Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal ... more Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal space bound by the photoreceptor (PR) outer segments and the processes of the retinal pigmented epithelium (RPE). IRBP binds retinoids, including 11-cis-retinal and all-trans-retinol. In this study, visual function for demanding visual tasks was assessed in IRBP knock-out (KO) mice. Surprisingly, IRBP KO mice showed no differences in scotopic critical flicker frequency (CFF) compared to wildtype (WT). However, they did have lower photopic CFF than WT. IRBP KO mice had reduced scotopic and photopic acuity and contrast sensitivity compared to WT. IRBP KO mice had a significant reduction in outer nuclear layer (ONL) thickness, PR outer and inner segment, and full retinal thickness (FRT) compared to WT. There were fewer cones in IRBP KO mice. Overall, these results confirm substantial loss of rods and significant loss of cones within 30 days. Absence of IRBP resulted in cone circuit damage, ...
Purpose: Desensitization in the rod cell of the mammalian retina is initiated when light-activate... more Purpose: Desensitization in the rod cell of the mammalian retina is initiated when light-activated rhodopsin is phosphorylated by the G protein-coupled receptor kinase (GRK), GRK1, often referred to as rhodopsin kinase. A distinct kinase that specifically phosphorylates cone opsins in a similar manner has not been identified in mammals. To determine the existence of a cone opsin kinase, RNA from the retinas of cone- and rod-dominant mammals was analyzed by PCR. Methods: RNA prepared from the retinas of two cone-dominant mammals, the thirteen-lined ground squirrel and the eastern chipmunk, and a rod-dominant mammal, the pig, was used to clone a new GRK family member by RT-PCR. The tissue distribution and localization of the kinase in retina were determined by Northern blot hybridization and in situ hybridization. The protein encoded by this cDNA was expressed in human embryonic kidney-293 (HEK-293) cells and compared with bovine GRK1 for its ability to phosphorylate bovine rhodopsin and to undergo autophosphorylation. Results: The cDNA cloned from ground squirrel contains an open reading frame encoding a 548 amino-acid protein. Sequence analysis indicates that this protein is orthologous to GRK7 recently cloned from O. latipes, the medaka fish. Partial cDNA fragments of GRK7 were also cloned from RNA prepared from eastern chipmunk and pig retinas. In situ hybridization demonstrated widespread labeling in the photoreceptor layer of the ground squirrel retina, consistent with expression in cones. Recombinant ground squirrel GRK7 phosphorylates bovine rhodopsin in a light-dependent manner and can be autophosphorylated, similar to bovine GRK1. Conclusions: These results indicate that cone- and rod-dominant mammals both express GRK7. The presence of this kinase in cones in the ground squirrel and its ability to phosphorylate rhodopsin suggests that it could function in cone cells as a cone opsin kinase.
1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied ... more 1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied by recording membrane current from individual cones in small pieces of retina. 2. Brief flashes of light produced transient reductions of the dark current; saturating response amplitudes were up to 67 pA. A flash strength of about 11,000 photons microns‐2 at lambda max was required to give a half‐saturating response. The stimulus‐response relation was well fitted by an exponential saturation curve. Responses below 20% of maximum behaved linearly. 3. The response to a dim flash in most cells had a time to peak of 20‐30 ms and resembled the impulse response of a series of five low‐pass filters. 4. The variance of the dim‐flash response amplitude put an upper limit of 80 fA on the size of the single photon response. Estimates based on the effective collecting area suggest the single photon response to be of the order of 10 fA. 5. Flash responses of squirrel cones usually lacked the undersho...
Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that e... more Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by mutations in over 70 genes. While most mutations in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some mutations cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in mouse model harboring the most prevalent RP59-associated DHDDS mutation (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are shorter than in the corresponding tissues of age-matched controls, as repo...
Advances in experimental medicine and biology, 2018
A visual response to flickering light requires complex retinal computation, and thus ERG measures... more A visual response to flickering light requires complex retinal computation, and thus ERG measures are an excellent test of retinal circuit fidelity. Critical flicker frequency (CFF) is the frequency at which the retinal response is no longer modulated. Traditionally, CFF is obtained with a series of steady flicker stimuli with increasing frequencies. However, this method is slow and susceptible to experimental drift and/or adaptational effects. The current study compares the steady flicker method to CFF measurements obtained using a frequency sweep protocol. We introduce a light source programmed to produce a linear sweep of frequencies in a single trial. Using the traditional steady flicker method and a criterion response of 3 μV, we obtained a scotopic CFF of 18.4 ± 0.66 Hz and a photopic CFF of 44.4 ± 1.67 Hz. Our sweep flicker method, used on the same animals, produces a waveform best analyzed by Fourier transform; wherein a 6.18 log μV2 threshold was found to yield CFF values e...
