Faculty Profiles - SAKAMOTO Hirotaka

Papers - SAKAMOTO Hirotaka

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Comparing nerve-mediated FGF signalling in the early initiation phase of organ regeneration across mutliple amphibian species. Reviewed International journal

Sakiya Yamamoto, Rena Kashimoto, Saya Furukawa, Hirotaka Sakamoto, Akira Satoh

Journal of experimental zoology. Part B, Molecular and developmental evolution 336 ( 7 ) 529 - 539 2021.11

Language：English Publishing type：Research paper (scientific journal)

Amphibians have a very high capacity for regeneration among tetrapods. This superior regeneration capability in amphibians can be observed in limbs, the tail, teeth, external gills, the heart, and some internal organs. The mechanisms underlying the superior organ regeneration capability have been studied for a long time. Limb regeneration has been investigated as the representative phenomenon for organ-level regeneration. In limb regeneration, a prominent difference between regenerative and nonregenerative animals after limb amputation is blastema formation. A regeneration blastema requires the presence of nerves in the stump region. Thus, nerve regulation is responsible for blastema induction, and it has received much attention. Nerve regulation in regeneration has been investigated using the limb regeneration model and newly established alternative experimental model called the accessory limb model. Previous studies have identified some candidate genes that act as neural factors in limb regeneration, and these studies also clarified related events in early limb regeneration. Consistent with the nervous regulation and related events in limb regeneration, similar regeneration mechanisms in other organs have been discovered. This review especially focuses on the role of nerve-mediated fibroblast growth factor in the initiation phase of organ regeneration. Comparison of the initiation mechanisms for regeneration in various amphibian organs allows speculation about a fundamental regenerative process.

DOI： 10.1002/jez.b.23093

Sexual Experience Induces the Expression of Gastrin-Releasing Peptide and Oxytocin Receptors in the Spinal Ejaculation Generator in Rats. International journal

Takumi Oti, Ryota Ueda, Ryoko Kumagai, Junta Nagafuchi, Takashi Ito, Tatsuya Sakamoto, Yasuhiko Kondo, Hirotaka Sakamoto

International Journal of Molecular Sciences 22 ( 19 ) 2021.9

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3-L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain-spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that >50% of dXL neurons were activated in the first ejaculation group and ~30% in the control and intromission groups in sexually naïve males. In contrast, in sexually experienced males, ~50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to ~30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord.

DOI： 10.3390/ijms221910362

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years. Reviewed International journal

Akito Otubo, Sho Maejima, Takumi Oti, Keita Satoh, Yasumasa Ueda, John F Morris, Tatsuya Sakamoto, Hirotaka Sakamoto

International Journal of Molecular Sciences 22 ( 17 ) 2021.8

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

DOI： 10.3390/ijms22179180

Systemic effects of oxytocin on male sexual activity via the spinal ejaculation generator in rats. Reviewed International journal

Takumi Oti, Tatsuya Sakamoto, Hirotaka Sakamoto*

Communicative & Integrative Biology 14 ( 1 ) 55 - 60 2021.3

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal)

Oxytocin is produced in the hypothalamus and stimulates uterine contraction and milk ejection. While many people consider oxytocin to be a female hormone, it is reported that, in men, the plasma oxytocin level increases markedly after ejaculation. However, this aspect of oxytocin physiology is poorly understood. The spinal ejaculation generator (SEG), which expresses the neuropeptide, gastrin-releasing peptide (GRP), can trigger ejaculation in rats. Therefore, we focused on systemic effects of oxytocin on the GRP/SEG neuron system in the lumbar spinal cord controlling sexual activity in male rats. We found that systemic administration of oxytocin significantly shortened the latency to the first mount, intromission and ejaculation during male copulatory behavior. In addition, the local oxytocin level in the lumbar cord was significantly higher in males than in females. Histological analysis showed that oxytocin-binding is apparent in spinal GRP/SEG neurons. We therefore conclude that oxytocin influences male sexual activity via the SEG.

