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The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a "complete photoregeneration ability," which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a "dark-active and light-inactivated" GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570 nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

Animal opsin-based pigments are light-activated G-protein-coupled receptors (GPCRs), which drive signal transduction cascades via G-proteins. Thousands of animal opsins have been identified, and molecular phylogenetic and biochemical analyses have revealed the unexpected diversity in selectivity of G-protein activation and photochemical property. Here we discuss the optogenetic potentials of diverse animal opsins, particularly recently well-characterized three non-canonical opsins, parapinopsin, peropsin, and LWS bistable opsin. Unlike canonical opsins such as vertebrate visual opsins that have been conventionally used for optogenetic applications, these opsins are bistable; opsin-based pigments do not release the chromophore retinal after light absorption, and the stable photoproducts revert to their original dark states upon subsequent light absorption. Parapinopsins have a “complete photoregeneration ability,” which allows a clear color-dependent regulation of signal transductions. On the other hand, peropsins serve as a “dark-active and light-inactivated” GPCR to regulate signal transductions in the opposite way compared with usual opsins. In addition, an LWS bistable opsin from a butterfly was revealed to be the longest wavelength-sensitive animal opsin with its absorption maximum at ~570 nm. The property-dependent optical regulations of signal transductions were demonstrated in mammalian cultured cells, showing potentials of new optogenetic tools.

In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency. Supplementary Information The online version contains supplementary material available at 10.1186/s40851-021-00171-1.

Significance Color discrimination in animals is considered to require opponent processing of signals from two or more opsins sensitive to different parts of the spectrum. We previously reported that lower vertebrate pineal organs, which discriminate between UV and visible light, employ a bistable opsin called parapinopsin that has two stable photointerconvertible states, a signaling-inactive state maximally sensitive to UV and a visible light-sensitive signaling-active photoproduct. Here, we present evidence that the photoequilibrium between these two states in the zebrafish pineal organ is dependent on the spectral composition of incident light, setting the opsin’s signaling activity according to color to allow parapinopsin alone to generate color opponency between UV and visible light at the level of single pineal photoreceptor cells., Lower vertebrate pineal organs discriminate UV and visible light. Such color discrimination is typically considered to arise from antagonism between two or more spectrally distinct opsins, as, e.g., human cone-based color vision relies on antagonistic relationships between signals produced by red-, green-, and blue-cone opsins. Photosensitive pineal organs contain a bistable opsin (parapinopsin) that forms a signaling-active photoproduct upon UV exposure that may itself be returned to the signaling-inactive “dark” state by longer-wavelength light. Here we show the spectrally distinct parapinopsin states (with antagonistic impacts on signaling) allow this opsin alone to provide the color sensitivity of this organ. By using calcium imaging, we show that single zebrafish pineal photoreceptors held under a background light show responses of opposite signs to UV and visible light. Both such responses are deficient in zebrafish lacking parapinopsin. Expressing a UV-sensitive cone opsin in place of parapinopsin recovers UV responses but not color opponency. Changes in the spectral composition of white light toward enhanced UV or visible wavelengths respectively increased vs. decreased calcium signal in parapinopsin-sufficient but not parapinopsin-deficient photoreceptors. These data reveal color opponency from a single kind of bistable opsin establishing an equilibrium-like mixture of the two states with different signaling abilities whose fractional concentrations are defined by the spectral composition of incident light. As vertebrate visual color opsins evolved from a bistable opsin, these findings suggest that color opponency involving a single kind of bistable opsin might have been a prototype of vertebrate color opponency.

