Neurotensin Co-Expressed in Orexin-Producing Neurons in the Lateral Hypothalamus Plays an Important Role in Regulation of Sleep/Wakefulness States

<div><p>Both orexin and neurotensin are expressed in the lateral hypothalamic area (LHA) and have been implicated in the regulation of feeding, motor activity and the reward system. A double label immunofluorescence and <i>in situ</i> hybridization studies showed that neurotensin colocalizes with orexin in neurons of the LHA. Pharmacological studies suggested that neurotensin excites orexin-producing neurons (orexin neurons) through activation of neurotensin receptor-2 (NTSR-2) and non-selective cation channels. <i>In situ</i> hybridization study showed that most orexin neurons express <i>neurotensin receptor-2</i> mRNA but not <i>neurotensin receptor-1</i> (<i>Ntsr-1</i>) mRNA. Immunohistochemical studies showed that neurotensin-immunoreactive fibers make appositions to orexin neurons. A neurotensin receptor antagonist decreased Fos expression in orexin neurons and wakefulness time in wild type mice when administered intraperitoneally. However, the antagonist did not evoke any effect on these parameters in orexin neuron-ablated mice. These observations suggest the importance of neurotensin in maintaining activity of orexin neurons. The evidence presented here expands our understanding of the regulatory mechanism of orexin neurons.</p></div

A neurotensin receptor antagonist SR142948 dose-dependently decreased wakefulness time and duration during the lights on period in wild-type mice, but not in orexin neuron-ablated mice.

<p>Hourly amounts (A, B) and average episode duration (C, D) of awake, non-REM, and REM sleep states (mean±SE) plotted over 4 hr after administration of saline (dotted line) or SR142948 (solid line) in wild-type (n = 27, 14 and 7 for saline, SR 1 mg/kg, and 3 mg/kg, respectively) (A, C) and <i>orexin/ataxin-3</i> mice (n = 14, 14 and 7 for saline, SR 1 mg/kg, and 3 mg/kg, respectively) (B, D). Data for the dark phase are displayed in shaded panels. SR142948 was administered at the start of light or dark periods (8:45 or 20:45).</p

Neurotensinergic fibers appose orexin neurons.

<p><b>A</b>, Double immunofluorescence study shows that many varicose terminals with neurotensin-like immunoreactivity (green) make appositions to orexin neurons (red) in the LHA (Left panels). Right panel, high power view of rectangular region in left panel. <b>B</b>, Photomicrograph showing distribution of Fos (black nuclear label) and orexin (brown label) expression in LHA. Scale bar indicates 40 µm. Upper panels, left panel, control (saline injection); right panel, SR142948 injection at 20:45. Animals were sacrificed at 21:45 and subjected to immunostaining. Lower panels, left panel, control (saline injection); right panel, SR142948 injection at 8:45. Animals were sacrificed at 9:45 and subjected to immunostaining. <b>C</b>, SR142948 administration decreased Fos immunoreactivity of orexin neurons, but not in MCH neurons.</p

A Single Nucleotide Polymorphism of the Neuropeptide B/W Receptor-1 Gene Influences the Evaluation of Facial Expressions

<div><p>Neuropeptide B/W receptor-1 (NPBWR1) is expressed in discrete brain regions in rodents and humans, with particularly strong expression in the limbic system, including the central nucleus of the amygdala. Recently, Nagata-Kuroiwa et al. reported that <em>Npbwr1</em>^−/− mice showed changes in social behavior, suggesting that NPBWR1 plays important roles in the emotional responses of social interactions.</p> <p>The human <em>NPBWR1</em> gene has a single nucleotide polymorphism at nucleotide 404 (404A>T; SNP rs33977775). This polymorphism results in an amino acid change, Y135F. The results of an in vitro experiment demonstrated that this change alters receptor function. We investigated the effect of this variation on emotional responses to stimuli of showing human faces with four categories of emotional expressions (anger, fear, happiness, and neutral). Subjects' emotional levels on seeing these faces were rated on scales of hedonic valence, emotional arousal, and dominance (V-A-D). A significant genotype difference was observed in valence evaluation; the 404AT group perceived facial expressions more pleasantly than did the 404AA group, regardless of the category of facial expression. Statistical analysis of each combination of [V-A-D and facial expression] also showed that the 404AT group tended to feel less submissive to an angry face than did the 404AA group. Thus, a single nucleotide polymorphism of NPBWR1 seems to affect human behavior in a social context.</p> </div

Phase shift of liver clock, insulin secretion, and clock gene expression in GPR120-deficient mice with tuna oil-containing diet.

<p>GPR120-deficient (KO) and wild-type (WT) mice were fed an AIN-93M diet tablet containing tuna oil at ZT0 for 2 days and bioluminescence rhythm was recorded after sacrifice on Day 3. The horizontal axis indicates projected Zeitgeber time (pZT) at the peak of the bioluminescence rhythm. ZT0 is lights-on time and ZT12 is lights-off time in the housing room prior to sacrifice. (A) de-trended data of expression rhythms of liver <i>PER2</i>::LUCIFERASE bioluminescence in mice under restricted-feeding (RF) of AIN-93M diet containing tuna or soybean oil. The horizontal line indicates time lapse. (B) magnitude of phase-delay by tuna or soybean oil in WT or KO mice. Values are expressed as mean ± SEM. **<i>P</i> < 0.01, (vs. WT SB group, Tukey-Kramer test). (C, D) on Day 2 under RF conditions, mice were fed AIN-93M containing tuna or soybean oil at ZT0, and sacrificed at 120 min to measure insulin and clock gene expression in the liver after feeding. (C) serum insulin levels. Values are expressed as mean ± SEM. *<i>P</i> < 0.05 *** <i>P</i> < 0.001 vs. WT SB group, ## <i>P</i> < 0.01 vs. WT SB (FF) group, Dunn test. (D) <i>Per2</i> gene expression. The relative levels of expression were normalized to <i>GAPDH</i>. *** <i>P</i> < 0.001 (vs. WT SB group), # <i>P</i> < 0.05 [vs. WT SB (FF) group Dunn test]. SB, soybean; FF, free feeding. Numbers in parentheses indicate the number of tested mice.</p

