ON Bipolar Cells Research Articles

TRPM1 Autoantibodies in Melanoma Patients Without Self-Reported Visual Symptoms.

PurposeMelanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma (CMM). Visual symptoms include night blindness, photopsia, and reduced-contrast sensitivity. An abnormal ERG b-wave and the presence of anti-bipolar cell autoantibodies, including autoantibodies reacting with the ON-bipolar cell TRPM1 channel, help to confirm the diagnosis. The goal of this study was to determine if CMM patients without visual symptoms also express anti-TRPM1 autoantibodies.MethodsSerum samples from 15 CMM patients were tested using three assays: immunofluorescent labeling of TRPM1-transfected HEK cells, immunofluorescent labeling of retinal sections from wild-type and TRPM1 knockout mice, and immunoblot detection of a bacterially produced recombinant TRPM1 peptide.ResultsSerum specimens from 5 of the 15 CMM patients without declared visual symptoms were positive for anti-TRPM1 autoantibodies in at least one of the three assays. One of 50 control sera from patients not known to have cancer was also weakly reactive with the TRPM1 peptide.ConclusionsAutoantibodies against TRPM1 are present in CMM patient sera without self-reported visual symptoms. Most patients had advanced (stage III and IV) disease and were undergoing aggressive treatments, including immunotherapy. It is unknown if immunotherapy affects the expression of TRPM1 autoantibodies. The presence of TRPM1 autoantibodies may predispose patients for MAR.

Course of loss of photoreceptor function and progressive Müller cell gliosis in rhodopsin P347L transgenic rabbits

Long living animal models of retinitis pigmentosa (RP) can provide important information on the retinal changes that occur at the late stages of photoreceptor degeneration. The rhodopsin Pro347Leu transgenic rabbit (P347L Tg) is a model of RP, and it has been used to analyze the functional and morphological changes in the retina following the degeneration of the photoreceptors. They have also been used to test newly-developed therapies to treat eyes with photoreceptor degeneration. However, assessments of the retinal changes in P347L Tg rabbits older than 1-year have not been reported even though the data are important for research on developing new therapies to restore vision at the end stages of RP. The purpose of this study was to determine the time course of the loss of photoreceptor function and the changes in the morphology of the retina of P347L Tg rabbits. The experiments were performed on 26 older P347L Tg rabbits. The results showed that the amplitudes of the ERGs of the P347L Tg rabbits gradually decreased and reached <10 μV between 30- and 54-months-of-age. Histological analysis at these later stages showed a loss of the photoreceptor layer, and OCT analysis showed absence of the layering of the retina. However, the thickness between the inner limiting membrane and the outer plexiform layer was about 1.7 times thicker than the corresponding thickness of WT rabbits in the OCT images. This thickening was caused by a marked gliosis of the entire retina which was confirmed by light and transmission electron microscopy. In addition, immunohistochemical analysis showed there was excessive staining of the glial fibrillary acid protein in the older P347L Tg rabbits although the rod ON bipolar cells and horizontal cells were still present in the inner nuclear layer. Our results indicate that the P347L Tg rabbit progressed to complete photoreceptor loss within 30- and 54-months-of-age and severe gliosis altered the morphology of the retina.

Gap Junctions in A8 Amacrine Cells Are Made of Connexin36 but Are Differently Regulated Than Gap Junctions in AII Amacrine Cells.

In the mammalian retina, amacrine cells represent the most diverse cell class and are involved in the spatio-temporal processing of visual signals in the inner plexiform layer. They are connected to bipolar, other amacrine and ganglion cells, forming complex networks via electrical and chemical synapses. The small-field A8 amacrine cell was shown to receive non-selective glutamatergic input from OFF and ON cone bipolar cells at its bistratified dendrites in sublamina 1 and 4 of the inner plexiform layer. Interestingly, it was also shown to form electrical synapses with ON cone bipolar cells, thus resembling the rod pathway-specific AII amacrine cell. In contrast to the AII cell, however, the electrical synapses of A8 cells are poorly understood. Therefore, we made use of the Ier5-GFP mouse line, in which A8 cells are labeled by GFP, to study the gap junction composition and frequency in A8 cells. We found that A8 cells form <20 gap junctions per cell and these gap junctions consist of connexin36. Connexin36 is present at both OFF and ON dendrites of A8 cells, preferentially connecting A8 cells to type 1 OFF and type 6 and 7 ON bipolar cells and presumably other amacrine cells. Additionally, we show that the OFF dendrites of A8 cells co-stratify with the processes of dopaminergic amacrine cells from which they may receive GABAergic input via GABAA receptor subunit α3. As we found A8 cells to express dopamine receptor D1 (but not D2), we also tested whether A8 cell coupling is modulated by D1 receptor agonists and antagonists as was shown for the coupling of AII cells. However, this was not the case. In summary, our data suggests that A8 coupling is differently regulated than AII cells and may even be independent of ambient light levels and serve signal facilitation rather than providing a separate neuronal pathway.

