Current understanding of photophobia, visual networks and headaches.

OBJECTIVE: To review clinical and pre-clinical evidence supporting the role of visual pathways, from the eye to the cortex, in the development of photophobia in headache disorders. BACKGROUND: Photophobia is a poorly understood light-induced phenomenon that emerges in a variety of neurological and ophthalmological conditions. Over the years, multiple mechanisms have been proposed to explain its causes; however, scarce research and lack of systematic assessment of photophobia in patients has made the search for answers quite challenging. In the field of headaches, significant progress has been made recently on how specific visual networks contribute to photophobia features such as light-induced intensification of headache, increased perception of brightness and visual discomfort, which are frequently experienced by migraineurs. Such progress improved our understanding of the phenomenon and points to abnormal processing of light by both cone/rod-mediated image-forming and melanopsin-mediated non-image-forming visual pathways, and the consequential transfer of photic signals to multiple brain regions involved in sensory, autonomic and emotional regulation. CONCLUSION: Photophobia phenotype is diverse, and the relative contribution of visual, trigeminal and autonomic systems may depend on the disease it emerges from. In migraine, photophobia could result from photic activation of retina-driven pathways involved in the regulation of homeostasis, making its association with headache more complex than previously thought.

Ocular Topical Anesthesia Does Not Attenuate Light-Induced Discomfort Using Blue and Red Light Stimuli.

Purpose: To investigate whether melanopsin-containing ophthalmic trigeminal ganglion cells provide significant input to mediate light-induced discomfort. This is done by studying the effect of ocular topical anesthesia on light-induced discomfort threshold to blue light and red light stimuli using a psychophysical approach. Method: Ten visually normal participants completed the experiment consisting of two trials: an anesthesia trial in which light stimuli were presented to both eyes following 0.5% proparacaine eye drops administration, and a placebo trial in which normal saline drops were used. In each trial, a randomized series of 280 blue and red light flashes were presented over seven intensity steps with 20 repetitions for each color and light intensity. Participants were instructed to report whether they perceived each stimulus as either "uncomfortably bright" or "not uncomfortably bright" by pressing a button. The proportion of "uncomfortable" responses was pooled to generate individual psychometric functions, from which 50% discomfort thresholds (defined as the light intensity at which the individuals perceived the stimulus to be uncomfortably bright/unpleasant 50% of the time) were calculated. Results: When blue light was presented, there was no significant difference in the light-induced discomfort thresholds between anesthesia and placebo trials (P = 0.44). Similarly, when red light was used, no significant difference in threshold values was found between the anesthesia and placebo trials (P = 0.28). Conclusions: Ocular topical anesthesia does not alter the light-induced discomfort thresholds to either blue or red light, suggesting that the melanopsin-containing ophthalmic trigeminal ganglion cells provide little or no significant input in mediating light-induced discomfort under normal physiologic conditions.

Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition.

We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the nonvisual G protein-coupled receptor melanopsin (Opsin 4; Opn4). Here we tested the hypothesis that nonvisual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G protein-coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2, as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs preconstricted with phenylephrine (PE), with a maximum response between 400 and 460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiological desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.

Absorption Characteristics of Vertebrate Non-Visual Opsin, Opn3.

Most animals possess multiple opsins which sense light for visual and non-visual functions. Here, we show spectral characteristics of non-visual opsins, vertebrate Opn3s, which are widely distributed among vertebrates. We successfully expressed zebrafish Opn3 in mammalian cultured cells and measured its absorption spectrum spectroscopically. When incubated with 11-cis retinal, zebrafish Opn3 formed a blue-sensitive photopigment with an absorption maximum around 465 nm. The Opn3 converts to an all-trans retinal-bearing photoproduct with an absorption spectrum similar to the dark state following brief blue-light irradiation. The photoproduct experienced a remarkable blue-shift, with changes in position of the isosbestic point, during further irradiation. We then used a cAMP-dependent luciferase reporter assay to investigate light-dependent cAMP responses in cultured cells expressing zebrafish, pufferfish, anole and chicken Opn3. The wild type opsins did not produce responses, but cells expressing chimera mutants (WT Opn3s in which the third intracellular loops were replaced with the third intracellular loop of a Gs-coupled jellyfish opsin) displayed light-dependent changes in cAMP. The results suggest that Opn3 is capable of activating G protein(s) in a light-dependent manner. Finally, we used this assay to measure the relative wavelength-dependent response of cells expressing Opn3 chimeras to multiple quantally-matched stimuli. The inferred spectral sensitivity curve of zebrafish Opn3 accurately matched the measured absorption spectrum. We were unable to estimate the spectral sensitivity curve of mouse or anole Opn3, but, like zebrafish Opn3, the chicken and pufferfish Opn3-JiL3 chimeras also formed blue-sensitive pigments. These findings suggest that vertebrate Opn3s may form blue-sensitive G protein-coupled pigments. Further, we suggest that the method described here, combining a cAMP-dependent luciferase reporter assay with chimeric opsins possessing the third intracellular loop of jellyfish opsin, is a versatile approach for estimating absorption spectra of opsins with unknown signaling cascades or for which absorption spectra are difficult to obtain.