DOI： 10.1080/19420889.2021.1902056

Metabotropic glutamate receptor subtype 7 is essential for ejaculation. Reviewed International journal

Miwako Masugi-Tokita, Keiji Tomita, Kenichi Kobayashi, Tetsuya Yoshida, Susumu Kageyama, Hirotaka Sakamoto, Akihiro Kawauchi

Molecular Neurobiology 57 ( 12 ) 5208 - 5218 2020.12

Language：English Publishing type：Research paper (scientific journal)

Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of the group III mGluRs, which are negatively coupled to adenylate cyclase via Gi/Go proteins and localized to presynaptic active zones of the mammalian central nervous system (CNS). To elucidate the mechanism of impaired reproductivity of mGluR7 knockout (KO) mice, we investigated sexual behavior in this line, which exhibits ejaculatory disorder, although with normal sexual motivation and erectile function. To identify the site of action within the CNS responsible for the effect of mGluR7 on ejaculation, we then used a para-chloroamphetamine (PCA)-induced ejaculation model. Intrathecal administration of the mGluR7-selective antagonist 6-(4-methoxyphenyl)-5-methyl-3-pyridin-4-ylisoxazolo[4,5-c]pyridin-4(5H)-one (MMPIP) into the lumbosacral spinal cord inhibited PCA-induced ejaculation. Immunohistochemistry revealed mGluR7-like immunoreactivity (LI) expressed in the same area where lumbar spinothalamic (LSt) cells regulate the parasympathetic ejaculatory pathway. At high magnification, the apposition of mGluR7-LI puncta and neuronal nitric oxide synthase (nNOS)-LI-positive putative parasympathetic preganglionic neurons was evident. These results indicate that mGluR7 in the lumbosacral spinal cord regulates ejaculation by potentiating the excitability of parasympathetic preganglionic neurons. The ejaculatory disorder is a major issue in the field of male reproductive function. Erectile dysfunction (ED) can be treated by phosphodiesterase type 5 inhibitors like sildenafil (Viagra®), but the ejaculatory disorder cannot. Lack of understanding of the ejaculatory mechanism hinders the development of therapies for ejaculatory problems. This study is the first to demonstrate that mGluR7 regulates ejaculation and the results provide insight into the mechanism of ejaculation as well as a strategy for future therapies to treat ejaculatory disorders in humans.

DOI： 10.1007/s12035-020-02090-2

Detection and characterization of estrogen receptor beta expression in the brain with newly developed transgenic mice. Reviewed International journal

Shoko Sagoshi, Sho Maejima, Masahiro Morishita, Satoshi Takenawa, Akito Otubo, Keiko Takanami, Tatsuya Sakamoto, Hirotaka Sakamoto, Shinji Tsukahara, Sonoko Ogawa

Neuroscience 438 182 - 197 2020.7

Language：English Publishing type：Research paper (scientific journal)

Two types of nuclear estrogen receptors, ERα and ERβ, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERβ expression, detailed anatomical distribution and neurochemical characteristics of ERβ expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERβ-RFPtg, in which RFP was inserted downstream of ERβ BAC promotor. We verified RFP signals as ERβ by confirming: (1) high ERβ mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERβ mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERβ-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERβ, and those expressing exclusively either ERα or ERβ. The majority of PVN and DRN cells expressed only ERβ-RFP. Further, ERβ-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERβ-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERβ-RFPtg mice can be a powerful tool for future studies on ERβ function in the estrogenic regulation of social behaviors.

DOI： 10.1016/j.neuroscience.2020.04.047

The phosphatidylcholine transfer protein StarD7 is important for myogenic differentiation in mouse myoblast C2C12 cells and human primary skeletal myoblasts. Reviewed International journal

Yasuhiro Horibata, Satomi Mitsuhashi, Hiroaki Shimizu, Sho Maejima, Hirotaka Sakamoto, Chieko Aoyama, Hiromi Ando, Hiroyuki Sugimoto

Scientific Reports 10 ( 1 ) 2845 - 2845 2020.2

Language：English Publishing type：Research paper (scientific journal)

StarD7 is a phosphatidylcholine (PC)-specific lipid transfer protein essential for the maintenance of mitochondrial PC composition, morphogenesis, and respiration. Here, we studied the role of StarD7 in skeletal myoblast differentiation using mouse myoblast C2C12 cells and human primary myoblasts. Immunofluorescence and immuno-electron microscopy revealed that StarD7 was distributed in the cytosol, inner mitochondria space, and outer leaflet of the outer mitochondrial membrane in C2C12 cells. Unlike human kidney embryonic cell line HEK293 cells, the mitochondrial proteinase PARL was not involved in the processing and maturation of StarD7 in C2C12 cells. StarD7 was constantly expressed during myogenic differentiation of C2C12 cells. The siRNA-mediated knockdown of StarD7 in C2C12 cells and human primary myoblasts significantly impaired myogenic differentiation and reduced the expression of myomaker, myomerger and PGC-1α. The reduction in mitochondrial PC levels and oxygen consumption rates, decreased expression of myomaker, myomerger and PGC-1α, as well as impaired myogenic differentiation, were completely restored when the protein was reintroduced into StarD7-knockout C2C12 cells. These results suggest that StarD7 is important for skeletal myogenesis in mammals.

