Connexin57 is expressed in dendro‐dendritic and axo‐axonal gap junctions of mouse horizontal cells and its distribution is modulated by light

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat‐mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57‐deficient mice. Significantly more connexin57‐positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57‐immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions. J. Comp. Neurol. 513:363–374, 2009. © 2009 Wiley‐Liss, Inc.     

Selective Cre-mediated gene deletion identifies connexin 43 as the main connexin channel supporting olfactory ensheathing cell networks

Many functions of glial cells depend on the formation of selective glial networks mediated by gap junctions formed by members of the connexin family. Olfactory ensheathing cells (OECs) are specialized glia associated with olfactory sensory neuron axons. Like other glia, they form selective networks, however, the connexins that support OEC connectivity in vivo have not been identified. We used an in vivo mouse model to selectively delete candidate connexin genes with temporal control from OECs and address the physiological consequences. Using this model, we effectively abolished the expression of connexin 43 (Cx43) in OECs in both juvenile and adult mice. Cx43-deleted OECs exhibited features consistent with the loss of gap junctions including reduced membrane conductance, largely reduced sensitivity to the gap junction blocker meclofenamic acid and loss of dye coupling. This indicates that Cx43, a typically astrocytic connexin, is the main connexin forming functional channels in OECs. Despite these changes in functional properties, the deletion of Cx43 deletion did not alter the density of OECs. The strategy used here may prove useful to delete other candidate genes to better understand the functional roles of OECs in vivo.     

Horizontal cell receptive fields are reduced in connexin57-deficient mice

Horizontal cells are coupled by gap junctions; the extensive coupling of the horizontal cells is reflected in their large receptive fields, which extend far beyond the dendritic arbor of the individual cell. In the mouse retina, horizontal cells express connexin57 (Cx57). Tracer coupling of horizontal cells is impaired in Cx57-deficient mice, which suggests that the receptive fields of Cx57-deficient horizontal cells might be similarly reduced. To test this hypothesis we measured the receptive fields of horizontal cells from wildtype and Cx57-deficient mice. First, we examined the synaptic connections between horizontal cells and photoreceptors: no major morphological alterations were found. Moreover, horizontal cell spacing and dendritic field size were unaffected by Cx57 deletion. We used intracellular recordings to characterize horizontal cell receptive fields. Length constants were computed for each cell using the cell's responses to concentric light spots of increasing diameter. The length constant was dependent on the intensity of the stimulus: increasing stimulus intensity reduced the length constant. Deletion of Cx57 significantly reduced horizontal cell receptive field size. Dark resting potentials were strongly depolarized and response amplitudes reduced in Cx57-deficient horizontal cells compared to the wildtype, suggesting an altered input resistance. This was confirmed by patch-clamp recordings from dissociated horizontal cells; mean input resistance of Cx57-deficient horizontal cells was 27% lower than that of wildtype cells. These data thus provide the first quantification of mouse horizontal cell receptive field size and confirm the unique role of Cx57 in horizontal cell coupling and physiology.     

Subcellular distribution of connexin45 in OFF bipolar cells of the mouse retina

In the mouse retina, connexin45 (Cx45) participates in the gap junction between ON cone bipolar cells and AII amacrine cells, which constitutes an essential element of the primary rod pathway. Although it has been shown that Cx45 is also expressed in OFF bipolar cells, its subcellular localization and functional role in these cells are unknown. Here, we analyzed the localization of Cx45 on OFF bipolar cells in the mouse retina. For this, we used wild-type mice and a transgenic mouse line that expressed, in addition to native Cx45, a fusion protein consisting of Cx45 and the enhanced green fluorescent protein (EGFP). Cx45-EGFP expression generates an EGFP signal at gap junctions containing Cx45. Combining immunohistochemistry with intracellular injections, we found that Cx45 was present on dendrites and axon terminals of all OFF bipolar cell types. Cx45 was not found at intersections of two terminal processes of the same type, suggesting that Cx45 might not form gap junctions between axon terminals of the same OFF bipolar cell type but rather might connect OFF bipolar cells to amacrine or ganglion cells. In OFF bipolar cell dendrites, Cx45 was found predominantly in the proximal outer plexiform layer (OPL), well below the cone pedicles. Cx45 did not colocalize with Cx36, which is found predominantly in the distal OPL. We conclude that Cx45 is expressed on OFF bipolar cell dendrites, presumably forming gap junctions with cells of the same type, and on OFF bipolar cell axon terminals, presumably forming heterologous gap junctions with other retinal neurons.     