Differential expression of PKCα and -β in the zebrafish retina.

The retina is a complex neural circuit, which processes and transmits visual information from light perceiving photoreceptors to projecting retinal ganglion cells. Much of the computational power of the retina rests on signal integrating interneurons, such as bipolar cells. Commercially available antibodies against bovine and human conventional protein kinase C (PKC) α and -β are frequently used as markers for retinal ON-bipolar cells in different species, despite the fact that it is not known which bipolar cell subtype(s) they actually label. In zebrafish (Danio rerio) five prkc genes (coding for PKC proteins) have been identified. Their expression has not been systematically determined. While prkcg is not expressed in retinal tissue, the other four prkc (prkcaa, prkcab, prkcba, prkcbb) transcripts were found in different parts of the inner nuclear layer and some as well in the retinal ganglion cell layer. Immunohistochemical analysis in adult zebrafish retina using fluorescent in situ hybridization and PKC antibodies showed an overlapping immunolabeling of ON-bipolar cells that are most likely of the BON s6 and BON s6L or RRod type. However, comparison of transcript expression with immunolabeling, implies that these antibodies are not specific for one single zebrafish conventional PKC, but rather detect a combination of PKC -α and -β variants.

SubILM Injection of AAV for Gene Delivery to the Retina.

Adeno-associated virus (AAV) has emerged as the vector of choice for delivering genes to the retina. Indeed, the first gene therapy to receive FDA approval in the United States is an AAV-based treatment for the inherited retinal disease, Leber congenital amaurosis-2. Voretigene neparvovec (Luxturna™) is delivered to patients via subretinal (SR) injection, an invasive surgical procedure that requires detachment of photoreceptors (PRs) from the retinal pigment epithelium (RPE). It has been reported that subretinal administration of vector under the cone-exclusive fovea leads to a loss of central retinal structure and visual acuity in some patients. Due to its technical difficulty and potential risks, alternatives to SR injection have been explored in primates. Intravitreally (Ivt) delivered AAV transduces inner retina and foveal cones, but with low efficiency. Novel AAV capsid variants identified via rational design or directed evolution have offered only incremental improvements, and have failed to promote pan-inner retinal transduction or significant outer retinal transduction beyond the fovea. Problems with retinal transduction by Ivt-delivered AAV include dilution in the vitreous, potential antibody-mediated neutralization of capsid in this nonimmune privileged space, and the presence of the inner limiting membrane (ILM), a basement membrane separating the vitreous from the neural retina. We have developed an alternative "subILM" injection method that overcomes all three hurdles. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. We have shown that subILM injection promotes more efficient retinal transduction by AAV than Ivt injection, and results in uniform and extensive transduction of retinal ganglion cells (RGCs) beneath the subILM bleb. We have also demonstrated transduction of Muller glia, ON bipolar cells, and photoreceptors by subILM injection. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes is greatly enhanced. Here we describe in detail methods for vector preparation, vector dilution, and subILM injection as performed in macaque (Macaca sp.).

Distribution and density of mixed-input ON bipolar cells of the goldfish (Carassius auratus) during growth.

Neurons are continuously produced at different rates and locations in the teleost retina. Goldfish rods are homogeneously distributed and maintain a stable density throughout growth, whereas little is known about their postsynaptic partners. We examined the distribution and density of mixed-input ON bipolar cells (ON mBCs) in 57 goldfish of various sizes by immunolabeling their retinas with an antibody against PKCα and counting PKCα-positive neurons in wholemounts. Cell densities were correlated with morphometric data for the same animals, and the spatial resolution of the ON mBC mosaic was calculated in each case. The distribution of ON mBCs is homogeneous throughout growth. For a 10-fold change in body size (i.e., from 20 to 200 mm), the total number of ON mBCs increases 2.8 times, while retinal area expands around 10 times. As a consequence, the density of ON mBCs in large fish falls to ∼1/3 of that of small animals, and intercellular spacing doubles. The eye and the lens become around three times larger from small to large fish. This causes the retinal magnification factor (and thereby the image projected onto retina) to augment by the same amount. Because the retinal magnification factor rises more than the intercellular spacing in the same animals, the spatial resolution of the ON mBC mosaic improves from 0.8 to 1.4 cycles/degree as the body size increases from 20 to 200 mm. As ON mBCs are mostly rod-driven, our results suggest that the scotopic acuity of the goldfish may improve as the animal grows.

