor eye is feasible for whole-eye transplantation.

High pH accelerates GABA deactivation on perch-rho1B receptors.

The ionotropic GABA(C) receptor, formed by GABA rho subunits, is known to be modulated by a variety of endogenous compounds, as well as by changes in pH. In this study, we explore the proton sensitivity of the GABA rho subunits cloned from the perch retina, and report a novel action of high pH on the homomeric receptor formed by one of the GABA rho subunits, the perch-rho(1B) subunit. Raising extracellular pH to 9.5 significantly accelerated GABA deactivation responses elicited from oocytes expressing the perch-rho(1B) subunit, and reduced its sensitivity to GABA. The change in the kinetics of the GABA-offset response occurred without altering the maximum response amplitude, and the reduced GABA sensitivity was independent of membrane potential. Although acidification of the extracellular solution also accelerated GABA deactivation for all other GABA rho receptors examined in this study, the effects of high pH were unique to the homomeric receptor formed by the perch-rho(1B) subunit. In addition, we found that, unlike the effects on the response to the naturally occurring full agonist GABA, the responses elicited by partial agonists (imidazole-4-acetic acid (I4AA) and beta-alanine) in the presence of the high pH solution showed a significant reduction in the maximum response amplitude. When considered in terms of a model describing the activation of GABA(C) receptors, in which pH can potentially affect either the binding affinity or the rate of channel closure, the results were consistent with the view that external alkalization reduces the gating efficiency of the receptor. To identify the proton sensitive domain(s) of the perch-rho(1B) receptor, chimeras were constructed by domain swapping with other perch-rho subunits. Analysis of the pH sensitivities of the various chimeric receptors revealed that the alkaline-sensitive residues are located in the N-terminal region of the perch-rho(1B) subunit.

Gap junctions remain open during cytochrome c-induced cell death: relationship of conductance to 'bystander' cell killing.

Previous reports have shown that gap junctions relay cell death in many cell types. However, changes in electrical coupling and their dynamics during cell death are poorly understood. We performed comprehensive studies of electrical coupling following induction of cell death by single-cell cytochrome c (cyC) injection in paired Xenopus oocytes. Cell death was rapidly induced by cyC in injected cells, and cell death was also observed in uninjected bystander cells electrically coupled to the cyC-injected oocytes. Gap junction currents either remained at pre-cyC injection levels or increased dramatically as the injected cell died. Nonjunctional currents increased in injected cells immediately following cyC injection; nonjunctional currents increased slowly in uninjected bystander cells. Bystander cell death occurred only when junctional conductance was approximately 6 muS. Both 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxy-methyl ester and Xestospongin C inhibited bystander cell death in pairs that had reached the death conductance threshold, suggesting that Ca(2+) and inositol 1,4,5 triphosphate are involved in the process.

The GABA rho1 subunit interacts with a cellular retinoic acid binding protein in mammalian retina.

Interactions between the intracellular domain of ligand-gated membrane receptors and cytoplasmic proteins play important roles in their assembly, clustering, and function. In addition, protein-protein interactions may provide an alternative mechanism by which neurotransmitters activate intracellular pathways. In this study, we report a novel interaction between the GABA rho1 subunit and cellular retinoic acid binding protein in mammalian retina that could serve as a link between the GABA signaling pathway and the control of gene expression in neurons. The interaction between the intracellular loop of the human GABA rho subunit and cellular retinoic acid binding protein was identified using a CytoTrap XR yeast two-hybrid system, and was further confirmed by co-precipitation of the human GABA rho subunit and cellular retinoic acid binding protein from baboon retinal samples. The cellular retinoic acid binding protein binding domain on the human rho1 subunit was located to the C-terminal region of human GABA rho subunit, and the interaction of the human GABA rho subunit with cellular retinoic acid binding protein could be antagonized by a peptide derived from within the binding domain of the rho1 subunit. Since cellular retinoic acid binding protein is a carrier protein for retinoic acid, we investigated the effect of GABA on retinoic acid activity in neuroblastoma cells containing endogenously expressed cellular retinoic acid binding protein. In the absence of the rho1 receptor, these cells showed enhanced neurite outgrowth when exposed to retinoic acid and GABA had no effect on their response to retinoic acid. In contrast, cells stably transfected with the human rho1 subunit showed a significantly reduced sensitivity to retinoic acid when exposed to GABA. These results suggest that the GABA receptor subunit effectively altered gene expression through its interaction with the cellular retinoic acid binding protein pathway.