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.     

Endogenous dopaminergic regulation of horizontal cell coupling in the mammalian retina

Horizontal cells in an isolated wholemount preparation of the mouse retina were injected with Lucifer yellow and neurobiotin to characterize both the pattern of gap junctional connectivity and its regulation by dopamine. The injected horizontal cells had a uniform morphology of a round cell body, a compact dendritic tree, and an axon, which could sometimes be traced to an expansive terminal system. The dendro-dendritic gap junctions between neighboring cells mediated both weak Lucifer yellow dye coupling and strong neurobiotin tracer coupling. The extent of the tracer coupling was decreased by either exogenous dopamine (100 microM) or cyclic adenosine monophosphate (cAMP) analogs and was significantly increased by the D1 antagonist SCH 23390 (10 microM). These results provide the first evidence in the mammalian retina that the gap junctions between horizontal cells are endogenously regulated by dopamine, which acts through D1 receptors to increase the intracellular cAMP. It has been proposed that the gap junctional coupling between horizontal cells is mediated by connexin 32 (Cx32), but the pattern and dopaminergic regulation of horizontal cell coupling were unaffected in Cx32-knockout mice, ruling out the possible involvement of Cx32. Every tracer-coupled horizontal cell showed calbindin immunoreactivity, and vice versa, providing strong evidence that the horizontal cells in the mouse retina comprise a single cell type. Like the axonless horizontal cells in other mammalian retinas, the axon-bearing horizontal cells in the mouse retina are coupled by gap junctions that are permeable to Lucifer yellow and dopamine sensitive, suggesting that the mouse horizontal cells have hybrid properties to compensate for the absence of axonless horizontal cells.     

Membrane‐associated guanylate kinase scaffolds organize a horizontal cell synaptic complex restricted to invaginating contacts with photoreceptors

Synaptic processes and plasticity of synapses are mediated by large suites of proteins. In most cases, many of these proteins are tethered together by synaptic scaffold proteins. Scaffold proteins have a large number and typically a variety of protein interaction domains that allow many different proteins to be assembled into functional complexes. Because each scaffold protein has a different set of protein interaction domains and a unique set of interacting partners, the presence of synaptic scaffolds can provide insight into the molecular mechanisms that regulate synaptic processes. In studies of rabbit retina, we found SAP102 and Chapsyn110 selectively localized in the tips of B‐type horizontal cell processes, where they contact cone and rod photoreceptors. We further identified some known SAP102 binding partners, kainate receptor GluR6/7 and inward rectifier potassium channel Kir2.1, closely associated with SAP102 in photoreceptor invaginations. The kainate receptor occupies a position distinct from that of the majority of AMPA receptors that dominate the horizontal cell postsynaptic response. GluR6/7 and Kir2.1 presumably are involved in synaptic processes that govern cell‐to‐cell communication and could both contribute in different ways to synaptic currents that mediate feedback signaling. Notably, we failed to find evidence for the presence of Cx57 or Cx59 that might be involved in ephaptic feedback signaling in this complex. The presence of SAP102 and its binding partners in both cone and rod invaginating synapses suggests that whatever mechanism is supported by this protein complex is present in both types of photoreceptors. J. Comp. Neurol. 525:850–867, 2017. © 2016 Wiley Periodicals, Inc.     