Measuring and using light in the melanopsin age.

Light is a potent stimulus for regulating circadian, hormonal, and behavioral systems. In addition, light therapy is effective for certain affective disorders, sleep problems, and circadian rhythm disruption. These biological and behavioral effects of light are influenced by a distinct photoreceptor in the eye, melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs), in addition to conventional rods and cones. We summarize the neurophysiology of this newly described sensory pathway and consider implications for the measurement, production, and application of light. A new light-measurement strategy taking account of the complex photoreceptive inputs to these non-visual responses is proposed for use by researchers, and simple suggestions for artificial/architectural lighting are provided for regulatory authorities, lighting manufacturers, designers, and engineers.

[Circadian clock and non-visual functions: the role of light in humans]. / Horloge circadienne et fonctions non visuelles : rôle de la lumière chez l'Homme.

Hormonal secretion, cognitive performance, motor activity, metabolic processes, the sleep wake cycle and, most recently shown, cell division and ADN repair show a 24 h rhythmicity that is driven by the circadian timing system (the biological clock). Their appropriate activity over the 24 h requires appropriate entrainment of the circadian clock, which is achieved through the synchronizing effects of ocular light exposure. The activation of melanopsin-expressing ganglion cells in the retina depends on timing, quality, intensity, and history of light exposure. Inappropriate lighting leads to inappropriate synchronization of the clock, and activation of non-visual functions (mood, wakefulness, cognition, etc.). In turn, a deficit of circadian entrainment to the 24 h is responsible for alterations of a large number of functions, and leads to altered sleep, wake, mood, neurobehavioral processes and cell division, but also to pathologies. The crucial role of the circadian clock and the nature of the non-visual functions activated by light give rise to the concept that light is a biological need fundamental to health. Without an appropriate light hygiene, the clock receives an odd tempo, and it is cacophony!

Complex interaction of circadian and non-circadian effects of light on mood: shedding new light on an old story.

In addition to its role in vision, light exerts strong effects on behavior. Its powerful role in the modulation of mood is well established, yet remains poorly understood. Much research has focused on the effects of light on circadian rhythms and subsequent interaction with alertness and depression. The recent discovery of a third photoreceptor, melanopsin, expressed in a subset of retinal ganglion cells, allows major improvement of our understanding of how photic information is processed. Light affects behavior in two ways, either indirectly through the circadian timing system, or directly through mechanisms that are independent of the circadian system. These latter effects have barely been studied in regard to mood, but recent investigations on the direct effects of light on sleep and alertness suggest additional pathways through which light could influence mood. Based on our recent findings, we suggest that light, via melanopsin, may exert its antidepressant effect through a modulation of the homeostatic process of sleep. Further research is needed to understand how these mechanisms interplay and how they contribute to the photic regulation of mood. Such research could improve therapeutic management of affective disorders and influence the management of societal lighting conditions.

Melanopsin contributions to irradiance coding in the thalamo-cortical visual system.

Photoreception in the mammalian retina is not restricted to rods and cones but extends to a subset of retinal ganglion cells expressing the photopigment melanopsin (mRGCs). These mRGCs are known to drive such reflex light responses as circadian photoentrainment and pupillomotor movements. By contrast, until now there has been no direct assessment of their contribution to conventional visual pathways. Here, we address this deficit. Using new reporter lines, we show that mRGC projections are much more extensive than previously thought and extend across the dorsal lateral geniculate nucleus (dLGN), origin of thalamo-cortical projection neurons. We continue to show that this input supports extensive physiological light responses in the dLGN and visual cortex in mice lacking rods+cones (a model of advanced retinal degeneration). Moreover, using chromatic stimuli to isolate melanopsin-derived responses in mice with an intact visual system, we reveal strong melanopsin input to the ∼40% of neurons in the LGN that show sustained activation to a light step. We demonstrate that this melanopsin input supports irradiance-dependent increases in the firing rate of these neurons. The implication that melanopsin is required to accurately encode stimulus irradiance is confirmed using melanopsin knockout mice. Our data establish melanopsin-based photoreception as a significant source of sensory input to the thalamo-cortical visual system, providing unique irradiance information and allowing visual responses to be retained even in the absence of rods+cones. These findings identify mRGCs as a potential origin for aspects of visual perception and indicate that they may support vision in people suffering retinal degeneration.

Spectral responses of the human circadian system depend on the irradiance and duration of exposure to light.

