Identification of oscillatory firing neurons associated with locomotion in the earthworm through synapse imaging.

We used FM imaging to identify neurons that receive sensory feedback from the body wall in a circuit for octopamine (OA)-evoked rhythmic locomotion in the earthworm, Eisenia fetida. We visualized synapses in which postsynaptic neurons receive the sensory feedback, by using FM1-43 dye to label the synapses of both motor and sensory pathways that are associated with locomotion, then clearing the motor pathway synapse labeling, and finally identifying the target synapses by distinguishing physiologically functional synapses through destaining using a high-K(+) solution. A pair of synaptic regions associated with the sensory feedback was found to be located two or three cell body-widths away from the midline, between the anterior parts of the roots of the second lateral nerves (LNs) at the segmental ganglia (SGs). Using conventional intracellular recording and dye loading of the cell bodies surrounding these synaptic regions, we identified a pair of bilateral neurons with cell bodies larger than those of other cells in these regions, and named them "Oscillatory firing neurons Projecting to Peripheral nerves" (OPPs). These had a bipolar shape and projected neurites to the ipsilateral first and third LNs, fired rhythmically, and had a burst timing synchronized with the motor pattern bursts from the ipsilateral first LNs. Current injection into an OPP caused firing in the ipsilateral first LNs, supporting the hypothesis that OPPs functionally project to the peripheral nerves. OPPs also sent neurites to the adjacent anterior and posterior SGs, suggesting connections with the adjacent segments. We conclude that FM imaging can be used to identify neurons involved in specific functions, and that OPPs are the first neurons to be associated with OA-induced locomotion in the earthworm.

Pretreatment of donor islets with the Na(+) /Ca(2+) exchanger inhibitor improves the efficiency of islet transplantation.

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2-3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN-γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca(2+) influx into ß cells through the Na(+) /Ca(2+) exchanger (NCX). Moreover, the hypoxia-induced ß cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.

Increase in interleukin-6 immediately after wheelchair basketball games in persons with spinal cord injury: preliminary report.

STUDY DESIGN: Case series. OBJECTIVES: To investigate the effects of wheelchair basketball game on plasma interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), C-reactive protein (CRP) and blood cell counts in persons with spinal cord injury (SCI). SETTING: The 2009 Mei-shin League of Wheelchair Basketball Games held at Wakayama, Japan. PARTICIPANTS: Five wheelchair basketball players with SCI voluntarily participated in this study. INTERVENTIONS: Blood samples were taken approximately 1 h before the player warm-up for the game and immediately after the game. MAIN OUTCOME MEASURES: IL-6, TNF-α, CRP and blood cell count were measured. RESULTS: Plasma IL-6 level and number of monocytes were significantly increased after the game, compared with pre-game measurements (P<0.05). No changes were observed in other measurements. There was a significant relationship between increased IL-6 levels and accumulated play duration. CONCLUSION: The lack of change in TNF-α and CRP levels suggested that the exercise-induced rise in IL-6 was not related to exercise-induced inflammatory response. Furthermore, the associated increase in the number of monocytes did not correlate with exercise-induced IL-6 changes, negating monocytes as the source of IL-6.

Plasma IL-6 levels during arm exercise in persons with spinal cord injury.

STUDY DESIGN: Non-randomized study. OBJECTIVES: Previous studies indicated that at least 2-h leg exercise at more than 60% maximum oxygen consumption (VO(2)max) increased plasma interleukin (IL)-6 in able-bodied (AB) subjects. The purpose of the present study was to compare IL-6 response to arm exercise in AB subjects and persons with spinal cord injury (SCI). SETTING: Wakayama Medical University in Japan. METHODS: Six subjects with SCI between T6 and T10 and seven AB subjects performed 2-h arm crank ergometer exercise at 60%VO(2)max. Plasma catecholamines, IL-6, tumor necrosis factor (TNF)-α and high-sensitivity C-reactive protein (hsCRP) were measured before exercise, 60-min exercise, immediately and 2 h after the completion of exercise. RESULTS: Arm exercise increased myoglobin and plasma IL-6 levels in SCI and AB (P<0.01), but there were no differences in them between the two groups throughout the study. Plasma levels creatine kinase, lactate dehydrogenase, TNF-α and hsCRP did not change throughout the study in both groups. CONCLUSION: These findings suggest neither significant muscle damage nor inflammatory response during exercise. The increase in plasma IL-6 in SCI was not unexpected, confirming that moderate intensity and relatively long-arm exercise is safe and beneficial for SCI subjects with regard to IL-6 excretion, as in AB subjects.

Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses. However, exogenous GRK7 in GRK7-tg animals led to lowered rod sensitivity, as occurs in cones, but surprisingly to slower response kinetics. Examination of responses to long series of very dim flashes suggested the possibility that the GRK7-tg rods generated two classes of single-photon response, perhaps corresponding to the interaction of activated rhodopsin with GRK1 (giving a standard response) or with GRK7(giving a very small response). Behavioural measurement of optokinetic responses (OKR) in intact GRK7-tg zebrafish larvae showed that the overall rod visual pathway was less sensitive, in accord with the lowered sensitivity of the rods. These results help provide an understanding for the molecular basis of the electrophysiological differences between cones and rods.

Regulatory mechanism for the stability of the meta II intermediate of pinopsin.

Pinopsin is a chicken pineal photoreceptive molecule with a possible role in photoentrainment of the circadian clock. Sequence comparison among members of the rhodopsin family has suggested that pinopsin might have properties more similar to cone visual pigments than to rhodopsin, but the lifetime of the physiologically active intermediate (meta II) of pinopsin is rather similar to that of metarhodopsin II, which is far more stable than meta II intermediates of cone visual pigments [Nakamura, A. et al., (1999) Biochemistry 38, 14738-14745]. In the present study, we investigated the amino acid residue(s) contributing to this unique property of pinopsin by using site-directed mutagenesis to pinopsin-specific structural features, (i) Ser171, (ii) Asn184, and (iii) the second extracellular loop two-amino acids shorter than that of cone visual pigments. The meta II stability of the 171/184 double mutant of pinopsin (S171R/N184D) is almost the same as that of wild-type pinopsin. In contrast, the meta II lifetime is markedly shortened (one third) by introduction of the third mutation (replacement of a six-amino acid stretch, 188-193, by the corresponding eight residues of chicken green-sensitive cone pigment) to the 171/184 double mutant of pinopsin. Consistently, meta II of the green-sensitive pigment mutant, in which the eight-amino acid stretch is inversely replaced by the corresponding six residues of pinopsin, is more stable than meta II of the wild-type pigment. These results strongly suggest that the specific sequence and/or the number of residues at amino acids 188-193 in pinopsin play an important role in the stabilization of the meta II intermediate.

Vertebrate ancient-long opsin: a green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells.

Nonretinal/nonpineal photosensitivity has been found in the brain of vertebrates, but the molecular basis for such a "deep brain" photoreception system remains unclear. We conducted an extensive search for brain opsin cDNAs of the zebrafish (Danio rerio), a useful animal model for genetic studies, and we have isolated a partial cDNA clone encoding an ortholog of vertebrate ancient (VA) opsin, the function of which is unknown. Subsequent characterization revealed the occurrence of two kinds of mRNAs encoding putative splicing variants, VA and VA-Long (VAL) opsin, the latter of which is a novel variant of the former. Both opsins shared a common core sequence in the membrane-spanning domains, but VAL-opsin had a C-terminal tail much longer than that of VA-opsin. Functional reconstitution experiments on the recombinant proteins showed that VAL-opsin with bound 11-cis-retinal is a green-sensitive pigment (lambdamax approximately 500 nm), whereas VA-opsin exhibited no photosensitivity even in the presence of 11-cis-retinal. Immunoreactivity specific to this functionally active VAL-opsin was localized at a limited number of cells surrounding the diencephalic ventricle of central thalamus, and these cells were distributed over approximately 200 micrometer along the rostrocaudal axis. Taken together with the previous study on the locus of the teleost brain photosensitivity (von Frisch K, 1911), it is strongly suggested that the VAL-positive cells in the zebrafish brain represent the deep brain photoreceptors. The VAL-specific immunoreactivity was also detected in a subset of non-GABAergic horizontal cells in the zebrafish retina. The existence of VAL-opsin, a new member of the rhodopsin superfamily, in these tissues may indicate its multiple roles in visual and nonvisual photosensory physiology.

Non-visual photoreception by a variety of vertebrate opsins.

Extraretinal photoreceptors in animals are involved in a variety of physiological functions such as photo-entrainment of circadian rhythm, photoperiodicity and body colour change. We have identified pinopsin in the chicken pineal gland as a typical 'non-visual' photoreceptive molecule. Pinopsin with bound 11-cis-retinal shows a blue-light sensitivity (lambda max = 468 nm), and it may play a role in synchronizing the phase of the endogenous circadian oscillator with an environmental dark-light cycle. Pinopsin is not a unique pineal opsin in animals. In the zebrafish, we have detected expression of two rhodopsin genes, the nucleotide sequences of which are very similar but distinct from each other. One is canonical rhodopsin expressed in the retina, and the other is expressed in the pineal gland. The latter gene is widely distributed among teleosts, and we named it 'exo-rhodopsin' after extraocular rhodopsin. On the other hand, our effort to identify the 'deep brain opsin' responsible for the photoperiodic gonadal response resulted in the identification of two kinds of opsins; pinopsin in the toad anterior preoptic nucleus and rhodopsin in the pigeon lateral septum. Both of these opsins are localized in the cerebrospinal fluid-contacting neurons in the brain of the two animals. We also identified VAL opsin in zebrafish retinal horizontal cells, which have not been considered as photoreceptive cells. It has become evident that animals employ a wide variety of photoreceptive molecules for 'non-visual' purposes.

