Centrins control chicken cone cell lipid droplet dynamics through lipid-droplet-localized SPDL1.

As evolutionarily conserved organelles, lipid droplets (LDs) carry out numerous functions and have various subcellular localizations in different cell types and species. In avian cone cells, there is a single apically localized LD. We demonstrated that CIDEA (cell death inducing DFFA like effector a) and microtubules promote the formation of the single LD in chicken cone cells. Centrins, which are well-known centriole proteins, target to the cone cell LD via their C-terminal calcium-binding domains. Centrins localize on cone cell LDs with the help of SPDL1-L (spindle apparatus coiled-coil protein 1-L), a previously uncharacterized isoform of the kinetochore-associated dynein adaptor SPDL1. The loss of CETN3 or overexpression of a truncated CETN1 abrogates the apical localization of the cone cell LD. Simulation analysis showed that multiple LDs or a single mispositioned LD reduces the light sensitivity. Collectively, our findings identify a role of centrins in the regulation of cone cell LD localization, which is important for the light sensitivity of cone cells.

Molecular bases of rod and cone differences.

In the vertebrate retina, rods and cones both detect light, but they differ in functional aspects such as light sensitivity and temporal resolution, and in some cell biological aspects. For functional aspects, both types of photoreceptors use a phototransduction cascade, consisting of a series of enzymatic reactions, to convert photon capture to an electrical signal. To understand the mechanisms underlying the functional differences between rods and cones at the molecular level, we compared biochemically, each of the reactions in the phototransduction cascades of rods and cones using the cells isolated and purified from carp retina. Although the cascade proteins are identical or are functionally similar between rods and cones, their activities together with their expression levels are mostly different. In general, reactions that generate a response are somewhat less effective in cones than in rods, but each of the reactions for termination and recovery of a response are much more effective in cones. These findings explain lower light sensitivity and briefer light responses in cones than in rods. In addition, our considerations suggest that a Ca2+-binding protein, S-modulin or recoverin, has a currently unnoticed role in shaping light responses. Upon comparison of the expression levels of proteins and/or mRNAs using purified cells, several proteins were found to be specifically or predominantly expressed in cones. These proteins will be of interest in future studies aimed at characterizing the differences between rods and cones.

PACAP Induces Light Aversion in Mice by an Inheritable Mechanism Independent of CGRP.

The neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) have emerged as mediators of migraine, yet the potential overlap of their mechanisms remains unknown. Infusion of PACAP, like CGRP, can cause migraine in people, and both peptides share similar vasodilatory and nociceptive functions. In this study, we have used light aversion in mice as a surrogate for migraine-like photophobia to compare CGRP and PACAP and ask whether CGRP or PACAP actions were dependent on each other. Similar to CGRP, PACAP induced light aversion in outbred CD-1 mice. The light aversion was accompanied by increased resting in the dark, but not anxiety in a light-independent open field assay. Unexpectedly, about one-third of the CD-1 mice did not respond to PACAP, which was not seen with CGRP. The responder and nonresponder phenotypes were stable, inheritable, and not sex linked, although there was a trend for greater responses among male mice. RNA-sequencing analysis of trigeminal ganglia yielded hierarchical clustering of responder and nonresponder mice and revealed a number of candidate genes, including greater expression of the Trpc5 and Kcnk12 ion channels and glycoprotein hormones and receptors in a subset of male responder mice. Importantly, an anti-PACAP monoclonal antibody could block PACAP-induced light aversion but not CGRP-induced light aversion. Conversely, an anti-CGRP antibody could not block PACAP-induced light aversion. Thus, we propose that CGRP and PACAP act by independent convergent pathways that cause a migraine-like symptom in mice.SIGNIFICANCE STATEMENT The relationship between the neuropeptides CGRP (calcitonin gene-related peptide) and PACAP (pituitary adenylate cyclase-activating polypeptide) in migraine is relevant given that both peptides can induce migraine in people, yet to date only drugs that target CGRP are available. Using an outbred strain of mice, we were able to show that most, but not all, mice respond to PACAP in a preclinical photophobia assay. Our finding that CGRP and PACAP monoclonal antibodies do not cross-inhibit the other peptide indicates that CGRP and PACAP actions are independent and suggests that PACAP-targeted drugs may be effective in patients who do not respond to CGRP-based therapeutics.

