Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin-2 (Syt2). Syt2 was localized in a population of small-field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF-layer and only a few lobular processes extending into the ON-layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2-labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ-aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from ∼200/mm(2) in peripheral retina to ∼1,400/mm(2) in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2-labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small-field amacrine cells closely resembling A8 amacrine cells and their cone-dominated bipolar cell input. J. Comp. Neurol. 521:709-724, 2013. © 2012 Wiley Periodicals, Inc.

Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABA(A) receptor subunit α2 (GABA(A)R α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(A)R α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(A)R α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(A)Rs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.

Protocadherins (Pcdhs) are thought to be key features of cell-type-specific synapse formation. Here we analyzed the expression pattern of Pcdh subunit β16 (β16) in the primate retina by applying antibodies against β16, different subunits of ionotropic glutamate receptors (GluRs), and cell-type-specific markers as well as by coimmunoprecipitation and Western blots. Immunocytochemical localization was analyzed by confocal microscopy and preembedding electron microscopy. In the outer plexiform layer (OPL) H1, but not H2, horizontal cells expressed β16 as revealed by the strong reduction of β16 at short-wavelength-sensitive cones. β16 colocalized with the GluR subunits GluR2-4 at horizontal cell dendritic tips and with GluR2-4 and GluR6/7 at the desmosome-like junctions. At the latter, these AMPA and kainate receptor subunits were found to be clustered within single synaptic hot spots. Additionally, β16-labeled dendritic tips of OFF cone bipolar cells appeared in triad-associated positions at the cone pedicle base, pointing to β16 expression by OFF midget or DB3 bipolar cells. In the inner plexiform layer, β16 was localized also postsynaptically at most of the glutamatergic synapses. Overall, we provide evidence for a cell-type-specific localization of β16 together with GluRs at defined postsynaptic sites and a coexistence of AMPA and kainate receptors within single synaptic hot spots. This study supports the hypothesis that β16 plays an important role in the formation and/or stabilization of specific glutamatergic synapses, whereas our in vivo protein biochemical results argue against the existence of protein complexes formed by β16 and GluRs.

The physiological aging process of the retina is accompanied by various and sometimes extensive changes: Macular degeneration, retinopathies and glaucoma are the most common findings in the elderly and can potentially lead to irreversible visual disablements up to blindness. To study the aging process and to identify possible therapeutic targets to counteract these diseases, the use of appropriate animal models is mandatory. Besides the most commonly used rodent species, a non-human primate, the common marmoset (Callithrix jacchus) emerged as a promising animal model of human aging over the last years. However, the visual aging process in this species is only partially characterized, especially with regard to retinal aberrations. Therefore, we assessed here for the first time potential changes in retinal morphology of the common marmoset of different age groups. By cell type specific immunolabeling, we analyzed different cell types and distributions, potential photoreceptor and ganglion cell loss, and structural reorganization. We detected no signs of age-related differences in staining patterns or densities of various cell populations. For example, there were no signs of photoreceptor degeneration, and there was only minimal sprouting of rod bipolar cells in aged retinas. Altogether, we describe here the maintenance of a stable neuronal architecture, distribution and number of different cell populations with only mild aberrations during the aging process in the common marmoset retina. These findings are in stark contrast to previously reported findings in rodent species and humans and deserve further investigations to identify the underlying mechanisms and possible therapeutic targets.

Although retinal organization is remarkably conserved, morphological anomalies can be found to different extents and varieties across animal species with each presenting unique characteristics and patterns of displaced and misplaced neurons. One of the most widely used non-human primates in research, the common marmoset (Callithrix jaccus) could potentially also be of interest for visual research, but is unfortunately not well characterized in this regard. Therefore, the aim of our study was to provide a first time description of structural retinal layering including morphological differences and distinctive features in this species. Retinas from animals (n = 26) of both sexes and different ages were immunostained with cell specific antibodies to label a variety of bipolar, amacrine and ganglion cells. Misplaced ganglion cells with somata in the outermost part of the inner nuclear layer and rod bipolar cells with axon terminals projecting into the outer plexiform layer instead of the inner plexiform layer independent of age or sex of the animals were the most obvious findings, whereas misplaced amacrine cells and misplaced cone bipolar axon terminals occurred to a lesser extent. With this first time description of developmental retinal errors over a wide age range, we provide a basic characterization of the retinal system of the common marmosets, which can be taken into account for future studies in this and other animal species. The finding of misplaced ganglion cells and misplaced bipolar cell axon terminals was not reported before and displays an anatomic variation worthwhile for future analyzes of their physiological and functional impact.

