Effects of Astaxanthin, Lutein, and Zeaxanthin on Eye-Hand Coordination and Smooth-Pursuit Eye Movement after Visual Display Terminal Operation in Healthy Subjects: A Randomized, Double-Blind Placebo-Controlled Intergroup Trial.

(1) Background: The impairment of eye-hand coordination and smooth-pursuit eye movement caused by visual display terminal (VDT) operation is thought to impair daily living activities, for which no effective methods are currently known. On the other hand, various food ingredients, including astaxanthin, lutein, and zeaxanthin, are known to help improve the eye health of VDT operators. This study aimed to test the hypothesis that the combination of astaxanthin, lutein, and zeaxanthin can prevent the impairment of eye-hand coordination and smooth-pursuit eye movement caused by VDT operation. (2) Methods: We conducted a randomized, placebo-controlled, parallel-group clinical trial. Healthy subjects who regularly worked with VDTs were randomly assigned to the active and placebo groups. All of the subjects took soft capsules containing 6 mg of astaxanthin, 10 mg of lutein, and 2 mg of zeaxanthin or placebo soft capsules once daily for eight weeks. We evaluated the eye-hand coordination, smooth-pursuit eye movements, and macular pigment optical density (MPOD) at 0, two, four, and eight weeks after soft-capsule intake. (3) Results: The active group showed significantly improved eye-hand coordination after VDT operation at eight weeks. However, there was no clear improvement in the effect of the supplementation on smooth-pursuit eye movements. The active group also showed a significant increase in MPOD levels. (4) Conclusions: Consumption of a supplement containing astaxanthin, lutein, and zeaxanthin mitigates the decline of eye-hand coordination after VDT operation.

Mathematical analysis of phototransduction reaction parameters 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) 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 essential for inducing faster inactivation of VPs in rods, and possibly also in cones.

Ablation of Ctrp9, Ligand of AdipoR1, and Lower Number of Cone Photoreceptors in Mouse Retina.

Purpose: C1q/TNF-related protein (CTRP) 9 is one of the adiponectin paralogs, and a genetic ablation of its receptor, AdipoR1, is known to cause retinal degeneration. The purpose of this study was to determine the role played by CTRP9 in the retina. Methods: The retinas of Ctrp9 gene knockout (KO) and wild type (WT) mice were examined by electroretinography (ERG), histology, RNA sequencing, and quantitative real-time PCR. Results: The amplitude of the photopic ERG elicited by the maximum stimulus intensity was smaller by 40% in the Ctrp9 KO mice than in WT mice at 8 weeks of age. However, the photopic ERGs was not reduced from 8 weeks to 6 months of age. The amplitudes of the scotopic ERGs were not reduced in the Ctrp9 KO mice at 8 weeks and 6 months of age. No distinct histological abnormalities were found in the retinal sections but the density of peanut agglutinin-stained cells in the retinal flat mount of KO mice was reduced to about 70% of that of WT mice. Genomewide RNA sequencing of the retina revealed the absence of the expression of CTRP9 in both KO and WT mice. RNA sequencing and quantitative real-time PCR analysis showed that the expressions of the transcripts of genes expressed in cones, Opn1sw, Opn1mw, Gnat2, and Cnga3, were reduced in the KO mice retina, however, the degree of expression of the transcripts in rods was not significantly reduced. Conclusions: CTRP9 is released ectopically from other tissues, and it regulates the number of cones in the mouse retinas.

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.

Gene delivery to cone photoreceptors by subretinal injection of rAAV2/6 in the mouse retina.

Adeno-associated virus (AAV) has been studied as a safe delivery tool for gene therapy of retinal blinding diseases such as Leber's congenital amaurosis (LCA). The tropism of recombinant AAV (rAAV) including its specificity and efficiency in targeting retinal cell types has been studied with native or engineered capsids, along with specific promoters. However, one of the rAAV serotypes, rAAV2/6, has not been well-studied based on a report of low infection efficiency in the retina. We investigated the tropism of several rAAVs by subretinal injection in the adult mouse and found that rAAV2/6 predominantly infected cone photoreceptors including the main spectral type. Our data suggest that subretinal injection with rAAV2/6 may provide both an efficacious and specific means of gene delivery to cone photoreceptors in murine retinas.