Selective amplification of ipRGC signals accounts for interictal photophobia in migraine.

Second only to headache, photophobia is the most debilitating symptom reported by people with migraine. While the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) are thought to play a role, how cone and melanopsin signals are integrated in this pathway to produce visual discomfort is poorly understood. We studied 60 people: 20 without headache and 20 each with interictal photophobia from migraine with or without visual aura. Participants viewed pulses of spectral change that selectively targeted melanopsin, the cones, or both and rated the degree of visual discomfort produced by these stimuli while we recorded pupil responses. We examined the data within a model that describes how cone and melanopsin signals are weighted and combined at the level of the retina and how this combined signal is transformed into a rating of discomfort or pupil response. Our results indicate that people with migraine do not differ from headache-free controls in the manner in which melanopsin and cone signals are combined. Instead, people with migraine demonstrate an enhanced response to integrated ipRGC signals for discomfort. This effect of migraine is selective for ratings of visual discomfort, in that an enhancement of pupil responses was not seen in the migraine group, nor were group differences found in surveys of other behaviors putatively linked to ipRGC function (chronotype, seasonal sensitivity, presence of a photic sneeze reflex). By revealing a dissociation in the amplification of discomfort vs. pupil response, our findings suggest a postretinal alteration in processing of ipRGC signals for photophobia in migraine.

Blue light activates pulvinar nuclei in longstanding idiopathic photophobia: A case report.

Numerous pathologies can contribute to photophobia. When considering light transduction alone, photophobia may be triggered through melanopsin pathways (non-image forming), rod and cone pathways (image-forming), or some combination of the two. We evaluated a 39 year old female patient with longstanding idiopathic photophobia that was exacerbated by blue light, and tested her by presenting visual stimuli in an event-related fMRI experiment. Analysis showed significantly greater activation in bilateral pulvinar nuclei, associated with the melanopsin intrinsically photosensitive retinal ganglion cell (ipRGC) visual pathway, and their activation is consistent with the patient's report that blue light differentially evoked photophobia. This appears to be the first demonstration of functional activation of the ipRGC pathway during photophobia in a patient.

Melanopsin-expressing retinal ganglion cells in aging and disease.

Melanopsin-expressing retinal ganglion cells (mRGCs) constitute a system in the mammalian retina used for irradiance detection, regulating non-image forming functions, such as photoentrainment of circadian rhythms, control of the pupillary light reflex, masking response, light-regulated melatonin secretion, and modulation of the sleep/wake cycle. There are five subtypes of mRGCs differentiated by morphology and function. Recent years of research on mRGCs have identified a broad number of neurodegenerative diseases in the eye and the brain with altered physiologic light responses, leading to disturbances of non-image forming light response(s). In this review, we briefly summarise the melanopsin system in the normal retina and discuss its role in connection to human aging (sleep/wake problems) and retinal pathology in Alzheimer and Parkinson diseases, diabetic retinopathy, mitochondrial optic neuropathies, glaucoma, retinitis pigmentosa, and in photophobia during migraine and in seasonal affective disorder (SAD). Finally, we discuss the diagnostic tools that are being used to differentiate retinal diseases involving the melanopsin system in the rods and cones from the inner versus the outer retina.

Photophobia and sensations of dryness in patients with migraine occur independent of baseline tear volume and improve following botulinum toxin A injections.

