Nyctalopin Expression in Retinal Bipolar Cells Restores Visual Function in a Mouse Model of Complete X-Linked Congenital Stationary Night Blindness

Mutations in the NYX gene that encodes the protein nyctalopin cause congenital stationary night blindness type 1. In no b-wave ( nob) mice, a mutation in Nyx results in a functional phenotype that includes the absence of the electroretinogram b-wave and abnormal spontaneous and light-evoked activity in retinal ganglion cells (RGCs). In contrast, there is no morphological abnormality in the retina at either the light or electron microscopic levels. These functional deficits suggest that nyctalopin is required for normal synaptic transmission between retinal photoreceptors and depolarizing bipolar cells (DBCs). However, the synaptic etiology and, specifically, the exact location and function of nyctalopin, remain uncertain. We show that nob DBCs fail to respond to exogenous application of the photoreceptor neurotransmitter, glutamate, thus demonstrating a postsynaptic deficit in photoreceptor to bipolar cell communication. To determine if postsynaptic expression of nyctalopin is necessary and sufficient to rescue the nob phenotype, we constructed transgenic mice that expressed an EYFP-nyctalopin fusion protein on the dendritic tips of the DBCs. Immunohistochemical and ultrastructural studies verified that fusion protein expression was limited to the DBC dendritic tips. Fusion gene expression in nob mice restored normal outer and inner visual function as determined by the electroretinogram and RGC spontaneous and evoked responses. Together, our data show that nyctalopin expression on DBC dendrites is required for normal function of the murine retina.

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Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness

Human Molecular Genetics ◽

10.1093/hmg/ddv341 ◽

2015 ◽

Vol 24 (21) ◽

pp. 6229-6239 ◽

Cited By ~ 34

Author(s):

Miranda L. Scalabrino ◽

Sanford L. Boye ◽

Kathryn M. H. Fransen ◽

Jennifer M. Noel ◽

Frank M. Dyka ◽

...

Keyword(s):

Mouse Model ◽

Visual Function ◽

Night Blindness ◽

Bipolar Cells ◽

Congenital Stationary Night Blindness ◽

Aav Vector

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Disinhibition of intrinsic photosensitive retinal ganglion cells in patients with X-linked congenital stationary night blindness

Graefe s Archive for Clinical and Experimental Ophthalmology ◽

10.1007/s00417-019-04319-w ◽

2019 ◽

Vol 257 (6) ◽

pp. 1207-1215 ◽

Cited By ~ 1

Author(s):

Andreas Schatz ◽

Carina Kelbsch ◽

Christina Zeitz ◽

Susanne Kohl ◽

Eberhart Zrenner ◽

...

Keyword(s):

Retinal Ganglion Cells ◽

Night Blindness ◽

Ganglion Cells ◽

Retinal Ganglion ◽

Congenital Stationary Night Blindness

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Glycine receptor immunoreactivity is localized at amacrine synapses in cat retina

Visual Neuroscience ◽

10.1017/s0952523800010397 ◽

1991 ◽

Vol 7 (6) ◽

pp. 611-618 ◽

Cited By ~ 36

Author(s):

Roberta G. Pourcho ◽

Michael T. Owczarzak

Keyword(s):

Ganglion Cells ◽

Glycine Receptor ◽

Plexiform Layer ◽

Amacrine Cells ◽

Bipolar Cells ◽

Electron Microscopic Level ◽

Inner Plexiform Layer ◽

Glycine Receptors ◽

Electron Microscopic ◽

Cat Retina

AbstractImmunocytochemical techniques were used to localize strychnine-sensitive glycine receptors in cat retina. Light microscopy showed staining in processes ramifying throughout the inner plexiform layer and in cell bodies of both amacrine and ganglion cells. At the electron-microscopic level, receptor immunoreactivity was seen to be clustered at sites postsynaptic to amacrine cells. In contrast, bipolar cells were neither presynaptic nor postsynaptic elements at sites of glycine receptor staining. Double-label studies verified the presence of glycine immunoreactivity in amacrine terminals presynaptic to glycine receptors. These findings support a role for glycine as an inhibitory neurotransmitter in amacrine cells.

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498. Optimization of rAAV Targets ON Bipolar Cells and Rescues the nobnyx Mouse Model of X-Linked Congenital Stationary Night Blindness

Molecular Therapy ◽

10.1016/s1525-0016(16)34107-7 ◽

2015 ◽

Vol 23 ◽

pp. S199

Author(s):

Miranda L. White ◽

Sanford L. Boye ◽

Frank M. Dyka ◽

Kathryn M. Fransen ◽

Charles N. de Leeuw ◽

...