Several studies have shown the importance of calcium channels in the development and/or maturation of synapses. The Ca(V)1.4(α(1F)) knockout mouse is a unique model to study the role of calcium channels in photoreceptor synapse formation. It features abnormal ribbon synapses and aberrant cone morphology. We investigated the expression and targeting of several key elements of ribbon synapses and analyzed the cone morphology in the Ca(V)1.4(α(1F)) knockout retina. Our data demonstrate that most abnormalities occur after eye opening. Indeed, scaffolding proteins such as Bassoon and RIM2 are properly targeted at first, but their expression and localization are not maintained in adulthood. This indicates that either calcium or the Ca(V)1.4 channel, or both are necessary for the maintenance of their normal expression and distribution in photoreceptors. Other proteins, such as Veli3 and PSD-95, also display abnormal expression in rods prior to eye opening. Conversely, vesicle related proteins appear normal. Our data demonstrate that the Ca(V)1.4 channel is important for maintaining scaffolding proteins in the ribbon synapse but less vital for proteins related to vesicular release. This study also confirms that in adult retinae, cones show developmental features such as sprouting and synaptogenesis. Overall we present evidence that in the absence of the Ca(V)1.4 channel, photoreceptor synapses remain immature and are unable to stabilize.

In the vertebrate retina, amacrine and ganglion cells represent the most diverse cell classes. They can be classified into different cell types by several features, such as morphology, light responses, and gene expression profile. Although birds possess high visual acuity (similar to primates that we used here for comparison) and tetrachromatic color vision, data on the expression of transcription factors in retinal ganglion cells of birds are largely missing. In this study, we tested various transcription factors, known to label subpopulations of cells in mammalian retinae, in two avian species: the common buzzard (Buteo buteo), a raptor with exceptional acuity, and the domestic pigeon (Columba livia domestica), a good navigator and widely used model for visual cognition. Staining for the transcription factors Foxp2, Satb1 and Satb2 labeled most ganglion cells in the avian ganglion cell layer. CtBP2 was established as marker for displaced amacrine cells, which allowed us to reliably distinguish ganglion cells from displaced amacrine cells and assess their densities in buzzard and pigeon. When we additionally compared the temporal and central fovea of the buzzard with the fovea of primates, we found that the cellular organization in the pits was different in primates and raptors. In summary, we demonstrate that the expression of transcription factors is a defining feature of cell types not only in the retina of mammals but also in the retina of birds. The markers, which we have established, may provide useful tools for more detailed studies on the retinal circuitry of these highly visual animals.

Detailed analysis of the synaptic inputs to the primate DB1 bipolar cell has been precluded by the absence of a suitable immunohistochemical marker. Here we demonstrate that antibodies for the EF-hand calcium-binding protein, secretagogin, strongly label the DB1 bipolar cell as well as a mixed population of GABAergic amacrine cells in the macaque retina. Using secretagogin as a marker, we show that the DB1 bipolar makes synaptic contact with both L/M as well as S-cone photoreceptors and only minimal contact with rod photoreceptors. Electron microscopy showed that the DB1 bipolar makes flat contacts at both triad-associated and nontriad-associated positions on the cone pedicle. Double labeling with various glutamate receptor subunit antibodies failed to conclusively determine the subunit composition of the glutamate receptors on DB1 bipolar cells. In the IPL, DB1 bipolar cell axon terminals expressed the glycine receptor, GlyRα1, at sites of contact with AII amacrine cells, suggesting that these cells receive input from the rod pathway.

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Whereas the retinal distributions of glycine receptor (GlyR) subunits alpha1, alpha2, and alpha3 have been mapped, the role of the alpha4 subunit in retinal circuitry remains unclear. A rabbit polyclonal antiserum was raised against a peptide that comprises the C-terminal 14 amino acids of the mouse GlyR alpha4 subunit. Using immunocytochemistry, we localized the alpha4 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by double-labeling sections for GlyR alpha4 and synaptic markers (bassoon, gephyrin). Double-labeling sections for GlyR alpha4 and the other GlyR alpha subunits shows that they are mostly clustered at different synapses; however, approximately 30% of the alpha4-containing synapses also express the alpha2 subunit. We also studied the pre- and postsynaptic partners at GlyR alpha4-containing synapses and found that displaced (ON-) cholinergic amacrine cells prominently expressed the alpha4 subunit. The density of GlyR alpha4-expressing synapses in wildtype, Glra1(ot/ot), and Glra3(-/-) mouse retinas did not differ significantly. Thus, there is no apparent compensation of the loss of alpha1 or alpha3 subunits by an upregulation of alpha4 subunit gene expression; however, the alpha2 subunit is moderately upregulated.