We investigated the etiology of decreased cone-driven vision in a light damage (LD) model of reti... more We investigated the etiology of decreased cone-driven vision in a light damage (LD) model of retinal degeneration. To induce slow, moderate degeneration, albino rats underwent low-intensity light exposure for 10 days. Electroretinography was utilized to assess physiologic function of the rod- and cone-driven retinal function in LD and control rats. Immunohistochemistry targeting cone arrestin allowed for quantification of cone density and for comparison of the decline in function. Photoreceptor loss was quantified by outer nuclear layer thickness decreases, as observed by optical coherence tomography and histology. The LD rats showed decreased rod- and cone-driven function with partial recovery 30 days after cessation of light exposure. In addition, LD rats showed decreased cone photoreceptor densities in the central retinal region compared to control rats. Our results demonstrate that the loss of cone-driven visual function induced by light damage is at least partially due to the d...
The full-field ERG is useful for index rod- or cone-mediated retinal function in rodent models of... more The full-field ERG is useful for index rod- or cone-mediated retinal function in rodent models of retinal degeneration. However, the relationship between the ERG response amplitudes and visually guided behavior, such as flicker detection, is not well understood. A comparison of ERG to behavioral responses in a light-damage model of retinal degeneration allows us to better understand the functional implications of electrophysiological changes. Flicker-ERG and behavioral responses to flicker were used to determine critical flicker frequency (CFF) under scotopic and photopic conditions before and up to 90 d after a 10-day period of low-intensity light damage. Dark- and light-adapted ERG flash responses were significantly reduced after light damage. The a-wave was permanently reduced, while the b-wave amplitude recovered over three weeks after light damage. There was a small, but significant dip in scotopic ERG CFF. Photopic behavioral CFF was slightly lower following light damage. The ...
Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal ... more Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycoprotein in the subretinal space bound by the photoreceptor (PR) outer segments and the processes of the retinal pigmented epithelium (RPE). IRBP binds retinoids, including 11-cis-retinal and all-trans-retinol. In this study, visual function for demanding visual tasks was assessed in IRBP knock-out (KO) mice. Surprisingly, IRBP KO mice showed no differences in scotopic critical flicker frequency (CFF) compared to wildtype (WT). However, they did have lower photopic CFF than WT. IRBP KO mice had reduced scotopic and photopic acuity and contrast sensitivity compared to WT. IRBP KO mice had a significant reduction in outer nuclear layer (ONL) thickness, PR outer and inner segment, and full retinal thickness (FRT) compared to WT. There were fewer cones in IRBP KO mice. Overall, these results confirm substantial loss of rods and significant loss of cones within 30 days. Absence of IRBP resulted in cone circuit damage, ...
Purpose: Desensitization in the rod cell of the mammalian retina is initiated when light-activate... more Purpose: Desensitization in the rod cell of the mammalian retina is initiated when light-activated rhodopsin is phosphorylated by the G protein-coupled receptor kinase (GRK), GRK1, often referred to as rhodopsin kinase. A distinct kinase that specifically phosphorylates cone opsins in a similar manner has not been identified in mammals. To determine the existence of a cone opsin kinase, RNA from the retinas of cone- and rod-dominant mammals was analyzed by PCR. Methods: RNA prepared from the retinas of two cone-dominant mammals, the thirteen-lined ground squirrel and the eastern chipmunk, and a rod-dominant mammal, the pig, was used to clone a new GRK family member by RT-PCR. The tissue distribution and localization of the kinase in retina were determined by Northern blot hybridization and in situ hybridization. The protein encoded by this cDNA was expressed in human embryonic kidney-293 (HEK-293) cells and compared with bovine GRK1 for its ability to phosphorylate bovine rhodopsin and to undergo autophosphorylation. Results: The cDNA cloned from ground squirrel contains an open reading frame encoding a 548 amino-acid protein. Sequence analysis indicates that this protein is orthologous to GRK7 recently cloned from O. latipes, the medaka fish. Partial cDNA fragments of GRK7 were also cloned from RNA prepared from eastern chipmunk and pig retinas. In situ hybridization demonstrated widespread labeling in the photoreceptor layer of the ground squirrel retina, consistent with expression in cones. Recombinant ground squirrel GRK7 phosphorylates bovine rhodopsin in a light-dependent manner and can be autophosphorylated, similar to bovine GRK1. Conclusions: These results indicate that cone- and rod-dominant mammals both express GRK7. The presence of this kinase in cones in the ground squirrel and its ability to phosphorylate rhodopsin suggests that it could function in cone cells as a cone opsin kinase.