Domain-specific distribution of gap junctions defines cellular coupling to establish a vascular relay in the retina

In the retina, diverse functions of neuronal gap junctions (GJs) have been established. However, the distribution and function of vascular GJs are less clear. Here in the mouse retina whole mounts, we combined structural immunohistochemical analysis and a functional assessment of cellular coupling with a GJ-permeable tracer Neurobiotin to determine distribution patterns of three major vascular connexins. We found that Cx43 was expressed in punctate fashion on astroglia, surrounding all types of blood vessels and in continuous string-like structures along endothelial cell contacts in specialized regions of the vascular tree. Specifically, these Cx43-positive strings originated at the finest capillaries and extended toward the feeding artery. As this structural arrangement promoted strong and exclusive coupling of pericytes and endothelial cells along the corresponding branch, we termed this region a “vascular relay.” Cx40 expression was found predominantly along the endothelial cell contacts of the primary arteries and did not overlap with Cx43-positive strings. At their occupied territories, Cx43 and Cx40 clustered with tight junctions and, to a lesser extent, with adhesion contacts, both key elements of the blood–retina barrier. Finally, Cx37 puncta were associated with the entire surface of both mural and endothelial cells across all regions of the vascular tree. This combinatorial analysis of vascular connexins and identification of the vascular relay region will serve as a structural foundation for future studies of neurovascular signaling in health and disease.     

Functional expression of connexin57 in horizontal cells of the mouse retina

Horizontal cells are interneurons of the vertebrate retina that exhibit strong electrical and tracer coupling but the identity of the channel-forming connexins has remained elusive. Here we show that horizontal cells of the mouse retina express connexin57 (Cx57). We have generated Cx57-deficient mice by replacing the Cx57 coding region with a lacZ reporter gene, expressed under control of the endogenous Cx57 promoter. These mice were fertile and showed no obvious anatomical or behavioural abnormalities. Cx57 mRNA was expressed in the retina of wild-type littermates but was absent from the retina of Cx57-deficient mice. Previously reported results that the Cx57 gene was very weakly expressed in several other mouse tissues turned out to be unspecific. Cx57 mRNA is abundantly expressed in the retina and weakly in the thymus of adult mice but absent in all other adult tissues tested, including brain. Furthermore, Cx57 is expressed in embryonic kidney at E16.5 to E18.5 days post-conception, as indicated by the pattern of lacZ expression. Within the retina, lacZ signals were assigned exclusively to horizontal cells based on co-localization with cell-type-specific marker proteins. Microinjection of Neurobiotin into horizontal cells of isolated retinae revealed less than 1% of tracer coupling in Cx57-deficient retinae compared with wild-type controls. Cx57 is the first connexin identified in mammalian horizontal cells and the first connexin whose expression is apparently restricted to only one type of neuron.     

Retinal pigment epithelial integrity is compromised in the developing albino mouse retina

In the developing murine eye, melanin synthesis in the retinal pigment epithelium (RPE) coincides with neurogenesis of retinal ganglion cells (RGCs). Disruption of pigmentation in the albino RPE is associated with delayed neurogenesis in the ventrotemporal retina, the source of ipsilateral RGCs, and a reduced ipsilateral RGC projection. To begin to unravel how melanogenesis and the RPE regulate RGC neurogenesis and cell subpopulation specification, we compared the features of albino and pigmented mouse RPE cells during the period of RGC neurogenesis (embryonic day, E, 12.5 to 18.5) when the RPE is closely apposed to developing RGC precursors. At E12.5 and E15.5, although albino and pigmented RPE cells express RPE markers Otx2 and Mitf similarly, albino RPE cells are irregularly shaped and have fewer melanosomes compared with pigmented RPE cells. The adherens junction protein P‐cadherin appears loosely distributed within the albino RPE cells rather than tightly localized on the cell membrane, as in pigmented RPE. Connexin 43 (gap junction protein) is expressed in pigmented and albino RPE cells at E13.5 but at E15.5 albino RPE cells have fewer small connexin 43 puncta, and a larger fraction of phosphorylated connexin 43 at serine 368. These results suggest that the lack of pigment in the RPE results in impaired RPE cell integrity and communication via gap junctions between RPE and neural retina during RGC neurogenesis. Our findings should pave the way for further investigation of the role of RPE in regulating RGC development toward achieving proper RGC axon decussation. J. Comp. Neurol. 524:3696–3716, 2016. © 2016 Wiley Periodicals, Inc.     