Contribution of Bipolar Cells of Cone ON and OFF Pathways to Electroretinograms Elicited by Ultraviolet and Middle Wavelength Stimuli

ABSTRACTPurpose: To determine the contribution of the ON and OFF cone bipolar cell pathways to the electroretinograms (ERGs) elicited by ultraviolet (UV) and middle wavelength light in mice.Materials and Methods: The experiments were performed on 8- to 10-week-old C57BL/6J mice. The ERGs elicited by single-flash and flickering UV light stimuli were compared to those elicited by green light stimuli under photopic conditions. Pharmacological agents were used to selectively block the ON and OFF pathways contributing to the ERGs. Saline was used as a control. The flicker ERGs elicited by UV light were compared to the ERGs elicited by green light after the injection of the pharmacological agents to determine the contribution of the cone ON and OFF pathways to the ERGs.Results: The photopic single-flash and flicker ERGs were more sensitive to the UV light stimuli than to those elicited by green light stimuli. The flicker ERG responses elicited by both UV and green light stimuli at stimulus frequencies lower than 15-Hz decreased after L-2-amino-4-phosphobutyric acid was injected. The ERGs elicited by UV light at 30-Hz and by green light at frequencies lower than 15-Hz decreased significantly after the intravitreal injection of cis-2, 3-piperidine-dicarboxylic acid. An analysis of the ON and OFF components of the flicker ERGs showed that there might be pharmacological differences between the UV light-sensitive responses and the green light-sensitive responses.Conclusions: These results suggest that the UV light-sensitive cones connect to both the ON and OFF bipolar cells differently than that of the green light-sensitive cones.

The M6 cell: A small-field bistratified photosensitive retinal ganglion cell.

We have identified a novel, sixth type of intrinsically photosensitive retinal ganglion cell (ipRGC) in the mouse-the M6 cell. Its spiny, highly branched dendritic arbor is bistratified, with dendrites restricted to the inner and outer margins of the inner plexiform layer, co-stratifying with the processes of other ipRGC types. We show that M6 cells are by far the most abundant ganglion cell type labeled in adult pigmented Cdh3-GFP BAC transgenic mice. A few M5 ipRGCs are also labeled, but no other RGC types were encountered. Several distinct subnuclei in the geniculate complex and the pretectum contain labeled retinofugal axons in the Cdh3-GFP mouse. These are presumably the principle central targets of M6 cells (as well as M5 cells). Projections from M6 cells to the dorsal lateral geniculate nucleus were confirmed by retrograde tracing, suggesting they contribute to pattern vision. M6 cells have low levels of melanopsin expression and relatively weak melanopsin-dependent light responses. They also exhibit strong synaptically driven light responses. Their dendritic fields are the smallest and most abundantly branched of all ipRGCs. They have small receptive fields and strong antagonistic surrounds. Despite deploying dendrites partly in the OFF sublamina, M6 cells appear to be driven exclusively by the ON pathway, suggesting that their OFF arbor, like those of certain other ipRGCs, may receive ectopic input from passing ON bipolar cells axons in the OFF sublayer.

A group I metabotropic glutamate receptor controls synaptic gain between rods and rod bipolar cells in the mouse retina.

