Faculty Profiles - SAKAMOTO Hirotaka

Papers - SAKAMOTO Hirotaka

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Neuropeptidergic control circuits in the spinal cord for male sexual behaviour: Oxytocin-gastrin-releasing peptide systems. Reviewed International journal

Takumi Oti, Hirotaka Sakamoto

Journal of neuroendocrinology 35 ( 9 ) e13324 2023.9

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

The neuropeptidergic mechanisms controlling socio-sexual behaviours consist of complex neuronal circuitry systems in widely distributed areas of the brain and spinal cord. At the organismal level, it is now becoming clear that "hormonal regulations" play an important role, in addition to the activation of neuronal circuits. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the "spinal ejaculation generator (SEG)." Oxytocin, long known as a neurohypophyseal hormone, is now known to be involved in the regulation of socio-sexual behaviors in mammals, ranging from social bonding to empathy. However, the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system remains unclear. Oxytocin is known to be synthesised mainly in hypothalamic neurons and released from the posterior pituitary into the circulation. Oxytocin is also released from the dendrites of the neurons into the hypothalamus where they have important roles in social behaviours via non-synaptic volume transmission. Because the most familiar functions of oxytocin are to regulate female reproductive functions including parturition, milk ejection, and maternal behaviour, oxytocin is often thought of as a "feminine" hormone. However, there is evidence that a group of parvocellular oxytocin neurons project to the lower spinal cord and control male sexual function in rats. In this report, we review the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system and effects of these neuropeptides on male sexual behaviour. Furthermore, we discuss the finding of a recently identified, localised "volume transmission" role of oxytocin in the spinal cord. Findings from our studies suggest that the newly discovered "oxytocin-mediated spinal control of male sexual function" may be useful in the treatment of erectile and ejaculatory dysfunction.

DOI： 10.1111/jne.13324

Endolysosomal TPCs regulate social behavior by controlling oxytocin secretion. Reviewed International coauthorship International journal

Lora L Martucci, Jean-Marie Launay, Natsuko Kawakami, Cécile Sicard, Nathalie Desvignes, Mbarka Dakouane-Giudicelli, Barbara Spix, Maude Têtu, Franck-Olivier Gilmaire, Sloane Paulcan, Jacques Callebert, Cyrille Vaillend, Franz Bracher, Christian Grimm, Philippe Fossier, Sabine de la Porte, Hirotaka Sakamoto, John Morris, Antony Galione, Sylvie Granon, José-Manuel Cancela

Proceedings of the National Academy of Sciences of the United States of America 120 ( 7 ) e2213682120 2023.2

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

Oxytocin (OT) is a prominent regulator of many aspects of mammalian social behavior and stored in large dense-cored vesicles (LDCVs) in hypothalamic neurons. It is released in response to activity-dependent Ca2+ influx, but is also dependent on Ca2+ release from intracellular stores, which primes LDCVs for exocytosis. Despite its importance, critical aspects of the Ca2+-dependent mechanisms of its secretion remain to be identified. Here we show that lysosomes surround dendritic LDCVs, and that the direct activation of endolysosomal two-pore channels (TPCs) provides the critical Ca2+ signals to prime OT release by increasing the releasable LDCV pool without directly stimulating exocytosis. We observed a dramatic reduction in plasma OT levels in TPC knockout mice, and impaired secretion of OT from the hypothalamus demonstrating the importance of priming of neuropeptide vesicles for activity-dependent release. Furthermore, we show that activation of type 1 metabotropic glutamate receptors sustains somatodendritic OT release by recruiting TPCs. The priming effect could be mimicked by a direct application of nicotinic acid adenine dinucleotide phosphate, the endogenous messenger regulating TPCs, or a selective TPC2 agonist, TPC2-A1-N, or blocked by the antagonist Ned-19. Mice lacking TPCs exhibit impaired maternal and social behavior, which is restored by direct OT administration. This study demonstrates an unexpected role for lysosomes and TPCs in controlling neuropeptide secretion, and in regulating social behavior.

DOI： 10.1073/pnas.2213682120

Footedness for scratching itchy eyes in rodents Reviewed International journal

Yukitoshi Katayama, Ayane Miura, Tatsuya Sakamoto, Keiko Takanami, Hirotaka Sakamoto

Proceedings of the Royal Society B: Biological Sciences 289 ( 1985 ) 2022.10

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

The neural bases of itchy eye transmission remain unclear compared with those involved in body itch. Here, we show in rodents that the gastrin-releasing peptide receptor (GRPR) of the trigeminal sensory system is involved in the transmission of itchy eyes. Interestingly, we further demonstrate a difference in scratching behaviour between the left and right hindfeet in rodents; histamine instillation into the conjunctival sac of both eyes revealed right-foot biased laterality in the scratching movements. Unilateral histamine instillation specifically induced neural activation in the ipsilateral sensory pathway, with no significant difference between the activations following left- and right-eye instillations. Thus, the behavioural laterality is presumably due to right-foot preference in rodents. Genetically modified rats with specific depletion of Grpr- expressing neurons in the trigeminal sensory nucleus caudalis of the medulla oblongata exhibited fewer and shorter histamine-induced scratching movements than controls and eliminated the footedness. These results taken together indicate that the Grpr -expressing neurons are required for the transmission of itch sensation from the eyes, but that foot preference is generated centrally. These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches to refractory pruritic eye disorders.

DOI： 10.1098/rspb.2022.1126

Other Link： https://royalsocietypublishing.org/doi/full-xml/10.1098/rspb.2022.1126

Neuronal pentraxin 2 is required for facilitating excitatory synaptic inputs onto spinal neurons involved in pruriceptive transmission in a model of chronic itch. Reviewed International coauthorship International journal

Kensho Kanehisa, Keisuke Koga, Sho Maejima, Yuto Shiraishi, Konatsu Asai, Miho Shiratori-Hayashi, Mei-Fang Xiao, Hirotaka Sakamoto, Paul F Worley, Makoto Tsuda

Nature Communications 13 ( 1 ) 2367 - 2367 2022.5

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

An excitatory neuron subset in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for pruriceptive transmission. Here, we show that glutamatergic excitatory inputs onto GRPR+ neurons are facilitated in mouse models of chronic itch. In these models, neuronal pentraxin 2 (NPTX2), an activity-dependent immediate early gene product, is upregulated in the dorsal root ganglion (DRG) neurons. Electron microscopy reveals that NPTX2 is present at presynaptic terminals connected onto postsynaptic GRPR+ neurons. NPTX2-knockout prevents the facilitation of synaptic inputs to GRPR+ neurons, and repetitive scratching behavior. DRG-specific NPTX2 expression rescues the impaired behavioral phenotype in NPTX2-knockout mice. Moreover, ectopic expression of a dominant-negative form of NPTX2 in DRG neurons reduces chronic itch-like behavior in mice. Our findings indicate that the upregulation of NPTX2 expression in DRG neurons contributes to the facilitation of glutamatergic inputs onto GRPR+ neurons under chronic itch-like conditions, providing a potential therapeutic target.

DOI： 10.1038/s41467-022-30089-x

A spinal microglia population involved in remitting and relapsing neuropathic pain. Reviewed International journal

Keita Kohno, Ryoji Shirasaka, Kohei Yoshihara, Satsuki Mikuriya, Kaori Tanaka, Keiko Takanami, Kazuhide Inoue, Hirotaka Sakamoto, Yasuyuki Ohkawa, Takahiro Masuda, Makoto Tsuda

Science (New York, N.Y.) 376 ( 6588 ) 86 - 90 2022.4

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

Neuropathic pain is often caused by injury and diseases that affect the somatosensory system. Although pain development has been well studied, pain recovery mechanisms remain largely unknown. Here, we found that CD11c-expressing spinal microglia appear after the development of behavioral pain hypersensitivity following nerve injury. Nerve-injured mice with spinal CD11c+ microglial depletion failed to recover spontaneously from this hypersensitivity. CD11c+ microglia expressed insulin-like growth factor-1 (IGF1), and interference with IGF1 signaling recapitulated the impairment in pain recovery. In pain-recovered mice, the depletion of CD11c+ microglia or the interruption of IGF1 signaling resulted in a relapse in pain hypersensitivity. Our findings reveal a mechanism for the remission and recurrence of neuropathic pain, providing potential targets for therapeutic strategies.

DOI： 10.1126/science.abf6805

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians. Reviewed International coauthorship International journal

Aoshi Kobayashi, Mayuko Hamada, Masa-Aki Yoshida, Yasuhisa Kobayashi, Naoaki Tsutsui, Toshio Sekiguchi, Yuta Matsukawa, Sho Maejima, Joseph J Gingell, Shoko Sekiguchi, Ayumu Hamamoto, Debbie L Hay, John F Morris, Tatsuya Sakamoto, Hirotaka Sakamoto

Science Advances 8 ( 9 ) eabk0331 2022.3

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

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

DOI： 10.1126/sciadv.abk0331

The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus Reviewed

Asuka Hirooka, Mayuko Hamada, Daiki Fujiyama, Keiko Takanami, Yasuhisa Kobayashi, Takumi Oti, Yukitoshi Katayama, Tatsuya Sakamoto, Hirotaka Sakamoto

Scientific Reports 11 ( 1 ) 2021.12

Authorship：Last author,　Corresponding author Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

<title>Abstract</title>Bombesin is a putative antibacterial peptide isolated from the skin of the frog, <italic>Bombina bombina</italic>. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (<italic>Xenopus tropicalis</italic>), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in <italic>Xenopus</italic>. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of <italic>Xenopus</italic>. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the <italic>‘gut-brain peptide’</italic> systems.

DOI： 10.1038/s41598-021-92528-x

Other Link： http://www.nature.com/articles/s41598-021-92528-x

Estrogens influence female itch sensitivity via the spinal gastrin-releasing peptide receptor neurons Reviewed International coauthorship

Keiko Takanami, Daisuke Uta, Ken Ichi Matsuda, Mitsuhiro Kawata, Earl Carstens, Tatsuya Sakamoto, Hirotaka Sakamoto

Proceedings of the National Academy of Sciences of the United States of America 118 ( 31 ) e2103536118 - e2103536118 2021.8

Authorship：Last author Publishing type：Research paper (scientific journal) Publisher：Proceedings of the National Academy of Sciences

There are sex differences in somatosensory sensitivity. Circulating estrogens appear to have a pronociceptive effect that explains why females are reported to be more sensitive to pain than males. Although itch symptoms develop during pregnancy in many women, the underlying mechanism of female-specific pruritus is unknown. Here, we demonstrate that estradiol, but not progesterone, enhances histamine-evoked scratching behavior indicative of itch in female rats. Estradiol increased the expression of the spinal itch mediator, gastrin-releasing peptide (GRP), and increased the histamine-evoked activity of itch-processing neurons that express the GRP receptor (GRPR) in the spinal dorsal horn. The enhancement of itch behavior by estradiol was suppressed by intrathecal administration of a GRPR blocker. In vivo electrophysiological analysis showed that estradiol increased the histamine-evoked firing frequency and prolonged the response of spinal GRP-sensitive neurons in female rats. On the other hand, estradiol did not affect the threshold of noxious thermal pain and decreased touch sensitivity, indicating that estradiol separately affects itch, pain, and touch modalities. Thus, estrogens selectively enhance histamine-evoked itch in females via the spinal GRP/GRPR system. This may explain why itch sensation varies with estrogen levels and provides a basis for treating itch in females by targeting GRPR.

DOI： 10.1073/pnas.2103536118

Other Link： https://syndication.highwire.org/content/doi/10.1073/pnas.2103536118

Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin. Reviewed International journal

Natsuko Kawakami, Akito Otubo, Sho Maejima, Ashraf H Talukder, Keita Satoh, Takumi Oti, Keiko Takanami, Yasumasa Ueda, Keiichi Itoi, John F Morris, Tatsuya Sakamoto, Hirotaka Sakamoto*

The Journal of Comparative Neurology 529 ( 7 ) 1372 - 1390 2021.5

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

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

DOI： 10.1002/cne.25026

In vivo electrophysiology of peptidergic neurons in deep layers of the lumbar spinal cord after optogenetic stimulation of hypothalamic paraventricular oxytocin neurons in rats. Reviewed International journal

Daisuke Uta*, Takumi Oti, Tatsuya Sakamoto, Hirotaka Sakamoto*

International Journal of Molecular Sciences 22 ( 7 ) 2021.3

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

The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain-spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior.

DOI： 10.3390/ijms22073400

Oxytocin influences male sexual activity via non-synaptic axonal release in the spinal cord Reviewed International journal

Takumi Oti, Keita Satoh, Daisuke Uta, Junta Nagafuchi, Sayaka Tateishi, Ryota Ueda, Keiko Takanami, Larry J. Young, Antony Galione, John F. Morris, Tatsuya Sakamoto, Hirotaka Sakamoto*

Current Biology 31 ( 1 ) 103 - 114.e5 2021.1

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

Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the "spinal ejaculation generator (SEG)." We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion-a localized volume transmission-to reach oxytocin receptors on GRP neurons and facilitate male sexual function.

DOI： 10.1016/j.cub.2020.09.089

Spinal astrocytes in superficial laminae gate brainstem descending control of mechanosensory hypersensitivity Reviewed International journal

Yuta Kohro, Tsuyoshi Matsuda, Kohei Yoshihara, Keita Kohno, Keisuke Koga, Ryuichi Katsuragi, Takaaki Oka, Ryoichi Tashima, Sho Muneta, Takuya Yamane, Shota Okada, Kazuya Momokino, Aogu Furusho, Kenji Hamase, Takumi Oti, Hirotaka Sakamoto, Kenichiro Hayashida, Ryosuke Kobayashi, Takuro Horii, Izuho Hatada, Hidetoshi Tozaki-Saitoh, Katsuhiko Mikoshiba, Verdon Taylor, Kazuhide Inoue, Makoto Tsuda

Nature Neuroscience 23 ( 11 ) 1376 - 1387 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Springer Science and Business Media LLC

Astrocytes are critical regulators of CNS function and are proposed to be heterogeneous in the developing brain and spinal cord. Here we identify a population of astrocytes located in the superficial laminae of the spinal dorsal horn (SDH) in adults that is genetically defined by Hes5. In vivo imaging revealed that noxious stimulation by intraplantar capsaicin injection activated Hes5+ SDH astrocytes via α1A-adrenoceptors (α1A-ARs) through descending noradrenergic signaling from the locus coeruleus. Intrathecal norepinephrine induced mechanical pain hypersensitivity via α1A-ARs in Hes5+ astrocytes, and chemogenetic stimulation of Hes5+ SDH astrocytes was sufficient to produce the hypersensitivity. Furthermore, capsaicin-induced mechanical hypersensitivity was prevented by the inhibition of descending locus coeruleus-noradrenergic signaling onto Hes5+ astrocytes. Moreover, in a model of chronic pain, α1A-ARs in Hes5+ astrocytes were critical regulators for determining an analgesic effect of duloxetine. Our findings identify a superficial SDH-selective astrocyte population that gates descending noradrenergic control of mechanosensory behavior.