DOI： 10.1038/s41598-020-59444-y

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1–24) or ACTH(1–13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus Reviewed

Alexa L. Thomas, Fumihiko Maekawa, Takaharu Kawashima, Hirotaka Sakamoto, Tatsuya Sakamoto, Perry Davis, Robert M. Dores

General and Comparative Endocrinology 256 50 - 56 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Academic Press Inc.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1 day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland
mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1–24) at concentrations ranging from 10−13 M to 10−6 M. As expected, MC2R required co-expression with MRAP1 for functional expression
whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1–24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1–24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1–24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1–13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

DOI： 10.1016/j.ygcen.2017.09.002

Other Link： http://orcid.org/0000-0002-6224-4591

The mineralo-corticoid receptor knockout in medaka is further validated by glucocorticoid receptor compensation Reviewed

Tatsuya Sakamoto, Madoka Yoshiki, Hirotaka Sakamoto

Scientific Data 4 924 - 928 2017.12

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

To study the critical role of mineralocorticoid signalling, we generated a constitutive mineralocorticoid receptor (MR)-knockout (KO) medaka as the first adult-viable MR-KO animal. This KO medaka displayed abnormal behaviours affected by visual stimuli. In contrast, the loss of MR did not result in overt phenotypic changes in osmoregulation, despite the well-known osmoregulatory functions of MR in mammals. Since glucocorticoid receptor (GR) has been suggested to compensate for loss of MR, we examined expression of duplicated GRs with markedly different ligand sensitivities, in various tissues. qRT-PCR results revealed that the absence of MR induced GR1 in the brain and eyes, but not in osmoregulatory organs. This reinforces the important functions of glucocorticoid signalling, but the minor role of mineralocorticoid signalling, in fish osmoregulation. Because both 11-deoxycorticosterone (DOC) and cortisol are ligands for MR, whereas GRs are specific to cortisol, GR1 signalling may compensate for the absence of cortisol-MR, rather than that of DOC-MR. Thus, this GR expression suggests that our MR-KO model can be used specifically to characterize DOC-MR signalling.

DOI： 10.1038/sdata.2017.189

Other Link： http://orcid.org/0000-0002-6224-4591

MORPHOLOGICAL AND MOLECULAR EVOLUTIONAL ANALYSES OF ITCH FOCUSED ON THE GASTRIN-RELEASING PEPTIDE SYSTEM IN MAMMALS Reviewed

Keiko Takanami, Keita Satoh, Kazuyoshi Murata, Tatsuya Sakamoto, Hirotaka Sakamoto

ACTA DERMATO-VENEREOLOGICA 97 ( 8 ) 1049 - 1050 2017.9

Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish Reviewed

Tatsuya Sakamoto, Madoka Yoshiki, Hideya Takahashi, Masayuki Yoshida, Yukiko Ogino, Toshitaka Ikeuchi, Tomoya Nakamachi, Norifumi Konno, Kouhei Matsuda, Hirotaka Sakamoto

Scientific Reports 6 37991 2016.11

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper-and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

DOI： 10.1038/srep37991

Postnatal development of the gastrin-releasing peptide system in the lumbosacral spinal cord controlling male reproductive function in rats Reviewed

Nao Katayama, Takumi Oti, Keiko Takanami, Tatsuya Sakamoto, Hirotaka Sakamoto*

PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES 92 ( 2 ) 69 - 75 2016.2

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN ACAD

A sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord, which projects to the lower spinal centers, controls erection and ejaculation in rats. However, little is known about the postnatal development of this system. In this study, we therefore examined the postnatal development of the male-dominant spinal GRP system and its sexual differentiation in rats using immunohistochemistry. Our results show that male-dominant expression of GRP is prominent from the onset of puberty and that sexually dimorphism persists into adulthood. These results suggest that androgen surge during male puberty plays an important role in the development and maintenance of the male-specific GRP function in the rat spinal cord.