The canonical mGluR6‐Trpm1 pathway that generates the sign‐inverting signal between photoreceptors and ON bipolar cells has been well described. However, one type of ON bipolar cell, the rod bipolar cell (RBC), additionally is thought to express the group I mGluRs whose function is unknown. We examined the role of group I mGluRs in mouse RBCs and here provide evidence that it controls synaptic gain between rods and RBCs. In dark‐adapted conditions, the mGluR1 antagonists LY367385 and (RS)‐1‐Aminoindan‐1,5‐dicarboxylic acid, but not the mGluR5 antagonist 2‐Methyl‐6‐(phenylethynyl)pyridine hydrochloride reduced the light‐evoked responses in RBCs indicating that mGluR1, but not mGluR5, serves to potentiate RBC responses. Perturbing the downstream phospholipase C (PLC)‐protein kinase C (PKC) pathway by inhibiting PLC, tightly buffering intracellular Ca2+, or preventing its release from intracellular stores reduced the synaptic potentiation by mGluR1. The effect of mGluR1 activation was dependent upon adaptation state, strongly increasing the synaptic gain in dark‐, but not in light‐adapted retinas, or in the presence of a moderate background light, consistent with the idea that mGluR1 activation requires light‐dependent glutamate release from rods. Moreover, immunostaining revealed that protein kinase Cα (PKC α) is more strongly expressed in RBC dendrites in dark‐adapted conditions, revealing an additional mechanism behind the loss of mGluR1 potentiation. In light‐adapted conditions, exogenous activation of mGluR1 with the agonist 3,5‐Dihydroxyphenylglycine increased the mGluR6 currents in some RBCs and decreased it in others, suggesting an additional action of mGluR1 that is unmasked in the light‐adapted state. Elevating intracellular free Ca2+, consistently resulted in a decrease in synaptic gain. Our results provide evidence that mGluR1 controls the synaptic gain in RBCs.

The rod signaling pathway in marsupial retinae.

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.

The role of Zhx2 transcription factor in bipolar cell differentiation during mouse retinal development

We found that the Zhx2 gene (whose product is known to act as a tumor suppressor in hepatocellular carcinoma) is expressed in embryonic retinal progenitors and in developing cone bipolar cells in the postnatal retina, as well as in Müller glia in the mature retina. To examine the functions of Zhx2 protein during retinal development, we performed loss- and gain-of-function analyses using a retinal explant culture system. We introduced a plasmid encoding Zhx2 shRNA into isolated mouse retinas at E17.5, and the retinas were cultured as explants. After 3 days of culture, proliferation was slightly enhanced, leading to retinas thicker than in the control, but this phenomenon was observed only transiently. After 14 days of the culture, the thickness and gross morphology of retinas expressing sh-Zhx2 were indistinguishable from those of the control. The numbers of rod cells, amacrine cells, and Müller glia were the same in both groups. However, although the total number of bipolar cells was the same, the experimental group saw an increased population of ON bipolar cells, and decreased numbers of a subset of OFF bipolar cells. We also examined the effects of overexpression of Zhx2. Although Zhx2 acts as a tumor suppressor, its overexpression in developing retinas did not lead to any discernible difference in retinal thickness, suggesting that proliferation activity was not affected. After 14 days of explant culture, the total number of bipolar cells decreased, and subset composition was altered. Taken together, these results suggest that Zhx2 plays roles in the regulation of bipolar cell subset fate determination during retinal development.

Revisiting cannabinoid receptor 2 expression and function in murine retina

The cannabinoid receptor CB2 plays a significant role in the regulation of immune function whereas neuronal expression remains a subject of contention. Multiple studies have described CB2 in retina and a recent study showed that CB2 deletion altered retinal visual processing. We revisited CB2 expression using immunohistochemistry and a recently developed CB2-eGFP reporter mouse. We examined the consequence of acute vs. prolonged CB2 deactivation on the electroretinogram (ERG) responses. We also examined lipidomics in CB2 knockout mice and potential changes in microglia using Scholl analysis.Consistent with a published report, in CB2 receptor knockout mice see an increased ERG scotopic a-wave, as well as stronger responses in dark adapted cone-driven ON bipolar cells and, to a lesser extent cone-driven ON bipolar cells early in light adaptation. Significantly, however, acute block with CB2 antagonist, AM630, did not mimic the results observed in the CB2 knockout mice whereas chronic (7 days) block did. Immunohistochemical studies show no CB2 in retina under non-pathological conditions, even with published antibodies. Retinal CB2–eGFP reporter signal is minimal under baseline conditions but upregulated by intraocular injection of either LPS or carrageenan. CB2 knockout mice see modest declines in a broad spectrum of cannabinoid-related lipids. The numbers and morphology of microglia were unaltered. In summary minimal CB2 expression is seen in healthy retina. CB2 appears to be upregulated under pathological conditions. Previously reported functional consequences of CB2 deletion are an adaptive response to prolonged blockade of these receptors. CB2 therefore impacts retinal signaling but perhaps in an indirect, potentially extra-ocular fashion.

