The non-visual opsins expressed in deep brain neurons projecting to the retina in lampreys

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.

constructed using an Ultra-low Input RNA Kit (Chrontech), and each cDNA library was sequenced by using Illumina HiSeq 2500. Analyses of fragments per kilobase of exon per million (FPKM) values of two parapinopsin genes were conducted using MASER pipelines that can serially process Trinity, Bowtie, eXpress and DEGseq output (http://cell-innovation.nig.ac.jp/maser/index_en.html).
Briefly, all sequenced raw reads were used for de novo assembly, and contigs containing each parapinopsin sequence and their FPKM values were obtained. The mRNA expression level of each portion is presented as a relative value obtained by dividing the FPKM value of the contig containing the sequence of each parapinopsin by the FPKM value of the contig containing the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene.

Expression and Spectrophotometry of P-opsin.
The expression of P-opsin in HEK293S cells and its purification was performed as described previously [1]. Briefly, cDNA of river lamprey P-opsin was tagged with the epitope sequence for the monoclonal antibody, Rho1D4 (ETSQVAPA). Tagged cDNA was inserted into the plasmid vector, pcDNA3.1 (Invitrogen). Expressed P-opsin was incubated overnight with excess 11-cis-retinal to reconstitute the pigment. P-opsin pigments were then extracted with a detergent, 1% dodecyl β-D-maltoside, in 50 mM HEPES buffer (pH 6.5) containing 140 mM NaCl (buffer A).
The absorption spectra of pigments were recorded at 10°C using a Shimadzu UV-2450 spectrophotometer (Shimadzu, Japan).
Parapinopsin is more abundantly distributed in the pineal organ-containing portion than other brain portions. bPPL is distributed in the diencephalon-, mesencephalon-, and pineal organ-containing portions and its mRNA level in each portion is much lower than parapinopsin in the pineal organ-containing portion. Relative values in each portion were calculated by dividing mRNA FPKM values of each opsin by GAPDH mRNA FPKM values as the internal standard. The level of parapinopsin in the pineal organ was referred to as 1.0. P: pineal organ, T: telencephalon, D: diencephalon, M: mesencephalon, R: rhombencephalon. In situ hybridisation with the bPPL antisense probe shows that bPPL is not expressed in the pineal organ (a) or the medial hypothalamus of the diencephalon (b). Previous immunohistochemical study on anti-cone opsin antibody suggested that a deep brain photoreceptor exists in the medial hypothalamus, such as the nucleus of the postoptic commissure [2]. Therefore, opsin(s) other than bPPL might be expressed in the medial hypothalamus of the diencephalon. Scale bar = 100 μm. HEK 293S cells expressing the river lamprey bPPL (a-c) and P-opsin (d-f) were treated with antisera against bPPL (a and d), P-opsin (b and e), and Rho1D4, a mouse monoclonal antibody against bovine rhodopsin C-terminal sequence (c and f). The bPPL or P-opsin C-terminus was tagged with Rho1D4 epitope sequence. Panels a1-f1 are anti-mouse IgG-treated images (green). Panels a2-f2 are anti-rabbit IgG-treated images (magenta). Panels a3-f3 are merged images in addition to the nuclear staining (blue). HEK293S cells expressing bPPL were immunostained by a rabbit polyclonal antiserum to bPPL and anti-rabbit IgG (a2). HEK293 cells expressing P-opsin were immunostained with anti-P-opsin antiserum and anti-mouse IgG (e1). Other combinations of first and secondary antibodies were immunonegative. Therefore, these antisera do not cross-react with other opsins. Scale bar = 50 µm. Localisation of P-opsin (a, e; green) and bPPL (b, f; magenta) in the river lamprey (a, b) and the brook lamprey (e, f) is shown in low magnification views. P-opsin (c, g; green) and bPPL (d, h; magenta) are localised in the cilia structures of the M5NS (arrowheads) in river (c, d) and brook (g, h) lampreys. Panels c, d, g and h are higher magnification views of the areas indicated as boxes in panels a, b, e and f, respectively. V: third ventricle. Scale bars = 200 μm (b, f), 20 μ m (d, h).  Figure S5. The difference absorption spectrum of P-opsin.
The detergent extract of P-opsin-expressing cells was irradiated with green light in the presence of 5 mM hydroxylamine at pH 6.5. The difference spectrum of before minus after irradiation shows that the absorption maximum of P-opsin is at around 500 nm.