DOI： 10.1038/s41593-020-00713-4

Other Link： http://www.nature.com/articles/s41593-020-00713-4

Degradation of mutant protein aggregates within the endoplasmic reticulum of vasopressin neurons. Reviewed International journal

Takashi Miyata, Daisuke Hagiwara, Yuichi Hodai, Tsutomu Miwata, Yohei Kawaguchi, Junki Kurimoto, Hajime Ozaki, Kazuki Mitsumoto, Hiroshi Takagi, Hidetaka Suga, Tomoko Kobayashi, Mariko Sugiyama, Takeshi Onoue, Yoshihiro Ito, Shintaro Iwama, Ryoichi Banno, Mami Matsumoto, Natsuko Kawakami, Nobuhiko Ohno, Hirotaka Sakamoto, Hiroshi Arima

iScience 23 ( 10 ) 101648 - 101648 2020.10

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

Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER.

DOI： 10.1016/j.isci.2020.101648

Vasopressin gene products are colocalised with corticotrophin-releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata). Reviewed International journal

Akito Otubo, Natsuko Kawakami, Sho Maejima, Yasumasa Ueda, John F Morris, Tatsuya Sakamoto, Hirotaka Sakamoto*

Journal of Neuroendocrinology 32 ( 8 ) e12875 2020.8

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

Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin-releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP-associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF- and AVP-producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription-polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII-immunoreactive (AVP-producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF-immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense-cored vesicles of CRF-containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic-pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co-packaged in neurosecretory vesicles in monkeys and are thus likely to be co-released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary.

DOI： 10.1111/jne.12875

Effects of Sex Steroids on the Spinal Gastrin-Releasing Peptide System Controlling Male Sexual Function in Rats Reviewed International journal

Takumi Oti, Keiko Takanami, Saya Ito, Takashi Ueda, Ken Ichi Matsuda, Mitsuhiro Kawata, Jintetsu Soh, Osamu Ukimura, Tatsuya Sakamoto, Hirotaka Sakamoto*

Endocrinology 159 ( 4 ) 1886 - 1896 2018.4

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

The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord controls male sexual function in rats. In contrast, in female rats, GRP neurons could scarcely be detected around puberty when circulating ovarian steroid hormones such as estradiol and progesterone levels are increasing. However, little information is available on feminizing or demasculinizing effects of ovarian steroids on the central nervous system in female puberty and adulthood. In this study, to visualize the spinal GRP neurons in vivo, we generated a GRP-promoter-Venus transgenic (Tg) rat line and studied the effects of the sex steroid hormones on GRP expression in the rat lumbar cord by examining the Venus fluorescence. In these Tg rats, the sexually dimorphic spinal GRP neurons controlling male sexual function were clearly labeled with Venus fluorescence. As expected, Venus fluorescence in the male lumbar cord was markedly decreased after castration and restored by chronic androgen replacement. Furthermore, androgen-induced Venus expression in the spinal cord of adult Tg males was significantly attenuated by chronic treatment with progesterone but not with estradiol. A luciferase assay using a human GRP-promoter construct showed that androgens enhance the spinal GRP system, and more strikingly, that progesterone acts to inhibit the GRP system via an androgen receptor-mediated mechanism. These results demonstrate that circulating androgens may play an important role in the spinal GRP system controlling male sexual function not only in rats but also in humans and that progesterone could be an important feminizing factor in the spinal GRP system in females during pubertal development.

DOI： 10.1210/en.2018-00043

Nemertean and phoronid genomes reveal lophotrochozoan evolution and the origin of bilaterian heads Reviewed

Yi-Jyun Luo, Miyuki Kanda, Ryo Koyanagi, Kanako Hisata, Tadashi Akiyama, Hirotaka Sakamoto, Tatsuya Sakamoto, Noriyuki Satoh

Nature Ecology and Evolution 2 ( 1 ) 141 - 151 2018.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Nature Publishing Group

Nemerteans (ribbon worms) and phoronids (horseshoe worms) are closely related lophotrochozoans - a group of animals including leeches, snails and other invertebrates. Lophotrochozoans represent a superphylum that is crucial to our understanding of bilaterian evolution. However, given the inconsistency of molecular and morphological data for these groups, their origins have been unclear. Here, we present draft genomes of the nemertean Notospermus geniculatus and the phoronid Phoronis australis, together with transcriptomes along the adult bodies. Our genome-based phylogenetic analyses place Nemertea sister to the group containing Phoronida and Brachiopoda. We show that lophotrochozoans share many gene families with deuterostomes, suggesting that these two groups retain a core bilaterian gene repertoire that ecdysozoans (for example, flies and nematodes) and platyzoans (for example, flatworms and rotifers) do not. Comparative transcriptomics demonstrates that lophophores of phoronids and brachiopods are similar not only morphologically, but also at the molecular level. Despite dissimilar head structures, lophophores express vertebrate head and neuronal marker genes. This finding suggests a common origin of bilaterian head patterning, although different heads evolved independently in each lineage. Furthermore, we observe lineage-specific expansions of innate immunity and toxin-related genes. Together, our study reveals a dual nature of lophotrochozoans, where conserved and lineage-specific features shape their evolution.

DOI： 10.1038/s41559-017-0389-y

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

A sexually dimorphic peptidergic system in the lower spinal cord controlling penile function in non-human primates Reviewed

T. Ito, T. Oti, K. Takanami, K. Satoh, Y. Ueda, T. Sakamoto, H. Sakamoto

Spinal Cord 56 ( 1 ) 57 - 62 2018

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

Study design: Experimental animal study. Objectives: Although a population of gastrin-releasing peptide (GRP) neurons in the lumbar spinal cord has an important role in erection and ejaculation in rats, little information exists on this GRP system in primates. To identify the male-specific GRP system in the primate spinal cord, we studied the lumbosacral cord in macaque monkeys as a non-human primate model. Setting: University laboratory in Japan. Methods: To determine the gene sequence of GRP precursors, the rhesus macaque monkey genomic sequence data were searched, followed by phylogenetic analysis. Subsequently, immunocytochemical analysis for GRP was performed in the monkey spinal cord. Results: We have used bioinformatics to identify the ortholog gene for GRP precursor in macaque monkeys. Phylogenetic analysis suggested that primate prepro-GRP is separated from that of other mammalian species and clustered to an independent branch as primates. Immunocytochemistry for GRP further demonstrated that male-dominant sexual dimorphism was found in the spinal GRP system in monkeys as in rodents. Conclusion: We have demonstrated in macaque monkeys that the GRP system in the lower spinal cord shows male-specific dimorphism and May have an important role in penile functions not only in rodents but also in primates.

DOI： 10.1038/sc.2017.105

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

Activation of supraoptic oxytocin neurons by secretin facilitates social recognition. Reviewed International journal

Yuki Takayanagi, Masahide Yoshida, Akihide Takashima, Keiko Takanami, Shoma Yoshida, Katsuhiko Nishimori, Ichiko Nishijima, Hirotaka Sakamoto, Takanori Yamagata, Tatsushi Onaka

Biological Psychiatry 81 ( 3 ) 243 - 251 2017.2

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE INC

BACKGROUND: Social recognition underlies social behavior in animals, and patients with psychiatric disorders associated with social deficits show abnormalities in social recognition. Oxytocin is implicated in social behavior and has received attention as an effective treatment for sociobehavioral deficits. Secretin receptor-deficient mice show deficits in social behavior. The relationship between oxytocin and secretin concerning social behavior remains to be determined. METHODS: Expression of c-Fos in oxytocin neurons and release of oxytocin from their dendrites after secretin application were investigated. Social recognition was examined after intracerebroventricular or local injection of secretin, oxytocin, or an oxytocin receptor antagonist in rats, oxytocin receptor-deficient mice, and secretin receptor-deficient mice. Electron and light microscopic immunohistochemical analysis was also performed to determine whether oxytocin neurons extend their dendrites into the medial amygdala. RESULTS: Supraoptic oxytocin neurons expressed the secretin receptor. Secretin activated supraoptic oxytocin neurons and facilitated oxytocin release from dendrites. Secretin increased acquisition of social recognition in an oxytocin receptor-dependent manner. Local application of secretin into the supraoptic nucleus facilitated social recognition, and this facilitation was blocked by an oxytocin receptor antagonist injected into, but not outside of, the medial amygdala. In the medial amygdala, dendrite-like thick oxytocin processes were found to extend from the supraoptic nucleus. Furthermore, oxytocin treatment restored deficits of social recognition in secretin receptor-deficient mice. CONCLUSIONS: The results of our study demonstrate that secretin-induced dendritic oxytocin release from supraoptic neurons enhances social recognition. The newly defined secretin-oxytocin system may lead to a possible treatment for social deficits.

DOI： 10.1016/j.biopsych.2015.11.021

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

Perinatal testosterone exposure is critical for the development of the male-specific sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that mediates erection and ejaculation Reviewed

Takumi Oti, Keiko Takanami, Nao Katayama, Tomoca Edey, Keita Satoh, Tatsuya Sakamoto, Hirotaka Sakamoto*

BIOLOGY OF SEX DIFFERENCES 7 ( 1 ) 4 2016.1

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

Background: In rats, a sexually dimorphic spinal gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord projects to spinal centers that control erection and ejaculation. This system controls the sexual function of adult males in an androgen-dependent manner. In the present study, we assessed the influence of androgen exposure on the spinal GRP system during a critical period of the development of sexual dimorphism.
Methods: Immunohistochemistry was used to determine if the development of the spinal GRP system is regulated by the perinatal androgen surge. We first analyzed the responses of neonates administered with anti-androgen flutamide. To remove endogenous androgens, rats were castrated at birth. Further, neonatal females were administered androgens during a critical period to evaluate the development of the male-specific spinal GRP system.
Results: Treatment of neonates with flutamide on postnatal days 0 and 1 attenuated the spinal GRP system during adulthood. Castrating male rats at birth resulted in a decrease in the number of GRP neurons and the intensity of neuronal GRP in the spinal cord during adulthood despite testosterone supplementation during puberty. This effect was prevented if the rats were treated with testosterone propionate immediately after castration. Moreover, treating female rats with androgens on the day of birth and the next day, masculinized the spinal GRP system during adulthood, which resembled the masculinized phenotype of adult males and induced a hypermasculine appearance.
Conclusions: The perinatal androgen surge plays a key role in masculinization of the spinal GRP system that controls male sexual behavior. Further, the present study provides potentially new approaches to treat sexual disorders of males.

DOI： 10.1186/s13293-016-0058-x

In vivo imaging of axonal transport of mitochondria in the diseased and aged mammalian CNS. Reviewed International journal

Yuji Takihara, Masaru Inatani, Kei Eto, Toshihiro Inoue, Alexander Kreymerman, Seiji Miyake, Shinji Ueno, Masatoshi Nagaya, Ayami Nakanishi, Keiichiro Iwao, Yoshihiro Takamura, Hirotaka Sakamoto, Keita Satoh, Mineo Kondo, Tatsuya Sakamoto, Jeffrey L Goldberg, Junichi Nabekura, Hidenobu Tanihara

Proceedings of the National Academy of Sciences of the United States of America 112 ( 33 ) 10515 - 20 2015.8

Language：English

The lack of intravital imaging of axonal transport of mitochondria in the mammalian CNS precludes characterization of the dynamics of axonal transport of mitochondria in the diseased and aged mammalian CNS. Glaucoma, the most common neurodegenerative eye disease, is characterized by axon degeneration and the death of retinal ganglion cells (RGCs) and by an age-related increase in incidence. RGC death is hypothesized to result from disturbances in axonal transport and in mitochondrial function. Here we report minimally invasive intravital multiphoton imaging of anesthetized mouse RGCs through the sclera that provides sequential time-lapse images of mitochondria transported in a single axon with submicrometer resolution. Unlike findings from explants, we show that the axonal transport of mitochondria is highly dynamic in the mammalian CNS in vivo under physiological conditions. Furthermore, in the early stage of glaucoma modeled in adult (4-mo-old) mice, the number of transported mitochondria decreases before RGC death, although transport does not shorten. However, with increasing age up to 23-25 mo, mitochondrial transport (duration, distance, and duty cycle) shortens. In axons, mitochondria-free regions increase and lengths of transported mitochondria decrease with aging, although totally organized transport patterns are preserved in old (23- to 25-mo-old) mice. Moreover, axonal transport of mitochondria is more vulnerable to glaucomatous insults in old mice than in adult mice. These mitochondrial changes with aging may underlie the age-related increase in glaucoma incidence. Our method is useful for characterizing the dynamics of axonal transport of mitochondria and may be applied to other submicrometer structures in the diseased and aged mammalian CNS in vivo.

DOI： 10.1073/pnas.1509879112

In vivo processing and release into the circulation of GFP fusion protein in arginine vasopressin enhanced GFP transgenic rats: response to osmotic stimulation Reviewed

Keita Satoh, Takumi Oti, Akiko Katoh, Yoichi Ueta, John F. Morris, Tatsuya Sakamoto, Hirotaka Sakamoto*

FEBS JOURNAL 282 ( 13 ) 2488 - 2499 2015.7

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

Arginine vasopressin (AVP) is a neurohypophysial hormone synthesized as a part of a prepropeptide precursor containing the signal peptide, AVP hormone, AVP-associated neurophysin II and copeptin in the hypothalamic neurosecretory neurons. A transgenic (Tg) rat line expressing the AVP-eGFP fusion gene has been generated. To establish the AVP-eGFP Tg rat as a unique model for an analysis of AVP dynamics invivo, we first examined the invivo molecular dynamics of the AVP-eGFP fusion gene, and then the release of GFP in response to physiological stimuli. Double immunoelectron microscopy demonstrated that GFP was specifically localized in neurosecretory vesicles of AVP neurons in this Tg rat. After stimulation of the posterior pituitary with high potassium we demonstrated the exocytosis of AVP neurosecretory vesicles containing GFP at the ultrastructural level. Biochemical analyses indicated that the AVP-eGFP fusion gene is subjected to invivo post-translational modifications like the native AVP gene, and is packaged into neurosecretory vesicles as a fusion protein: copeptin(1-14)-GFP. Moreover, GFP release into the circulating blood appeared to be augmented after osmotic stimulation, like native AVP. Thus, here we show for the first time the invivo molecular processing of the AVP-eGFP fusion gene and stimulated secretion after osmotic stimulation in rats. Because GFP behaved like native AVP in the hypothalamo-pituitary axis, and in particular was released into the circulation in response to a physiological stimulus, the AVP-eGFP Tg rat model appears to be a powerful tool for analyzing neuroendocrine systems at the organismal level.