The spread of apoptosis through gap-junctional channels in BHK cells transfected with Cx32.

Programmed cell death (apoptosis) occurs both during normal development and as a result of various pathological conditions. An in vitro system was used to explore the transmission of death signals from apoptotic cells to cells with which they were coupled via gap junctions. Confluent cultures of baby hamster kidney (BHK) cells, stably transfected with the gap-junctional protein connexin32, were scrape loaded with cytochrome C (cyC), a mitochondria-derived apoptotic agent, to introduce the protein into cells injured by the cut. The cultures were subsequently analyzed for the presence of activated caspases, the distribution of TUNEL staining, and the binding of annexin V. Although cyC is too large to traverse the gap junctional channel, each of the assays revealed that apoptosis had spread from dying cells at the margin of the scrape to otherwise healthy neighboring cells to which they were coupled. This "bystander effect" was significantly reduced in the presence of agents that block gap junctional intercellular communication.

Cobalt ions inhibit negative feedback in the outer retina by blocking hemichannels on horizontal cells.

In goldfish, negative feedback from horizontal cells to cones shifts the activation function of the Ca2+ current of the cones to more negative potentials. This shift increases the amount of Ca2+ flowing into the cones, resulting in an increase in glutamate release. The increased glutamate release forms the basis of the feedback-mediated responses in second-order neurons, such as the surround-induced responses of bipolar cells and the spectral coding of horizontal cells. Low concentrations of Co2+ block these feedback-mediated responses in turtle retina. The mechanism by which this is accomplished is unknown. We studied the effects of Co2+ on the cone/horizontal network of goldfish retina and found that Co2+ greatly reduced the feedback-mediated responses in both cones and horizontal cells in a GABA-independent way. The reduction of the feedback-mediated responses is accompanied by a small shift of the Ca2+ current of the cones to positive potentials. We have previously shown that hemichannels on the tips of the horizontal cell dendrites are involved in the modulation of the Ca2+ current in cones. Both the absence of this Co2+-induced shift of the Ca2+ current in the absence of a hemichannel conductance and the sensitivity of Cx26 hemichannels to low concentrations of Co2+ are consistent with a role for hemichannels in negative feedback from horizontal cells to cones.

Responses of small- and large-field bipolar cells to GABA and glycine.

Morphologically distinct subtypes of retinal bipolar cells transmit information along parallel pathways to convey different aspects of the visual scene, but the synaptic mechanisms that regulate signal transmission are largely unknown. The all-rod retina of skate provides a comparatively simple system in which to correlate bipolar cell morphology with responses to the inhibitory neurotransmitters GABA and glycine. Two subtypes of bipolar cells can be identified when isolated in culture: large-field bipolar cells with extensive dendritic arbors, and small-field bipolar cells with one or two dendritic branches. Under voltage-clamp, glycine elicited significant current responses from small-field cells, but not from large-field bipolar cells. Although all bipolar cells displayed GABA-activated chloride currents mediated by both GABA(A) and GABA(C) receptors, the small-field bipolar cells showed a significantly greater contribution from GABA(A) receptors. The results of the present study reveal for the first time that the relative expression of the two classes of GABA receptor on each bipolar cell type correlates with cell morphology and the presence of the glycine receptor.

Identification and functional expression of HCx31.9, a novel gap junction gene.

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.

The rhodopsin cycle is preserved in IRBP "knockout" mice despite abnormalities in retinal structure and function.