研究グループは、奈良女子大学の研究グループと共同で、哺乳類以外の脊椎動物において「第3の目」とも呼ばれる松果体の光感覚のメカニズムについて、脊椎動物の中で「原始的」とも呼べる特徴を多く残し、進化研究で重要視されてきた円口類ヤツメウナギを用いて解析しました。その結果、異なる2種類の光受容タンパク質をそれぞれ含む2種類の光受容細胞を用いて色を感じるという、松果体色感覚の新規のメカニズム(2細胞システム)を発見しました。本研究グループは、魚類と爬虫類の松果体では、ヤツメウナギの場合とは異なり、上述の2種類の光受容タンパク質が一つの細胞にセットになって存在する1細胞システムにより色検出していることを既に明らかにしています。今回の研究成果から、強い光の下では、1細胞システムの方が2細胞システムよりも明瞭な色検出が可能であることが明らかになり、現存する魚類や爬虫類がもつ1細胞システムは、もともと目と同じように2つの光受容タンパク質が別々の2細胞に別れていた2細胞システムが1つの細胞中に融合したかのような進化を遂げた結果である可能性を示しています。すなわち、より多くの細胞を巻き込んで進化したとされる目の色検出のメカニズムの進化とは異なり、2細胞から1細胞の方向に進化したことを初めて提唱するものです。これは、松果体色検出についてこれまでの常識を覆すとともに、進化のみならず未解明の機能を探る上で重要な発見といえます。, Background: Pineal-related organs in cyclostomes, teleosts, amphibians, and reptiles exhibit color opponency, generating antagonistic neural responses to different wavelengths of light and thereby sensory information about its "color". Our previous studies suggested that in zebrafish and iguana pineal-related organs, a single photoreceptor cell expressing both UV-sensitive parapinopsin and green-sensitive parietopsin generates color opponency in a "one-cell system." However, it remains unknown to what degree these opsins and the single cell-based mechanism in the pineal color opponency are conserved throughout non-mammalian vertebrates. / Results: We found that in the lamprey pineal organ, the two opsins are conserved but that, in contrast to the situation in other vertebrate pineal-related organs, they are expressed in separate photoreceptor cells. Intracellular electrophysiological recordings demonstrated that the parietopsin-expressing photoreceptor cells with Go-type G protein evoke a depolarizing response to visible light. Additionally, spectroscopic analyses revealed that parietopsin with 11-cis 3-dehydroretinal has an absorption maximum at ~570 nm, which is in approximate agreement with the wavelength (~560 nm) that produces the maximum rate of neural firing in pineal ganglion cells exposed to visible light. The vesicular glutamate transporter is localized at both the parietopsin- and parapinopsin-expressing photoreceptor terminals, suggesting that both types of photoreceptor cells use glutamate as a transmitter. Retrograde tracing of the pineal ganglion cells revealed that the terminal of the parietopsin-expressing cells is located close enough to form a neural connection with the ganglion cells, which is similar to our previous observation for the parapinopsin-expressing photoreceptor cells and the ganglion cells. In sum, our observations point to a "two-cell system" in which parietopsin and parapinopsin, expressed separately in two different types of photoreceptor cells, contribute to the generation of color opponency in the pineal ganglion cells. / Conclusion: Our results indicate that the jawless vertebrate, lamprey, employs a system for color opponency that differes from that described previously in jawed vertebrates. From a physiological viewpoint, we propose an evolutionary insight, the emergence of pineal "one-cell system" from the ancestral "multiple (two)-cell system," showing the opposite evolutionary direction to that of the ocular color opponency.

【概要】下等な脊椎動物は脳内でも光を受容し、脳内の光受容細胞には視覚以外の機能に関わる光受容タンパク質(非視覚オプシン)が発現しています。これまでに、非視覚オプシンの一種であるパラピノプシンは紫外線に感度が高く、ヤツメウナギや硬骨魚類において、松果体と呼ばれる脳器官での波長(色)識別を担うことを見出し、脳内光受容のカギとなる非視覚オプシンであると考えられました。この論文では、ヤツメウナギにおいて、最近見出されたパラピノプシンに類似したオプシンが、パラピノプシンとは異なる分子特性と脳内分布を示すことを明らかにしました。このパラピノプシン類似オプシンは、紫から青に対して高い感度を持ち、効率的に光情報を伝達することが明らかになりました。興味深いことに、2種類の日本産ヤツメウナギでは、このオプシンは、中脳のシェーバーのM5核と呼ばれる脳深部領域に発現し、緑色光に高い感度を持つ別のオプシンと同じ細胞に共局在していました。つまり、この脳領域が紫色から緑色の光の受容に関わると考えられました。さらに、これらのオプシンを発現する脳内光受容細胞と網膜との間に神経接続が認められたことから、網膜の機能が脳の光受容によって制御されている可能性を示唆する初めての発見と言えます。, In lower vertebrates, brain photoreceptor cells express vertebrate-specific non-visual opsins. We previously revealed that a pineal-related organ-specific opsin, parapinopsin, is UV-sensitive and allows pineal wavelength discrimination in lampreys and teleost. The Australian pouched lamprey was recently reported as having two parapinopsin-related genes. We demonstrate that a parapinopsin-like opsin from the Japanese river lamprey exhibits different molecular properties and distribution than parapinopsin. This opsin activates Gi-type G protein in a mammalian cell culture assay in a light-dependent manner. Heterologous action spectroscopy revealed that the opsin forms a violet to blue-sensitive pigment. Interestingly, the opsin is co-localised with green-sensitive P-opsin in the cells of the M5 nucleus of Schober (M5NS) in the mesencephalon of the river and brook lamprey. Some opsins-containing cells of the river lamprey have cilia and others an axon projecting to the retina. The opsins of the brook lamprey are co-localised in the cilia of centrifugal neurons projecting to the retina, suggesting that cells expressing the parapinopsin-like opsin and P-opsin are sensitive to violet to green light. Moreover, we found neural connections between M5NS cells expressing the opsins and the retina. These findings suggest that the retinal activity might be modulated by brain photoreception.