Retinas of macaque monkeys were immunostained for glycogen phosphorylase (glypho). Glypho was localized to regular and displaced amacrine cells. Their processes occupied two narrow strata within the inner plexiform layer (IPL). The labeling pattern is reminiscent of cholinergic amacrine cells; however, double immunostaining of the retinas for choline acetyltransferase and glypho revealed two different cell populations. Intracellular injection of DiI showed that glypho-immunoreactive amacrine cells are wide-field amacrine cells with straight, radially oriented, and sparsely branched dendrites. The density of the cells increased from approximately 70/mm(2) in the peripheral retina to approximately 700/mm(2) in the central retina. The regular glypho-immunoreactive amacrine cells branch in sublamina 2 of the IPL, where they receive input from OFF-cone bipolar cells. The displaced cells branch in sublamina 3/4 and receive input from ON-cone bipolar cells. This suggests that the regular cells are OFF-cells and the displaced cells are ON-cells. The cells express gamma-aminobutyric acid (GABA)-like immunoreactivity and receive glycinergic input through synapses expressing preferentially the glycine receptor alpha2 subunit. The close proximity of the dendritic strata of glypho-immunoreactive amacrine cells, cholinergic amacrine cells, and direction-selective ganglion cells suggests a possible role of the cells in the generation of direction-selective light responses of the monkey retina.

Amacrine cells comprise ∼ 30 morphological types in the mammalian retina. The synaptic connectivity and function of a few γ-aminobutyric acid (GABA)ergic wide-field amacrine cells have recently been studied; however, with the exception of the rod pathway-specific AII amacrine cell, the connectivity of glycinergic small-field amacrine cells has not been investigated in the mouse retina. Here, we studied the morphology and connectivity pattern of the small-field A8 amacrine cell. A8 cells in mouse retina are bistratified with lobular processes in the ON sublamina and arboreal dendrites in the OFF sublamina of the inner plexiform layer. The distinct bistratified morphology was first visible at postnatal day 8, reaching the adult shape at P13, around eye opening. The connectivity of A8 cells to bipolar cells and ganglion cells was studied by double and triple immunolabeling experiments by using various cell markers combined with synaptic markers. Our data suggest that A8 amacrine cells receive glutamatergic input from both OFF and ON cone bipolar cells. Furthermore, A8 cells are coupled to ON cone bipolar cells by gap junctions, and provide inhibitory input via glycine receptor (GlyR) subunit α1 to OFF cone bipolar cells and to ON A-type ganglion cells. Measurements of spontaneous glycinergic postsynaptic currents and GlyR immunolabeling revealed that A8 cells express GlyRs containing the α2 subunit. The results show that the bistratified A8 cell makes very similar synaptic contacts with cone bipolar cells as the rod pathway-specific AII amacrine cell. However, unlike AII cells, A8 amacrine cells provide glycinergic input to ON A-type ganglion cells.

Parallel processing of an image projected onto the retina starts at the first synapse, the cone pedicle, and each cone feeds its light signal into a minimum of eight different bipolar cell types. Hence, the morphological classification of bipolar cells is a prerequisite for analyzing retinal circuitry. Here we applied common bipolar cell markers to the cone-dominated ground squirrel retina, studied the labeling by confocal microscopy and electron microscopy, and compared the resulting bipolar cell types with those of the mouse (rod dominated) and primate retina. Eight different cone bipolar cell types (three OFF and five ON) and one rod bipolar cell were distinguished. The major criteria for classifying the cells were their immunocytochemical identity, their dendritic branching pattern, and the shape and stratification level of their axons in the inner plexiform layer (IPL). Immunostaining with antibodies against Gγ13, a marker for ON bipolar cells, made it possible to separate OFF and ON bipolars. Recoverin-positive OFF bipolar cells partly overlapped with ON bipolar axon terminals at the ON/OFF border of the IPL. Antibodies against HCN4 labeled the S-cone selective (bb) bipolar cell. The calcium-binding protein CaB5 was expressed in two OFF and two ON cone bipolar cell types, and CD15 labeled a widefield ON cone bipolar cell comparable to the DB6 in primate.