Adeno Associated Virus (AAV) as a Tool for Clinical and Experimental Delivery of Target Genes into the Mammalian Retina.

With an increasing number of identified causative genes, the widespread use of gene therapy is quickly becoming feasible. Once a target gene is selected, it is important to have a cell delivery method that is both specific and efficient. Cell type specificity and high efficiency is particularly important for the treatment of retinal degeneration, since viruses are efficient gene delivery vehicles for the nervous system, but often bring with them non-specific infections. In this review, we focus on adeno-associated virus (AAV). Over the last few decades, AAV has become a leading choice for safe gene delivery, in part due to its replication deficiency in cells without a helper virus. Here, we summarize the tropism of recombinant AAV (rAAV) for various types of mammalian retinal neurons in relation to capsid serotype and administration method. We also include our recent findings on an AAV serotype that AAV was specifically infected mouse cone photoreceptors when delivered by subretinal administration.

Evaluation of critical flicker-fusion frequency measurement methods using a touchscreen-based visual temporal discrimination task in the behaving mouse.

The critical flicker-fusion frequency (CFF), defined as the frequency at which a flickering light is indistinguishable from a continuous light, is a useful measure of visual temporal resolution. The mouse CFF has been studied by electrophysiological approaches such as recordings of the electroretinogram (ERG) and the visually evoked potential (VEP), but it has not been measured behaviorally. Here we estimated the mouse CFF by using a touchscreen based operant system. The test with ascending series of frequencies and that with randomized frequencies resulted in about 17 and 14 Hz, respectively, as the frequency which could not be distinguished from steady lights. Since the ascending method of limits tend to overestimate the threshold than the descending method, we estimated the mouse CFF to be about 14 Hz. Our results highlight usefulness of the operant conditioning method in measurement of the mouse visual temporal resolution.

Different Activity Patterns in Retinal Ganglion Cells of TRPM1 and mGluR6 Knockout Mice.

TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamily, is the visual transduction channel downstream of metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells (BCs). Human TRPM1 mutations are associated with congenital stationary night blindness (CSNB). In both TRPM1 and mGluR6 KO mouse retinas, OFF but not ON BCs respond to light stimulation. Here we report an unexpected difference between TRPM1 knockout (KO) and mGluR6 KO mouse retinas. We used a multielectrode array (MEA) to record spiking in retinal ganglion cells (RGCs). We found spontaneous oscillations in TRPM1 KO retinas, but not in mGluR6 KO retinas. We performed a structural analysis on the synaptic terminals of rod ON BCs. Intriguingly, rod ON BC terminals were significantly smaller in TRPM1 KO retinas than in mGluR6 KO retinas. These data suggest that a deficiency of TRPM1, but not of mGluR6, in rod ON bipolar cells may affect synaptic terminal maturation. We speculate that impaired signaling between rod BCs and AII amacrine cells (ACs) leads to spontaneous oscillations. TRPM1 and mGluR6 are both essential components in the signaling pathway from photoreceptors to ON BC dendrites, yet they differ in their effects on the BC terminal and postsynaptic circuitry.

[Electrophysiological Analysis of Retinal Oscillation in Normal and Degenerated Retina].

　The vertebrate retina is one of the most sophisticated parts of the nervous system. It comprises five classes of neurons and one glial type cell. During development, but prior to a vertebrate's eyes opening, retinal circuits are refined by endogenous neural activity. Characteristic patterns of activity, including oscillatory activity, occur in the normal retina, whereas distinctive alternative patterns occur in abnormal retinas. In this paper, we first describe the electrophysiological and spike sorting methods used to study retinal oscillations. Next, we describe the mechanisms and functions of oscillation in the normal retina. Finally, we characterize the distinctive oscillations and abnormal spontaneous activities in the degenerative retina.