Many animals capture light information via opsin-based pigments. Several thousands of opsins have been identified thus far and the opsin family is divided into eight groups. Members belonging to four out of the eight groups have been elucidated to couple to transducin, Go, Gs, and Gq, respectively, in photoreceptor cells. Accumulated evidence suggests a novel classification of the animal phototransductions, cyclic nucleotide signaling mediated by transducin, Go or Gs in ciliary photoreceptor cells and phosphoinositol signaling mediated by Gq in rhabdomeric photoreceptor cells. Varied opsin-based pigments are spectroscopically classified into two types, bleaching and bistable pigments; that is, the photoproduct of vertebrate visual pigments dissociates its chromophore retinal over time and bleaches, whereas most other opsin-based pigments convert to a stable photoproduct, which can revert to original dark state by subsequent light absorption. Mutational analyses of the both types of pigments implied that during molecular evolution of the vertebrae visual pigments, displacement of the counterion, important amino acid residue for visible light absorption of opsin-based pigment, resulted in not only unique bleaching property but also acquisition of red-sensitive visual pigment and higher G-protein activation ability generated by larger light-induced conformational change of the pigment. Interestingly, a bleaching pigment rhodopsin and parapinopsin, which closely relates to the vertebrate visual pigment but has a bistable nature, couple to visual arrestin and β arrestin, respectively, in the lamprey pineal organ, suggesting the bleaching property also might facilitate the evolution of visual arrestin which is specialized for vertebrate visual function. WIREs Membr Transp Signal 2012, 1:104–111. doi: 10.1002/wmts.6 For further resources related to this article, please visit the WIREs website.

Background Recent genome projects of various animals have uncovered an unexpectedly large number of opsin genes, which encode protein moieties of photoreceptor molecules, in most animals. In visual systems, the biological meanings of this diversification are clear; multiple types of visual opsins with different spectral sensitivities are responsible for color vision. However, the significance of the diversification of non-visual opsins remains uncertain, in spite of the importance of understanding the molecular mechanism and evolution of varied non-visual photoreceptions. Results Here, we investigated the diversification of the pineal photopigment parapinopsin, which serves as the UV-sensitive photopigment for the pineal wavelength discrimination in the lamprey, linking it with other pineal photoreception. Spectroscopic analyses of the recombinant pigments of the two teleost parapinopsins PP1 and PP2 revealed that PP1 is a UV-sensitive pigment, similar to lamprey parapinopsin, but PP2 is a blue-sensitive pigment, with an absorption maximum at 460–480 nm, showing the diversification of non-visual pigment with respect to spectral sensitivity. We also found that PP1 and PP2 exhibit mutually exclusive expressions in the pineal organs of three teleost species. By using transgenic zebrafish in which these parapinopsin-expressing cells are labeled, we found that PP1-expressing cells basically possess neuronal processes, which is consistent with their involvement in wavelength discrimination. Interestingly, however, PP2-expressing cells rarely possess neuronal processes, raising the possibility that PP2 could be involved in non-neural responses rather than neural responses. Furthermore, we found that PP2-expressing cells contain serotonin and aanat2, the key enzyme involved in melatonin synthesis from serotonin, whereas PP1-expressing cells do not contain either, suggesting that blue-sensitive PP2 is instead involved in light-regulation of melatonin secretion. Conclusions In this paper, we have clearly shown the different molecular properties of duplicated non-visual opsins by demonstrating the diversification of parapinopsin with respect to spectral sensitivity. Moreover, we have shown a plausible link between the diversification and its physiological impact by discovering a strong candidate for the underlying pigment in light-regulated melatonin secretion in zebrafish; the diversification could generate a new contribution of parapinopsin to pineal photoreception. Current findings could also provide an opportunity to understand the “color” preference of non-visual photoreception. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0174-9) contains supplementary material, which is available to authorized users.