Keyword(s):

Mouse Model ◽

Night Blindness ◽

Bipolar Cells ◽

Congenital Stationary Night Blindness

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Identification of a new mutant allele, Grm6nob7, for complete congenital stationary night blindness

Visual Neuroscience ◽

10.1017/s0952523815000012 ◽

2015 ◽

Vol 32 ◽

Cited By ~ 5

Author(s):

HAOHUA QIAN ◽

RUI JI ◽

RONALD G. GREGG ◽

NEAL S. PEACHEY

Keyword(s):

Night Blindness ◽

Frameshift Mutation ◽

Metabotropic Glutamate Receptor ◽

Bipolar Cells ◽

Human Condition ◽

Congenital Stationary Night Blindness ◽

Base Pairs ◽

Reading Frame ◽

Mouse Mutants ◽

The Impact

AbstractElectroretinogram (ERG) studies identified a new mouse line with a normal a-wave but lacking the b-wave component. The ERG phenotype of this new allele, nob7, matched closely that of mouse mutants for Grm6, Lrit3, Trpm1, and Nyx, which encode for proteins expressed in depolarizing bipolar cells (DBCs). To identify the underlying mutation, we first crossed nob7 mice with Grm6nob3 mutants and measured the ERGs in offspring. All the offspring lacked the b-wave, indicating that nob7 is a new allele for Grm6: Grm6nob7. Sequence analyses of Grm6nob7 cDNAs identified a 28 base pair insertion between exons 8 and 9, which would result in a frameshift mutation in the open reading frame that encodes the metabotropic glutamate receptor 6 (Grm6). Sequencing both the cDNA and genomic DNA from exon 8 and intron 8, respectively, from the Grm6nob7 mouse revealed a G to A transition at the last position in exon 8. This mutation disrupts splicing and the normal exon 8 is extended by 28 base pairs, because splicing occurs 28 base pairs downstream at a cryptic splice donor. Consistent with the impact of the resulting frameshift mutation, there is a loss of mGluR6 protein (encoded by Grm6) from the dendritic tips of DBCs in the Grm6nob7 retina. These results indicate that Grm6nob7 is a new model of the complete form of congenital stationary night blindness, a human condition that has been linked to mutations of GRM6.

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Synaptic circuits for irradiance coding by intrinsically photosensitive retinal ganglion cells

10.1101/442954 ◽

2018 ◽

Cited By ~ 3

Author(s):

Shai Sabbah ◽

Carin Papendorp ◽

Elizabeth Koplas ◽

Marjo Beltoja ◽

Cameron Etebari ◽

...

Keyword(s):

Retinal Ganglion Cells ◽

Ganglion Cells ◽

Bipolar Cells ◽

Retinal Ganglion ◽

Electron Microscopic ◽

Synaptic Circuits ◽

Ribbon Synapses ◽

High Pass ◽

Excitatory Drive ◽

Rod Bipolar Cells

SummaryWe have explored the synaptic networks responsible for the unique capacity of intrinsically photosensitive retinal ganglion cells (ipRGCs) to encode overall light intensity. This luminance signal is crucial for circadian, pupillary and related reflexive responses light. By combined glutamate-sensor imaging and patch recording of postsynaptic RGCs, we show that the capacity for intensity-encoding is widespread among cone bipolar types, including OFF types.Nonetheless, the bipolar cells that drive ipRGCs appear to carry the strongest luminance signal. By serial electron microscopic reconstruction, we show that Type 6 ON cone bipolar cells are the dominant source of such input, with more modest input from Types 7, 8 and 9 and virtually none from Types 5i, 5o, 5t or rod bipolar cells. In conventional RGCs, the excitatory drive from bipolar cells is high-pass temporally filtered more than it is in ipRGCs. Amacrine-to-bipolar cell feedback seems to contribute surprisingly little to this filtering, implicating mostly postsynaptic mechanisms. Most ipRGCs sample from all bipolar terminals costratifying with their dendrites, but M1 cells avoid all OFF bipolar input and accept only ectopic ribbon synapses from ON cone bipolar axonal shafts. These are remarkable monad synapses, equipped with as many as a dozen ribbons and only one postsynaptic process.