Synaptic Transfer between Rod and Cone Pathways Mediated by AII Amacrine Cells in the Mouse Retina

To understand computation in a neural circuit requires a complete synaptic connectivity map and a thorough grasp of the information-processing tasks performed by the circuit. Here, we dissect a microcircuit in the mouse retina in which scotopic visual information (i.e., single photon events, luminance, contrast) is encoded by rod bipolar cells (RBCs) and distributed to parallel ON and OFF cone bipolar cell (CBC) circuits via the AII amacrine cell, an inhibitory interneuron. Serial block-face electron microscopy (SBEM) reconstructions indicate that AIIs preferentially connect to one OFF CBC subtype (CBC2); paired whole-cell patch-clamp recordings demonstrate that, depending on the level of network activation, AIIs transmit distinct components of synaptic input from single RBCs to downstream ON and OFF CBCs. These findings highlight specific synaptic and circuit-level features that allow intermediate neurons (e.g., AIIs) within a microcircuit to filter and propagate information to downstream neurons.

Bistratified starburst amacrine cells in Sox2 conditional knockout mouse retina display ON and OFF responses.

Cell-intrinsic factors, in conjunction with environmental signals, guide migration, differentiation, and connectivity during early development of neuronal circuits. Within the retina, inhibitory starburst amacrine cells (SBACs) comprise ON types with somas in the ganglion cell layer (GCL) and dendrites stratifying narrowly in the inner half of the inner plexiform layer (IPL) and OFF types with somas in the inner nuclear layer (INL) and dendrites stratifying narrowly in the outer half of the IPL. The transcription factor Sox2 is crucial to this subtype specification. Without Sox2, many ON-type SBACs destined for the GCL settle in the INL while many that reach the GCL develop bistratified dendritic arbors. This study asked whether ON-type SBACs in Sox2-conditional knockout retinas exhibit selective connectivity only with ON-type bipolar cells or their bistratified morphology allows them to connect to both ON and OFF bipolar cells. Physiological data demonstrate that these cells receive ON and OFF excitatory inputs, indicating that the ectopically stratified dendrites make functional synapses with bipolar cells. The excitatory inputs were smaller and more transient in Sox2-conditional knockout compared with wild type; however, inhibitory inputs appeared largely unchanged. Thus dendritic stratification, rather than cellular identification, may be the major factor that determines ON vs. OFF connectivity. NEW & NOTEWORTHY Conditional knockout of the transcription factor Sox2 during early embryogenesis converts a monostratifying starburst amacrine cell into a bistratifying starburst cell. Here we show that these bistratifying starburst amacrine cells form functional synaptic connections with both ON and OFF bipolar cells. This suggests that normal ON vs. OFF starburst connectivity may not require distinct molecular specification. Proximity alone may be sufficient to allow formation of functional synapses.

Phenotypic characterization of complete CSNB in the inbred research beagle: how common is CSNB in research and companion dogs?

Although congenital stationary night blindness (CSNB) has been described in a Japanese beagle dog research colony, certain clinical correlates with human CSNB have not yet been described, nor has an estimate of frequency of the condition been made in inbred and outbred beagle populations. A beagle with CSNB obtained from a commercial research dog supplier in the USA and matched control dogs (n = 3) underwent examination, refraction, ocular imaging, assessment of visual navigation ability and detailed electroretinography (ERG). Retrospective review of ERGs in two independent groups of inbred (n = 15 and 537, respectively) and one group of outbred dogs (n = 36) was used to estimate CSNB frequency in these populations. In the affected dog, there were absent dark-adapted b-waves in response to dim-light flashes, severely reduced dark-adapted b-waves in response to bright-light flashes, and normal light-adapted b-waves with a-waves that had broadened troughs. Long-flash ERGs confirmed a markedly reduced b-wave with a preserved d-wave, consistent with cone ON-bipolar cell dysfunction. There was evidence of normal rod photoreceptor a-wave dark adaptation, and rapid light adaptation. In the wider beagle populations, five inbred beagles had a b/a wave ratio of < 1 in dark-adapted bright-flash ERG, whereas no outbred beagles had ERGs consistent with CSNB. The identified dog had clinical findings consistent with complete type CSNB, similar to that described in the Japanese colony. CSNB appears to be a rare disorder in the wider beagle population, although its detection could confound studies that use retinal function as an outcome measure in research dogs, necessitating careful baseline studies to be performed prior to experimentation.