DOI： 10.1111/febs.13291

Androgen regulates the sexually dimorphic gastrin-releasing peptide system in the lumbar spinal cord that mediates male sexual function Reviewed

Hirotaka Sakamoto*, Keiko Takanami, Damian G. Zuloaga, Ken-ichi Matsuda, Cynthia L. Jordan, S. Marc Breedlove, Mitsuhiro Kawata

ENDOCRINOLOGY 150 ( 8 ) 3672 - 3679 2009.8

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

A collection of neurons in the upper lumbar spinal cord of male rats projects to the lower lumbar spinal cord, releasing gastrin-releasing peptide (GRP) onto somatic and autonomic centers known to regulate male sexual reflexes such as erection and ejaculation. Because these reflexes are androgen dependent, we asked whether manipulating levels of androgen in adult rats would affect GRP expression in this spinal center. We found that castration resulted, 28 d later, in a profound decrease in the expression of GRP in the spinal cord, as reflected in immunocytochemistry and competitive ELISA for the protein as well as real-time quantitative PCR for the transcript. These effects were prevented if the castrates were treated with testosterone propionate. Genetically male (XY) rats with the dysfunctional testicular feminization allele for the androgen receptor (AR) displayed GRP mRNA and protein levels in the spinal cord similar to those of females, indicating that androgen normally maintains the system through AR. We saw no effect of castration or the testicular feminization allele on expression of the receptor for GRP in the spinal cord, but castration did reduce expression of AR transcripts within the spinal cord as revealed by real-time quantitative PCR and Western blots. Taken together, these results suggest that androgen signaling plays a pivotal role in the regulation of GRP expression in male lumbar spinal cord. A greater understanding of how androgen modulates the spinal GRP system might lead to new therapeutic approaches to male sexual dysfunction. (Endocrinology 150: 3672-3679, 2009)

DOI： 10.1210/en.2008-1791

Stress affects a gastrin-releasing peptide system in the spinal cord that mediates sexual function: Implications for psychogenic erectile dysfunction Reviewed

Hirotaka Sakamoto*, Ken-Ichi Matsuda, Damian G. Zuloaga, Nobuko Nishiura, Keiko Takanami, Cynthia L. Jordan, S. Marc Breedlove, Mitsuhiro Kawata

PLoS ONE 4 ( 1 ) e4276 2009.1

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

Background: Many men suffering from stress, including post-traumatic stress disorder (PTSD), report sexual dysfunction, which is traditionally treated via psychological counseling. Recently, we identified a gastrin-releasing peptide (GRP) system in the lumbar spinal cord that is a primary mediator for male reproductive functions.
Methodology/Principal Findings: To ask whether an acute severe stress could alter the male specific GRP system, we used a single-prolonged stress (SPS), a putative rat model for PTSD in the present study. Exposure of SPS to male rats decreases both the local content and axonal distribution of GRP in the lower lumbar spinal cord and results in an attenuation of penile reflexes in vivo. Remarkably, pharmacological stimulation of GRP receptors restores penile reflexes in SPS-exposed males, and induces spontaneous ejaculation in a dose-dependent manner. Furthermore, although the level of plasma testosterone is normal 7 days after SPS exposure, we found a significant decrease in the expression of androgen receptor protein in this spinal center.
Conclusions/Significance: We conclude that the spinal GRP system appears to be a stress-vulnerable center for male reproductive functions, which may provide new insight into a clinical target for the treatment of erectile dysfunction triggered by stress and psychiatric disorders.

DOI： 10.1371/journal.pone.0004276

Sexually dimorphic gastrin releasing peptide system in the spinal cord controls male reproductive functions Reviewed

Hirotaka Sakamoto*, Ken-Ichi Matsuda, Damian G. Zuloaga, Hisayuki Hongu, Etsuko Wada, Keiji Wada, Cynthia L. Jordan, S. Marc Breedlove, Mitsuhiro Kawata

Nature Neuroscience 11 ( 6 ) 634 - 636 2008.6

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

Neurons in the upper lumbar spinal cord project axons containing gastrin-releasing peptide (GRP) to innervate lower lumbar regions controlling erection and ejaculation. This system is vestigial in female rats and in males with genetic dysfunction of androgen receptors, but in male rats, pharmacological stimulation of spinal GRP receptors restores penile reflexes and ejaculation after castration. GRP offers new avenues for understanding potential therapeutic approaches to masculine reproductive dysfunction.

DOI： 10.1038/nn.2126

Expression of G protein-coupled receptor-30, a g protein-coupled membrane estrogen receptor, in oxytocin neurons of the rat paraventricular and supraoptic nuclei Reviewed

Hirotaka Sakamoto*, Ken-Ichi Matsuda, Koji Hosokawa, Mayumi Nishi, John F. Morris, Eric R. Prossnitz, Mitsuhiro Kawata

ENDOCRINOLOGY 148 ( 12 ) 5842 - 5850 2007.12

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

The regulatory actions of estrogens on magnocellular oxytocin (OT) neurons of the paraventricular and supraoptic nuclei are well documented. Although the expression and distribution of nuclear estrogen receptor-beta, but not estrogen receptor-alpha, in the OT neuron has been described, the nuclear receptors may not explain all aspects of estrogen function in the hypothalamic OT neuron. Recently a G protein-coupled receptor (GPR) for estrogens, GPR30, has been identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we therefore investigated the expression and localization of GPR30 in magnocellular OT neurons to understand the mode of rapid estrogen actions within these neurons. Here we show that, in the paraventricular nucleus and supraoptic nucleus, GPR30 is expressed in magnocellular OT neurons at both mRNA and protein levels but is not expressed in vasopressin neurons. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was localized mainly in the Golgi apparatus of the neurons but could not be detected at the cell surface. In addition, the expression of GPR30 is also detected in the neurohypophysis. These results suggest that GPR30 may serve primarily as a nongenomic transducer of estrogen actions in the hypothalamo-neurohypophyseal system.

DOI： 10.1210/en.2007-0436

Keratinocyte-driven dermal collagen formation in the axolotl skin

Ayaka Ohashi, Hirotaka Sakamoto, Junpei Kuroda, Yohei Kondo, Yasuhiro Kamei, Shigenori Nonaka, Saya Furukawa, Sakiya Yamamoto, Akira Satoh

Nature Communications 16 ( 1 ) 2025.2

Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla. Reviewed International journal

Takanori Ikenaga, Aoshi Kobayashi, Akihisa Takeuchi, Kentaro Uesugi, Takanobu Maezawa, Norito Shibata, Tatsuya Sakamoto, Hirotaka Sakamoto

Zoological Science 41 ( 3 ) 281 - 289 2024.6

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

Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system "brain" rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized 'central' nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms.

DOI： 10.2108/zs230082

Membrane-Targeted palGFP Predominantly Localizes to the Plasma Membrane but not to Neurosecretory Vesicle Membranes in Rat Oxytocin Neurons. Reviewed International journal

Hirotaka Sakamoto, Ayumu Inutsuka

Acta histochemica et cytochemica 57 ( 2 ) 85 - 88 2024.4

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

Recent advances in viral vector technology, specifically using adeno-associated virus (AAV) vectors, have significantly expanded possibilities in neuronal tracing. We have utilized the Cre/loxP system in combination with AAV techniques in rats to explore the subcellular localization of palmitoylation signal-tagged GFP (palGFP) in oxytocin-producing neurosecretory neurons. A distinctive branching pattern of single axons was observed at the level of the terminals in the posterior pituitary. Despite challenges in detecting palGFP signals by fluorescent microscopy, immunoelectron microscopy demonstrated predominant localization on the plasma membrane, with a minor presence on the neurosecretory vesicle membrane. These findings suggest that membrane-anchored palGFP may undergo exocytosis, translocating from the plasma membrane to the neurosecretory vesicle membrane. In this study, we observed characteristic axon terminal structures in the posterior pituitary of oxytocin neurons. This study indicates the importance of understanding the plasma membrane-specific sorting system in neuronal membrane migration and encourages future studies on the underlying mechanisms.

DOI： 10.1267/ahc.24-00001

Model systems for discovering evolutionary singularity of bilaterian physiological regulation: lessons from studies on simple/primitive flatworms Reviewed International journal

Shunsuke Mori, Aoshi Kobayashi, Hirotaka Sakamoto, Mayuko Hamada, Tatsuya Sakamoto, Ryo Nakamura

Biophysics and Physicobiology 21 e211012 2024.3

Function of gastrin-releasing peptide receptors in ocular itch transmission in the mouse trigeminal sensory system Reviewed International journal

Keiko Takanami, Masaya Kuroiwa, Ren Ishikawa, Yuji Imai, Akane Oishi, Midori Hashino, Yasushi Shimoda, Hirotaka Sakamoto, Tsuyoshi Koide

Frontiers in Molecular Neuroscience 16 1280024 2023.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Frontiers Media SA

The prevalence of allergic conjunctivitis in itchy eyes has increased constantly worldwide owing to environmental pollution. Currently, anti-allergic and antihistaminic eye drops are used; however, there are many unknown aspects about the neural circuits that transmit itchy eyes. We focused on the gastrin-releasing peptide (GRP) and GRP receptor (GRPR), which are reportedly involved in itch transmission in the spinal somatosensory system, to determine whether the GRP system is involved in itch neurotransmission of the eyes in the trigeminal sensory system. First, the instillation of itch mediators, such as histamine (His) and non-histaminergic itch mediator chloroquine (CQ), exhibited concentration-dependent high levels of eye scratching behavior, with a significant sex differences observed in the case of His. Histological analysis revealed that His and CQ significantly increased the neural activity of GRPR-expressing neurons in the caudal part of the spinal trigeminal nucleus of the medulla oblongata in GRPR transgenic mice. We administered a GRPR antagonist or bombesin-saporin to ablate GRPR-expressing neurons, followed by His or CQ instillation, and observed a decrease in CQ-induced eye-scratching behavior in the toxin experiments. Intracisternal administration of neuromedin C (NMC), a GRPR agonist, resulted in dose-dependent excessive facial scratching behavior, despite the absence of an itch stimulus on the face. To our knowledge, this is the first study to demonstrate that non-histaminergic itchy eyes were transmitted centrally via GRPR-expressing neurons in the trigeminal sensory system, and that NMC in the medulla oblongata evoked facial itching.

DOI： 10.3389/fnmol.2023.1280024

Amyloid deposition through endocytosis in vascular endothelial cells. Reviewed International journal

Seiji Nishikage, Akira Fujisawa, Hiromi Endoh, Hirotaka Sakamoto, Tomohide Suzuki, Maki Kanzawa, Shinichi Ishii, Mitsumasa Okano, Eriko Nitta, Kimikazu Yakushijin, Hidesaku Asakura, Kandai Nozu, Ryo Nitta, Yoshio Katayama, Kazuhiko Sakaguchi

Experimental hematology 129 104129 2023.11

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

No mechanistic lead is known for establishing AL amyloid deposits in organs. We here report an electron microscopic (EM) analysis in a case of intestinal AL amyloidosis before initiating treatment for amyloidosis. The dense deposits of amyloid fibrils are concentrated around the small blood vessels in the submucosal area of intestinal tissue. Surprisingly, we observed endothelial cells (ECs) of blood vessels containing plenty of endocytotic (pinocytotic) and transcytotic vesicles at the luminal side and above the basement membrane, indicating the one-way active trafficking of either the immunoglobulin (Ig) light chain or preassembled amyloid fibrils from the luminal side of ECs to the extraluminal area of ECs. Immunoelectron microscopy displayed that the immuno-gold signals were observed in the vascular cavity and the subendothelial area of amyloid deposits. However, there is no sign of an Ig light chain in pinocytotic vesicles. Therefore, the intestinal ECs may actively pump out mainly the preassembled amyloid fibrils (not light chains) from the blood stream into the subendothelial area as a physiologic function.

DOI： 10.1016/j.exphem.2023.11.003

Chronic corticosterone exposure evokes itch hypersensitivity and sexual dysfunction in male rats: Relationship between the two distinct gastrin-releasing peptide systems in the spinal cord Reviewed International journal

Keiko Takanami, Makoto Morishita, Tatsuya Sakamoto, Hirotaka Sakamoto

General and Comparative Endocrinology 339 114289 - 114289 2023.8

Spinal Transection Switches the Effect of Metabotropic Glutamate Receptor Subtype 7 from the Facilitation to Inhibition of Ejaculation. Reviewed International journal

Miwako Masugi-Tokita, Shigehisa Kubota, Kenichi Kobayashi, Tetsuya Yoshida, Susumu Kageyama, Hirotaka Sakamoto, Akihiro Kawauchi

Neuroscience 509 10 - 19 2023.1

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

Metabotropic glutamate receptor subtype 7 (mGluR7) is a member of the group III mGluRs, which localize to presynaptic active zones of the central nervous system. We previously reported that mGluR7 knockout (KO) mice exhibit ejaculatory disorders, although they have normal sexual motivation. We hypothesized that mGluR7 regulates ejaculation by potentiating the excitability of the neural circuit in the lumbosacral spinal cord, because administration of the mGluR7-selective antagonist into that region inhibits drug-induced ejaculation. In the present study, to elucidate the mechanism of impaired ejaculation in mGluR7 KO mice, we eliminated the influence of the brain by spinal transection (spinalization). Unexpectedly, sexual responses of male mGluR7 KO mice were stronger than those of wild-type mice after spinalization. Histological examination indicated that mGluR7 controls sympathetic neurons as well as parasympathetic neurons. In view of the complexity of its synaptic regulation, mGluR7 might control ejaculation by multi-level and multi-modal mechanisms. Our study provides insight into the mechanism of ejaculation as well as a strategy for future therapies to treat ejaculatory disorders in humans.