[A Potential Mechanism for Spontaneous Oscillation in the Abnormal Retina].

　Rhythmic neural activities are observed in many brain regions, and these are considered to play an important role in neural information processing. On the other hand, distinct rhythmic neural activities emerge under several pathological conditions, suggesting that rhythmic neural activity has a close relation to brain function and dysfunction. In many pathological cases, the intrinsic property of unusual rhythm generation in a neuron or a neuronal network is prevented under normal conditions, but released by the pathological condition. Therefore, it may be useful to explore which conditions determine rhythm generation in order to understand the mechanisms of brain function/dysfunction. The pathological retina in retinal degeneration exhibits rhythmic neural activity not observed in the healthy retina. In this review, we first provide a brief introduction to the possible mechanisms of rhythm generation in a neural system. Then we introduce experimental evidence of rhythm generation in the pathological retina, as well as two hypotheses regarding this mechanism. Finally, we raise several issues to be solved for the further understanding of pathological rhythm generation.

Expression of Quaking RNA-Binding Protein in the Adult and Developing Mouse Retina.

Quaking (QKI), which belongs to the STAR family of KH domain-containing RNA-binding proteins, functions in pre-mRNA splicing, microRNA regulation, and formation of circular RNA. QKI plays critical roles in myelinogenesis in the central and peripheral nervous systems and has been implicated neuron-glia fate decision in the brain; however, neither the expression nor function of QKI in the neural retina is known. Here we report the expression of QKI RNA-binding protein in the developing and mature mouse retina. QKI was strongly expressed by Müller glial cells in both the developing and adult retina. Intriguingly, during development, QKI was expressed in early differentiating neurons, such as the horizontal and amacrine cells, and subsequently in later differentiating bipolar cells, but not in photoreceptors. Neuronal expression was uniformly weak in the adult. Among QKI isoforms (5, 6, and 7), QKI-5 was the predominantly expressed isoform in the adult retina. To study the function of QKI in the mouse retina, we examined quakingviable(qkv) mice, which have a dysmyelination phenotype that results from deficiency of QKI expression and reduced numbers of mature oligodendrocytes. In homozygous qkv mutant mice (qkv/qkv), the optic nerve expression levels of QKI-6 and 7, but not QKI-5 were reduced. In the retina of the mutant homozygote, QKI-5 levels were unchanged, and QKI-6 and 7 levels, already low, were also unaffected. We conclude that QKI is expressed in developing and adult Müller glia. QKI is additionally expressed in progenitors and in differentiating neurons during retinal development, but expression weakened or diminished during maturation. Among QKI isoforms, we found that QKI-5 predominated in the adult mouse retina. Since Müller glial cells are thought to share properties with retinal progenitor cells, our data suggest that QKI may contribute to maintaining retinal progenitors prior to differentiation into neurons. On the other hand, the expression of QKI in different retinal neurons may suggest a role in neuronal cell type specific fate determination and maturation. The data raises the possibility that QKI may function in retinal cell fate determination and maturation in both glia and neurons.

Reproducing retinal rod bipolar cell light response by mathematical model including neurotransmitter receptors.

Detailed mathematical model of retinal cells is useful for the quantitative understanding of the subcellular processes of the visual system. Retinal bipolar cells receive information from photoreceptor cells, horizontal cells and amacrine cells, thus it can be considered as information integration system. Despite its importance, bipolar cell model including inputs and outputs has not been proposed. In this paper, we propose a rod bipolar cell model which can reproduce voltage response of light. The model includes TRPM1 channel which receives signal from photoreceptor cells, GABA channel which receives signal from surrounding amacrine cells, and cell body model which is based on the model proposed by Ishihara et al. The model was evaluated with several light signals, where experimentally obtained photoreceptor cell responses were used as TRPM1 channel input. Resulting bipolar cell membrane potential showed good agreement with the reported experimental results.