In humans, modulation of circadian rhythms by light is thought to be mediated primarily by melanopsin-containing retinal ganglion cells, not rods or cones. Melanopsin cells are intrinsically blue light-sensitive but also receive input from visual photoreceptors. We therefore tested in humans whether cone photoreceptors contribute to the regulation of circadian and neuroendocrine light responses. Dose-response curves for melatonin suppression and circadian phase resetting were constructed in subjects exposed to blue (460 nm) or green (555 nm) light near the onset of nocturnal melatonin secretion. At the beginning of the intervention, 555-nm light was equally effective as 460-nm light at suppressing melatonin, suggesting a significant contribution from the three-cone visual system (lambda(max) = 555 nm). During the light exposure, however, the spectral sensitivity to 555-nm light decayed exponentially relative to 460-nm light. For phase-resetting responses, the effects of exposure to low-irradiance 555-nm light were too large relative to 460-nm light to be explained solely by the activation of melanopsin. Our findings suggest that cone photoreceptors contribute substantially to nonvisual responses at the beginning of a light exposure and at low irradiances, whereas melanopsin appears to be the primary circadian photopigment in response to long-duration light exposure and at high irradiances. These results suggest that light therapy for sleep disorders and other indications might be optimized by stimulating both photoreceptor systems.

Functional expression, targeting and Ca2+ signaling of a mouse melanopsin-eYFP fusion protein in a retinal pigment epithelium cell line.

Melanopsin, first discovered in Xenopus melanophores, is now established as a functional sensory photopigment of the intrinsically photosensitive retinal ganglion cells. These ganglion cells drive circadian rhythm and pupillary adjustments through projection to the brain. Melanopsin shares structural similarities with all known opsins. Comprehensive characterization of melanopsin with respect to its spectral properties, photochemical cascade and signaling partners requires a suitable recombinant system and high expression levels. This combination has not yet been described. To address this issue, we have expressed recombinant mouse melanopsin in several cell lines. Using enhanced yellow fluorescent protein (eYFP) as a visualization tag, expression was observed in all cell lines. Confocal microscopy revealed that melanopsin was properly routed to the plasma membrane only in retinal pigment epithelium (RPE)-derived D407 cells and in human embryonic kidney (HEK) cells. Further, we performed intracellular calcium measurements in order to probe the melanopsin signaling activity of this fusion protein. Transfected cells were loaded with the calcium indicator Fura2-AM. Upon illumination, an immediate but transient calcium response was observed in HEK as well as in D407 cells, while mock-transfected cells showed no calcium response under identical conditions. Supplementation with 11-cis retinal or all-trans retinal enhanced the response. After prolonged illumination the cells became desensitized. Thus, RPE-derived cells expressing recombinant melanopsin may constitute a suitable system for the study of the structural and functional characteristics of melanopsin.

Regulation of melanopsin expression.

Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light-detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non-imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin-like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non-neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light-responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.

Light-induced phase shift in the Syrian hamster (Mesocricetus auratus) is attenuated by the PACAP receptor antagonist PACAP6-38 or PACAP immunoneutralization.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.

Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice.

In the mammalian retina, a small subset of retinal ganglion cells (RGCs) are intrinsically photosensitive, express the opsin-like protein melanopsin, and project to brain nuclei involved in non-image-forming visual functions such as pupillary light reflex and circadian photoentrainment. We report that in mice with the melanopsin gene ablated, RGCs retrograde-labeled from the suprachiasmatic nuclei were no longer intrinsically photosensitive, although their number, morphology, and projections were unchanged. These animals showed a pupillary light reflex indistinguishable from that of the wild type at low irradiances, but at high irradiances the reflex was incomplete, a pattern that suggests that the melanopsin-associated system and the classical rod/cone system are complementary in function.

Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals.

In mammals the retina contains photoactive molecules responsible for both vision and circadian photoresponse systems. Opsins, which are located in rods and cones, are the pigments for vision but it is not known whether they play a role in circadian regulation. A subset of retinal ganglion cells with direct projections to the suprachiasmatic nucleus (SCN) are at the origin of the retinohypothalamic tract that transmits the light signal to the master circadian clock in the SCN. However, the ganglion cells are not known to contain rhodopsin or other opsins that may function as photoreceptors. We have found that the two blue-light photoreceptors, cryptochromes 1 and 2 (CRY1 and CRY2), recently discovered in mammals are specifically expressed in the ganglion cell and inner nuclear layers of the mouse retina. In addition, CRY1 is expressed at high level in the SCN and oscillates in this tissue in a circadian manner. These data, in conjunction with the established role of CRY2 in photoperiodism in plants, lead us to propose that mammals have a vitamin A-based photopigment (opsin) for vision and a vitamin B2-based pigment (cryptochrome) for entrainment of the circadian clock.