DOI： 10.1016/j.neuroscience.2022.11.012

Mating experiences with the same partner enhanced mating activities of naïve male medaka fish. Reviewed International journal

Masahiro Daimon, Takafumi Katsumura, Hirotaka Sakamoto, Satoshi Ansai, Hideaki Takeuchi

Scientific reports 12 ( 1 ) 19665 - 19665 2022.11

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

Mating experience shapes male mating behavior across species, from insects, fish, and birds, to rodents. Here, we investigated the effect of multiple mating experiences on male mating behavior in "naïve" (defined as sexually inexperienced) male medaka fish. The latency to mate with the same female partner significantly decreased after the second encounter, whereas when the partner was changed, the latency to mate was not decreased. These findings suggest that mating experiences enhanced the mating activity of naïve males for the familiar female, but not for an unfamiliar female. In contrast, the mating experiences of "experienced" (defined as those having mated > 7 times) males with the same partner did not influence their latency to mate. Furthermore, we identified 10 highly and differentially expressed genes in the brains of the naïve males after the mating experience and revealed 3 genes that are required for a functional cascade of the thyroid hormone system. Together, these findings suggest that the mating experience of naïve male medaka fish influences their mating behaviors, with neural changes triggered by thyroid hormone activation in the brain.

DOI： 10.1038/s41598-022-23871-w

Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition Reviewed International journal

Yuki Sato, Mugiho Shigematsu, Maria Shibata-Kanno, Sho Maejima, Chie Tamura, Hirotaka Sakamoto

Development 150 ( 11 ) dev201275 2022.9

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：Cold Spring Harbor Laboratory

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical HSCs, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in HECs and that aquaporin-1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding, and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence indicating that morphological segregation of HSCs from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses.

DOI： 10.1101/2022.09.03.506460

Evolutionary differentiation of androgen receptor ohnologs is responsible for the development of androgen-dependent unique sexual characteristics in a teleost fish Reviewed International coauthorship International journal

Yukiko Ogino, Satoshi Ansai, Eiji Watanabe, Masaki Yasugi, Yukitoshi Katayama, Hirotaka Sakamoto, Keigo Okamoto, Kataaki Okubo, Yasuhiro Yamamoto, Ikuyo HARA, Touko Yamazaki, Ai Kato, Yasuhiro Kamei, Kiyoshi Naruse, Kohei Ohta, Hajime Ogino, Tatsuya Sakamoto, Shinichi Miyagawa, TOMOMI SATO, Gen Yamada, Michael Baker, Taisen Iguchi

Nature Communications 14 ( 1 ) 1428 2022.7

Language：English Publishing type：Research paper (scientific journal) Publisher：Research Square Platform LLC

Abstract

Teleost fishes exhibit complex unique sexual characteristics, such as fin enlargement and courtship display, in response to androgens. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analysed medaka (Oryzias latipes) mutants deficient in androgen receptor ohnologs (ara and arb) generated by the teleost-specific whole-genome duplication event (TSGD). We discovered that both ar ohnologs are not required for spermatogenesis and appear to be functionally redundant for courtship display in males, while both copies were necessary for their reproductive success; ara was required for tooth enlargement and behavioural attractiveness, while arb for male-specific fin morphogenesis and sexual motivation. We further showed that the differences in both the transcription of the two ars, cellular localisation of their encoded proteins and their downstream genetic programs could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in the teleost lineage in two different ways: First through the loss of their roles in spermatogenesis and second through the gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

DOI： 10.21203/rs.3.rs-1848626/v1

Other Link： https://www.researchsquare.com/article/rs-1848626/v1.html

Characterization of the expression of gastrin-releasing peptide and its receptor in the trigeminal and spinal somatosensory systems of Japanese macaque monkeys: Insight into humans. Reviewed International coauthorship International journal

Keiko Takanami, Takumi Oti, Yasuhisa Kobayashi, Koki Hasegawa, Takashi Ito, Naoaki Tsutsui, Yasumasa Ueda, Earl Carstens, Tatsuya Sakamoto, Hirotaka Sakamoto

The Journal of Comparative Neurology 530 ( 16 ) 2804 - 2819 2022.6

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

Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.

DOI： 10.1002/cne.25376

Lattice-patterned collagen fibers and their dynamics in axolotl skin regeneration Reviewed International journal

Rena Kashimoto, Saya Furukawa, Sakiya Yamamoto, Yasuhiro Kamei, Joe Sakamoto, Shigenori Nonaka, Tomonobu M. Watanabe, Tatsuya Sakamoto, Hirotaka Sakamoto, Akira Satoh

iScience 25 ( 7 ) 104524 - 104524 2022.6

Nanobody-Based RFP-Dependent Cre Recombinase for Selective Anterograde Tracing in RFP-Expressing Transgenic Animals Reviewed International journal

Ayumu Inutsuka, Sho Maejima, Hiroyuki Mizoguchi, Ryosuke Kaneko, Rei Nomura, Keiko Takanami, Hirotaka Sakamoto, Tatsushi Onaka

Communications Biology 5 ( 1 ) 979 2022.1

Language：English Publishing type：Research paper (scientific journal) Publisher：Research Square Platform LLC

Abstract

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. GFP-dependent gene regulation utilizes these lines to manipulate gene expression; however, its application has been limited to fluorescent proteins derived from Aequorea jellyfish. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs (mCherry, mRFP1) are different from those for dimeric RFP (tdTomato). We confirmed target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors including Cre-DOR, target RFP, and reporter GFP vector. We found highly selective GFP expression in RFP-positive cortical neurons with 93.5 ± 0.6% of GFP-positive cells being mRFP1-positive. In estrogen receptor-beta (Esr2)-mRFP1 mice, we confirmed that Cre-DOR can be used for selective expression of membrane-bound GFP in the paraventricular nucleus of the hypothalamus. The neural projection from Esr2-expressing neurons in the hypothalamic paraventricular nucleus to the posterior pituitary was visualized by Cre-DOR. In gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we similarly achieved anterograde tracing of Grpr-expressing neurons in the medial amygdala and found that they are projecting axons to the posterior bed nucleus of the stria terminalis. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can increase or decrease Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.

DOI： 10.21203/rs.3.rs-1234462/v1

Other Link： https://www.researchsquare.com/article/rs-1234462/v1.html

Behavioural osmoregulation during land invasion in fish: Prandial drinking and wetting of the dry skin. Reviewed International journal

Yukitoshi Katayama, Takehiro Tsukada, Susumu Hyodo, Hirotaka Sakamoto, Tatsuya Sakamoto

PLoS ONE 17 ( 12 ) e0277968 2022

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

Osmoregulatory behaviours should have evolutionarily modified for terrestrialisation of vertebrates. In mammals, sensations of buccal food and drying have immediate effects on postprandial thirst to prevent future systemic dehydration, and is thereby considered to be 'anticipatory thirst'. However, it remains unclear whether such an anticipatory response has been acquired in the non-tetrapod lineage. Using the mudskipper goby (Periophthalmus modestus) as a semi-terrestrial ray-finned fish, we herein investigated postprandial drinking and other unique features like full-body 'rolling' over on the back although these behaviours had not been considered to have osmoregulatory functions. In our observations on tidal flats, mudskippers migrated into water areas within a minute after terrestrial eating, and exhibited rolling behaviour with accompanying pectoral-fin movements. In aquarium experiments, frequency of migration into a water area for drinking increased within a few minutes after eating onset, without systemic dehydration. During their low humidity exposure, frequency of the rolling behaviour and pectoral-fin movements increased by more than five times to moisten the skin before systemic dehydration. These findings suggest anticipatory responses which arise from oral/gastrointestinal and cutaneous sensation in the goby. These sensation and motivation seem to have evolved in distantly related species in order to solve osmoregulatory challenges during terrestrialisation.

DOI： 10.1371/journal.pone.0277968

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

Identification of CNS Neurons Innervating the Levator Ani and Ventral Bulbospongiosus Muscles in Male Rats Reviewed

Amy D. Dobberfuhl, Takumi Oti, Hirotaka Sakamoto, Lesley Marson

JOURNAL OF SEXUAL MEDICINE 11 ( 3 ) 664 - 677 2014.3

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

IntroductionThe pelvic striated muscles play an important role in mediating erections and ejaculation, and together these muscles compose a tightly coordinated neuromuscular system that is androgen sensitive and sexually dimorphic.
AimTo identify spinal and brains neurons involved in the control of the levator ani (LA) and bulbospongiosus (BS) in the male adult and preadolescent rat.
MethodsRats were anesthetized, and the transsynaptic retrograde tracer pseudorabies virus (PRV) was injected into the LA muscle of adults or the ventral BS muscle in 30-day-old rats. After 3-5 days rats were sacrificed, and PRV-labeled neurons in the spinal cords and brains were identified using immunohistochemistry. The presence of gastrin-releasing peptide (GRP) in the lumbar spinal neurons was examined.
Main Outcomes MeasuresThe location and number of PRV-labeled neurons in the spinal cord and brain and GRP colocalization in the lumbar spinal cord.
ResultsPRV-labeled spinal interneurons were found distributed throughout T11-S1 of the spinal cord, subsequent to dorsal medial motoneuron infection. The majority of spinal interneurons were found in the lumbosacral spinal cord in the region of the dorsal gray commissure and parasympathetic preganglionic neurons. Preadolescent rats had more PRV-labeled spinal interneurons at L5-S1 where the motoneurons were located but relatively less spread rostrally in the spinal cord compared with adults. Lumbar spinothalmic neurons in medial gray of L3-L4 co-localized PRV and GRP. In the brain consistent labeling was seen in areas known to be involved in male sexual behavior including the ventrolateral medulla, hypothalamic paraventricular nucleus, and medial preoptic area.
ConclusionCommon spinal and brain pathways project to the LA and BS muscles in the rat suggesting that these muscles act together to coordinate male sexual reflexes. Differences may exist in the amount of synaptic connections/neuronal pathways in adolescents compared with adults. Dobberfuhl AD, Oti T, Sakamoto H, and Marson L. Identification of CNS neurons innervating the levator ani and ventral bulbospongiosus muscles in male rats. J Sex Med 2014;11:664-677.

DOI： 10.1111/jsm.12418

Androgen regulates development of the sexually dimorphic gastrin-releasing peptide neuron system in the lumbar spinal cord: Evidence from a mouse line lacking androgen receptor in the nervous system Reviewed

Hirotaka Sakamoto, Kazuhiro Saito, Clarisse Marie-Luce, Kalina Raskin, Takumi Oti, Keita Satoh, Kei Tamura, Tatsuya Sakamoto, Sakina Mhaouty-Kodja

NEUROSCIENCE LETTERS 558 109 - 114 2014.1

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

Androgens including testosterone, organize the nervous system as well as masculine external and internal genitalia during the perinatal period. Androgen organization involves promotion of masculine body features, usually by acting through androgen receptors (ARs). We have recently demonstrated that the gastrin-releasing peptide (GRP) system in the lumbar spinal cord also mediates spinal centers promoting penile reflexes during male sexual behavior in rats. Testosterone may induce sexual differentiation of this spinal GRP system during development and maintain its activation in adulthood. In the present study, we examined the role of ARs in the nervous system regulating the development of the sexually dimorphic GRP system. For this purpose, we used a conditional mouse line selectively lacking the AR gene in the nervous system. AR foxed males carrying (mutants) or not (controls) the nestin-Cre transgene were castrated in adulthood and supplemented with physiological amounts of testosterone. Loss of AR expression in the nervous system resulted in a significant decrease in the number of GRP neurons compared to control littermates. Consequently, the intensity of GRP axonal projections onto the lower lumbar and upper sacral spinal cord was greater in control males than in mutant males. These results suggest that ARs expressed in the nervous system play a significant role in the development of the GRP system in the male lumbar spinal cord. The AR-deletion mutation may attenuate sexual behavior and activity of mutant males via spinal GRP system-mediated neural mechanisms. (C) 2013 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2013.10.068

Sexually dimorphic nuclei in the spinal cord control male sexual functions Reviewed

Hirotaka Sakamoto

Frontiers in Neuroscience 8 ( 8 ) 184 2014

Authorship：Lead author,　Last author,　Corresponding author Language：English Publisher：Frontiers Research Foundation

Lower spinal cord injuries frequently cause sexual dysfunction in men, including erectile dysfunction and an ejaculation disorder. This indicates that the important neural centers for male sexual function are located within the lower spinal cord. It is interesting that the lumbar spinal segments contain several neural circuits, showing a clear sexually dimorphism that, in association with neural circuits of the thoracic and sacral spinal cord, are critical in expressing penile reflexes during sexual behavior. To date, many sex differences in the spinal cord have been discovered. Interestingly, most of these are male dominant. Substantial evidence of sexually dimorphic neural circuits in the spinal cord have been reported in many animal models, but major issues remain unknown. For example, it is not known how the different circuits cooperatively function during male sexual behavior. In this review, therefore, the anatomical and functional significance of the sexually dimorphic nuclei in the spinal cord corresponding to the expression of male sexual behavior is discussed. © 2014 Sakamoto.

DOI： 10.3389/fnins.2014.00184

The gastrin-releasing peptide receptor (GRPR) in the spinal cord as a novel pharmacological target Reviewed

Keiko Takanami, Hirotaka Sakamoto

CURRENT NEUROPHARMACOLOGY 12 ( 5 ) 434 - 443 2014

Authorship：Last author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：BENTHAM SCIENCE PUBL LTD

Gastrin-releasing peptide (GRP) is a mammalian neuropeptide that acts through the G protein-coupled receptor, GRP receptor (GRPR). Increasing evidence indicates that GRPR-mediated signaling in the central nervous system plays an important role in many physiological processes in mammals. Additionally, we have recently reported that the GRP system within the lumbosacral spinal cord not only controls erection but also triggers ejaculation in male rats. This system of GRP neurons is sexually dimorphic, being prominent in male rats but vestigial or absent in females. It is suggested that the sexually dimorphic GRP/GRPR system in the lumbosacral spinal cord plays a critical role in the regulation of male sexual function. In parallel, it has been reported that the somatosensory GRP/GRPR system in the spinal cord contributes to the regulation of itch specific transmission independently of the pain transmission. Interestingly, these two distinct functions in the same spinal region are both regulated by the neuropeptide, GRP. In this report, we review findings on recently identified GRP/GRPR systems in the spinal cord. These GRP/GRPR systems in the spinal cord provide new insights into pharmacological treatments for psychogenic erectile dysfunction as well as for chronic pruritus.

