Asymmetric Distributions of Achromatic Bipolar Cells in the Mouse Retina

In the retina, evolutionary changes can be traced in the topography of photoreceptors. The shape of the visual streak depends on the height of the animal and its habitat, namely, woods, prairies, or mountains. Also, the distribution of distinct wavelength-sensitive cones is unique to each animal. For example, UV and green cones reside in the ventral and dorsal regions in the mouse retina, respectively, whereas in the rat retina these cones are homogeneously distributed. In contrast with the abundant investigation on the distribution of photoreceptors and the third-order neurons, the distribution of bipolar cells has not been well understood. We utilized two enhanced green fluorescent protein (EGFP) mouse lines, Lhx4-EGFP (Lhx4) and 6030405A18Rik-EGFP (Rik), to examine the topographic distributions of bipolar cells in the retina. First, we characterized their GFP-expressing cells using type-specific markers. We found that GFP was expressed by type 2, type 3a, and type 6 bipolar cells in the Rik mice and by type 3b, type 4, and type 5 bipolar cells in the Lhx4 mice. All these types are achromatic. Then, we examined the distributions of bipolar cells in the four cardinal directions and three different eccentricities of the retinal tissue. In the Rik mice, GFP-expressing bipolar cells were more highly observed in the nasal region than those in the temporal retina. The number of GFP cells was not different along with the ventral-dorsal axis. In contrast, in the Lhx4 mice, GFP-expressing cells occurred at a higher density in the ventral region than in the dorsal retina. However, no difference was observed along the nasal-temporal axis. Furthermore, we examined which type of bipolar cells contributed to the asymmetric distributions in the Rik mice. We found that type 3a bipolar cells occurred at a higher density in the temporal region, whereas type 6 bipolar cells were denser in the nasal region. The asymmetricity of these bipolar cells shaped the uneven distribution of the GFP cells in the Rik mice. In conclusion, we found that a subset of achromatic bipolar cells is asymmetrically distributed in the mouse retina, suggesting their unique roles in achromatic visual processing.

Two Functional Classes of Rod Bipolar Cells in the Healthy and Degenerated Optogenetically Treated Murine Retina

Bipolar cells have become successful targets for optogenetic gene therapies that restore vision after photoreceptor degeneration. However, degeneration was shown to cause changes in neuronal connectivity and protein expression, which may impact the quality of synthetically restored signaling. Further, the expression of an optogenetic protein may alter passive membrane properties of bipolar cells affecting signal propagation. We here investigated the passive membrane properties of rod bipolar cells in three different systems, the healthy retina, the degenerated retina, and the degenerated retina expressing the optogenetic actuator Opto-mGluR6. We found that, based on the shape of their current-voltage relations, rod bipolar cells in healthy and degenerated retinas form two clear functional groups (type 1 and type 2 cells). Depolarizing the membrane potential changed recorded current-voltage curves from type 1 to type 2, confirming a single cell identity with two functional states. Expression of Opto-mGluR6 did not alter the passive properties of the rod bipolar cell. With progressing degeneration, dominant outward rectifying currents recorded in type 2 rod bipolar cells decreased significantly. We demonstrate that this is caused by a downregulation of BK channel expression in the degenerated retina. Since this BK conductance will normally recover the membrane potential after RBCs are excited by open TRPM1 channels, a loss in BK will decrease high-pass filtering at the rod bipolar cell level. A better understanding of the changes of bipolar cell physiology during retinal degeneration may pave the way to optimize future treatment strategies of blindness.

Temporal and Isoform-Specific Expression of CTBP2 Is Evolutionarily Conserved Between the Developing Chick and Human Retina

Complex transcriptional gene regulation allows for multifaceted isoform production during retinogenesis, and novel isoforms transcribed from a single locus can have unlimited potential to code for diverse proteins with different functions. In this study, we explored the CTBP2/RIBEYE gene locus and its unique repertoire of transcripts that are conserved among vertebrates. We studied the transcriptional coregulator (CTBP2) and ribbon synapse-specific structural protein (RIBEYE) in the chicken retina by performing comprehensive histochemical and sequencing analyses to pinpoint cell and developmental stage-specific expression of CTBP2/RIBEYE in the developing chicken retina. We demonstrated that CTBP2 is widely expressed in retinal progenitors beginning in early retinogenesis but becomes limited to GABAergic amacrine cells in the mature retina. Inversely, RIBEYE is initially epigenetically silenced in progenitors and later expressed in photoreceptor and bipolar cells where they localize to ribbon synapses. Finally, we compared CTBP2/RIBEYE regulation in the developing human retina using a pluripotent stem cell derived retinal organoid culture system. These analyses demonstrate that similar regulation of the CTBP2/RIBEYE locus during chick and human retinal development is regulated by different members of the K50 homeodomain transcription factor family.