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Parvalbumin-immunoreactive amacrine cells of macaque retina

Visual Neuroscience ◽

10.1017/s0952523809090075 ◽

2009 ◽

Vol 26 (3) ◽

pp. 287-296 ◽

Cited By ~ 14

Author(s):

KATHRYN E. KLUMP ◽

AI-JUN ZHANG ◽

SAMUEL M. WU ◽

DAVID W. MARSHAK

Keyword(s):

Light Intensity ◽

Ganglion Cells ◽

Amacrine Cells ◽

Morphological Type ◽

Golgi Method ◽

Bipolar Cells ◽

Spatial Density ◽

Ultrastructural Studies ◽

Aii Amacrine Cells ◽

Aii Amacrine

AbstractA number of authors have observed amacrine cells containing high levels of immunoreactive parvalbumin in primate retinas. The experiments described here were designed to identify these cells morphologically, to determine their neurotransmitter, to record their light responses, and to describe the other cells that they contact. Macaque retinas were fixed in paraformaldehyde and labeled with antibodies to parvalbumin and one or two other markers, and this double- and triple-labeled material was analyzed by confocal microscopy. In their morphology and dendritic stratification patterns, the parvalbumin-positive cells closely resembled the knotty type 2 amacrine cells described using the Golgi method in macaques. They contained immunoreactive glycine transporter, but not immunoreactive γ-aminobutyric acid, and therefore, they use glycine as their neurotransmitter. Their spatial density was relatively high, roughly half that of AII amacrine cells. They contacted lobular dendrites of AII cells, and they are expected to be presynaptic to AII cells based on earlier ultrastructural studies. They also made extensive contacts with axon terminals of OFF midget bipolar cells whose polarity cannot be predicted with certainty. A macaque amacrine cell of the same morphological type depolarized at the onset of increments in light intensity, and it was well coupled to other amacrine cells. Previously, we described amacrine cells like these that contacted OFF parasol ganglion cells and OFF starburst amacrine cells. Taken together, these findings suggest that one function of these amacrine cells is to inhibit the transmission of signals from rods to OFF bipolar cells via AII amacrine cells. Another function may be inhibition of the OFF pathway following increments in light intensity.

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Localization of AMPA-selective glutamate receptor subunits in the cat retina: A light- and electron-microscopic study

Visual Neuroscience ◽

10.1017/s0952523899161121 ◽

1999 ◽

Vol 16 (1) ◽

pp. 169-177 ◽

Cited By ~ 33

Author(s):

PU QIN ◽

ROBERTA G. POURCHO

Keyword(s):

Glutamate Receptor ◽

Ganglion Cells ◽

Plexiform Layer ◽

Amacrine Cells ◽

Bipolar Cells ◽

Morphological Evidence ◽

Inner Plexiform Layer ◽

Electron Microscopic ◽

Cat Retina ◽

Cone Bipolar Cells

The distribution of AMPA-selective glutamate receptor subunits was studied in the cat retina using antisera against GluR1 and GluR2/3. Both antisera were localized in postsynaptic sites in the outer plexiform layer (OPL) as well as the inner plexiform layer (IPL). Immunoreactivity for GluR1 was seen in a subpopulation of OFF cone bipolar cells and a number of amacrine and ganglion cells. Within the IPL, processes staining for GluR1 received input from OFF and ON cone bipolar cells but not from rod bipolars. Labeling for GluR2/3 was seen in horizontal cells, an occasional cone bipolar cell, and numerous amacrine and ganglion cells. In the IPL, GluR2/3 staining was postsynaptic to cone bipolar cells in both sublaminae. AII amacrine cells which receive rod bipolar input were also labeled for GluR2/3. With both antisera, staining was limited to a single member of the bipolar dyad complex, providing morphological evidence for functional diversity in glutamatergic pathways.

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Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness

10.1101/2021.10.04.462588 ◽

2021 ◽

Author(s):

Keiko Miyadera ◽

Evelyn Santana ◽

Karolina Roszak ◽

Sommer Iffrig ◽

Meike Visel ◽

...

Keyword(s):

Gene Therapy ◽

Night Blindness ◽

Canine Model ◽

Bipolar Cells ◽

Large Animal Model ◽

Transduction Efficiency ◽

Large Animal ◽

Congenital Stationary Night Blindness ◽

Scotopic Vision ◽

Large Animal Models

AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling.

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