DOI： 10.2174/1570159X12666140923201432

Potential roles of arginine-vasotocin in the regulation of aggressive behavior in the mudskipper (Periophthalmus modestus) Reviewed

Nao Kagawa, Yudai Nishiyama, Kanoko Kato, Hideya Takahashi, Yasuhisa Kobayashi, Hirotaka Sakamoto, Tatsuya Sakamoto

General and Comparative Endocrinology 194 257 - 263 2013.12

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

The hypothalamic hormones, arginine-vasotocin (VT) and isotocin (IT), play central roles in osmoregulation and in the regulation of social behaviors including aggressive behavior in many vertebrates including fish. Here, we examined whether these hormones are associated with aggressive behavior in the mudskipper (Periophthalmus modestus). The mudskipper is an amphibious fish, which lives in the brackish water of river mouths and displays unique aggressive behavior. Upon introduction to each other in an experimental tank with aquatic and terrestrial areas, a pair of males can be classified as aggressive dominant or submissive subordinate based on the frequency of their aggressive acts, which is significantly higher in dominant male. Additionally, the length of stay in terrestrial area of dominant was longer than that of the subordinate. The latter remained in aquatic area almost throughout the period of behavioral observation. The expression of brain VT mRNA was significantly higher in subordinate than in dominant, whereas neither IT mRNA expression nor plasma cortisol level differed between subordinate and dominant male. On the other hand, an intracerebroventricular injection of VT increased aggressive behaviors in mudskippers. In addition to known roles of VT in mediation of aggressive behavior, these results may shed light on the role of endogenous VT toward water migration in submissive mudskippers. The amphibious fish is a valuable experimental model to observe the relationship between effects of central VT on the osmoregulation and social behavioral regulation in vertebrates. © 2013.

DOI： 10.1016/j.ygcen.2013.09.023

Ultrahigh voltage electron microscopy links neuroanatomy and neuroscience/neuroendocrinology. Reviewed

Sakamoto H, Kawata M

Anatomy Research International 2012 948704 2012.5

Brain-spinal cord neural circuits controlling male sexual function and behavior Reviewed

Hirotaka Sakamoto

NEUROSCIENCE RESEARCH 72 ( 2 ) 103 - 116 2012.2

Language：English Publisher：ELSEVIER IRELAND LTD

Men and women exhibit differences in sexual behavior. This indicates that neural circuits within the central nervous system (CNS) that control sexual behavior differ between the sexes, although differences in behavior are also influenced by sociocultural and hormonal factors. Sexual differentiation of the body and brain occurs during the embryonic and neonatal periods in humans and persists into adulthood with relatively low plasticity. Male sexual behavior is complex and depends on intrinsic and extrinsic factors, including olfactory, somatosensory and visceral cues. Many advances in our understanding of sexually dimorphic neural circuits have been achieved in animal models, but major issues are yet to be resolved. This review summarizes the sexually dimorphic nuclei controlling male sexual function in the rodent CNS and focuses on the interactions of the brain-spinal cord neural networks controlling male sexual behavior. Possible factors that relate findings from animal studies to human behavior are also discussed. (C) 2011 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2011.11.002

Rapid signaling of steroid hormones in the vertebrate nervous system Reviewed

Hirotaka Sakamoto, Hideya Takahashi, Ken-Ichi Matsuda, Mayumi Nishi, Keiko Takanami, Maho Ogoshi, Tatsuya Sakamoto, Mitsuhiro Kawata

FRONTIERS IN BIOSCIENCE-LANDMARK 17 996 - 1019 2012.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：FRONTIERS IN BIOSCIENCE INC

Steroid hormones easily cross the blood-brain barrier because of their physicochemical lipid solubility. The hormones act through nuclear receptor-mediated mechanisms and modulate gene transcription. In contrast to their genomic actions, the non-genomic rapid action of steroid hormones, acting via various types of membrane-associated receptors, reveals pharmacological properties that are distinct from the actions of the intracellular nuclear receptors. As a result, non-genomic rapid actions have gained increased scientific interest. However, insight into the phylogenic and/or comparative actions of steroids in the brain is still poorly understood. In this review, we summarize recent findings concerning the rapid, non-genomic signaling of steroid hormones in the vertebrate central nervous system, and we discuss (using a comparative view from fish to mammals) recently published data regarding the mechanism underlying physiology and behavior.

DOI： 10.2741/3970

Immunohistochemical Analysis of the Effects of Estrogen on Intraarticular Neurogenic Inflammation in a Rat Anterior Cruciate Ligament Transection Model of Osteoarthritis Reviewed

Atsuhiko Yoshida, Toru Morihara, Ken-ichi Matsuda, Hirotaka Sakamoto, Yuji Arai, Yoshikazu Kida, Mitsuhiro Kawata, Toshikazu Kubo

CONNECTIVE TISSUE RESEARCH 53 ( 3 ) 197 - 206 2012

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

Synovitis is considered as one of the factors associated with the pathogenesis of osteoarthritis (OA). There is currently a significant amount of research linking estrogen deficiencies with the development of OA in estrogen-deficient women, including postmenopausal women; however, the exact etiology remains unclear. Various neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), have been shown to contribute to synovitis in OA joints, and the influence of estrogen on the expressions of SP and CGRP in the synovium of OA joints has been noted. After ovariectomy (OVX) followed by estradiol (E2) replacement, 24 female rats were divided into three groups: OVX group, OVX + E2 replacement group (E2 group), and a sham group. All rats underwent transection of the anterior cruciate ligament at the same time. After 30 days, the histological findings of knee joints by hematoxylin-eosin staining and immunofluorescence staining of protein gene product 9.5 (pan-neuronal marker), SP, and CGRP were compared among experimental groups. The degree of synovitis in the OVX group was higher than in the E2 and sham groups. No significant differences in the density of protein gene product 9.5-immunoreactive nerve fibers were observed among the three experimental groups, but the density of SP- or CGRP-immunoreactive nerve fibers in the OVX group was significantly higher than in the E2 and sham groups. These findings suggest that estrogen partly regulates intraarticular neurogenic inflammation in OA joints by modulating the expressions of neuropeptides in the synovium.

DOI： 10.3109/03008207.2011.628059

Corticosteroids stimulate the amphibious behavior in mudskipper: Potential role of mineralocorticoid receptors in teleost fish Reviewed

Tatsuya Sakamoto, Chie Mori, Shogo Minami, Hideya Takahashi, Tsukasa Abe, Daisuke Ojima, Maho Ogoshi, Hirotaka Sakamoto

PHYSIOLOGY & BEHAVIOR 104 ( 5 ) 923 - 928 2011.10

Authorship：Last author Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

It has long been held that cortisol, a glucocorticoid in many vertebrates, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. However, 11-deoxycorticosterone (DOC) has been identified as a specific endogenous ligand for the teleostean mineralocorticoid receptor (MR). Furthermore, the expressions of MR mRNA are modest in the osmoregulatory organs, but considerably higher in the brain of most teleosts. These recent findings suggest that the mineralocorticoid system (DOC/MR) may carry out some behavioral functions in fish. To test this possibility, we examined the effects of cortisol and DOC administration in the amphibious behavior in mudskipper (Periophthalmus modestus) in vivo. It was found that mudskippers remained in the water for an increased period of time when they were immersed into 5 mu M DOC or cortisol for 8 h. Additionally, an exposure to 25 mu M DOC for 4 to 8 h caused a decreased migratory frequency of mudskippers to the water, reflected a tendency to remain in the water. It was further observed that after 8 h of intracerebroventricular (ICV) injection with 0.3 pmol DOC or cortisol the staying period in the water increased in fish. The migratory frequency was decreased after ICV DOC injection which indicated that fishes stayed in the water. Concurrent ICV injections of cortisol with RU486 [a specific glucocorticoid-receptor (GR) antagonist] inhibited only the partial effects of cortisol. Together with no changes in the plasma DOC concentrations under terrestrial conditions, these results indicate the involvement of brain MRs as cortisol receptors in the preference for an aquatic habitat of mudskippers. Although the role of GR signaling cannot be excluded in the aquatic preference, our data further suggest that the MR may play an important role in the brain dependent behaviors of teleost fish. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.physbeh.2011.06.002

The gastrin-releasing peptide system in the spinal cord mediates masculine sexual function Reviewed

Hirotaka Sakamoto*

ANATOMICAL SCIENCE INTERNATIONAL 86 ( 1 ) 19 - 29 2011.3

Authorship：Lead author,　Last author,　Corresponding author Language：English Publisher：SPRINGER

The lumbar spinal segments are of particular interest because they are sexually dimorphic and contain several neuronal circuits that are important in eliciting male sexual responses such as erection and ejaculation. Gastrin-releasing peptide (GRP) is a member of the bombesin-like peptide family first isolated from the porcine stomach. A collection of neurons in the lumbar spinal cord (L3-L4 level) of male rats projects to the lower lumbar spinal cord (L5-L6 level), releasing GRP onto somatic and autonomic centers known to regulate male sexual reflexes. All these target neurons express and localize specific receptors for GRP. This system of GRP neurons is sexually dimorphic, being prominent in male rats but vestigial in females. The system is completely feminine in genetically XY rats with a dysfunctional androgen receptor gene, demonstrating the androgen-dependent nature of the dimorphism. Pharmacological stimulation of GRP receptors in this spinal region remarkably restores sexual reflexes in castrated male rats. Exposure of male rats to a severe traumatic stress decreases the local content and the axonal distribution of GRP in the lumbar spinal cord and results in an attenuation of penile reflexes in vivo. Administration of a specific agonist for GRP receptors restores penile reflexes in the traumatic stress-exposed male rats. This review summarizes findings on this recently identified spinal GRP system, which may be vulnerable to stress, that controls male reproductive function. The identification of a male-specific neuronal system regulating sexual functions offers new avenues for potential therapeutic approaches to masculine reproductive dysfunction.

DOI： 10.1007/s12565-010-0097-z

Biosynthesis, mode of action, and functional significance of neurosteroids in the purkinje cell Reviewed

Kazuyoshi Tsutsui, Kazuyoshi Ukena, Hirotaka Sakamoto, Shin-Ichiro Okuyama, Shogo Haraguchi

Frontiers in Endocrinology 2 ( OCT ) 61 2011

Language：English

The brain has traditionally been considered to be a target site of peripheral steroid hormones. In addition to this classical concept, we now know that the brain has the capacity to synthesize steroids de novo from cholesterol, the so-called "neurosteroids." In the middle 1990s, the Purkinje cell, an important cerebellar neuron, was identified as a major site for neurosteroid formation in the brain of mammals and other vertebrates. This discovery has provided the opportunity to understand neuronal neurosteroidogenesis in the brain. In addition, biological actions of neurosteroids are becoming clear by the studies using the Purkinje cell, an excellent cellular model, which is known to play an important role in memory and learning processes. Based on the studies on mammals over the past decade, it is considered that the Purkinje cell actively synthesizes progesterone and estradiol from cholesterol during neonatal life, when cerebellar neuronal circuit formation occurs. Both progesterone and estradiol promote dendritic growth, spinogenesis, and synaptogenesis via each cognate nuclear receptor in the developing Purkinje cell. Such neurosteroid actions mediated by neurotrophic factors may contribute to the formation of cerebellar neuronal circuit during neonatal life. 3α,5α-Tetrahydroprogesterone (allopregnanolone), a progesterone metabolite, is also synthesized in the cerebellum and considered to act as a survival factor of Purkinje cells in the neonate. This review summarizes the current knowledge regarding the biosynthesis, mode of action, and functional significance of neurosteroids in the Purkinje cell during development in terms of synaptic formation of cerebellar neuronal networks. © 2011 Tsutsui, Ukena, Sakamoto, Okuyama and Haraguchi.

DOI： 10.3389/fendo.2011.00061

The neurobiology of psychogenic erectile dysfunction in the spinal cord Reviewed

Hirotaka Sakamoto*

JOURNAL OF ANDROLOGY 31 ( 6 ) 519 - 526 2010.11

Authorship：Lead author,　Last author,　Corresponding author Language：English Publisher：AMER SOC ANDROLOGY, INC

We recently reported a previously unknown peptider gic system within the lumbosacral spinal cord that uses gastrin releasing peptide (GRP) to trigger erection and ejaculation in male rats Many men suffering from stress including posttraumatic stress disorder (PTSD) and major depressive disorder report sexual dysfunction Sexual dysfunction in men suffering from stress and major depressive disorder is traditionally treated via psychological counseling To determine whether acute severe stress could alter the male specific GRP system, we used single prolonged stress (SPS) exposure in a putative rat model for PTSD Exposure of male rats to SPS decreases the local content and the axonal distribution of GRP in the lower lumbar spinal cord and results in an attenuation of penile reflexes in vivo Pharmacological stimulation of GRP receptors remarkably restores penile reflexes in SPS exposed male rats and in castrated male rats The administration of a GRP agonist to these animal models interestingly induces spontaneous ejaculation in a dose dependent manner Furthermore although the circulating level of androgens is normal 1 week after SPS exposure there is a significant decrease in the expression of androgen receptor protein in lumbar segments 3 and 4 of the spinal cord This might make the spinal center less responsive to androgens In this report I review findings on a recently identified spinal GRP system that could be vulnerable to stress and that controls male reproductive function This system provides new insights into the clinical treatment of psychogenic erectile dysfunction triggered by stress and psychiatric disorders

DOI： 10.2164/jandrol.110.010041

Induction of the arginine vasopressin-enhanced green fluorescent protein fusion transgene in the rat locus coeruleus Reviewed

Miwako Todoroki, Yoichi Ueta, Hiroaki Fujihara, Hiroki Otsubo, Minori Shibata, Hirofumi Hashimoto, Mizuki Kobayashi, Hirotaka Sakamoto, Mitsuhiro Kawata, Govindan Dayanithi, David Murphy, Hisanori Hiro, Ken Takahashi, Shoji Nagata

STRESS-THE INTERNATIONAL JOURNAL ON THE BIOLOGY OF STRESS 13 ( 4 ) 281 - 292 2010.7

Language：English Publishing type：Research paper (scientific journal) Publisher：INFORMA HEALTHCARE

We examined the effects of intracerebroventricular (i.c.v.) administration of colchicine on the expression of the arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene in rats. In rats administered i.c.v. vehicle ( control), eGFP fluorescence was observed in the supraoptic nucleus ( SON), the magnocellular division of the paraventricular nucleus (PVN), the suprachiasmatic nucleus (SCN), the median eminence ( ME) and the posterior pituitary. Two days after i.c.v. administration of colchicine, eGFP fluorescence was markedly increased in the SON, the magnocellular and parvocellular divisions of the PVN, the SCN, the ME and the locus coeruleus (LC). Immunohistochemical staining for eGFP confirmed the distribution of fluorescence in both groups. In the colchicines-administered groups, immunohistochemistry for tyrosine hydroxylase (TH) revealed that the eGFP fluorescence was co-localised with TH-immunoreactivity in the LC. Similarly, in situ hybridization histochemistry for eGFP mRNA revealed a significant increase in gene expression in the LC, the SON and the PVN 12-48 h after administration of colchicine. Our results indicate that the synthesis of AVP-eGFP is upregulated in noradrenergic neurones in the LC after colchicine administration. This implies that AVP and noradrenaline, originating from LC neurones, might play a role in response to chronic stress.