Retinal Development and Pathophysiology in Kcnj13 Knockout Mice

Purpose: We constructed and characterized knockout and conditional knockout mice for KCNJ13, encoding the inwardly rectifying K+ channel of the Kir superfamily Kir7.1, mutations in which cause both Snowflake Vitreoretinal Degeneration (SVD) and Retinitis pigmentosa (RP) to further elucidate the pathology of this disease and to develop a potential model system for gene therapy trials.Methods: A Kcnj13 knockout mouse line was constructed by inserting a gene trap cassette expressing beta-galactosidase flanked by FRT sites in intron 1 with LoxP sites flanking exon two and converted to a conditional knockout by FLP recombination followed by crossing with C57BL/6J mice having Cre driven by the VMD2 promoter. Lentiviral replacement of Kcnj13 was driven by the EF1a or VMD2 promoters.Results: Blue-Gal expression is evident in E12.5 brain ventricular choroid plexus, lens, neural retina layer, and anterior RPE. In the adult eye expression is seen in the ciliary body, RPE and choroid. Adult conditional Kcnj13 ko mice show loss of photoreceptors in the outer nuclear layer, inner nuclear layer thinning with loss of bipolar cells, and thinning and disruption of the outer plexiform layer, correlating with Cre expression in the overlying RPE which, although preserved, shows morphological disruption. Fundoscopy and OCT show signs of retinal degeneration consistent with the histology, and photopic and scotopic ERGs are decreased in amplitude or extinguished. Lentiviral based replacement of Kcnj13 resulted in increased ERG c- but not a- or b- wave amplitudes.Conclusion: Ocular KCNJ13 expression starts in the choroid, lens, ciliary body, and anterior retina, while later expression centers on the RPE with no/lower expression in the neuroretina. Although KCNJ13 expression is not required for survival of the RPE, it is necessary for RPE maintenance of the photoreceptors, and loss of the photoreceptor, outer plexiform, and outer nuclear layers occur in adult KCNJ13 cKO mice, concomitant with decreased amplitude and eventual extinguishing of the ERG and signs of retinitis pigmentosa on fundoscopy and OCT. Kcnj13 replacement resulting in recovery of the ERG c- but not a- and b-waves is consistent with the degree of photoreceptor degeneration seen on histology.

Cell Type-Selective Loss of Peroxisomal β-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2−/− mice. In conclusion, the early photoreceptor death in global Mfp2−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

A Promising Combination: PACAP and PARP Inhibitor Have Therapeutic Potential in Models of Diabetic and Hypertensive Retinopathies

Diabetes and hypertension are complex pathologies with increasing prevalence nowadays. Their interconnected pathways are frequently manifested in retinopathies. Severe retinal consequences and their tight connections as well as their possible treatments are particularly important to retinal research. In the present, work we induced diabetes with streptozotocin in spontaneously hypertensive rats and treated them either with PACAP or olaparib and alternatively with both agents. Morphological and immunohistochemical analyses were carried out to describe cell-specific changes during pathologies and after different treatments. Diabetes and hypertension caused massive structural and cellular changes especially when they were elicited together. Hypertension was crucial in the formation of ONL and OPL damage while diabetes caused significant differences in retinal thickness, OPL thickness and in the cell number of the GCL. In diabetes, double neuroprotective treatment ameliorated changes of calbindin-positive cells, rod bipolar cells and dopaminergic amacrine cells. Double treatment was curative in hypertensive diabetic rat retinas, especially in the case of rod bipolar and parvalbumin-positive cells compared to untreated or single-treated retinas. Our results highlighted the promising therapeutic benefits of olaparib and PACAP in these severe metabolic retinal disorders.

Origins of direction selectivity in the primate retina

From mouse to primate, there is a striking discontinuity in our current understanding of the neural coding of motion direction. In non-primate mammals, directionally selective cell types and circuits are a signature feature of the retina, situated at the earliest stage of the visual process1,2. In primates, by contrast, direction selectivity is a hallmark of motion processing areas in visual cortex3,4, but has not been found in the retina, despite significant effort5,6. Here we combined functional recordings of light-evoked responses and connectomic reconstruction to identify diverse direction-selective cell types in the macaque monkey retina with distinctive physiological properties and synaptic motifs. This circuitry includes an ON-OFF ganglion cell type, a spiking, ON-OFF poly-axonal amacrine cell and the starburst amacrine cell, all of which show direction selectivity. Moreover, we found unexpectedly that macaque starburst cells possess a strong, non-GABAergic, antagonistic surround mediated by input from excitatory bipolar cells that is critical for the generation of radial motion sensitivity in these cells. Our findings open a new door to investigation of a novel circuitry that computes motion direction in the primate visual system.

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.