Immunocytochemical discrimination of distinct bipolar cell types in the mouse retina is a prerequisite for analyzing retinal circuitry in wild-type and transgenic mice. Here we demonstrate that among the more than 10 anatomically defined mouse bipolar cell types, type 4 bipolar cells are specifically recognized by anti-calsenilin antibodies. Axon terminals in the inner plexiform layer are not readily identifiable because calsenilin is also expressed in a subset of amacrine and ganglion cells. In contrast, in the outer plexiform layer calsenilin immunoreactivity allows the analysis of photoreceptor to type 4 bipolar cell contacts. A dense plexus of calsenilin-positive dendrites makes several basal contacts at cone pedicles. An individual calsenilin-positive bipolar cell contacts five to seven cones. In addition, some calsenilin-positive dendrites contact rod photoreceptors. On average we counted 10 rod spherule contacts per type 4 bipolar cell, and approximately 10% of rods contacted type 4 bipolar cells. We suggest that type 4 bipolar cells, together with the recently described type 3a and b cells, provide an alternative and direct route from rods to OFF cone bipolar cells. In the Bassoon DeltaEx4/5 mouse, a mouse mutant that shows extensive remodeling of the rod system including sprouting of horizontal and rod bipolar cells into the outer nuclear layer due to impaired synaptic transmission, we found that in addition mixed-input (type 3 and 4) OFF bipolar cells sprout to ectopic sites. In contrast, true cone-selective type 1 and 2 OFF cone bipolar cells did not show sprouting in the Bassoon mouse mutant.

Recent studies suggested that different types of OFF bipolar cells express specific types of ionotropic (AMPA or kainate) glutamate receptors (GluRs) at their contacts with cone pedicles. However, the question of which GluR type is expressed by which type of OFF bipolar cell in primate retina is still open. In this study, the expression of AMPA and kainate receptor subunits at the dendritic tips of flat (OFF) midget bipolar (FMB) cells was analyzed in the retina of the common marmoset, Callithrix jacchus. We used preembedding electron microscopy and double immunofluorescence with subunit-specific antibodies. The FMB cells were labeled with antibodies against the carbohydrate epitope CD15. Cone pedicles were identified with peanut agglutinin. Immunoreactivity for the GluR1 subunit and for CD15 is preferentially located at triad-associated flat contacts. Furthermore, the large majority of GluR1 immunoreactive puncta is localized at the dendritic tips of FMB cells. These results suggest that FMB cells express the AMPA receptor subunit GluR1. In contrast, the kainate receptor subunit GluR5 is not colocalized with the dendritic tips of FMB cells or with the GluR1 subunit. Immunoreactive puncta for the GluR1 subunit are found at all M/L-cone pedicles but are only rarely associated with S-cone pedicles. This is consistent with our recent findings in marmoset retina that FMB cells do not contact S-cone pedicles. The presence of GluR5 clusters at S-cone pedicles indicates that in primate retinas OFF bipolar cells expressing kainate receptor subunits receive some S-cone input.

The functional roles and synaptic features of horizontal cells in the mammalian retina are still controversial. Evidence exists for feedback signaling from horizontal cells to cones and feed-forward signaling from horizontal cells to bipolar cells, but the details of the latter remain elusive. Here, immunohistochemistry and confocal microscopy were used to analyze the expression patterns of the SNARE protein syntaxin-4, the GABA receptor subunits α1 and ρ, and the cation-chloride cotransporters NKCC and KCC2 in the outer plexiform layer of primate retina. In macaque retina, as observed previously in other species, syntaxin-4 was expressed on dendrites and axon terminals of horizontal cells at cone pedicles and rod spherules. At cones, syntaxin-4 appeared densely clustered in two bands, at horizontal cell dendritic tips and at the level of desmosome-like junctions. Interestingly, in the lower band where horizontal cells may synapse directly onto bipolar cells, syntaxin-4 was highly enriched beneath short-wavelength sensitive (S) cones and colocalized with calbindin, a marker for HII horizontal cells. The enrichment at S-cones was not observed in either mouse or ground squirrel. Furthermore, high amounts of both GABA receptor and cation-chloride cotransporter subunits were found beneath primate S-cones. Finally, while syntaxin-4 was expressed by both HI and HII horizontal cell types, the intense clustering and colocalization with calbindin at S-cones indicated an enhanced expression in HII cells. Taken together, GABA receptors beneath cone pedicles, chloride transporters, and syntaxin-4 are putative constituents of a synaptic set of proteins which would be required for a GABA-mediated feed-forward pathway via horizontal cells carrying signals directly from cones to bipolar cells.