DOI： 10.3109/10253890903383406

Aldosterone-Sensitive Nucleus Tractus Solitarius Neurons Regulate Sensitivity of the Baroreceptor Reflex in High Sodium-Loaded Rats Reviewed

Tomoharu Masuda, Yasutoshi Hirabara, Yusuke Nakamura, Akiko Chishaki, Mai Tsuruhisa, Masayuki Miyakawa, Kenji Honda, Ryo Saito, Hirotaka Sakamoto, Mitsuhiro Kawata, Yukio Takano

JOURNAL OF PHARMACOLOGICAL SCIENCES 112 ( 4 ) 482 - 486 2010.4

Language：English Publishing type：Research paper (scientific journal) Publisher：JAPANESE PHARMACOLOGICAL SOC

We examined the role of aldosterone-sensitive neurons in the nucleus tractus solitarius (NTS) in the arterial baroreceptor reflex (baroreflex) function. Baroreflex sensitivity was induced by phenylephrine in high sodium-loaded rats and was significantly reduced. This baroreflex sensitivity was reversed by microinjection of the mineralocorticoid receptor (MR) antagonist eplerenone into the NTS. 11 beta-Hydroxysteroid dehydrogenase type 2 neurons and MR were also identified in the NTS. These data suggest that the aldosterone-sensitive neurons in the NTS may have an important role in baroreflex function.

DOI： 10.1254/jphs.09362SC

Expression of G protein-coupled receptor 30 in the spinal somatosensory system Reviewed

Keiko Takanami, Hirotaka Sakamoto, Ken-Ichi Matsuda, Koji Hosokawa, Mayumi Nishi, Eric R. Prossnitz, Mitsuhiro Kawata

BRAIN RESEARCH 1310 17 - 28 2010.1

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCIENCE BV

Estrogens were originally identified as the primary sex steroid hormones in females and regulators of reproductive function and sexual behavior, but it has long been suggested that estrogens also have local effects on the somatosensory system at the spinal cord level. it is well known that the effects of estrogens are mediated by nuclear estrogen receptors (ERs) through genomic action, but recently a membrane-bound G protein-coupled receptor, GPR30, was identified as a non-genomic estrogen receptor. in this study we investigated the presence and localization of GPR30 in the rat spinal cord and dorsal root ganglion (DRG) in comparison with ER alpha. Using immunohistochemistry and in situ hybridization, we showed the expression of GPR30 in DRG neurons in male and female rats at mRNA and protein levels without specific sexual difference. A dense accumulation of GPR30 immunoreactivity was observed in the outer layer of the spinal dorsal horn, and selective spinal dorsal rhizotomy revealed that GPR30 was transported from the DRG to terminals located in the spinal dorsal horn. GPR30 expression was downregulated in DRG neurons of ovariectomized female rats. The spinal somatosensory system might be modulated by estradiol via putative membrane ER, GPR30-mediated mechanism. (C) 2009 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.brainres.2009.11.004

High-Voltage Electron Microscopy Reveals Direct Synaptic Inputs from a Spinal Gastrin-Releasing Peptide System to Neurons of the Spinal Nucleus of Bulbocavernosus Reviewed

Hirotaka Sakamoto*, Tatsuo Arii, Mitsuhiro Kawata

ENDOCRINOLOGY 151 ( 1 ) 417 - 421 2010.1

Authorship：Lead author,　Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

The spinal nucleus of bulbocavernosus (SNB) is a sexually dimorphic motor nucleus located in the anterior horn of the fifth and sixth lumbar segments of the spinal cord that plays a significant role in male sexual function. We recently found that a sexually dimorphic expression of gastrin-releasing peptide (GRP) in the lumbar spinal cord regulates male copulatory reflexes. Although it is reported that these systems are both profoundly regulated by circulating androgen levels in male rats, no direct evidence has been reported regarding GRP synaptic inputs onto SNB motoneurons. The aim of the current study was to determine the axodendritic synaptic inputs of spinal GRP neurons to SNB motoneurons. Immunoelectron microscopy, combined with a retrograde tracing technique using high-voltage electron microscopy (HVEM), provided a three-dimensional visualization of synaptic contacts from the GRP system in the lumbar spinal cord onto SNB motoneurons. HVEM analysis clearly demonstrated that GRP-immunoreactive axon terminals directly contact dendrites that extend into the dorsal gray commissure from the SNB. These HVEM findings provide an ultrastructural basis for understanding how the spinal GRP system regulates male sexual behavior. (Endocrinology 151: 417-421, 2010)

DOI： 10.1210/en.2009-0485

Neurobiology of PTSD: Effects on the spinal cord

Hirotaka Sakamoto, S. Marc Breedlove, Mitsuhiro Kawata

Neurobiology of Post-Traumatic Stress Disorder 107 - 118 2010

Language：English Publishing type：Part of collection (book) Publisher：Nova Science Publishers, Inc.

Many men suffering from stress, including post-traumatic stress disorder (PTSD), report sexual dysfunction, which is traditionally treated via psychological counseling. Recently, we identified a gastrin-releasing peptide (GRP) system in the lumbar spinal cord that is a primary mediator for male reproductive functions. To ask whether acute severe stress could alter the male-specific GRP system, we used a single-prolonged stress (SPS), a putative rat model for PTSD. Exposure of male rats to SPS decreases both the local content and axonal distribution of GRP in the lower lumbar spinal cord and results in an attenuation of penile reflexes in vivo. Remarkably, pharmacological stimulation of GRP receptors restores penile reflexes in SPS-exposed males, and induces spontaneous ejaculation in a dose-dependent manner. Furthermore, although the level of plasma testosterone is normal one week after SPS exposure, there is a significant decrease in the expression of androgen receptor protein in this spinal center, which may make the spinal center less responsive to circulating androgens. We conclude that the spinal GRP system for male reproductive function is vulnerable to stress, which may provide new insights into clinical treatment of erectile dysfunction triggered by stress and psychiatric disorders. © 2010 Nova Science Publishers, Inc. All rights reserved.

Characterization of In Vivo Effects of Platelet-Rich Plasma and Biodegradable Gelatin Hydrogel Microspheres on Degenerated Intervertebral Discs Reviewed

Kazuhide Sawamura, Takumi Ikeda, Masateru Nagae, Shin-ichi Okamoto, Yasuo Mikami, Hitoshi Hase, Kazuya Ikoma, Tetsuya Yamada, Hirotaka Sakamoto, Ken-ichi Matsuda, Yasuhiko Tabata, Mitsuhiro Kawata, Toshikazu Kubo

TISSUE ENGINEERING PART A 15 ( 12 ) 3719 - 3727 2009.12

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT, INC

We have previously shown that administration of platelet-rich plasma-impregnated gelatin hydrogel microspheres (PRP-GHMs) into a degenerated intervertebral disc (IVD) markedly suppresses progression of IVD degeneration. In the current study, we characterized the in vivo effects of PRP-GHM treatment in a degenerated IVD model in rabbit. On magnetic resonance images, the IVD height was significantly greater after treatment with PRP-GHMs compared with phosphate-buffered saline-impregnated GHMs, PRP without GHMs, and needle puncture only. Water content was also preserved in PRP-GHM-treated IVDs. Consistent with this observation, the mRNA expression of proteoglycan core protein and type II collagen was significantly higher after PRP-GHM treatment compared with other treatment groups. No proliferating cells were found in the nucleus pulposus and inner annulus fibrosus in any groups, but the number of apoptotic cells in the nucleus pulposus after PRP-GHM treatment was significantly lower than that after other treatments. These results provide an improved understanding of the therapeutic effects of PRP-GHM treatment of degenerated IVDs.

DOI： 10.1089/ten.tea.2008.0697

バゾプレッシン(AVP)-eGFPトランスジェニックラットにおける青斑核での特異的なAVP-eGFPの合成

轟美和子, 上田陽一, 藤原広明, 大坪弘樹, 柴田美雅, 橋本弘史, 小林瑞, 坂本浩隆, 河田光博

日本病態生理学会雑誌 18 ( 2 ) 39 - 39 2009.12

In Vivo Effects of Ovarian Steroid Hormones on the Expressions of Estrogen Receptors and the Composition of Extracellular Matrix in the Anterior Cruciate Ligament in Rats Reviewed

Atsuhiko Yoshida, Toru Morihara, Yoshiteru Kajikawa, Yuji Arai, Yasushi Oshima, Toshikazu Kubo, Ken-ichi Matsuda, Hirotaka Sakamoto, Mitsuhiro Kawata

CONNECTIVE TISSUE RESEARCH 50 ( 2 ) 121 - 131 2009

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Female athletes have a significantly higher rate of anterior cruciate ligament (ACL) injury than their male counterparts. Sex steroid hormones are considered to have an influence as risk factors for female ACL injuries. We hypothesized that estrogen and progesterone have specific and synergistic influences on the composition of extracellular matrix in ACL. By ovariectomy (OVX) followed by subcutaneous estradiol (E2) and/or progesterone (P4) replacement, 40 female rats were divided into 5 groups: E2, P4, combined E2 and P4 (EP), OVX control, and sham group. After 30 days, using undecalcified sections of knee joints in conjunction with immunofluorescence staining of estrogen receptor and (ER and ER), collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP), the immunoreactivities of these proteins in two distinct parts of ACL, proximal and middle portions, were compared semiquantitatively among experimental groups. By E2 replacement, the expressions of ER in ACL fibroblasts were elevated compared to the OVX group. At the proximal portion, the immunoreactivities of type 1 collagen by E2 replacement, type 3 collagen by P4 replacement, and COMP by E2 or P4 replacement were significantly reduced. At the middle portion, the immunoreactivity of type 3 collagen was significantly elevated by E2 replacement. However, no differences were observed between the sham and OVX groups. These findings suggest that ACL is ER-dependent and that ovarian hormones alter ligament tissue composition, especially at the proximal portion. Female hormonal influences are partly involved in the biological properties of ACL.

DOI： 10.1080/03008200802531287

Androgen action in the brain and spinal cord for the regulation of male sexual behaviors Reviewed

Ken-ichi Matsuda, Hirotaka Sakamoto, Mitsuhiro Kawata

CURRENT OPINION IN PHARMACOLOGY 8 ( 6 ) 747 - 751 2008.12

Language：English Publisher：ELSEVIER SCI LTD

Circulating levels of androgens determine the sexual differentiation of the brain and spinal cord at a critical period, Although estradiol, which is converted from testosterone by aromatase action, can explain the cytological basis for the sexual dimorphism, androgen has its own regulatory mechanism to promote male-specific behavior through receptors. The central nervous system (CNS) employs a sex-specific neuronal network involving peptides and steroids for the expression of the sexual phenotype. Elucidation of the molecular mechanism along with the neuroanatomical background should guide the development of novel pharmacotherapeutics for sexual behavior dysfunction.

DOI： 10.1016/j.coph.2008.08.010

Expression and intracellular distribution of the G protein-coupled receptor 30 in rat hippocampal formation Reviewed

KenIchi Matsuda, Hirotaka Sakamoto, Hiroko Mori, Koji Hosokawa, Akeo Kawamura, Minoru Itose, Mayumi Nishi, Eric R. Prossnitz, Mitsuhiro Kawata

NEUROSCIENCE LETTERS 441 ( 1 ) 94 - 99 2008.8

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER IRELAND LTD

Although the expression and distribution of nuclear estrogen receptors in the hippocampus has been described, it has been proposed that the nuclear receptors may not explain all aspects of estrogen function in the hippocampus. Recently, a G protein-coupled receptor for estrogen, GPR30, was identified as a membrane-localized estrogen receptor in several cancer cell lines. In this study, we examined the expression and intracellular distribution of GPR30 in the rat hippocampal formation. We found expression of GPR30 in pyramidal cells of CA1-3 and granule cells of the dentate gyrus at both mRNA and protein levels. Specific markers for intracellular organelles and immunoelectron microscopy revealed that GPR30 was mainly localized to the Golgi apparatus and partially in the endoplasmic reticulum of the neuron but could not detect the protein at the cell surface. Expression levels were not different among male, female in proestrus and female in estrus at the adult stage, but were higher in newborn male than newborn female. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2008.05.108

Platelet-rich plasma enhances the initial mobilization of circulation-derived cells for tendon healing Reviewed

Yoshiteru Kajikawa, Toru Morihara, Hirotaka Sakamoto, Ken-Ichi Matsuda, Yasushi Oshima, Atsuhiko Yoshida, Masateru Nagae, Yuji Arai, Mitsuhiro Kawata, Toshikazu Kubo

JOURNAL OF CELLULAR PHYSIOLOGY 215 ( 3 ) 837 - 845 2008.6

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

Circulation-derived cells play a crucial role in the healing processes of tissue. In early phases of tendon healing processes, circulation-derived cells temporarily exist in the wounded area to initiate the healing process and decrease in number with time. We assumed that a delay of time-dependent decrease in circulation-derived cells could improve the healing of tendons. In this study, we injected platelet-rich plasma (PRP) containing various kinds of growth factors into the wounded area of the patellar tendon, and compared the effects on activation of circulation-derived cells and enhancement of tendon healing with a control group (no PRP injection). To follow the circulation-derived cells, we used a green fluorescent protein (GFP) chimeric rat expressing GFP in the circulating cells and bone marrow cells. In the PRP group, the numbers of GFP-positive cells and heat-shock protein (HSP47; collagen-specific molecular chaperone)-positive cells were significantly higher than in the control group at 3 and 7 days after injury. At the same time, the immunoreactivity for types I and III collagen was higher in the PRP group than in the control group at early phase of tendon healing. These findings suggest that locally injected PRP is useful as an activator of circulation-derived cells for enhancement of the initial tendon healing process.