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland.

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.

Chimeric nature of pinopsin between rod and cone visual pigments.

Chicken pineal pinopsin is the first example of extra-retinal opsins, but little is known about its molecular properties as compared with retinal rod and cone opsins. For characterization of extra-retinal photon signaling, we have developed an overexpression system providing a sufficient amount of purified pinopsin. The recombinant pinopsin, together with similarly prepared chicken rhodopsin and green-sensitive cone pigment, was subjected to photochemical and biochemical analyses by using low-temperature spectroscopy and the transducin activation assay. At liquid nitrogen temperature (-196 degrees C), we detected two kinds of photoproducts, bathopinopsin and isopinopsin, having their absorption maxima (lambda(max)) at 527 and approximately 440 nm, respectively, and we observed complete photoreversibility among pinopsin, bathopinopsin, and isopinopsin. A close parallel of the photoreversibility to the rhodopsin system strongly suggests that light absorbed by pinopsin triggers the initial event of cis-trans isomerization of the 11-cis-retinylidene chromophore. Upon warming, bathopinopsin decayed through a series of photobleaching intermediates: lumipinopsin (lambda(max) 461 nm), metapinopsin I (460 nm), metapinopsin II (385 nm), and metapinopsin III (460 nm). Biochemical and kinetic analyses showed that metapinopsin II is a physiologically important photoproduct activating transducin. Detailed kinetic analyses revealed that the formation of metapinopsin II is as fast as that of a chicken cone pigment, green, but that the decay process of metapinopsin II is as slow as that of the rod pigment, rhodopsin. These results indicate that pinopsin is a new type of pigment with a chimeric nature between rod and cone visual pigments in terms of the thermal behaviors of the meta II intermediate. Such a long-lived active state of pinopsin may play a role in the pineal-specific phototransduction process.

A novel Go-mediated phototransduction cascade in scallop visual cells.

Scallop retinas contain ciliary photoreceptor cells that respond to light by hyperpolarization like vertebrate rods and cones, but the response is generated by a different phototransduction cascade from those of rods and cones. To elucidate the cascade, we investigated a visual pigment and a G-protein functioning in the hyperpolarizing cell. Sequencing of cDNAs and in situ hybridization experiments showed that the hyperpolarizing cells express a novel subtype of visual pigment, which showed significant differences in amino acid sequence from other visual pigments. Cloning cDNA genes of G-protein and immunohistochemical analysis revealed the presence of an alpha subunit of a Go type G-protein, 83% identical in amino acid sequence to mammalian Go(alpha) in the nervous system, in the photoreceptive region of the cells. The results demonstrate that a novel, Go-mediated, phototransduction cascade is present in the hyperpolarizing cells. The phototransduction cascade in the scallop hyperpolarizing cell provides an alternative system to investigate Go-mediated transduction pathways in the nervous system. Molecular phylogenetic analysis strongly suggests that the Go-mediated phototransduction system emerged before the divergence of animals into vertebrate and invertebrate in the course of evolution.

Water and peptide backbone structure in the active center of bovine rhodopsin.

Difference FTIR spectra in the conversion of rhodopsin or isorhodopsin to bathorhodopsin were recorded for recombinant wild-type and E113Q bovine rhodopsins. Differences in various vibrational modes between E113Q and the wild-type proteins whose Schiff bases interact with chloride and Glu113, respectively, were analyzed. Water molecules in rhodopsin that change upon formation of bathorhodopsin are detected by a change in frequency of the O-H stretching vibration from 3538 to 3525 cm(-1). This change in the wild-type protein is absent in E113Q. One or a few water molecules are therefore suggested to be located in the proximity of Glu113, the counterion of the Schiff base. Another water vibration at 3564 cm(-1), which is shifted to 3542 cm(-1) in bathorhodopsin in the wild type, persists in E113Q but with approximately 5-cm(-1) shift toward higher frequency. This is due to water molecules that may be located at a site somewhat more remote from Glu113. Structural changes of some peptide carbonyls and amides are also absent in E113Q. On the other hand, the E113Q protein shows shifts of the N-H+ stretching vibrational band, that is probably due to the protonated Schiff base, upon conversion of rhodopsin to bathorhodopsin. No corresponding changes were observed in the wild type. We propose a model in which a water molecule interacts with Glu113, the protonated Schiff base, and peptide carbonyls, and amides. These residues undergo structural changes upon formation of bathorhodopsin.

Single amino acid residue as a functional determinant of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin.