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.     

Melanopsin ganglion cell outer retinal dendrites: Morphologically distinct and asymmetrically distributed in the mouse retina

A small population of retinal ganglion cells expresses the photopigment melanopsin and function as autonomous photoreceptors. They encode global luminance levels critical for light‐mediated non‐image forming visual processes including circadian rhythms and the pupillary light reflex. There are five melanopsin ganglion cell subtypes (M1–M5). M1 and displaced M1 (M1d) cells have dendrites that ramify within the outermost layer of the inner plexiform layer. It was recently discovered that some melanopsin ganglion cells extend dendrites into the outer retina. Outer Retinal Dendrites (ORDs) either ramify within the outer plexiform layer (OPL) or the inner nuclear layer, and while present in the mature retina, are most abundant postnatally. Anatomical evidence for synaptic transmission between cone photoreceptor terminals and ORDs suggests a novel photoreceptor to ganglion cell connection in the mammalian retina. While it is known that the number of ORDs in the retina is developmentally regulated, little is known about the morphology, the cells from which they originate, or their spatial distribution throughout the retina. We analyzed the morphology of melanopsin‐immunopositive ORDs in the OPL at different developmental time points in the mouse retina and identified five types of ORDs originating from either M1 or M1d cells. However, a pattern emerges within these: ORDs from M1d cells are generally longer and more highly branched than ORDs from conventional M1 cells. Additionally, we found ORDs asymmetrically distributed to the dorsal retina. This morphological analysis provides the first step in identifying a potential role for biplexiform melanopsin ganglion cell ORDs.     

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.     

Connexin36 is required for gap junctional coupling of most ganglion cell subtypes in the mouse retina

Converging evidence indicates that electrical synaptic transmission via gap junctions plays a crucial role in signal processing in the retina. In particular, amacrine and ganglion cells express numerous gap junctions, resulting in extensive electrical networks in the proximal retina. Both connexin36 (Cx36) and connexin45 (Cx45) subunits are widely distributed in the inner plexiform layer (IPL) and therefore are likely contribute to gap junctions formed by a number of ganglion cell subtypes. In the present study, we used the gap junction‐permeant tracer Neurobiotin to compare the coupling pattern of different ganglion cell subtypes in wild‐type (WT) and Cx36 knockout (KO) mouse retinas. We found that homologous ganglion‐to‐ganglion cell coupling was lost for two subtypes after deletion of Cx36, whereas two other ganglion cell subtypes retained homologous coupling in the KO mouse. In contrast, deletion of Cx36 resulted in a partial or complete loss of ganglion‐to‐amacrine cell heterologous coupling in 9 of 10 ganglion cell populations studied. Overall, our results indicate that Cx36 is the predominant subunit of gap junctions in the proximal mouse retina, expressed by most ganglion cell subtypes, and thereby likely plays a major role in the concerted activity generated by electrical synapses. J. Comp. Neurol. 518:911–927, 2010. © 2009 Wiley‐Liss, Inc.     

Expression and distribution of trophoblast glycoprotein in the mouse retina

We recently identified the leucine-rich repeat (LRR) adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKCα-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal LRR domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled RBC dendrites in the outer plexiform layer and axon terminals in the IPL, as well as a putative amacrine cell with their cell bodies in the inner nuclear layer (INL) and a dense layer in the middle of the inner plexiform layer (IPL). In live transfected HEK293 cells, TPBG was localized to the plasma membrane with the N-terminal LRR domain facing the extracellular space. TPBG immunofluorescence in RBCs was strongly altered by the loss of TRPM1 in the adult retina, with significantly less dendritic and axon terminal labeling in TRPM1 knockout compared to wild type, despite no change in total TPBG detected by immunoblotting. During retinal development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, LRR proteins have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.     