DOI： 10.1002/jcp.21368

Expression, localization and possible actions of 25-Dx, a membrane-associated putative progesterone-binding protein, in the developing Purkinje cell of the cerebellum: A new insight into the biosynthesis, metabolism and multiple actions of progesterone as a neurosteroid Reviewed

Hirotaka Sakamoto, Kazuyoshi Ukena, Mitsuhiro Kawata, Kazuyoshi Tsutsui

CEREBELLUM 7 ( 1 ) 18 - 25 2008.3

Authorship：Lead author,　Corresponding author Language：English Publisher：SPRINGER

Neurosteroids are now known to be steroids that are synthesized de novo from cholesterol in the central and peripheral nervous systems of vertebrates through mechanisms at least partly independent of peripheral steroidogenic glands, such as the adrenal and gonads. A series of our studies have demonstrated that the rat Purkinje cell, a cerebellar neuron, actively produces progesterone de novo from cholesterol only during neonatal life and progesterone promotes dendritic growth, spinogenesis and synaptogenesis via its nuclear receptor in this neuron. Thus the Purkinje cell serves as an excellent cellular model for understanding the formation of cerebellar neuronal circuit in relation to genomic neurosteroid actions. Recently, we have further found that Purkinje cells express the putative membrane progesterone receptor, 25-Dx in rats. By immunocytochemistry, the expression of 25-Dx was localized in the Purkinje cell and external granule cell layer. RT-PCR and Western immunoblot analyses revealed the expressions of 25-Dx and its mRNA in the rat cerebellum, which increased during neonatal life. Therefore, progesterone would promote dendritic growth, spinogenesis and synaptogenesis via 25-Dx as well as its nuclear receptor in the Purkinje cell in the neonate. Because the subcellular localization of 25-Dx was associated with membrane structures of the endoplasmic reticulum and Golgi, 25-Dx may also play a role in the regulation of neurosteroidogenesis in the developing Purkinje cell. Here we summarize the advances made in our understanding of the expression, localization and its possible actions of 25-Dx in the developing Purkinje cell.

DOI： 10.1007/s12311-008-0007-2

Behavior of host and graft cells in the early remodeling process of rotator cuff defects in a transgenic animal model Reviewed

Yoshio Iwata, Toru Morihara, Hisakazu Tachiiri, Yoshiteru Kajikawa, Atsuhiko Yoshida, Yuji Arai, Daisaku Tokunaga, Hirotaka Sakamoto, Ken-ichi Matsuda, Masao Kurokawa, Mitsuhiro Kawata, Toshikazu Kubo

JOURNAL OF SHOULDER AND ELBOW SURGERY 17 ( 1 ) 101S - 107S 2008.1

Language：English Publishing type：Research paper (scientific journal) Publisher：MOSBY-ELSEVIER

Autologous tissue graft is one of the treatment options for a large rotator cuff defect. To develop appropriate strategies for enhanced solid graft integration at the bone-tendon interface and tendon-tendon interface, clarifying the fate of the graft and host cells that contribute to repair and remodeling is necessary. We have developed a new grafting model using green fluorescent protein-transgenic rats and wild-type rats to simulate autologous transplantation for examining the behavior of the host and graft cells in the remodeling process after tendon grafting. We found that the host cells commenced proliferation in the graft at 1 day after grafting. The host cells infiltrated into the graft from the subacromial synovium, proximal tendon, and bone-tendon insertion. The number of graft-derived cells decreased with time. Our result clearly demonstrated that host cells, rather than graft cells, were essential for rotator cuff remodeling after tendon grafting for rotator cuff defect.

DOI： 10.1016/j.jse.2007.07.008

Mode of action and functional significance of estrogen-inducing dendritic growth, spinogenesis, and synaptogenesis in the developing purkinje cell Reviewed

Katsunori Sasahara, Hanako Shikimi, Shogo Haraguchi, Hirotaka Sakamoto, Shin-ichiro Honda, Nobuhiro Harada, Kazuyoshi Tsutsui

JOURNAL OF NEUROSCIENCE 27 ( 28 ) 7408 - 7417 2007.7

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC NEUROSCIENCE

Neurosteroids are synthesized de novo from cholesterol in the brain. To understand neurosteroid action in the brain, data on the regio- and temporal-specific synthesis of neurosteroids are needed. Recently, we identified the Purkinje cell as an active neurosteroidogenic cell. In rodents, this neuron actively produces several neurosteroids including estradiol during neonatal life, when cerebellar neuronal circuit formation occurs. Estradiol may be involved in cerebellar neuronal circuit formation through promoting neuronal growth and neuronal synaptic contact, because the Purkinje cell expresses estrogen receptor-beta (ER beta). To test this hypothesis, in this study we examined the effects of estradiol on dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell using neonatal wild-type (WT) mice or cytochrome P450 aromatase knock-out (ArKO) mice. Administration of estradiol to neonatal WT or ArKO mice increased dendritic growth, spinogenesis, and synaptogenesis in the Purkinje cell. In contrast, WT mice treated with tamoxifen, an ER antagonist, or ArKO mice exhibited decreased Purkinje dendritic growth, spinogenesis, and synaptogenesis at the same neonatal period. To elucidate the mode of action of estradiol, we further examined the expression of brain-derived neurotrophic factor ( BDNF) in response to estrogen actions in the neonate. Estrogen administration to neonatal WT or ArKO mice increased the BDNF level in the cerebellum, whereas tamoxifen decreased the BDNF level in WT mice similar to ArKO mice. BDNF administration to tamoxifen-treated WT mice increased Purkinje dendritic growth. These results indicate that estradiol induces dendritic growth, spinogenesis, and synaptogenesis in the developing Purkinje cell via BDNF action during neonatal life.

DOI： 10.1523/JNEUROSCI.0710-07.2007

GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing Reviewed

Yoshiteru Kajikawa, Toru Morihara, Nobuyoshi Watanabe, Hirotaka Sakamoto, Ken-Ichi Matsuda, Masashi Kobayash, Yasushi Oshima, Atsuhiko Yoshida, Mitsuhiro Kawata, Toshikazu Kubo

JOURNAL OF CELLULAR PHYSIOLOGY 210 ( 3 ) 684 - 691 2007.3

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

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP-positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation-derived cells but not tendon-derived cells. Tendon-derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation-derived cells had decreased in number and had been replaced by the tendon-derived cells. These findings suggest that circulation-derived and tendon-derived cells contribute to the healing of tendons in different periods as part of a biphasic process.

DOI： 10.1002/jcp.20876

Intervertebral disc regeneration using platelet-rich plasma and biodegradable gelatin hydrogel microspheres Reviewed

Masateru Nagae, Takumi Ikeda, Yasuo Mikami, Hitoshi Hase, Hitoshi Ozawa, Ken-Ichi Matsuda, Hirotaka Sakamot, Yasuhiko Tabata, Mitsuhiro Kawata, Toshikazu Kubo

TISSUE ENGINEERING 13 ( 1 ) 147 - 158 2007.1

Language：English Publishing type：Research paper (scientific journal) Publisher：MARY ANN LIEBERT INC

This study evaluated the regenerative effects of platelet-rich plasma (PRP) for the degenerated intervertebral disc (IVD) in vivo. After induction of IVD degeneration in rabbits, we prepared PRP by centrifuging blood obtained from these rabbits. These PRP were injected into the nucleus pulposus (NP) of the degenerated IVDs after impregnation into gelatin hydrogel microspheres that can immobilize PRP growth factors physiochemically and release them in a sustained manner with the degradation of the microspheres. As controls, microspheres impregnated with phosphate-buffered saline (PBS) and PRP without microspheres were similarly injected. Histologically, notable progress in IVD degeneration with time courses was observed in the PBS control, PRP-only, and sham groups. In contrast, progress was remarkably suppressed over the 8-week period in the PRP group. Moreover, in immunohistochemistry, intense immunostaining for proteoglycan in the NP and inner layer of the annulus fibrosus was observed 8 weeks after administration of PRP-impregnated microspheres. Almost all microspheres were indistinct 8 weeks after the injection, and there were no apparent side effects in this study. Our results suggest that the combined administration of PRP and gelatin hydrogel microspheres into the IVD may be a promising therapeutic modality for IVD degeneration.

DOI： 10.1089/ten.2006.0042

Intrauterine proximity to male fetuses affects the morphology of the sexually dimorphic nucleus of the preoptic area in the adult rat brain Reviewed

M Pei, K Matsuda, H Sakamoto, M Kawata

EUROPEAN JOURNAL OF NEUROSCIENCE 23 ( 5 ) 1234 - 1240 2006.3

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

Previous studies on polytocous rodents have revealed that the fetal intrauterine position influences its later anatomy, physiology, reproductive performance and behavior. To investigate whether the position of a fetus in the uterus modifies the development of the brain, we examined whether the structure of the sexually dimorphic nucleus of the preoptic area (SDN-POA) of rat brains accorded to their intrauterine positions. Brain sections of adult rats gestated between two male fetuses (2M) and between two female fetuses (2F) in the uterus were analysed for their immunoreactivity to calbindin-D28k, which is a marker of the SDN-POA. The SDN-POA volume of the 2M adult males was greater than that of the 2F adult males, whereas the SDN-POA volume of the 2M and 2F adult females showed no significant difference. This result indicated that contiguous male fetuses have a masculinizing effect on the SDN-POA volume of the male. To further examine whether the increment of SDN-POA volume in adulthood was due to exposure to elevated steroid hormones during fetal life, concentrations of testosterone and 17 beta-estradiol in the brain were measured with 2M and 2F fetuses during gestation, respectively. On gestation day 21, the concentrations of testosterone and 17 beta-estradiol in the brain were significantly higher in the 2M male rats as compared with the 2F male rats. The results suggested that there was a relationship between the fetal intrauterine position, hormone transfer from adjacent fetuses and the SDN-POA volume in adult rat brains.

DOI： 10.1111/j.1460-9568.2006.04661.x

Stress and corticosteroid receptors Reviewed

Mitsuhiro Kawata, Mayumi Nishi, Ken-Ichi Matsuda, Hirotaka Sakamoto, Cui Honghai, Takanori Yoshii

PTSD: Brain Mechanisms and Clinical Implications 29 - 36 2006

Language：English Publishing type：Part of collection (book) Publisher：Springer Japan

The hypothalamo-pituitary-adrenal (HPA) axis is the main hormonal system involvedin stress response. Neurons in the hypothalamic paraventricular nucleus secreteadrenocorticotrophic hormone-releasing hormone (CRF) and vasopressin,which then synergistically induce the secretion of adrenocorticotrophic hormone(ACTH) from corticotrophs in the anterior pituitary, activating the secretion of theglucocorticoids from the adrenal cortex. Glucocorticoids (Cortisol in human andcorticosterone in rodents) readily enter the brain from the circulating blood andbind to their receptors to induce the secretion of CRF and ACTH for feedback regulationto keep homeostasis through the HPA axis. Over the past decades there hasbeen increasing evidence supporting a critical role for the hippocampus in stressresponse (McEwen 1999). Neurons in the hippocampus express receptors for circulatingglucocorticoids, and animal models of repeated stress caused dendritic atrophyin CAS pyramidal neurons (Woolley et al. 1990
Watanabe et al. 1992
Magarinosand McEwen 1995
Sousa et al. 2000). In the primate prominent pyramidalneuron examples of damage such as dendritic atrophy and pyknotic changes wereobserved in the CAS area of animals subjected to repeated social stress and longtermtreatment of Cortisol (Uno et al. 1989, Sapolsky et al. 1990). These results suggestthat dendritic atrophy of the pyramidal neurons may be one of the factors contributingto the clinically observed decrease of hippocampal volume in humans sufferingfrom stress-related disorders including the posttraumatic stress disorder(PTSD) (Bremner et al. 1995). Transsynaptic inhibitory projection of the hippocampusto hypothalamic paraventricular nucleus has been demonstrated. Therefore, thehippocampus has been the focus of the stress response not only due to its susceptibilityto stress-related damages but also to its negative feedback regulation of thestress response via the HPA axis (Herman and Cullinan 1997). In contrast to thehippocampus, little has been known about how stress affects the amygdala and thenature of its role in the stress response. Recent evidence has demonstrated that theamygdala also plays a critical role in stress response and activation of the HPA axis(Le Doux 1994). It has been suggested that hippocampal plasticity mediates cogni-tive aspects of behavioral impairments caused by stress, whereas changes in theamygdala are more likely to contribute to the affective aspects of stress disorderswith memory consolidation.

DOI： 10.1007/4-431-29567-4_3

Dendritic growth in response to environmental estrogens in the developing Purkinje cell in rats Reviewed

H Shikimi, H Sakamoto, Y Mezaki, K Ukena, K Tsutsui

NEUROSCIENCE LETTERS 364 ( 2 ) 114 - 118 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

The cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated recently that the Purkinje cell actively produces sex steroids, such as estradiol and progesterone, de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs. We have further demonstrated that both estradiol and progesterone promote the growth of Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development. On the other hand, environmental estrogens, such as octylphenol (OP), bisphenol A (BPA), and nonylphenol (NP) are thought to mimic the action of estrogen in the developing central nervous system. Therefore, in this study, the effect of these environmental estrogens on the growth of Purkinje cells was examined in vivo using newborn rats. OP and BPA promoted a dose-dependent dendritic outgrowth of the Purkinje cell but did not affect its soma and cell number. The stimulatory effect of OP and BPA on Purkinje dendritic growth was induced by an injection of 500 mug/day into the cerebrospinal fluid for 4 days and blocked by the estrogen receptor antagonist tamoxifen. However, there was no significant effect of NP on any Purkinje cell morphology. These results suggest that the environmental estrogens, OP and BPA, promote Purkinje dendritic growth during neonatal life. This effect may be mediated by estrogen receptor in the Purkinje cell. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2004.04.023

Organizing actions of neurosteroids in the Purkinje neuron Reviewed

K Tsutsui, H Sakamoto, H Shikimi, K Ukena

NEUROSCIENCE RESEARCH 49 ( 3 ) 273 - 279 2004.7

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

It is becoming clear that steroids can be synthesized de novo by the brain of vertebrates. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific biosynthesis in particular sites of the brain at particular times. Therefore our studies for this exciting area of neuroscience research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This neuron actively synthesizes progesterone and estradiol de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs dramatically. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently the actions of progesterone and estradiol during cerebellar development have become clear by a series of our studies using an excellent Purkinje cellular model. These neurosteroids promote dendritic growth, spinogenesis and synaptogenesis via each receptor in the Purkinje cell. Here we summarize the advances made in our understanding of organizing actions of neurosteroids in the Purkinje cell, an important brain neuron. (C) 2004 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/j.neures.2004.03.006

Synapse formation in response to estrogen synthesized de novo in the developing Purkinje cell.