BACKGROUND: To evaluate the efficacy of botulinum toxin A (BoNT-A) in reducing photophobia and dry eye symptoms in individuals with chronic migraine. Additionally, we aimed to evaluate tear film volume as a potential contributor to symptoms in these patients. METHODS: Retrospective review of 76 patients who received BoNT-A for chronic migraine between 23 August 2017 and 13 December 2017 at the Miami Veterans Affairs Medical Center Neurotoxin Clinic. Demographic data and all comorbidities were queried via chart review. Standardised validated surveys were administered to assess symptoms prior to and after BoNT-A injection. Preinjection tear volumes were obtained using the phenol red thread (PRT) test. RESULTS: Preinjection migraine, photophobia and dry eye symptom scores were all significantly correlated, p<0.05, and none were associated with preinjection PRT results. After BoNT-A, improvements in migraine, photophobia and dry eye symptoms were also significantly correlated, p<0.05 and similarly did not associate with preinjection PRT results. Photophobia scores significantly improved following BoNT-A, while dry eye symptoms significantly improved in those with severe symptoms at baseline (DEQ-5 score ≥12), p=0.027. In logistic regression analysis of all individuals with dry eye symptoms (DEQ-5 ≥6), individuals with more severe dry eye symptoms were more likely improve, OR 1.27, 95% CI 1.06 to 1.51, p<0.01. CONCLUSIONS: BoNT-A significantly improved photophobia in patients being treated for migraine and also improved dry eye symptoms in patients with severe symptoms at baseline, independent of baseline tear film volume. These improvements may be due to modulation of shared trigeminal neural pathways.

Impact of long-term daylight deprivation on retinal light sensitivity, circadian rhythms and sleep during the Antarctic winter.

Long-term daylight deprivation such as during the Antarctic winter has been shown to lead to delayed sleep timing and sleep fragmentation. We aimed at testing whether retinal sensitivity, sleep and circadian rest-activity will change during long-term daylight deprivation on two Antarctic bases (Concordia and Halley VI) in a total of 25 healthy crew members (mean age: 34 ± 11y; 7f). The pupil responses to different light stimuli were used to assess retinal sensitivity changes. Rest-activity cycles were continuously monitored by activity watches. Overall, our data showed increased pupil responses under scotopic (mainly rod-dependent), photopic (mainly L-/M-cone dependent) as well as bright-blue light (mainly melanopsin-dependent) conditions during the time without direct sunlight. Circadian rhythm analysis revealed a significant decay of intra-daily stability, indicating more fragmented rest-activity rhythms during the dark period. Sleep and wake times (as assessed from rest-activity recordings) were significantly delayed after the first month without sunlight (p < 0.05). Our results suggest that during long-term daylight deprivation, retinal sensitivity to blue light increases, whereas circadian rhythm stability decreases and sleep-wake timing is delayed.

Clinical implications of the melanopsin-based non-image-forming visual system.

Since the discovery of the non-image-forming visual system, tremendous research efforts have been dedicated to understanding its mechanisms and functional roles. Original functions associated with the melanopsin system include the photoentrainment of circadian sleep-wake cycles and the pupillary light reflex. Recent findings, however, suggest a much broader involvement of this system in an array of physiologic responses to light. This newfound insight into the underlying function of the non-image-forming system has revealed the many connections to human pathology and attendant disease states, including seasonal affective disorder, migraine, glaucoma, inherited mitochondrial optic neuropathy, and sleep dysregulation of aging. In this review, the authors discuss in detail the clinical implications of the melanopsin system.

Fine-tuning light sensitivity in the starry flounder (Platichthys stellatus) retina: Regional variation in photoreceptor cell morphology and opsin gene expression.

Vertebrate color vision relies on the differential expression of visual pigment proteins (opsins) in cone photoreceptors of the retina. The diversity of opsins and their retinal expression patterns appear greatest for animals that experience variable light habitats, as is the case with flatfishes. Yet, opsin repertoires and expression patterns in this group of fishes are poorly described. Here, we unveil the visual opsin expression patterns of juvenile starry flounder (Platichthys stellatus) and describe the localization of cone types, their visual pigments and opsin expression. Juvenile starry flounder express eight opsins (Rh1, Sws1, Sws2A1, Sws2A2, Sws2B, Rh2A1, Rh2A2, Lws) and possess a corresponding number of photoreceptor visual pigments, with peak absorbance ranging from 369 to 557 nm. Retinal (vitamin A1) was the only chromophore detected in the retina. Intraretinal variation in opsin abundance consisted of greater expression of both RH2, and lesser expression of SWS1 and both SWS2A, opsin transcripts in the dorsal compared to the ventral retina. Overall cone density was greater in the dorsal retina which was also characterized by a larger proportion of unequal double cones compared with the ventral retina. Together, our results suggest that large opsin repertoires serve to optimize visual function under variable light environments by differential expression of opsin subsets with retinal location.