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

The primary rod pathway in mammals contains gap junctions between AII amacrine cells and ON cone bipolar cells which relay the rod signal into the cone pathway under scotopic conditions. Two gap junctional proteins, connexin36 (Cx36) and connexin45 (Cx45), appear to play a pivotal role in this pathway because lack of either protein leads to an impairment of visual transmission under scotopic conditions. To investigate whether these connexins form heterotypic gap junctions between ON cone bipolar and AII amacrine cells, we used newly developed Cx45 antibodies and studied the cellular and subcellular distribution of this protein in the mouse retina. Specificity of the Cx45 antibodies was determined, among others, by Western blot and immunostaining of mouse heart, where Cx45 is abundantly expressed. In mouse retina, Cx45 immunosignals were detected in both plexiform layers and the ganglion cell layer. Double staining for Cx45 and Cx36 revealed a partial overlap in the punctate patterns in the ON sublamina of the inner plexiform layer of the retina. We quantified the distributions of these two connexins in the ON sublamina, and detected 30% of the Cx45 signals to be co-localized with or in close apposition to Cx36 signals. Combining immunostaining and intracellular dye injection revealed an overlap or tight association of Cx36 and Cx45 signals on the terminals of injected AII amacrine and two types of ON cone bipolar cells. Our results provide direct evidence for heterotypic gap junctions composed of Cx36 and Cx45 between AII amacrine and certain types of ON cone bipolar cells.     

Connexin expression in the retina

Here, we review recent results from our laboratory on connexin expression in mouse retina in the context of previous results with other vertebrate species. In mouse retina, four different connexin proteins were detected by immunoblot and immunofluorescence: connexin (Cx)-36, -37, -43 and -45. Cx36 and Cx45 immunoreactive signals were found in the inner and outer plexiform layer, both of which are known to show interneuronal gap junctions. Cx43 was detected in the ganglion cell layer, presumably in astrocytes, where it appeared to be colocalized with glial fibrillary acid protein. Cx37 was expressed in retinal endothelial cells. Additionally, Cx26, -31, -32 and -40 mRNAs were detected in retina by RT–PCR but none of the corresponding proteins were found. In order to exclude cross reactions of the corresponding antibodies, retinae from targeted connexin-deficient mice (Cx31 −/−, Cx32 −/− and Cx40 −/−) were used as negative controls for immunoblot and immunofluorescence analyses of wild-type retina. Further detailed investigation of cell type specific connexin expression in the mouse retina will be necessary for functional analyses of targeted mouse mutants with defects in connexins expressed in retinal neuronal cells.     

Single-cell RNA sequencing study of retinal immune regulators identified CD47 and CD59a expression in photoreceptors—Implications in subretinal immune regulation

The neuroretina is protected by its own defense system, that is microglia and the complement system. Under normal physiological conditions, microglial activation is tightly regulated by the neurons although the underlying mechanism remains elusive. Using published single-cell RNA sequencing data sets, we found that immune regulatory molecules including CD200, CD47, CX3CL1, TGFβ, and complement inhibitor CD59a are expressed by various retinal neurons. Importantly, we found that photoreceptors express higher levels of CD47 and CD59a, which was further confirmed in cultured 661W cells, WERI-Rb1 cells, and microdissected photoreceptors from human eyes. The expression of CD59a mRNA in 661W cells was upregulated by TNFα and hypoxia, whereas LPS, hypoxia, and IL-4 upregulated CD47 mRNA expression in 661W cells. Immunofluorescence staining detected strong CD59a immunoreactivity in the outer nuclear layer, inner/outer segments, and discrete staining in ganglion cell layer (GCL), inner plexiform layer (IPL), and outer plexiform layer. The expression of CD59a in photoreceptors was increased in the detached retina, but decreased in retinas from experimental autoimmune uveoretinitis (EAU) mice. In EAU retina, CD59a was highly expressed by active immune cells. CD47 was detected in GCL, IPL, and inner nuclear layer and some photoreceptors. The expression of CD47 in photoreceptors was also increased in the detached retina but decreased in EAU retina. In a coculture system, 661W enhanced arginase-1 and reduced IL-6 mRNA expression in BV2 microglial cells. Our results suggest that photoreceptors express immune regulatory molecules and may have the potential to regulate immune activation in the outer retina/subretinal space under pathophysiological conditions.