H. Shikimi, H. Sakamoto, K. Ukena, K. Tsutsui

In: Trends in Comparative Endocrinology, 319 - 321 2004

Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell Reviewed

H Sakamoto, Y Mezaki, H Shikimi, K Ukena, K Tsutsui

ENDOCRINOLOGY 144 ( 10 ) 4466 - 4477 2003.10

Authorship：Lead author Language：English Publishing type：Research paper (scientific journal) Publisher：ENDOCRINE SOC

Neurosteroids are synthesized de novo in the brain, and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the Purkinje cell possesses intranuclear receptor for progesterone and actively produces progesterone de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs dramatically. We have further demonstrated that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis via its receptor in this neuron in the neonate. On the other hand, estrogen may also play an important role in the process of cerebellar cortical formation, because the neonatal rat Purkinje cell possesses estrogen receptor (ER) beta. However, estrogen formation in the neonatal cerebellum is still unclear. In this study, we therefore analyzed the biosynthesis and action of estrogen in Purkinje cells during neonatal life. RT-PCR-Southern and in situ hybridization analyses showed that Purkinje cells expressed the key enzyme of estrogen formation, cytochrome P450 aromatase, in neonatal rats. A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate were significantly higher than those in the prepuberty and adult. Both in vitro and in vivo studies with newborn rats showed that estradiol promoted dose-dependent dendritic growth of Purkinje cells. Estradiol also increased the density of Purkinje dendritic spines. These effects were inhibited by the ER antagonist tamoxifen. These results suggest that estradiol in the developing Purkinje cell promotes dendritic growth and spinogenesis via ERbeta in this neuron. Estradiol as well as progesterone may contribute to the growth of Purkinje cells during the cerebellar cortical formation.

DOI： 10.1210/en.2003-0307

Biosynthesis and action of neurosteroids in the cerebellar Purkinje neuron Reviewed

K Tsutsui, H Sakamoto, K Ukena

JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 85 ( 2-5 ) 311 - 321 2003.6

Language：English Publishing type：Research paper (scientific journal) Publisher：PERGAMON-ELSEVIER SCIENCE LTD

The brain is considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Therefore, our studies for this exciting area of brain research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently, genomic and nongenomic actions of neurosteroids have become clear by a series of our studies using an excellent Purkinje cellular model. On the basis of these findings, we summarize the advances made in our understanding of biosynthesis and action of neurosteroids in the cerebellar Purkinje cell. (C) 2003 Elsevier Ltd. All rights reserved.

DOI： 10.1016/S0960-0760(03)00229-2

Neonatal expression of progesterone receptor isoforms in the cerebellar Purkinje cell in rats Reviewed

H Sakamoto, H Shikimi, K Ukena, K Tsutsui

NEUROSCIENCE LETTERS 343 ( 3 ) 163 - 166 2003.6

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. Progesterone synthesized de novo in developing PCs participates in the promotion of dendritic growth, spinogenesis and synaptogenesis in this neuron and such organizing actions may contribute to the formation of the cerebellar neuronal circuit during rat neonatal life. Progesterone receptors (PR) occur as two isoforms (PR-A and PR-B) derived from a single gene. To clarify the mode of organizing actions of progesterone, therefore, we examined the expression of these PR isoforms in the rat cerebellum during development. Western immunoblot analysis revealed that both PR isoforms were expressed highly in the cerebellum during neonatal life and the expression decreased thereafter. PR-like immunoreactivity was localized primarily in PCs in the neonatal cerebellum. Thus, progesterone may act directly on PCs via PR isoforms to promote its dendritic growth, spinogenesis and synaptogenesis. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

DOI： 10.1016/S0304-9340(03)000362-8

A novel aspect of the cerebellum: biosynthesis of neurosteroids in the Purkinje cell Reviewed

K Tsutsui, H Sakamoto, K Ukena

CEREBELLUM 2 ( 3 ) 215 - 222 2003

Language：English Publishing type：Research paper (scientific journal) Publisher：TAYLOR & FRANCIS LTD

Peripheral steroid hormones act on brain tissues through intracellular receptor-mediated mechanisms to regulate several important brain neuronal functions. The brain is therefore considered to be a target site of steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To analyze neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Such information is crucial to developing hypotheses predicting the potential roles of particular neurosteroids in the developing and adult brains. Thus our studies for this exciting area of brain research have focused on the biosynthesis of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently genomic and nongenomic actions of neurosteroids have been suggested by a series of our studies using an excellent Purkinje cellular model. Here we summarize the advances made in our understanding of biosynthesis of neurosteroids in the cerebellar Purkinje cell.

DOI： 10.1080/14734220310016169

総説:ニューロンが合成するニューロステロイドのノンゲノミック作用とゲノミック作用

筒井和義, 坂本浩隆, 食見花子, 浮穴和義

生殖内分泌学会雑誌 8 19 - 26 2003

総説:ニューロステロイドのシナプス形成誘導作用

筒井和義, 坂本浩隆, 浮穴和義, 古川康雄

生体の科学 54 ( 2 ) 101 - 108 2003

Dendritic spine formation in response to progesterone synthesized de novo in the developing Purkinje cell in rats Reviewed

H Sakamoto, K Ukena, K Tsutsui

NEUROSCIENCE LETTERS 322 ( 2 ) 111 - 115 2002.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ELSEVIER SCI IRELAND LTD

The cerebellar Purkinje cell (PC) is a typical site for neurosteroid formation. We have demonstrated that this neuron possesses intranuclear receptor for progesterone and actively synthesizes progesterone de novo from cholesterol only during rat neonatal life, when the formation of the cerebellar cortex occurs dramatically. In this study, we therefore analyzed the effect of progesterone on dendritic spine formation of the PC. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone increases the density of PC dendritic spines in a dose-dependent manner. This effect was blocked by the progesterone receptor antagonist, RU486. Furthermore, trilostane, a specific inhibitor of progesterone synthesis, inhibited the increase of spine density. These results suggest that progesterone can promote dendritic spine formation, and endogenous progesterone synthesized de novo in the developing PC may induce such an effect. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Novel cerebellar function: Neurosteroids in the purkinje neuron and their genomic and nongenomic actions

K Tsutsui, K Ukena, H Sakamoto

NEUROPLASTICITY, DEVELOPMENT, AND STEROID HORMONE ACTION 101 - 116 2002

Activity and localization of 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase in the zebrafish central nervous system Reviewed

H Sakamoto, K Ukena, K Tsutsui

JOURNAL OF COMPARATIVE NEUROLOGY 439 ( 3 ) 291 - 305 2001.10

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

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3 beta -hydroxysteroid dehydrogenase/Delta (5)-Delta (4)-isomerase (3 beta HSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3 beta HSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3 beta HSD. By using Western immunoblotting with the polyclonal. antiserum. directed against purified bovine adrenal 3 beta HSD, a 3 beta HSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3 beta HSD-like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic. areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3 beta HSD-like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3 beta HSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3 beta HSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3 beta HSD expression in the fish CNS. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/cne.1351

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

Developmental changes in galanin in lumbosacral sympathetic ganglionic neurons innervating the avian uterine oviduct and galanin induction by sex steroids Reviewed

T Ubuka, H Sakamoto, D Li, K Ukena, K Tsutsui

JOURNAL OF ENDOCRINOLOGY 170 ( 2 ) 357 - 368 2001.8

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC ENDOCRINOLOGY

We recently found lumbosacral sympathetic ganglionic galanin neurons innervating the quail uterine oviduct. Galaninergic innervation of the uterine muscle may be essential for avian oviposition, as galanin evoked oviposition through a mechanism of induction of vigorous uterine contraction. The questions arising from these findings are: what changes occur in galanin expression in the sympathetic ganglionic galanin neuron during development, and what is the hormonal Factor(s) that induces galanin expression in this neuron? Therefore, the present study examined the developmental changes in galanin of the quail sympathetic ganglionic neuron and uterus, and the effect of administration of ovarian sex steroids on galanin induction. Immature birds reared under long-day photoperiods from 4 weeks of age demonstrated progressive increases in galanin levels both per unit ganglionic protein (concentration) and per ganglia (content) concurrent with ganglionic development during weeks 4-13. The uterine galanin content and uterine weight also increased progressively during the same period, but the galanin concentration in the uterus at 4 weeks was high due to the much smaller tissue mass. Immunocytochemical analysis with anti-galanin serum showed that immunoreactive ganglionic cells were few and small at 4 weeks and increased progressively thereafter. Administration of oestradiol-17 beta to immature birds at 3 weeks of age for I week increased both the galanin concentration and content in the ganglia without ganglionic growth. A marked increase in galanin-immunoreactive ganglionic cells was detected following oestradiol treatment. In contrast, progesterone increased ganglionic galanin levels, but the effects were low. Expression of the mRNAs encoding oestrogen receptor-alpha and -beta (ER alpha and ER beta) in the ganglionic tissue was verified by RT-PCR/Southern blot analysis. Immunocytochemical staining with anti-ER serum further revealed an intense immunoreaction restricted to the nucleus of ganglionic neurons.
These results suggest that ovarian sex steroids, in particular oestradiol-17 beta, contribute as hormonal factors to galanin induction, which takes place in the lumbosacral sympathetic ganglionic neurons innervating avian uterine oviduct during development. Oestradiol may act directly on this ganglionic neuron through intra-nuclear receptor-mediated mechanisms to induce galanin.

DOI： 10.1677/joe.0.1700357

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

Sympathetic ganglionic galanin neurons evoke avian oviposition by innervating the uterine oviduct: A novel neuronal mechanism of avian oviposition

H Sakamoto, T Ubuka, C Kohchi, K Ukena, K Tsutsui

PERSPECTIVE IN COMPARATIVE ENDOCRINOLOGY: UNITY AND DIVERSITY 647 - 653 2001

Language：English Publishing type：Research paper (international conference proceedings) Publisher：MEDIMOND S R L

We recently isolated an oviposition-inducing peptide that was identified as avian galanin from mature quail uterine oviducts. This peptide was localized in neuronal fibers terminating in the uterus, and its receptors were also observed in the uterus. To understand the control mechanism of avian oviposition by galanin, we identified the neurons that synthesize galanin and project to the uterus in mature quails. Retrograde labeling with neurobiotin from the uterus revealed that lumbosacral sympathetic ganglionic neurons projected to the uterine muscle. Colocalization of neurobiotin with galanin-like substance was further confirmed by the ultrastructural immunocytochemistry. Expressions of galanin and its mRNA in the neurons were further confirmed by competitive enzyme-linked immunosorbent assay and Northern blot analysis. These results suggest that lumbosacral sympathetic ganglionic neurons project to the uterine muscle and produce galanin in mature quails.

Novel brain function: biosynthesis and actions of neurosteroids in neurons Reviewed

K Tsutsui, K Ukena, M Usui, H Sakamoto, M Takase

NEUROSCIENCE RESEARCH 36 ( 4 ) 261 - 273 2000.4

Language：English Publisher：ELSEVIER IRELAND LTD

Peripheral steroid hormones act on brain tissues through intracellular receptor-mediated mechanisms to regulate several important brain neuronal functions. Therefore, the brain is considered to be a target site of steroid hormones. However, it is now established that the brain itself also synthesizes steroids de novo from cholesterol. The pioneering discovery of Baulicu and his colleagues, using mammals, and our studies with non-mammals have opened the door of a new research field. Such steroids synthesized in the brain are called neurosteroids. Because certain structures in vertebrate brains have the capacity to produce neurosteroids identification of neurosteroidogenic cells in the brain is essential to understand the physiological role of neurosteroids in brain functions. Glial cells are generally accepted to be the major site for neurosteroid formation. but the concept of neurosteroidogenesis in brain neurons has up to now been uncertain. We recently demonstrated neuronal neurosteroidogenesis in the blain and indicated that the Purkinje cell, a typical cerebellar neuron, actively synthesizes several neurosteroids de novo from cholesterol in both mammals and non-mammals. Pregnenolone sulfate, one of neurosteroids synthesized in the Purkinje neuron, may contribute to some important events in the cerebellum by modulating neurotransmission. Progesterone, produced as a neurosteroid in this neuron only during neonatal life, may be involved in the promotion of neuronal and glial growth and neuronal synaptic contact ill the cerebellum. More recently, biosynthesis and actions of neurosteroids in pyramidal neurons of the hippocampus were also demonstrated. These serve an excellent model for the study of physiological roles of neurosteroids in the brain, because both cerebellar Purkinje neurons and hippocampal neurons play an important role in memory and learning. This paper summarizes the advances made in our understanding of neurosteroids, produced in neurons, and their actions. (C) 2000 Elsevier Science ireland Ltd and the Japan Neuroscience Society. All rights reserved.

DOI： 10.1016/S0168-0102(99)00132-7

DOI： 10.1016/s0168-0102(99)00132-7

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

新しく見いだされた鳥類産卵の神経制御機構:微小脳が合成する産卵誘起ペプチドとその作用機構

筒井和義, 李丹, 坂本浩隆, 浮穴和義

日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology 96 17 - 22 2000.2

総説 : 神経ステロイドによるシナプス可塑性調節

筒井和義, 浮穴和義, 坂本浩隆

特集:脳のシナプス Brain Medical 12 298 - 306 2000

Reproduction strategy in the minute brain of birds. Egg laying peptide and its mechanism of action.

TSUTSUI Kazuyoshi, LI Dan, SAKAMOTO Hirotaka, UKENA Kazuyoshi

Dobutsu seiri 16 ( 3 ) 191 - 199 1999.9

Posthatching development of spinal motoneurons in the angelfish Pterophyllum scalare Reviewed

M Yoshida, M Fudoji, H Sakamoto, K Uematsu

BRAIN BEHAVIOR AND EVOLUTION 53 ( 4 ) 180 - 186 1999.4

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

We investigated the posthatching developmental sequence of spinal motoneurons innervating the axial muscles in the teleost angelfish, Pterophyllum scalare, by means of retrograde labeling with horseradish peroxidase. Two discrete types of spinal motoneurons, primary-type motoneurons and secondary motoneurons were labeled in a temporally different sequence during the course of larval development. These two types of motoneurons were morphologically distinguishable from one other. Primary-type motoneurons are generated by day 1 posthatching and do not increase in number over the observed period (to day 12 posthatching). In contrast, the secondary motoneurons increase in number through posthatching day 3. Differentiation of the spinal motoneurons appears to be nearly complete a few days before the onset of free swimming. In addition, the data suggest that the differentiation of secondary motoneurons precedes the development of the red muscle that is to be innervated by the motoneurons.

DOI： 10.1159/000006593

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

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