Mathematical Analysis of Electrical Responses in Photoreceptor Cells Reveals Distinct Chemical Control of Visual Signal Transduction in Rods and Cones.

Retinal photoreceptor cells, rods and cones, convert photons of light into chemical and electrical signals as the first step of the visual transduction cascade. Although the chemical processes in the phototransduction system are very similar to each other in these photoreceptors, the light sensitivity and time resolution of the photoresponse in rods are functionally different than those in the photoresponses of cones. To systematically investigate how photoresponses are divergently regulated in rods and cones, we have developed a detailed mathematical model on the basis of the Hamer model. The current model successfully reconstructed light intensity-, ATP- and GTP-dependent changes in concentrations of phosphorylated visual pigments (VPs), activated transducins (Tr*s) and phosphodiesterases (PDEs), as well as cyclic nucleotide-gated currents (ICNG) in rods and cones. In comparison to rods, the lower light sensitivity of cones was attributed not only to the lower affinity of activated VPs for Trs but also to the faster desensitization of the VPs. The assumption of an intermediate inactive state, MIIi, in the thermal decay of activated VPs was pivotal for inducing faster inactivation of VPs. In addition to the faster inactivation of VPs, calculating a faster rate of RGS9 intervention for PDE-induced Tr* inactivation in cones was indispensable for simulating the electrical waveforms of the light intensity-dependent ICNG at higher temporal resolution in experimental systems in vivo.

Circadian regulation of vertebrate cone photoreceptor function

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as Recoverins, Arrestins, Opsin kinases, and Regulator of G-protein signaling that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm-dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. Functional rhythmicity persists in continuous darkness, and it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.

Abstract P-17: Photophysical Properties of Freely Diffusing and Immobilized Fluorescent Conjugate Based on Calcium-Binding Protein Recoverin and Alexa647 Dye

Background: Recoverin is a calcium sensor membrane-associated protein that inhibits rhodopsin kinase thereby participating in the regulation of visual transduction. Here we examined calcium-induced conformational changes in recoverin conjugated with fluorescent dye Alexa647. Methods: Photophysical properties of immobilized and freely diffusing recoverin were investigated using fluorescence lifetime imaging microscopy and fluorescence emission spectroscopy. In solution, the formation and dissociation of the Ca2+-recoverin complex manifested as changes in Alexa647 spectra and the lifetime. In contrast, immobilization of recoverin on the microscopy glass via biotin–NeutrAvidin–biotinylated polyethylene glycol (PEG) tether inhibited changes in fluorescent signal. That can be provided by PEG as it prevented the calcium-induced changes in spectrum and lifetime of recoverin-bound Alexa647 in solution. The use of another immobilization facilitator, bovine serum albumin (BSA), did not affect calcium-induced changes in fluorescence of the conjugate in solution but produced the matrix, which was ineffective in recoverin immobilization. Results: Microscale thermophoresis demonstrated that biotinylated recoverin interacted with NeutrAvidin in solution indicating that immobilization affinity depended mainly on the geometry of the glass coating surface. Conclusion: Our results highlight the challenge of specific protein immobilization that does not affect protein functionality. By the example of recoverin, we showed that the employment of two common immobilization facilitators, PEG and BSA, yielded surfaces with different space geometry, which differently affect NeutrAvidin-based immobilization affinity as well as Ca2+-dependent conformational changes of the biotinylated protein.

Circadian Regulation of Vertebrate Cone Photoreceptor Function

Eukaryotes generally display a circadian rhythm as an adaption to the reoccurring day/night cycle. This is particularly true for visual physiology that is directly affected by changing light conditions. Here we investigate the influence of the circadian rhythm on the expression and function of visual transduction cascade regulators in diurnal zebrafish and nocturnal mice. We focused on regulators of shut-off kinetics such as recoverins, arrestins, opsin kinases, and GTPase-accelerating protein that have direct effects on temporal vision. Transcript as well as protein levels of most analyzed genes show a robust circadian rhythm dependent regulation, which correlates with changes in photoresponse kinetics. Electroretinography demonstrates that photoresponse recovery in zebrafish is delayed in the evening and accelerated in the morning. This physiological rhythmicity is mirrored in visual behaviors, such as optokinetic and optomotor responses. Functional rhythmicity persists in continuous darkness, it is reversed by an inverted light cycle and disrupted by constant light. This is in line with our finding that orthologous gene transcripts from diurnal zebrafish and nocturnal mice are often expressed in an anti-phasic daily rhythm.

