Neurotensin co-expressed in orexinproducing neurons in the lateral hypothalamus plays an important role in regulation of sleep/wakefulness states

13301甲第4095号博士（医学）金沢大学博士論文本文Full 以下に掲載：PLOS ONE 8(4) pp.e62391-e62391 2013. PLOS. 共著者：Furutani Naoki, Hondo Mari, Kageyama Haruaki, Tsujino Natsuko, Mieda Michihiro, Yanagisawa Masashi, Shioda Seiji, Sakurai Takesh

Activation of Bombesin receptor subtype-3 influences activity of orexin neurons by both direct and indirect pathways

金沢大学医薬保健研究域医学系The neuropeptides orexin A and orexin B (also known as hypocretin 1 and hypocretin 2), produced in lateral hypothalamic neurons, are critical regulators of feeding behavior, the reward system, and sleep/wake states. Orexin-producing neurons (orexin neurons) are regulated by various factors involved in regulation of energy homeostasis and sleep/wakefulness states. Bombesin receptor subtype 3 (BRS3) is an orphan receptor that might be implicated in energy homeostasis and is highly expressed in the hypothalamus. However, the neural pathway by which BRS3 regulates energy homeostasis is largely unknown. We examined whether BRS3 is involved in the regulation of orexin neurons. Using a calcium imaging method, we found that a selective BRS3 agonist [Ac-Phe-Trp-Ala-His-(τBzl)-Nip-Gly-Arg-NH2] increased the intracellular calcium concentration of orexin neurons. However, intracellular recordings from slice preparations revealed that the BRS3 agonist hyperpolarized orexin neurons. The BRS3 agonist depolarized orexin neuron in the presence of tetrodotoxin. Moreover, in the presence of GABA receptor blockers, picrotoxin and CGP55845, the BRS3 agonist induced depolarization and increased firing frequency. Additionally, double-label in situ hybridization study revealed that Brs3 mRNA was expressed in almost all orexin neurons and many cells around these neurons. These findings suggest that the BRS3 agonist indirectly inhibited orexin neurons through GABAergic input and directly activated orexin neurons. Inhibition of activity of orexin neurons through BRS3 might be an important pathway for regulation of feeding and sleep/wake states. This pathway might serve as a novel target for the treatment of obesity. © 2010 Springer Science+Business Media, LLC

Le FortⅠガタ ホネキリジュツ ニオケル ポリ L ニュウサン ポリグリコールサン キョウジュウゴウタイ プレート ノ アンテイセイ

This paper verified the stability of poly-L-Lactic/polyglycolic acid bone fixation devices in Le Fort I osteotomy. The subjects of the study were 40 patients (33 females and 7 males) who underwent Le Fort I osteotomy with fixation by poly-L-Lactic/polyglycolic acid bone fixation devices (plates and screws). The subjects were divided into two groups according to their skeletal deformity pattern: skeletal Class II and Class III. Lateral cephalometric radiographs were measured and compared for the absolute magnitude of skeletal relapse from pretreatment to immediately postoperative to 6 months and 1 year after surgery. The X-axis applied the Sella-Nasion line, and the Y-axis passed through Sella perpendicular to the X-axis. The evaluation factors were A-point, Prosthion, and posterior nasal spine (PNS). In both groups, statistical analysis showed no significant differences in postoperative skeletal stability. In conclusion, poly-L-Lactic/polyglycolic acid bone fixation devices can provide postoperative stability for Le Fort I osteotomy

Orexin neurons receive glycinergic innervations.