In spite of the high similarity in amino acid sequence between rod visual pigment rhodopsin and gecko blue-sensitive pigment (gecko blue), not only the spectral sensitivities but also the thermal decay rates of the meta II- and III-intermediates are noticeably different from one another [Kojima et al. (1995) Biochemistry 34, 1096-1106]. In order to identify the protein region(s) that contain(s) key residue being responsible for the functional difference, we constructed six chimerical mutants derived from gecko blue and bovine rhodopsin, with the aid of protein production in a human embryonic kidney cell line (293S). While the absorption maximum of every mutant was located in between gecko blue (466 nm) and bovine rhodopsin (500 nm), a large blue-shift (18 nm) was observed when the helices I-III of rhodopsin were replaced with those of gecko blue. A time resolved spectroscopic study demonstrated that this replacement also accelerated the decay rate of the meta II-intermediate. The decay of the meta III-intermediate of the mutants became faster as the compartment of gecko blue was increased. Thus, the faster decay of the meta II-intermediate of gecko blue is largely attributed to residues within helices I-III, while the decay of the meta III-intermediate apparently depends on the overall structure of the protein.

Purification and low temperature spectroscopy of gecko visual pigments green and blue.

We purified two kinds of visual pigments, gecko green and gecko blue, from retinas of Tokay geckos (Gekko gekko) by two steps of column chromatography, and investigated their photobleaching processes by means of low temperature spectroscopy. Absorption maxima of gecko green and blue solubilized in a mixture of 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CHAPS) and phosphatidylcholine were 522 and 465 nm, respectively, which are close to those observed in the photoreceptor cells. Low temperature spectroscopy identified six intermediates in the photobleaching process of gecko green; batho (lambda max = 569 nm), BL (lambda max = 519 nm), lumi (507 nm), meta I (approximately 486 nm), meta II (approximately 384 nm), and meta III intermediates (approximately 500 nm). In contrast to the high similarity in amino acid sequence between gecko green and iodopsin [Kojima, D., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6841-6845], the batho-green did not revert thermally to original gecko green but converts to the next intermediate. The photobleaching process of gecko blue was investigated by low temperature spectroscopy, and three intermediates, meta I (lambda max = approximately 470 nm), meta II (lambda max = approximately 370 nm) and meta III (lambda max = approximately 475 nm), were identified. A comparative study on the thermal behavior of meta intermediates revealed that the thermal stability of meta II intermediate of both of the gecko visual pigments is lower than that of metarhodopsin II. The result supports the idea that both the gecko visual pigments are cone-type ones.

Cone visual pigments are present in gecko rod cells.

The Tokay gecko (Gekko gekko), a nocturnal lizard, has two kinds of visual pigments, P467 and P521. In spite of the pure-rod morphology of the photoreceptor cells, the biochemical properties of P521 and P467 resemble those of iodopsin (the chicken red-sensitive cone visual pigment) and rhodopsin, respectively. We have found that the amino acid sequence of P521 deduced from the cDNA was very similar to that of iodopsin. In addition, P467 has the highest homology with the chicken green-sensitive cone visual pigment, although it also has a relatively high homology with rhodopsins. These results give additional strength to the transmutation theory of Walls [Walls, G. L. (1934) Am. J. Ophthalmol. 17, 892-915], who proposed that the rod-shaped photoreceptor cells of lizards have been derived from ancestral cone-like photoreceptors. Apparently amino acid sequences of visual pigments are less changeable than the morphology of the photoreceptor cells in the course of evolution.

Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments.

The chicken retina contains rhodopsin (a rod visual pigment) and four kinds of cone visual pigments. The primary structures of chicken red (iodopsin) and rhodopsin have been determined previously. Here we report isolation of three cDNA clones encoding additional pigments from a chicken retinal cDNA library. Based on the partial amino acid sequences of the purified chicken visual pigments together with their biochemical and spectral properties, we have identified these clones as encoding the chicken green, blue, and violet visual pigments. Chicken violet was very similar to human blue not only in absorption maximum (chicken violet, 415 nm; human blue, 419 nm) but also in amino acid sequence (80.6% identical). Interestingly, chicken green was more similar (71-75.1%) than any other known cone pigment (42.0-53.7%) to vertebrate rhodopsins. The fourth additional cone pigment, chicken blue, had relatively low similarity (39.3-54.6%) in amino acid sequence to those of the other vertebrate visual pigments. A phylogenetic tree of vertebrate visual pigments constructed on the basis of amino acid identity indicated that an ancestral visual pigment evolved first into four groups (groups L, S, M1, and M2), each of which includes one of the chicken cone pigments, and that group Rh including vertebrate rhodopsins diverged from group M2 later. Thus, it is suggested that the gene for scotopic vision (rhodopsin) has evolved out of that for photopic vision (cone pigments). The divergence of rhodopsin from cone pigments was accompanied by an increase in negative net charge of the pigment.