Lysosomal Trafficking Regulator (LYST).

Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections.

Measurement of visual sensitivity in migraine: Validation of two scales and correlation with visual cortex activation.

OBJECTIVE: The objectives of this article are to compare interictal and ictal visual sensitivity between migraine and controls using two published questionnaires, and to correlate responses with a physiologic measure of visual cortex activation. METHODS: Migraine with (MWA, n = 51) and without (MwoA, n = 45) aura and control individuals (n = 45) were enrolled and underwent BOLD fMRI with a visual stimulus. The visual discomfort score (VDS) assessed interictal and the migraine photophobia score (MPS) assessed ictal visual sensitivity. RESULT: VDS was significantly higher both in MWA and MwoA vs controls (both p = 0.0001). MPS was greater in MWA vs MwoA (p = 0.008). Ictal and interictal visual sensitivity strongly correlated in MWA (p = 0.004) but not MwoA patients (p = 0.12). BOLD activation in visual cortex was greater in MWA vs controls (2.7% vs 2.3%, p = 0.003) but similar between MwoA and controls. Increasing VDS was associated with greater BOLD signal change in MWA (p = 0.03) but not MwoA (p = 0.65) or controls (p = 0.53). MPS did not correlate with BOLD activation in either group. CONCLUSION: Increased interictal visual sensitivity is present both in MWA and MwoA. However, the correlation with ictal visual sensitivity and with cortical hyper-responsivity varies between MWA and MwoA, suggesting underlying differences between groups.

Photophobia and abnormally sustained pupil responses in a mouse model of bradyopsia.

PURPOSE: Mutations in the RGS9 gene cause the visual disorder bradyopsia, which includes difficulty adapting to changes in light and photophobia. The purpose of this study was to determine whether lack of Rgs9 also caused photophobia-like behavior in Rgs9 knockout (Rgs9-/-) mice and to identify useful diagnostic measures of Rgs9 dysfunction. METHODS: We measured two behavioral responses to light and the pupillary light reflex to determine the form and basis of photophobia in Rgs9-/- mice. RESULTS: Rgs9-/- mice spent less time than wild-type mice in both dim and bright light. The mice also showed increased sensitivity to light in negative masking behavior, with a half maximal response at 0.08 lux (0.01 µW·cm(-2)) in Rgs9-/- mice compared to 5.0 lux (0.85 µW·cm(-2)) in wild-type mice. These behaviors were not due to increased anxiety or increased pupil size causing more light to enter the eye. Rather, constriction of the pupil showed that Rgs9-/- mice had an abnormally sustained response to light across multiple irradiance measurement pathways. CONCLUSIONS: Rgs9-/- mice recapitulate a photophobia phenotype of bradyopsia, and the pupil light reflex identifies a simple means to screen for irradiance measurement abnormalities in bradyopsia and potentially other genetic disorders involving photophobia.

EML1 (CNG-modulin) controls light sensitivity in darkness and under continuous illumination in zebrafish retinal cone photoreceptors.

The ligand sensitivity of cGMP-gated (CNG) ion channels in cone photoreceptors is modulated by CNG-modulin, a Ca(2+)-binding protein. We investigated the functional role of CNG-modulin in phototransduction in vivo in morpholino-mediated gene knockdown zebrafish. Through comparative genomic analysis, we identified the orthologue gene of CNG-modulin in zebrafish, eml1, an ancient gene present in the genome of all vertebrates sequenced to date. We compare the photoresponses of wild-type cones with those of cones that do not express the EML1 protein. In the absence of EML1, dark-adapted cones are ∼5.3-fold more light sensitive than wild-type cones. Previous qualitative studies in several nonmammalian species have shown that immediately after the onset of continuous illumination, cones are less light sensitive than in darkness, but sensitivity then recovers over the following 15-20 s. We characterize light sensitivity recovery in continuously illuminated wild-type zebrafish cones and demonstrate that sensitivity recovery does not occur in the absence of EML1.