Depolarizing bipolar cell dysfunction due to a Trpm1 point mutation.

Mutations in TRPM1 are found in humans with an autosomal recessive form of complete congenital stationary night blindness (cCSNB). The Trpm1(-/-) mouse has been an important animal model for this condition. Here we report a new mouse mutant, tvrm27, identified in a chemical mutagenesis screen. Genetic mapping of the no b-wave electroretinogram (ERG) phenotype of tvrm27 localized the mutation to a chromosomal region that included Trpm1. Complementation testing with Trpm1(-/-) mice confirmed a mutation in Trpm1. Sequencing identified a nucleotide change in exon 23, converting a highly conserved alanine within the pore domain to threonine (p.A1068T). Consistent with prior studies of Trpm1(-/-) mice, no anatomical changes were noted in the Trpm1(tvrm27/tvrm27) retina. The Trpm1(tvrm27/tvrm27) phenotype is distinguished from that of Trpm1(-/-) by the retention of TRPM1 expression on the dendritic tips of depolarizing bipolar cells (DBCs). While ERG b-wave amplitudes of Trpm1(+/-) heterozygotes are comparable to wild type, those of Trpm1(+/tvrm27) mice are reduced by 32%. A similar reduction in the response of Trpm1(+/tvrm27) DBCs to LY341495 or capsaicin is evident in whole cell recordings. These data indicate that the p.A1068T mutant TRPM1 acts as a dominant negative with respect to TRPM1 channel function. Furthermore, these data indicate that the number of functional TRPM1 channels at the DBC dendritic tips is a key factor in defining DBC response amplitude. The Trpm1(tvrm27/tvrm27) mutant will be useful for elucidating the role of TRPM1 in DBC signal transduction, for determining how Trpm1 mutations impact central visual processing, and for evaluating experimental therapies for cCSNB.

mGluR6 deletion renders the TRPM1 channel in retina inactive.

In darkness, glutamate released from photoreceptors activates the metabotropic glutamate receptor 6 (mGluR6) on retinal ON bipolar cells. This activates the G protein G(o), which then closes transient receptor potential melastatin 1 (TRPM1) channels, leading to cells' hyperpolarization. It has been generally assumed that deleting mGluR6 would render the cascade inactive and the ON bipolar cells constitutively depolarized. Here we show that the rod bipolar cells in mGluR6-null mice were hyperpolarized. The slope conductance of the current-voltage curves and the current noise were smaller than in wild type. Furthermore, while in wild-type rod bipolar cells, TRPM1 could be activated by local application of capsaicin; in null cells, it did not. These results suggest that the TRPM1 channel in mGluR6-null rod bipolar cells is inactive. To explore the reason for this lack of activity, we tested if mGluR6 deletion affected expression of cascade components. Immunostaining for G protein subunit candidates Gα(o), Gß(3), and Gγ(13) showed no significant changes in their expression or distribution. Immunostaining for TRPM1 in the dendritic tips was greatly reduced, but the channel was still present in the soma and primary dendrites of mGluR6-null bipolar cells, where a certain fraction of TRPM1 appears to localize to the plasma membrane. Consequently, the lack of TRPM1 activity in the null retina is unlikely to be due to failure of the channels to localize to the plasma membrane. We speculate that, to be constitutively active, TRPM1 channels in ON bipolar cells have to be in a complex, or perhaps require an unidentified factor.

miR-124a is required for hippocampal axogenesis and retinal cone survival through Lhx2 suppression.