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation

Orexin neurons receive glycinergic innervations

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation本堂茉莉 Hondo, Mari 博士学位論文 Doctor thesi

Orexin Neurons Receive Glycinergic Innervations

Glycine, a nonessential amino-acid that acts as an inhibitory neurotransmitter in the central nervous system, is currently used as a dietary supplement to improve the quality of sleep, but its mechanism of action is poorly understood. We confirmed the effects of glycine on sleep/wakefulness behavior in mice when administered peripherally. Glycine administration increased non-rapid eye movement (NREM) sleep time and decreased the amount and mean episode duration of wakefulness when administered in the dark period. Since peripheral administration of glycine induced fragmentation of sleep/wakefulness states, which is a characteristic of orexin deficiency, we examined the effects of glycine on orexin neurons. The number of Fos-positive orexin neurons markedly decreased after intraperitoneal administration of glycine to mice. To examine whether glycine acts directly on orexin neurons, we examined the effects of glycine on orexin neurons by patch-clamp electrophysiology. Glycine directly induced hyperpolarization and cessation of firing of orexin neurons. These responses were inhibited by a specific glycine receptor antagonist, strychnine. Triple-labeling immunofluorescent analysis showed close apposition of glycine transporter 2 (GlyT2)-immunoreactive glycinergic fibers onto orexin-immunoreactive neurons. Immunoelectron microscopic analysis revealed that GlyT2-immunoreactive terminals made symmetrical synaptic contacts with somata and dendrites of orexin neurons. Double-labeling immunoelectron microscopy demonstrated that glycine receptor alpha subunits were localized in the postsynaptic membrane of symmetrical inhibitory synapses on orexin neurons. Considering the importance of glycinergic regulation during REM sleep, our observations suggest that glycine injection might affect the activity of orexin neurons, and that glycinergic inhibition of orexin neurons might play a role in physiological sleep regulation

Role of Asp193 in chromophore-protein interaction of pharaonis phoborhodopsin (sensory rhodopsin II).

Pharaonis phoborhodopsin (ppR; also pharaonis sensory rhodopsin II, psRII) is a receptor of the negative phototaxis of Natronobacterium pharaonis. By spectroscopic titration of D193N and D193E mutants, the pKa of the Schiff base was evaluated. Asp193 corresponds to Glu204 of bacteriorhodopsin (bR). The pKa of the Schiff base (SBH+) of D193N was ~10.1-10.0 (at XH+) and ~11.4-11.6 (at X) depending on the protonation state of a certain residue (designated by X) and independent of Cl, whereas those of the wild type and D193E were >12. The pKa values of XH+ were ~11.8-11.2 at the state of SB, 10.5 at SBH+ state in the presence of Cl, and 9.6 at SBH+ without Cl. These imply the presence of a long-range interaction in the extracellular channel. Asp193 was suggested to be deprotonated in the present dodecyl-maltoside (DDM) solubilized wild-type ppR, which is contrary to Glu204 of bR. In the absence of salts, the irreversible denaturation of D193N (but not the wild type and D193E) occurred via a metastable state, into which the addition of Cl reversed the intact pigment. This suggests that the negative charge at residue 193, which can be substituted by Cl, is necessary to maintain the proper conformation in the DDM-solubilized ppR

Fish oil accelerates diet-induced entrainment of the mouse peripheral clock via GPR120

The circadian peripheral clock is entrained by restricted feeding (RF) at a fixed time of day, and insulin secretion regulates RF-induced entrainment of the peripheral clock in mice. Thus, carbohydrate-rich food may be ideal for facilitating RF-induced entrainment, although the role of dietary oils in insulin secretion and RF-induced entrainment has not been described. The soybean oil component of standard mouse chow was substituted with fish or soybean oil containing docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA). Tuna oil (high DHA/EPA), menhaden oil (standard), and DHA/EPA dissolved in soybean oil increased insulin secretion and facilitated RF-induced phase shifts of the liver clock as represented by the bioluminescence rhythms of PER2::LUCIFERASE knock-in mice. In this model, insulin depletion blocked the effect of tuna oil and fish oil had no effect on mice deficient for GPR120, a polyunsaturated fatty acid receptor. These results suggest food containing fish oil or DHA/EPA is ideal for adjusting the peripheral clock