Enhancing performance while avoiding damage: a contribution of macular pigment.

PURPOSE: To compare action spectra for visual discomfort in the fovea and the parafovea and to determine the effect of macular pigment (MP). METHODS: Visual discomfort thresholds to lights from 440 to 600 nm were obtained for six young (<35 y), visually normal subjects with a wide range of MP densities (0.10-0.71 at 30' eccentricity). Foveal and parafoveal conditions were assessed. Discomfort thresholds were also obtained for xenon-white light (partially absorbed by MP), and a broadband yellow (outside the absorption band of MP). MP was measured psychophysically using heterochromatic flicker photometry (HFP). RESULTS: For the parafovea, discomfort sensitivity (1/threshold) increased sharply with decreasing wavelength for all subjects. Commensurate with a subject's MP level, MP significantly reduced visual discomfort to short wavelengths (including xenon-white light) for central viewing. CONCLUSIONS: MP simultaneously reduces visual discomfort and protects from light damage at short wavelengths. As a result, MP increases the range of safe and comfortable light levels. Because higher light levels enable improved visual sensitivity for fine detail, these findings indicate that the spectral absorption properties and spatial distribution of MP combine to protect the retina while enhancing visual performance. The action spectrum for visual discomfort closely matches the risk for acute light damage to the retinal pigment epithelium, and it is consistent with a major influence from the intrinsically photosensitive retinal ganglion cells containing melanopsin. We suggest that MP interacts with nonimage-forming retinal input to achieve the dual outcomes of visual discomfort reduction and protection from light damage.

Pituitary adenylate cyclase-activating polypeptide plays a key role in nitroglycerol-induced trigeminovascular activation in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (PAC1, VPAC) are present in sensory neurons and vascular smooth muscle. PACAP infusion was found to trigger migraine-like headache in humans and we showed its central pro-nociceptive function in several mouse pain models. Nitroglycerol (NTG)-induced pathophysiological changes were investigated in this study in PACAP gene-deleted (PACAP(-/-)) and wildtype (PACAP(+/+)) mice. Chemical activation of the trigeminovascular system was induced by 10 mg/kg i.p. NTG. Light-aversive behavior was determined in a light-dark box, meningeal microcirculation by laser Doppler blood perfusion scanning and the early neuronal activation marker c-Fos with immunohistochemistry. NTG-induced photophobia both in the early (0-30 min) and late phases (90-120 min) due to direct vasodilation and trigeminal sensitization, respectively, was significantly reduced in PACAP(-/-) mice. Meningeal blood flow increased by 30-35% during 4 h in PACAP(+/+) mice, but only a 5-10% elevation occurred from the second hour in PACAP(-/-) ones. The number of c-Fos expressing cells referring to neuronal activation in the trigeminal ganglia and nucleus caudalis significantly increased 4h after NTG in PACAP(+/+), but not in PACAP(-/-) animals. Similar PAC1 receptor immunostaining was detected in both groups, which did not change 4 h after NTG treatment. PACAP-38 (300 µg/kg, i.p.) produced photophobia similarly to NTG and 30% meningeal vasodilatation for 30 min in PACAP(+/+), but not in PACAP(-/-) mice. It significantly increased neural activation 4h later in the trigeminal ganglia of both groups, but in the nucleus caudalis of only the PACAP(+/+) mice. We provide the first experimental results that PACAP is a pivotal mediator of trigeminovascular activation/sensitization and meningeal vasodilation related to migraine.

Photoreactivity of the occipital cortex measured by functional magnetic resonance imaging-blood oxygenation level dependent in migraine patients and healthy volunteers: pathophysiological implications.