Visual (and probably also magnetic) signal processing starts at the first synapse, at which photoreceptors contact different types of bipolar cells, thereby feeding information into different processing channels. In the chicken retina, 15 and 22 different bipolar cell types have been identified based on serial electron microscopy and single-cell transcriptomics, respectively. However, immunohistochemical markers for avian bipolar cells were only anecdotally described so far. Here, we systematically tested 12 antibodies for their ability to label individual bipolar cells in the bird retina and compared the eight most suitable antibodies across distantly related species, namely domestic chicken, domestic pigeon, common buzzard, and European robin, and across retinal regions. While two markers (GNB3 and EGFR) labeled specifically ON bipolar cells, most markers labeled in addition to bipolar cells also other cell types in the avian retina. Staining pattern of four markers (CD15, PKCα, PKCβ, secretagogin) was species-specific. Two markers (calbindin and secretagogin) showed a different expression pattern in central and peripheral retina. For the chicken and European robin, we found slightly more ON bipolar cell somata in the inner nuclear layer than OFF bipolar cell somata. In contrast, OFF bipolar cells made more ribbon synapses than ON bipolar cells in the inner plexiform layer of these species. Finally, we also analyzed the photoreceptor connectivity of selected bipolar cell types in the European robin retina. In summary, we provide a catalog of bipolar cell markers for different bird species, which will greatly facilitate analyzing the retinal circuitry of birds on a larger scale.

In the vertebrate retina, several dozens of parallel channels relay information about the visual world to the brain. These channels are represented by the different types of retinal ganglion cells (RGCs), whose responses are rendered selective for distinct sets of visual features by various mechanisms. These mechanisms can be roughly grouped into synaptic interactions and cell-intrinsic mechanisms, with the latter including dendritic morphology as well as ion channel complement and distribution. Here, we investigate how strongly ion channel complement can shape RGC output by comparing two mouse RGC types, the well-described ON alpha cell and a little-studied ON cell that is EGFP-labelled in the Igfbp5 mouse line and displays an unusual selectivity for stimuli with high contrast. Using patch-clamp recordings and computational modelling, we show that a higher activation threshold and a pronounced slow inactivation of the voltage-gated Na+ channels contribute to the distinct contrast tuning and transient responses in ON Igfbp5 RGCs, respectively. In contrast, such a mechanism could not be observed in ON alpha cells. This study provides an example for the powerful role that the last stage of retinal processing can play in shaping RGC responses.

The retinal rod pathway, featuring dedicated rod bipolar cells (RBCs) and AII amacrine cells, has been intensely studied in placental mammals. Here, we analyzed the rod pathway in a nocturnal marsupial, the South American opossum Monodelphis domestica to elucidate whether marsupials have a similar rod pathway. The retina was dominated by rods with densities of 338,000-413,000/mm². Immunohistochemistry for the RBC-specific marker protein kinase Cα (PKCα) and the AII cell marker calretinin revealed the presence of both cell types with their typical morphology. This is the first demonstration of RBCs in a marsupial and of the integration of RBCs and AII cells in the rod signaling pathway. Electron microscopy showed invaginating synaptic contacts of the PKCα-immunoreactive bipolar cells with rods; light microscopic co-immunolabeling for the synaptic ribbon marker CtBP2 confirmed dominant rod contacts. The RBC axon terminals were mostly located in the innermost stratum S5 of the inner plexiform layer (IPL), but had additional side branches and synaptic varicosities in strata S3 and S4, with S3-S5 belonging to the presumed functional ON sublayer of the IPL, as shown by immunolabeling for the ON bipolar cell marker Gγ13. Triple-immunolabeling for PKCα, calretinin and CtBP2 demonstrated RBC synapses onto AII cells. These features conform to the pattern seen in placental mammals, indicating a basically similar rod pathway in M. domestica. The density range of RBCs was 9,900-16,600/mm2, that of AII cells was 1,500-3,260/mm2. The numerical convergence (density ratio) of 146-156 rods to 4.7-6.0 RBCs to 1 AII cell is within the broad range found among placental mammals. For comparison, we collected data for the Australian nocturnal dunnart Sminthopsis crassicaudata, and found it to be similar to M. domestica, with rod-contacting PKCα-immunoreactive bipolar cells that had axon terminals also stratifying in IPL strata S3-S5.

Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.