In the vertebrate retina, vision is initiated and maintained by the photolysis and regeneration, respectively, of light-sensitive pigments in the disk membranes of the photoreceptor outer segments. This cyclical process depends on an exchange of retinoids between the photoreceptors and the retinal pigment epithelium (RPE). There is a great deal of indirect evidence that the transport of retinoids between these cellular compartments is mediated by the interphotoreceptor retinoid-binding protein (IRBP), a large glycoprotein synthesized in the photoreceptors and extruded into the interphotoreceptor matrix (IPM) that fills the subretinal space. Nevertheless, a number of in vitro experiments have demonstrated that an intermembranous transfer of retinoids can occur through an aqueous medium independent of any retinoid-binding protein. This led to the suggestion that IRBP may play the more passive role of an extracellular buffer, serving to prevent the degradation and potentially cytotoxic effects of free retinoids when large amounts are released into the IPM. We have studied the structural and functional properties of transgenic mice in which homologous recombination was used to delete the IRBP gene. Light- and electron-microscopic examination of the retinas of "knockout" (IRBP-/-) mice revealed a significant loss of photoreceptor nuclei, and profound changes in the structure and organization of the receptor outer segments. Consistent with these observations, electroretinographic recordings showed a marked reduction in response amplitude for both rod- and cone-mediated potentials. However, despite the histological and electrophysiological changes, there was no evidence of gross abnormalities in the visual cycle. After bleaching a significant fraction of the available rhodopsin, electroretinogram amplitude and rhodopsin density gradually increased toward their pre-bleach levels, and the rates of recovery were even more rapid than those seen in wild-type (IRBP+/+) mice.

Functional properties, developmental regulation, and chromosomal localization of murine connexin36, a gap-junctional protein expressed preferentially in retina and brain.

Retinal neurons of virtually every type are coupled by gap-junctional channels whose pharmacological and gating properties have been studied extensively. We have begun to identify the molecular composition and functional properties of the connexins that form these 'electrical synapses,' and have cloned several that constitute a new subclass (gamma) of the connexin family expressed predominantly in retina and brain. In this paper, we present a series of experiments characterizing connexin36 (Cx36), a member of the gamma subclass that was cloned from a mouse retinal cDNA library. Cx36 has been localized to mouse chromosome 2, in a region syntenic to human chromosome 5, and immunocytochemistry showed strong labeling in the ganglion cell and inner nuclear layers of the mouse retina. Comparison of the developmental time course of Cx36 expression in mouse retina with the genesis of the various classes of retinal cells suggests that the expression of Cx36 occurs primarily after cellular differentiation is complete. Because photic stimulation can affect the gap-junctional coupling between retinal neurons, we determined whether lighting conditions might influence the steady state levels of Cx36 transcript in the mouse retina. Steady-state levels of Cx36 transcript were significantly higher in animals reared under typical cyclic-light conditions; exposure either to constant darkness or to continuous illumination reduced the steady-state level of mRNA approximately 40%. Injection of Cx36 cRNA into pairs of Xenopus oocytes induced intercellular conductances that were relatively insensitive to transjunctional voltage, a property shared with other members of the gamma subclass of connexins. Like skate Cx35, mouse Cx36 was unable to form heterotypic gap-junctional channels when paired with two other rodent connexins. In addition, mouse Cx36 failed to form voltage-activated hemichannels, whereas both skate and perch Cx35 displayed quinine-sensitive hemichannel activity. The conservation of intercellular channel gating contrasts with the failure of Cx36 to make hemichannels, suggesting that the voltage-gating mechanisms of hemichannels may be distinct from those of intact intercellular channels.

A single amino acid in the second transmembrane domain of GABA rho subunits is a determinant of the response kinetics of GABAC receptors.