AIN-93M diet chow containing various fish oils or DHA/EPA dissolved in soybean oil and RF-induced phase shifts of the liver and SCN circadian clock.

<p>(A, C) mice were fed an AIN-93M diet tablet containing various substituted oils at ZT0 for 2 days or 7 days, and bioluminescence rhythm was recorded after sacrifice on Day 3 or Day 8. The horizontal axis indicates projected Zeitgeber time (pZT) at the peak of the bioluminescence rhythm. ZT0 is lights-on time and ZT12 is lights-off time in the housing room prior to sacrifice. (A) experimental protocol. RF was applied for 2 days at ZT0. (B) de-trended expression rhythms of the liver PER2::LUCIFERASE bioluminescence in mice under RF of an AIN-93M diet containing tuna oil (arrows) or soybean oil (arrow heads) for 2 days at ZT0. The horizontal line indicates time lapse. (C) magnitude of phase delay by fish oil-containing AIN-93M diet chow for 2 days at ZT0. A control experiment was prepared under free feeding conditions (closed triangle, FF). SB (7 days) shows the magnitude of the phase shifts by 7-day RF of soybean oil-containing AIM-93M at ZT0–ZT4 (Values are expressed as mean ± SEM. **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (vs. SB, control chow, Dunn test). #<i>P</i> < 0.05, ###<i>P</i> < 0.001 [vs. SB, (FF), Dunn test]. (D) magnitude of phase-delay or phase-advance by DHA/EPA-containing AIN-93M diet chow. SB, soybean; FF, free-feeding. Numbers in parentheses indicate the number of tested mice. Values are expressed as mean ± SEM. *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 (vs. SB, control chow, Tukey-Kramer test).</p

AIN-93M diet chow containing tuna oil and DHA/EPA and RF-induced insulin secretion.

<p>(A, B) on Day 2 under RF conditions, mice were fed an AIN-93M diet containing tuna, menhaden, soybean, DHA/EPA-containing soybean oil or without oil at ZT0; mice were sacrificed at 60 min, 120 min, or 240 min after feeding of fish oil (A) and 120 min after feeding of DHA/EPA (B). Serum insulin levels were measured. SB, soybean; w/o oil, without oil. Numbers in parentheses indicate the number of tested mice. Values are expressed as mean ± SEM. *<i>P</i> < 0.05 (vs. SB control chow, Tukey-Kramer test).</p

Effect of oral administration of fish oil or soybean oil containing DHA and/or EPA alone at daytime under free-feeding conditions on phase shifts and insulin level.

<p>(A) magnitude of phase shifts induced by tuna oil or soybean oil containing DHA and/or EPA administration at ZT0 for 2 days under free-feeding conditions. Tuna oil, soybean oil, DHA- or EPA-containing oil was administered at 0.034 ml/10 g BW for 2 days, and mice were sacrificed to examine the phase of bioluminescence of liver on Day 3. (B) tuna oil, menhaden oil, or DHA-containing oil was administered at 0.034 ml/10 g BW at ZT0 under free-feeding conditions, and mice were sacrificed 2 h after injection to examine serum insulin level. *<i>P</i> < 0.05 (vs. SB group, Tukey-Kramer test). Numbers in parentheses indicate the number of tested mice.</p

AIN-93M diet chow containing tuna oil and RF-induced phase-delay of the liver circadian clock and serum insulin in streptozotocin-pretreated mice.

<p>(A) bioluminescence rhythm recorded in the liver of STZ-treated mice fed AIN-93M diet chow containing tuna or soybean oil at ZT0 for 2 days. The horizontal axis indicates pZT at the peak of the bioluminescence rhythm. (B) on Day 2 under RF conditions, STZ-treated mice were fed AIN-93M containing tuna or soybean oil at ZT0 and sacrificed 120 min after feeding. Serum insulin levels were measured. Numbers in parentheses indicate the number of tested mice. Values are expressed as mean ± SEM. **<i>P</i> < 0.01 (vs. STZ-treated group, Tukey-Kramer test).</p

AIN-93M diet chow containing various fish oils and DHA/EPA and the RF-induced increase of <i>Per2</i> gene expression in the liver.

<p>(A, B) on Day 2 under RF conditions, mice were fed AIN-93M containing tuna, menhaden, soybean, DHA/EPA-containing soybean oil or without oil at ZT0; mice were sacrificed at 60 min, 120 min, and 240 min after feeding of fish oil (A) and 120 min after feeding of DHA/EPA (B). <i>Per2</i> gene expression was measured. The relative levels of expression were normalized to <i>GAPDH</i>. SB, soybean; w/o oil, without oil. Numbers in parentheses indicate the number of tested mice. Values are expressed as mean ± SEM. *<i>P</i> < 0.05 (vs. SB control chow, Tukey-Kramer test).</p