1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied ... more 1. Visual transduction in photoreceptors of the ground squirrel, Citellus lateralis, was studied by recording membrane current from individual cones in small pieces of retina. 2. Brief flashes of light produced transient reductions of the dark current; saturating response amplitudes were up to 67 pA. A flash strength of about 11,000 photons microns‐2 at lambda max was required to give a half‐saturating response. The stimulus‐response relation was well fitted by an exponential saturation curve. Responses below 20% of maximum behaved linearly. 3. The response to a dim flash in most cells had a time to peak of 20‐30 ms and resembled the impulse response of a series of five low‐pass filters. 4. The variance of the dim‐flash response amplitude put an upper limit of 80 fA on the size of the single photon response. Estimates based on the effective collecting area suggest the single photon response to be of the order of 10 fA. 5. Flash responses of squirrel cones usually lacked the undersho...
Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that e... more Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by mutations in over 70 genes. While most mutations in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some mutations cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in mouse model harboring the most prevalent RP59-associated DHDDS mutation (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are shorter than in the corresponding tissues of age-matched controls, as repo...
Advances in experimental medicine and biology, 2018
A visual response to flickering light requires complex retinal computation, and thus ERG measures... more A visual response to flickering light requires complex retinal computation, and thus ERG measures are an excellent test of retinal circuit fidelity. Critical flicker frequency (CFF) is the frequency at which the retinal response is no longer modulated. Traditionally, CFF is obtained with a series of steady flicker stimuli with increasing frequencies. However, this method is slow and susceptible to experimental drift and/or adaptational effects. The current study compares the steady flicker method to CFF measurements obtained using a frequency sweep protocol. We introduce a light source programmed to produce a linear sweep of frequencies in a single trial. Using the traditional steady flicker method and a criterion response of 3 μV, we obtained a scotopic CFF of 18.4 ± 0.66 Hz and a photopic CFF of 44.4 ± 1.67 Hz. Our sweep flicker method, used on the same animals, produces a waveform best analyzed by Fourier transform; wherein a 6.18 log μV2 threshold was found to yield CFF values e...
We investigated the etiology of decreased cone-driven vision in a light damage (LD) model of reti... more We investigated the etiology of decreased cone-driven vision in a light damage (LD) model of retinal degeneration. To induce slow, moderate degeneration, albino rats underwent low-intensity light exposure for 10 days. Electroretinography was utilized to assess physiologic function of the rod- and cone-driven retinal function in LD and control rats. Immunohistochemistry targeting cone arrestin allowed for quantification of cone density and for comparison of the decline in function. Photoreceptor loss was quantified by outer nuclear layer thickness decreases, as observed by optical coherence tomography and histology. The LD rats showed decreased rod- and cone-driven function with partial recovery 30 days after cessation of light exposure. In addition, LD rats showed decreased cone photoreceptor densities in the central retinal region compared to control rats. Our results demonstrate that the loss of cone-driven visual function induced by light damage is at least partially due to the d...
The full-field ERG is useful for index rod- or cone-mediated retinal function in rodent models of... more The full-field ERG is useful for index rod- or cone-mediated retinal function in rodent models of retinal degeneration. However, the relationship between the ERG response amplitudes and visually guided behavior, such as flicker detection, is not well understood. A comparison of ERG to behavioral responses in a light-damage model of retinal degeneration allows us to better understand the functional implications of electrophysiological changes. Flicker-ERG and behavioral responses to flicker were used to determine critical flicker frequency (CFF) under scotopic and photopic conditions before and up to 90 d after a 10-day period of low-intensity light damage. Dark- and light-adapted ERG flash responses were significantly reduced after light damage. The a-wave was permanently reduced, while the b-wave amplitude recovered over three weeks after light damage. There was a small, but significant dip in scotopic ERG CFF. Photopic behavioral CFF was slightly lower following light damage. The ...
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Papers by Timothy Kraft