The rho subunits that constitute the gamma-aminobutyric acid (GABA)C receptors of retinal neurons form a unique subclass of ligand-gated chloride channels that give rise to sustained GABA-evoked currents that exhibit slow offset (deactivation) kinetics. We exploited this property to examine the molecular mechanisms that govern the disparate response kinetics and pharmacology of perch GABA rho1B and rho2A subunits expressed in Xenopus oocytes. Using a combination of domain swapping and site-directed mutagenesis, we identified the residues at amino acid position 320 in the second transmembrane domain as an important determinant of the receptor kinetics of GABAC receptors. When the site contains a proline residue, as in wild-type rho1 subunits, the receptor deactivates slowly; when serine occupies the site, as in wild-type rho2 subunits, the time course of deactivation is more rapid. In addition, we found that the same site also altered the pharmacology of GABA rho receptors, e.g., when the serine residue of the rho2A receptor was changed to proline, the response of the mutant receptor to imidazole-4-acetic acid (I4AA) mimicked that of the rho1B receptor. However, despite gross changes in receptor pharmacology, the apparent binding affinity for the drug was not significantly altered. These findings provide further evidence that the second transmembrane domain is involved in the gating mechanism that governs the response properties of the various rho receptor subunits. It is noteworthy that the proline residue in native rho1 subunits and the serine residue of rho2 subunits are well conserved in all species, a good indication that the presence of multiple GABA rho subunits serves to generate GABAC receptors that display the wide range of response kinetics observed on various types of retinal neurons.

Functional characteristics of skate connexin35, a member of the gamma subfamily of connexins expressed in the vertebrate retina.

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the gamma subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials >/= 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the alpha or beta subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 microm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1. 6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.

Response kinetics and pharmacological properties of heteromeric receptors formed by coassembly of GABA rho- and gamma 2-subunits.

Two of the gamma-aminobutyric acid (GABA) receptors, GABAA and GABAC, are ligand-gated chloride channels expressed by neurons in the retina and throughout the central nervous system. The different subunit composition of these two classes of GABA receptor result in very different physiological and pharmacological properties. Although little is known at the molecular level as to the subunit composition of any native GABA receptor, it is thought that GABAC receptors are homomeric assemblies of rho-subunits. However, we found that the kinetic and pharmacological properties of homomeric receptors formed by each of the rho-subunits cloned from perch retina did not resemble those of the GABAC receptors on perch bipolar cells. Because both GABAA and GABAC receptors are present on retinal bipolar cells, we attempted to determine whether subunits of these two receptor classes are capable of interacting with each other. We report here that, when coexpressed in Xenopus oocytes, heteromeric (rho 1B gamma 2) receptors formed by coassembly of the rho 1B-subunit with the gamma 2-subunit of the GABAA receptor displayed response properties very similar to those obtained with current recordings from bipolar cells. In addition to being unresponsive to bicuculline and diazepam, the time-constant of deactivation, and the sensitivities to GABA, picrotoxin and zinc closely approximated the values obtained from the native GABAC receptors on bipolar cells. These results provide the first direct evidence of interaction between GABA rho and GABAA receptor subunits. It seems highly likely that coassembly of GABAA and rho-subunits contributes to the molecular organization of GABAC receptors in the retina and perhaps throughout the nervous system.

Molecular and pharmacological properties of GABA-rho subunits from white perch retina.

Five gamma-aminobutyric acid (GABA)-rho subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-rho subunits cloned from mammalian retinas; two clones (perch-rho 1A and perch-rho 1B) were in the rho 1 family, two (perch-rho 2A and perch-rho 2B) were in the rho 2 family, and one clone has been tentatively identified as a perch-rho 3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (rho 3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-rho subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-rho 1B subunits were more sensitive to GABA than the receptors formed by other GABA-rho subunits, the dose-response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current-voltage curve for rho 2 receptors was approximately linear, whereas the responses from rho 1 receptors showed outward rectification. A further division in the properties of the GABA-rho subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type rho receptors and a partial agonist on the B-type rho receptors. These results suggest that there is a large diversity of GABAC receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA rho subunits, that exhibit different functional properties.