Mice with mutations in Trpm1, a gene in the locus of 15q13.3 microdeletion syndrome, display pronounced hyperactivity and decreased anxiety-like behavior

The 15q13.3 microdeletion syndrome is a genetic disorder characterized by a wide spectrum of psychiatric disorders that is caused by the deletion of a region containing 7 genes on chromosome 15 (MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7). The contribution of each gene in this syndrome has been studied using mutant mouse models, but no single mouse model recapitulates the whole spectrum of human 15q13.3 microdeletion syndrome. The behavior of Trpm1−/− mice has not been investigated in relation to 15q13.3 microdeletion syndrome due to the visual impairment in these mice, which may confound the results of behavioral tests involving vision. We were able to perform a comprehensive behavioral test battery using Trpm1 null mutant mice to investigate the role of Trpm1, which is thought to be expressed solely in the retina, in the central nervous system and to examine the relationship between TRPM1 and 15q13.3 microdeletion syndrome. Our data demonstrate that Trpm1−/− mice exhibit abnormal behaviors that may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduced anxiety-like behavior, abnormal social interaction, attenuated fear memory, and the most prominent phenotype of Trpm1 mutant mice, hyperactivity. While the ON visual transduction pathway is impaired in Trpm1−/− mice, we did not detect compensatory high sensitivities for other sensory modalities. The pathway for visual impairment is the same between Trpm1−/− mice and mGluR6−/− mice, but hyperlocomotor activity has not been reported in mGluR6−/− mice. These data suggest that the phenotype of Trpm1−/− mice extends beyond that expected from visual impairment alone. Here, we provide the first evidence associating TRPM1 with impairment of cognitive function similar to that observed in phenotypes of 15q13.3 microdeletion syndrome.

Biochemistry and physiology of zebrafish photoreceptors

All vertebrates share a canonical retina with light-sensitive photoreceptors in the outer retina. These photoreceptors are of two kinds: rods and cones, adapted to low and bright light conditions, respectively. They both show a peculiar morphology, with long outer segments, comprised of ordered stacks of disc-shaped membranes. These discs host numerous proteins, many of which contribute to the visual transduction cascade. This pathway converts the light stimulus into a biological signal, ultimately modulating synaptic transmission. Recently, the zebrafish (Danio rerio) has gained popularity for studying the function of vertebrate photoreceptors. In this review, we introduce this model system and its contribution to our understanding of photoreception with a focus on the cone visual transduction cascade.

Mice with mutations in Trpm1, a gene within the locus of 15q13.3 microdeletion syndrome, display pronounced hyperactivity and decreased anxiety-like behavior.

15q13.3 microdeletion syndrome is a genetic disorder caused by a deletion of a region containing seven genes on chromosome 15, MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7, and characterized by a wide spectrum of psychiatric disorders. The contribution of each gene in this syndrome has been studied using mutant mouse models, but no single mouse model recapitulates the whole spectrum of human 15q13.3 microdeletion syndrome. The behavior of Trpm1−/− mice with relation to 15q13.3 microdeletion syndrome has not been investigated due to the visual impairment in these mice, which may confound the results of behavioral tests that involve vision. We have now performed a comprehensive behavioral test battery in Trpm1 null mutant mice to demonstrate the role of Trpm1, which is thought to be solely expressed in the retina, in central nervous system and to examine the relationship of TRPM1 and 15q13.3 microdeletion syndrome. Our data indicate abnormal behavior of Trpm1−/− mice which may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduction of anxiety-like behavior, abnormality of social interaction, attenuation in fear memory, and hyperactivity, which is the most prominent phenotype of Trpm1 mutant mice. While the ON visual transduction pathway is impaired in Trpm1−/− mice, we did not detect compensatory high sensitivities for other sensory modalities. Although Trpm1−/− mice share the same pathway for visual impairment with mGluR6-/- mice, hyperlocomotor activity has not been reported in mGluR6-/- mice. These data suggest that the phenotype of Trpm1−/− mice extends beyond that expected from visual impairment alone. This is the first evidence to associate TRPM1 with impairment of cognitive function similar to that found in the phenotypes of 15q13.3 microdeletion syndrome.

Mice with Mutations in Trpm1, a Gene within the locus of 15q13.3 Microdeletion Syndrome, Display Pronounced Hyperactivity and Decreased Anxiety-Like Behavior.

15q13.3 microdeletion syndrome is a genetic disorder caused by a deletion of a region containing seven genes on chromosome 15, MTMR10, FAN1, TRPM1, MIR211, KLF13, OTUD7A, and CHRNA7, and characterized by a wide spectrum of psychiatric disorders. The contribution of each gene in this syndrome has been studied using mutant mouse models, but the phenotypes of these mice do not account for human phenotypes and the results are still controversial. The behavior of Trpm1−/− mice with relation to 15q13.3 microdeletion syndrome has not been investigated due to the visual impairment in these mice, which may confound the results of behavior tests that involve vision. We have now applied a comprehensive behavioral test battery to examine the relationship of TRPM1 and 15q13.3 microdeletion syndrome by using Trpm1 null mutant mice. Our data indicate abnormal behavior of Trpm1−/− mice which may explain some phenotypes of 15q13.3 microdeletion syndrome, including reduction of anxiety behavior, abnormality of social interaction, attenuation in fear memory, and hyperactivity, which is the most prominent phenotype of Trpm1 mutant mice. While the ON visual transduction pathway is impared in Trpm1−/− mice, we did not detect compensatory high sensitivities for other sensory modalities. Although Trpm1−/− mice share the same pathway for visual impairment with mGluR6−/− mice, hyperlocomotion activity has not been reported in mGluR6−/− mice. These data suggest that the phenotype of Trpm1−/− mice extends beyond that expected from visual impairment alone. This is the first evidence to associate TRPM1 with impairment of cognitive function similar to that found in the phenotypes of 15q13.3 microdeletion syndrome.