Feasibility study for a glutamate driven subretinal prosthesis: local subretinal application of glutamate on blind retina evoke network-mediated responses in different types of ganglion cells

Objective. A feasibility study for a transmitter based subretinal prosthesis, generating visual responses in blind mouse retina is presented. Approach. Degenerated rd1 mouse retina were stimulated in subretinal configuration by local glutamate (Glu) or NMDA application via micropipettes (~1.5 μm) and thereby the outer retinal activity was recorded by calcium-imaging or the ganglion cell (GC) activity was recorded by the multi-electrode array system. The network mediated activation of GC via bipolar cells was approved by the administration of Glu receptor blockers. Main results. Data of the degenerated and blind rd1 mouse retina reveals that the outer retina is Glu sensitive and that the subretinal Glu stimulation promotes network mediated GC responses. Analysis of the spatial activity-spread indicates that the Glu induced cell activation radius in the outer retina (~12.5 μm) and postsynaptically activated GC (~40 μm) is focal to the stimulation pipette tip. Moreover, the application of NMDA in subretinal space also evoked network mediated GC responses. The Glu-activated GC were identified as ON–OFF, OFF and two ON cells types. Significance. This study evaluates the prerequisite for the function of a transmitter based implant, that after the loss of the photoreceptors, the remnant blind retinal network is Glu sensitive and functional, positively. The differential activation of ON (hyperpolarisation) and OFF (depolarisation) bipolar cells by transmitter Glu is a unique feature and of high interest for retinal implants. Therefore, the respective bipolar cell types could only be driven by glutamatergic stimulation accurately and not by electrical stimulation. The preserved functionality of the blind retina at the onset of complete blindness is motivating to continue research on a transmitter-based prosthesis. Since the artificial Glu stimulation mimics the natural retinal input, early implantation of a Glu-prosthesis might delay the devastating retinal remodelling positively, due to the neuronal-plasticity.

Pregnancy-Associated Plasma Protein-aa Regulates Photoreceptor Synaptic Development to Mediate Visually Guided Behavior.

To guide behavior, sensory systems detect the onset and offset of stimuli and process these distinct inputs via parallel pathways. In the retina, this strategy is implemented by splitting neural signals for light onset and offset via synapses connecting photoreceptors to ON and OFF bipolar cells, respectively. It remains poorly understood which molecular cues establish the architecture of this synaptic configuration to split light-onset and light-offset signals. A mutant with reduced synapses between photoreceptors and one bipolar cell type, but not the other, could reveal a critical cue. From this approach, we report a novel synaptic role for pregnancy-associated plasma protein aa (pappaa) in promoting the structure and function of cone synapses that transmit light-offset information. Electrophysiological and behavioral analyses indicated pappaa mutant zebrafish have dysfunctional cone-to-OFF bipolar cell synapses and impaired responses to light offset, but intact cone-to-ON bipolar cell synapses and light-onset responses. Ultrastructural analyses of pappaa mutant cones showed a lack of presynaptic domains at synapses with OFF bipolar cells. pappaa is expressed postsynaptically to the cones during retinal synaptogenesis and encodes a secreted metalloprotease known to stimulate insulin-like growth factor 1 (IGF1) signaling. Induction of dominant-negative IGF1 receptor expression during synaptogenesis reduced light-offset responses. Conversely, stimulating IGF1 signaling at this time improved pappaa mutants' light-offset responses and cone presynaptic structures. Together, our results indicate Pappaa-regulated IGF1 signaling as a novel pathway that establishes how cone synapses convey light-offset signals to guide behavior.SIGNIFICANCE STATEMENT Distinct sensory inputs, like stimulus onset and offset, are often split at distinct synapses into parallel circuits for processing. In the retina, photoreceptors and ON and OFF bipolar cells form discrete synapses to split neural signals coding light onset and offset, respectively. The molecular cues that establish this synaptic configuration to specifically convey light onset or offset remain unclear. Our work reveals a novel cue: pregnancy-associated plasma protein aa (pappaa), which regulates photoreceptor synaptic structure and function to specifically transmit light-offset information. Pappaa is a metalloprotease that stimulates local insulin-like growth factor 1 (IGF1) signaling. IGF1 promotes various aspects of synaptic development and function and is broadly expressed, thus requiring local regulators, like Pappaa, to govern its specificity.