BACKGROUND: The brain of migraineurs is hyperexcitable, particularly the occipital cortex, which is probably hypersensitive to light. Photophobia or hypersensitivity to light may be accounted for by an increased excitability of trigeminal, the visual pathways, and the occipital cortex. OBJECTIVE: To study light sensitivity and photophobia by assessing the response to light stimuli with functional magnetic resonance imaging-blood oxygenation level dependent (fMRI-BOLD) of the occipital cortex in migraineurs and in controls. Also, to try to decipher the contribution of the occipital cortex to photophobia and whether the cortical reactivity of migraineurs may be part of a constitutional (defensive) mechanism or represents an acquired (sensitization) phenomenon. METHODS: Nineteen patients with migraine (7 with aura and 12 without aura) and 19 controls were studied with fMRI-BOLD during 4 increasing light intensities. Eight axial image sections of 0.5 cm that covered the occipital cortex were acquired for each intensity. We measured the extension and the intensity of activation for every light stimuli. Photophobia was estimated according to a 0 to 3 semiquantitative scale of light discomfort. RESULTS: Migraineurs had a significantly higher number of fMRI-activated voxels at low (320.4 for migraineurs [SD = 253.9] and 164.3 for controls [SD = 102.7], P = .027) and medium-low luminance levels (501.2 for migraineurs [SD = 279.5] and 331.1 for controls [SD = 194.3], P = .034) but not at medium-high (579.5 for migraineurs [SD = 201.4] and 510.2 for controls [SD = 239.5], P = .410) and high light stimuli (496.2 for migraineurs [SD = 216.2] and 394.7 for controls [SD = 240], P = .210). No differences were found with respect to the voxel activation intensity (amplitude of the BOLD wave) between migraineurs and controls (8.98 [SD = 2.58] vs 7.99 [SD = 2.57], P = .25; 10.82 [SD = 3.27] vs 9.81 [SD = 3.19], P = .31; 11.90 [SD = 3.18] vs 11.06 [SD = 2.56], P = .62; 11.45 [SD = 2.65] vs 10.25 [SD = 2.22], P = .16). Light discomfort was higher in the group of migraineurs at all the intensities tested, but there was no correlation with the number of activated voxels in the occipital cortex and photophobia. Repetitive light stimuli failed to demonstrate a lack of habituation in migraineurs. CONCLUSIONS: Migraineurs during interictal periods showed hyperxcitability of the visual cortex with a wider photoresponsive area, the underlying mechanism probably being dual: constitutional-defensive and acquired-sensitizating.

Induction of multiple photophobic behaviors in a transgenic mouse sensitized to CGRP.

Migraine is a complex neurological disorder with a significant impact on patients and society. Clinical and preclinical studies have established the neuropeptide calcitonin gene-related peptide (CGRP) as a key player in migraine and other neurovascular headaches. To study the role of CGRP in these disorders, we have characterized the photophobic phenotype of nestin/hRAMP1 mice, a transgenic model with genetically engineered increased sensitivity to CGRP. These mice have increased nervous system expression of a regulatory subunit of the CGRP receptor, human receptor activity-modifying receptor (hRAMP1). We have previously demonstrated that nestin/hRAMP1 mice display a light-aversive behavior that is greatly enhanced by CGRP and blocked by a CGRP receptor antagonist used to treat migraine. Here we have compared their behavior in two different experimental setups with testing chambers of different sizes and light intensities as well as in complete darkness. We demonstrated similar degrees of light aversion in nestin/hRAMP1 mice with 1000 and 50 lux. To control for other possible factors driving nestin/hRAMP1 mice to the dark zone, we tested them in the absence of any light, and they showed identical behavior as littermates. Furthermore, both nestin/hRAMP1 and control mice have decreased motility in response to CGRP in the dark, but not the light side of the chamber. Our findings confirm the robust CGRP-induced light-aversive phenotype of nestin/hRAMP1 mice, which can be a surrogate of photophobia, and validates its usefulness as a model of migraine and other disorders associated with photophobia.