MicroRNA-124a (miR-124a) is the most abundant microRNA expressed in the vertebrate CNS. Despite past investigations into the role of miR-124a, inconsistent results have left the in vivo function of miR-124a unclear. We examined the in vivo function of miR-124a by targeted disruption of Rncr3 (retinal non-coding RNA 3), the dominant source of miR-124a. Rncr3(-/-) mice exhibited abnormalities in the CNS, including small brain size, axonal mis-sprouting of dentate gyrus granule cells and retinal cone cell death. We found that Lhx2 is an in vivo target mRNA of miR-124a. We also observed that LHX2 downregulation by miR-124a is required for the prevention of apoptosis in the developing retina and proper axonal development of hippocampal neurons. These results suggest that miR-124a is essential for the maturation and survival of dentate gyrus neurons and retinal cones, as it represses Lhx2 translation.

A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites.

Expression of channels to specific neuronal sites can critically impact their function and regulation. Currently, the molecular mechanisms underlying this targeting and intracellular trafficking of transient receptor potential (TRP) channels remain poorly understood, and identifying proteins involved in these processes will provide insight into underlying mechanisms. Vision is dependent on the normal function of retinal depolarizing bipolar cells (DBCs), which couple a metabotropic glutamate receptor 6 to the TRP melastatin 1 (TRPM1) channel to transmit signals from photoreceptors. We report that the extracellular membrane-attached protein nyctalopin is required for the normal expression of TRPM1 on the dendrites of DBCs in mus musculus. Biochemical and genetic data indicate that nyctalopin and TRPM1 interact directly, suggesting that nyctalopin is acting as an accessory TRP channel subunit critical for proper channel localization to the synapse.

TRPM1: a vertebrate TRP channel responsible for retinal ON bipolar function.

The transient receptor potential (TRP) channels affect essential functions widely in sensory systems of various species, both invertebrates and vertebrates. The channel protein encoded by the trp gene, the first identified TRP superfamily molecule, is known to mediate the Drosophila light response. A vertebrate TRP channel playing a crucial role in the visual system has not yet been discovered, although numerous studies have revealed primal functions of TRP superfamily molecules in various sensory systems other than vision. In the retina, which is the entry tissue in the vertebrate visual pathway, the transduction cation channel in ON bipolar cells has been elusive, despite intensive investigation by many researchers over a long period of time. Recent studies finally revealed that TRPM1, the first member of the melanoma-related transient receptor potential (TRPM) subfamily to be discovered, is a visual transduction channel in retinal ON bipolar cells. This review covers the significant discoveries on the physiological function and regulatory mechanism of the TRPM1 channel in retinal ON bipolar cells and the association of human TRPM1 mutations with congenital stationary night blindness.

TRPM1 mutations are associated with the complete form of congenital stationary night blindness.

PURPOSE: To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations in patients with congenital stationary night blindness (CSNB). METHODS: We analyzed four different Japanese patients with complete CSNB in whom previous molecular examination revealed no mutation in either nyctalopin (NYX) or glutamate receptor, metabotropic 6 (GRM6). The ophthalmologic examination included best-corrected visual acuity, refraction, biomicroscopy, ophthalmoscopy, fundus photography, Goldmann kinetic perimetry, color vision tests, and electroretinography (ERG). Exons 2 through 27 and the exon-intron junction regions of human TRPM1 were sequenced. RESULTS: Five different mutations in human TRPM1 were identified. Mutations were present in three unrelated patients with complete CSNB. All three patients were compound heterozygotes. Fundus examination revealed no abnormalities other than myopic changes, and the single bright-flash, mixed rod-cone ERG showed a "negative-type" configuration with a reduced normal a-wave and a significantly reduced b-wave amplitude. Our biochemical and cell biologic analyses suggest that the two identified IVS mutations lead to abnormal TRPM1 protein production, and imply that the two identified missense mutations lead to the mislocalization of the TRPM1 protein in bipolar cells (BCs). CONCLUSIONS: Human TRPM1 mutations are associated with the complete form of CSNB in Japanese patients, suggesting that TRPM1 plays an essential role in mediating the photoresponse in ON BCs in humans as well as in mice.

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade.

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.