A Large Endoplasmic Reticulum-Resident Pool of TRPM1 in Retinal ON-Bipolar Cells.

The chemical signal of light onset, a decrease in glutamate release from rod and cone photoreceptors, is processed by a postsynaptic G protein signaling cascade in ON-bipolar cells (BPCs). The metabotropic glutamate receptor mGluR6, along with other cascade elements, is localized synaptically at the BPC dendritic tips. The effector ion channel protein transient receptor potential melastatin-1 (TRPM1), in contrast, is located not only at the dendritic tips but also in BPC bodies and axons. Little is known about the intracellular localization of TRPM1, or its trafficking route to the dendritic tip plasma membrane. Recombinant TRPM1 expressed in mammalian cells colocalized with endoplasmic reticulum (ER) markers, with little or none detected at the plasma membrane. In mouse retina, somatic TRPM1 was similarly intracellular, and not at the plasma membrane. Labeling of ER membranes by expression of a fluorescent marker showed that in BPCs the ER extends into axons and dendrites, but not dendritic tips. In cell bodies, TRPM1 colocalized with the ER, and not with the Golgi apparatus. Fluorescence protease protection (FPP) assays with TRPM1-GFP fusions in heterologous cells revealed that the N and C termini are both accessible to the cytoplasm, consistent with the transmembrane domain topology of related TRP channels. These results indicate that the majority of TRPM1 is present in the ER, from which it can potentially be transported to the dendritic tips as needed for ON light responses. The excess of ER-resident TRPM1 relative to the amount needed at the dendritic tips suggests a potential new function for TRPM1 in the ER.

Comparison between albino and pigmented rabbit ERGs

Pigmented and albino rabbits are commonly used in visual research; however, the lack of pigment in the eyes may affect retinal responses. Here, we compare and describe the differences of retinal function between pigmented (English Butterfly) and albino (New Zealand) rabbits. Electroretinograms were recorded in pigmented and albino rabbits in the dark-adapted eye, in the light-adapted eye and for four temporal frequencies in the light-adapted eye. The implicit time and amplitude of the a- and b-waves were analyzed, as well as the amplitude and phase of the first harmonic component of the photopic flicker response. Albino rabbits presented significantly larger amplitudes for both a- and b-waves at all intensities and frequencies. The intensity-response function of the scotopic b-wave also showed that the albino retina is more sensitive than the pigmented retina and the larger flicker amplitudes found in the albino group also revealed post-receptoral changes specifically related to cone pathways. The larger amplitude of albino receptoral and post-receptoral activities might be attributed to greater availability of light due to scatter and reflection at the retinal layer, and as the differences in response amplitudes between the groups increase with flicker frequency, we suggest that ON bipolar cells recover faster in the albino group, suggesting that this might be a mechanism to explain the higher temporal resolution for albinos compared to the pigmented group.

Pivotal roles of Fezf2 in differentiation of cone OFF bipolar cells and functional maturation of cone ON bipolar cells in retina

During development of the retina, common retinal progenitor cells give rise to six classes of neurons that subsequently further diversify into more than 55 subtypes of neuronal subtypes. Here, we have investigated the expression and function of Fezf2, Fez zinc finger family of protein, in the developing mouse retina. Expression of Fezf2 transcripts was strongly observed in the embryonic retinal progenitors at E14.5 and declined quickly in subsequent development of retina. Then, in postnatal stage at around day 8, Fezf2 was transiently expressed then declined again. Loss-of-function analysis using retinas from mice in which Fezf2 coding region was substituted with β-galactosidase showed that Fezf2 is expressed in a subset of cone OFF bipolar cells and required for their differentiation. Using electroretinogram, we found that Fezf2 knockout retina exhibited significantly reduced photopic b-wave, suggesting functional abnormality of cone ON bipolar cells. Furthermore, reduced expression of synaptic protein Trpm1 and structural alteration of ON bipolar cell invagination, both of which affected cone photoreceptor terminal synaptic activity, was identified by transmission electron microscopy and immunohistochemistry, respectively. Taken together, our results show that Fezf2 is indispensable in differentiation of bipolar precursors into cone OFF bipolar cells and in functional maturation of cone ON bipolar cells during development of mouse retina. These results contribute to our understanding of how diversity of neuronal subtypes and hence specificity of neuronal connections are established in the retina by intrinsic cues.