Pineal organs of lower vertebrates contain several kinds of photosensitive molecules, opsins that are suggested to be involved in different light-regulated physiological functions. We previously reported that parapinopsin is an ultraviolet (UV)-sensitive opsin that underlies hyperpolarization of the pineal photoreceptor cells of lower vertebrates to achieve pineal wavelength discrimination. Although, parapinopsin is phylogenetically close to vertebrate visual opsins, it exhibits a property similar to invertebrate visual opsins and melanopsin: the photoproduct of parapinopsin is stable and reverts to the original dark states, demonstrating the nature of bistable pigments. Therefore, it is of evolutionary interest to identify a phototransduction cascade driven by parapinopsin and to compare it with that in vertebrate visual cells. Here, we showed that parapinopsin is coupled to vertebrate visual G protein transducin in the pufferfish, zebrafish, and lamprey pineal organs. Biochemical analyses demonstrated that parapinopsins activated transducin in vitro in a light-dependent manner, similar to vertebrate visual opsins. Interestingly, transducin activation by parapinopsin was provoked and terminated by UV- and subsequent orange-lights irradiations, respectively, due to the bistable nature of parapinopsin, which could contribute to a wavelength-dependent control of a second messenger level in the cell as a unique optogenetic tool. Immunohistochemical examination revealed that parapinopsin was colocalized with Gt2 in the teleost, which possesses rod and cone types of transducin, Gt1, and Gt2. On the other hand, in the lamprey, which does not possess the Gt2 gene, in situ hybridization suggested that parapinopsin-expressing photoreceptor cells contained Gt1 type transducin GtS, indicating that lamprey parapinopsin may use GtS in place of Gt2. Because it is widely accepted that vertebrate visual opsins having a bleaching nature have evolved from non-bleaching opsins similar to parapinopsin, these results implied that ancestral bistable opsins might acquire coupling to the transducin-mediated cascade and achieve light-dependent hyperpolarizing response of the photoreceptor cells.

Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.

Pineal and related organs are major extraocular photoreceptors in non-mammalian vertebrates. The pineal organ contains several types of photoreceptor cells, which contribute to regulating light-dependent melatonin secretion and the neural light response, including irradiance detection and wavelength discrimination. Visual opsins and pineal-specific opsins have been identified from the pineal and related organs in a wide variety of non-mammalian vertebrates. Pinopsin and parapinopsin are key opsins for understanding melatonin secretion in the chicken pineal organ and wavelength discrimination in the lamprey pineal organ, respectively. Interestingly, parapinopsin has the molecular characteristics of both vertebrate and invertebrate opsin-based pigments, making it an important photopigment for understanding the molecular evolution of vertebrate visual opsins. In this chapter, we discuss the opsin-based pigments functioning in the pineal and related organs with a focus on parapinopsin.

SUMMARY In the pineal organ, two types of ganglion cell exhibit antagonistic chromatic responses to UV and green light, and achromatic responses to visible light. In this study, we histologically characterized UV-sensitive photoreceptor cells that contain a unique non-visual UV pigment, lamprey parapinopsin, in order to elucidate the neural network that is associated with antagonistic chromatic responses. These characteristics were compared with those of lamprey rhodopsin-containing cells, most of which are involved in achromatic responses. RT-PCR analysis revealed that lamprey parapinopsin was expressed in the pineal organ but not in the retina, unlike lamprey rhodopsin,which was expressed in both. Lamprey parapinopsin and lamprey rhodopsin were immunohistochemically localized in the dorsal and ventral regions of the pineal organ, respectively. The two pigments were localized in distinct photoreceptor cells throughout the pineal organ, namely the dorsal and ventral regions as well as the peripheral region, which corresponds to the dorso-ventral border region. The ratio of the number of lamprey parapinopsin-containing cells to lamprey rhodopsin-containing cells around the peripheral region was higher than in the central region. Electron-microscopic analysis revealed that lamprey parapinopsin-containing dorsal cells have outer segments and synaptic ribbons similar to those of ventral photoreceptor cells. However, unlike lamprey rhodopsin-containing cells, lamprey parapinopsin-containing cells connected with each other in a wide area of dorsal and peripheral portions and made direct contact with ganglion cells,mainly in the peripheral portion. These results suggest that UV light information captured by lamprey parapinopsin-containing photoreceptor cells is converged and directly transmitted to chromatic-type ganglion cells in the peripheral region to generate antagonistic chromatic responses.