DOI： 10.2183/pjab.92.69

Comparative Anatomy of Gastrin-releasing Peptide Pathways in the Trigeminal Sensory System of Mouse and the Asian House Musk Shrew Suncus murinus Reviewed

Keiko Takanami, Kaihei Inoue, Hiroki Mukai, Kei Tamura, Takamichi Jogahara, Sen-ichi Oda, Mitsuhiro Kawata, Tatsuya Sakamoto, Hirotaka Sakamoto*

ACTA HISTOCHEMICA ET CYTOCHEMICA 49 ( 6 ) 181 - 190 2016

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：JAPAN SOC HISTOCHEMISTRY & CYTOCHEMISTRY

Gastrin-releasing peptide (GRP) has recently been identified as an itch-signaling molecule in the primary afferents and spinal cord of rodents. However, little information exists on the expression and localization of GRP in the trigeminal somatosensory system other than in rats. We examined the generality of the trigeminal GRP system in mammals using two distinct species, suncus as a model of specialized placental mammals known to have a well-developed trigeminal sensory system and mice as a representative small laboratory animal. We first analyzed the gross morphology of the trigeminal somatosensory system in suncus to provide a brainstem atlas on which to map GRP distribution. Immunohistochemical analyses showed that 8% of trigeminal ganglion neurons in suncus and 6% in mice expressed GRP. Expression was restricted to cells with smaller somata. The GRP-containing fibers were densely distributed in the superficial layers of the caudal part of the trigeminal spinal nucleus (Vc) but rare in the rostral parts, both in suncus and mice. Expression of GRP receptor mRNA and protein was also detected in the Vc of suncus. Taken together, these results suggest that the trigeminal GRP system mediating itch sensation is conserved in mammals.

DOI： 10.1267/ahc.16030

Three-dimensional visualization of multiple synapses in thick sections using high-voltage electron microscopy in the rat spinal cord Reviewed

Data in Brief 4 566 - 570 2015.9

Authorship：Last author,　Corresponding author Language：English Publisher：Elsevier Inc.

This data article contains complementary figure and movies (Supplementary Movies 1-3) related to the research article entitled, "Effective synaptome analysis of itch-mediating neurons in the spinal cord: a novel immunohistochemical methodology using high-voltage electron microscopy" [7]. It is important to show the synaptic connections at the ultrastructural level to understand the neural circuit, which requires the three-dimensional (3-D) analyses in the electron microscopy. Here, we applied a new sample preparation method, a high-contrast en bloc staining according to the protocol of the National Center for Microscopy and Imaging Research (NCMIR), University of California, San Diego, CA, USA to high-voltage electron microscopy (HVEM) tomography in order to examine the 3-D chemical neuroanatomy of the rat spinal cord. Pre-embedding immunoelectron microscopy was used in this study. HVEM has an excellent potential to directly visualize the ultrastructures in semi-thin sections (~5. μm thick), and we have successfully visualized many itch-mediating synaptic connections and neural networks in the spinal cord using "HVEM tomography". Moreover, the methodology used in this study is simple and can be applied in multiple ways. This is an important contribution to ultrastructural investigations of the central nervous system in the present post-genomic age.

DOI： 10.1016/j.dib.2015.07.005

Oxytocin and the gastrin-releasing peptide system in the spinal cord: Implications for male sexual problems Reviewed

Sakamoto H*, Oti T

Interdisciplinary Information Sciences 21 ( 3 ) 235 - 242 2015.9

Authorship：Lead author,　Corresponding author Language：English Publisher：Tohoku University

Neural circuits underlying male sexual function comprise several nuclei located in the brain and spinal cord. We have previously demonstrated in rats that the gastrin-releasing peptide (GRP) system influences spinal centers promoting penile reflexes. Moreover, a group of oxytocin (OXT) neurons, situated in the parvocellular part of the paraventricular nucleus of the hypothalamus, project into the spinal cord and control penile reflexes. Therefore, it has been hypothesized that OXT is transported by long descending paraventriculospinal pathways and activates proerectile spinal centers. Consequently, we have shown that in rats, axonal distribution of OXT in the lumbar spinal cord exhibits a male-dominant sexual dimorphism. Furthermore, OXT binding is observed in the spinal GRP neurons. Thus, OXT axons may secrete OXT from spinal axonal terminals and regulate male sexual function via an OXT receptor-mediated mechanism in spinal GRP neurons. Future studies should address the relationship between the hypothalamic OXT and spinal GRP systems. Identification of the male-specific brain-spinal cord neural circuit that regulates male sexual behavior may provide new avenues for therapeutic approaches to masculine reproductive dysfunction, including erectile dysfunction and/or ejaculation disorder.

DOI： 10.4036/iis.2015.B.08

Other Link： https://jlc.jst.go.jp/DN/JLC/20015114165?from=CiNii

Osmotic/ionic status of body fluids in the euryhaline cephalopod suggest possible parallel evolution of osmoregulation Reviewed

Tatsuya Sakamoto, Satoshi Ogawa, Yudai Nishiyama, Chiaki Akada, Hideya Takahashi, Taro Watanabe, Hiroyuki Minakata, Hirotaka Sakamoto

SCIENTIFIC REPORTS 5 14469 2015.9

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：NATURE PUBLISHING GROUP

Acclimation from marine to dilute environments constitutes among the dramatic evolutionary transitions in the history of life. Such adaptations have evolved in multiple lineages, but studies of the blood/hemolymph homeostasis mechanisms are limited to those using evolutionarily advanced Deuterostome (chordates) and Ecdysozoa (crustaceans). Here, we examined hemolymph homeostasis in the advanced Lophotrochozoa/mollusc, the other unexplored taxa, and its possible regulation by the vasopressin/oxytocin superfamily peptides known to be implicated in fluid homeostasis in Chordata and Arthropoda. The hemolymph osmotic and ionic status in the euryhaline cephalopod (Octopus ocellatus) following transfer from 30-ppt normal seawater to 20 ppt salinity indicate hyperosmo- and hyperionoregulatory abilities for more than 1 week, as in crustaceans and teleost fish. While ventilation frequency decreased by 1 day, Na+/K+-ATPase activity, which has been generally implicated in ion transport, was induced in two of the eight posterior gills after 1 week. In addition, the octopuses were intravenously injected with 1 or 100 ng/g octopressin or cephalotocin, which are Octopus vasopressin/oxytocin orthologs. After 1 day, octopressin, but not cephalotocin, decreased the hemolymph osmolality and Ca concentrations, as well as urinary Na concentrations. These data provide evidence for possible parallel evolution in hyperionoregulatory mechanisms and coordination by conserved peptides.

DOI： 10.1038/srep14469

Neurohypophysial Hormones Regulate Amphibious Behaviour in the Mudskipper Goby Reviewed

Tatsuya Sakamoto, Yudai Nishiyama, Aoi Ikeda, Hideya Takahashi, Susumu Hyodo, Nao Kagawa, Hirotaka Sakamoto

PLOS ONE 10 ( 7 ) e0134605 2015.7

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：PUBLIC LIBRARY SCIENCE

The neurohypophysial hormones, arginine vasotocin and isotocin, regulate both hydromineral balance and social behaviors in fish. In the amphibious mudskipper, Periophthalmus modestus, we previously found arginine-vasotocin-specific regulation of aggressive behavior, including migration of the submissive subordinate into water. This migration also implies the need for adaptation to dehydration. Here, we examined the effects of arginine vasotocin and isotocin administration on the amphibious behavior of individual mudskippers in vivo. The mudskippers remained in the water for an increased period of time after 1-8 h of intracerebroventricular (ICV) injection with 500 pg/g arginine vasotocin or isotocin. The 'frequency of migration' was decreased after ICV injection of arginine vasotocin or isotocin, reflecting a tendency to remain in the water. ICV injections of isotocin receptor antagonist with arginine vasotocin or isotocin inhibited all of these hormonal effects. In animals kept out of water, mRNA expression of brain arginine vasotocin and isotocin precursors increased 3- and 1.5-fold, respectively. Given the relatively wide distribution of arginine vasotocin fibres throughout the mudskipper brain, induction of arginine vasotocin and isotocin under terrestrial conditions may be involved also in the preference for an aquatic habitat as ligands for brain isotocin receptors.

DOI： 10.1371/journal.pone.0134605

Effective synaptome analysis of itch-mediating neurons in the spinal cord: A novel immunohistochemical methodology using high-voltage electron microscopy Reviewed

Keita Satoh, Keiko Takanami, Kazuyoshi Murata, Mitsuhiro Kawata, Tatsuya Sakamoto, Hirotaka Sakamoto

599 86 - 91 2015.7

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Transmission electron microscopy (TEM) is used for three-dimensional (3-D) analysis of synaptic connections in neuroscience research. However, 3-D reconstruction of the synapses using serial ultrathin sections is a powerful but tedious approach requiring advanced technical skills. High-voltage electron microscopy (HVEM) allows examination of thicker sections of biological specimens due to the increased penetration of the more accelerated electrons, which is useful to analyze the 3-D structure of biological specimens. However, it is still difficult to visualize the neural networks and synaptic connections in 3-D using HVEM because of insufficient and non uniform heavy metal staining in the membranous structures in semi-thin sections. Here, we present the successful chemical 3-D neuroanatomy of the rat spinal dorsal horn at the ultrastructural level as a first step for effective synaptome analysis by applying a high-contrast en bloc staining method to immune-HVEM tomography. Our new approach made it possible to examine many itch-mediating synaptic connections and neural networks in the spinal cord simultaneously using HVEM tomography. This novel 3-D electron microscopy is very useful for the analysis of synaptic structure and the chemical neuroanatomy at the 3-D ultrastructural level. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2015.05.031

Quality Control of Photosystem II: Direct Imaging of the Changes in the Thylakoid Structure and Distribution of FtsH Proteases in Spinach Chloroplasts under Light Stress Reviewed

Miho Yoshioka-Nishimura, Daisuke Nanba, Takashi Takaki, Chikako Ohba, Nodoka Tsumura, Noriko Morita, Hirotaka Sakamoto, Kazuyoshi Murata, Yasusi Yamamoto

PLANT AND CELL PHYSIOLOGY 55 ( 7 ) 1255 - 1265 2014.7

Language：English Publishing type：Research paper (scientific journal) Publisher：OXFORD UNIV PRESS

Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids.

DOI： 10.1093/pcp/pcu079

Distribution of Gastrin-Releasing Peptide in the Rat Trigeminal and Spinal Somatosensory Systems Reviewed

Keiko Takanami, Hirotaka Sakamoto, Ken Ichi Matsuda, Keita Satoh, Takashi Tanida, Shunji Yamada, Kaihei Inoue, Takumi Oti, Tatsuya Sakamoto, Mitsuhiro Kawata

JOURNAL OF COMPARATIVE NEUROLOGY 522 ( 8 ) 1858 - 1873 2014.6

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-BLACKWELL

Gastrin-releasing peptide (GRP) has recently been identified as an itch-specific neuropeptide in the spinal sensory system in mice, but there are no reports of the expression and distribution of GRP in the trigeminal sensory system in mammals. We characterized and compared GRP-immunoreactive (ir) neurons in the trigeminal ganglion (TG) with those in the rat spinal dorsal root ganglion (DRG). GRP immunoreactivity was expressed in 12% of TG and 6% of DRG neurons and was restricted to the small- and medium-sized type cells. In both the TG and DRG, many GRP-ir neurons also expressed substance P and calcitonin gene-related peptide, but not isolectin B-4. The different proportions of GRP and transient receptor potential vanilloid 1 double-positive neurons in the TG and DRG imply that itch sensations via the TG and DRG pathways are transmitted through distinct mechanisms. The distribution of the axon terminals of GRP-ir primary afferents and their synaptic connectivity with the rat trigeminal sensory nuclei and spinal dorsal horn were investigated by using light and electron microscopic histochemistry. Although GRP-ir fibers were rarely observed in the trigeminal sensory nucleus principalis, oralis, and interpolaris, they were predominant in the superficial layers of the trigeminal sensory nucleus caudalis (Vc), similar to the spinal dorsal horn. Ultrastructural analysis revealed that GRP-ir terminals contained clear microvesicles and large dense-cored vesicles, and formed asymmetric synaptic contacts with a few dendrites in the Vc and spinal dorsal horn. These results suggest that GRP-dependent orofacial and spinal pruriceptive inputs are processed mainly in the superficial laminae of the Vc and spinal dorsal horn. J. Comp. Neurol. 522:1858-1873, 2014. (c) 2013 Wiley Periodicals, Inc.

DOI： 10.1002/cne.23506

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