Junko Hara

Genetic Ablation of Orexin Neurons in Mice Results in Narcolepsy, Hypophagia, and Obesity

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.

Hypothalamic orexin neurons regulate arousal according to energy balance in mice

Mammals respond to reduced food availability by becoming more wakeful and active, yet the central pathways regulating arousal and instinctual motor programs (such as food seeking) according to homeostatic need are not well understood. We demonstrate that hypothalamic orexin neurons monitor indicators of energy balance and mediate adaptive augmentation of arousal in response to fasting. Activity of isolated orexin neurons is inhibited by glucose and leptin and stimulated by ghrelin. Orexin expression of normal and ob/ob mice correlates negatively with changes in blood glucose, leptin, and food intake. Transgenic mice, in which orexin neurons are ablated, fail to respond to fasting with increased wakefulness and activity. These findings indicate that orexin neurons provide a crucial link between energy balance and arousal.

Difference in obesity phenotype between orexin-knockout mice and orexin neuron-deficient mice with same genetic background and environmental conditions

Orexins are a pair of neuropeptides expressed by a population of neurons located in the lateral hypothalamic area (LHA). Prepro-orexin- or orexin receptor type 2-deficient animals exhibit a phenotype remarkably similar to the human sleep disorder, narcolepsy, which is characterized by sleep/wakefulness fragmentation. Human narcolepsy is known to be associated with metabolic abnormalities, including an increased frequency of obesity and non-insulin-dependent diabetes mellitus. Complex disruption of energy homeostasis in orexin neuron-deficient transgenic mice (orexin/ataxin-3 mice) is also manifested as late-onset obesity despite eating less. Here, we report that the development of obesity in orexin neuron-ablated narcoleptic mice is critically dependent on their genetic background and environmental factors, and the phenotype is different from that of prepro-orexin knockout mice even under the same genetic background and environmental factors, suggesting that factors that co-localize in orexin neurons might have important roles in the regulation of energy homeostasis. Our observation also suggests that the obesity observed in orexin neuron-deficient narcolepsy is dependent on the genetic background and environmental factors.

Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia

Stress-induced analgesia (SIA) is a key component of the defensive behavioral fight-or-flight response. Although the neural substrates of SIA are incompletely understood, previous studies have implicated the hypocretin/orexin (Hcrt) and nociceptin/orphanin FQ (N/OFQ) peptidergic systems in the regulation of SIA. Using immunohistochemistry in brain tissue from wild-type mice, we identified N/OFQ-containing fibers forming synaptic contacts with Hcrt neurons at both the light and electron microscopic levels. Patch clamp recordings in GFP-tagged mouse Hcrt neurons revealed that N/OFQ hyperpolarized, decreased input resistance, and blocked the firing of action potentials in Hcrt neurons. N/OFQ postsynaptic effects were consistent with opening of a G protein-regulated inwardly rectifying K+ (GIRK) channel. N/OFQ also modulated presynaptic release of GABA and glutamate onto Hcrt neurons in mouse hypothalamic slices. Orexin/ataxin-3 mice, in which the Hcrt neurons degenerate, did not exhibit SIA, although analgesia was induced by i.c.v. administration of Hcrt-1. N/OFQ blocked SIA in wild-type mice, while coadministration of Hcrt-1 overcame N/OFQ inhibition of SIA. These results establish what is, to our knowledge, a novel interaction between the N/OFQ and Hcrt systems in which the corticotropin-releasing factor and N/OFQ systems coordinately modulate the Hcrt neurons to regulate SIA.

Thyrotropin-Releasing Hormone Increases Behavioral Arousal through Modulation of Hypocretin/Orexin Neurons

Thyrotropin-releasing hormone (TRH) has previously been shown to promote wakefulness and to induce arousal from hibernation. Expression of TRH-R1 (TRH receptor 1) is enriched in the tuberal and lateral hypothalamic area (LHA), brain regions in which the hypocretin/orexin (Hcrt) cells are located. Because the Hcrt system is implicated in sleep/wake control, we hypothesized that TRH provides modulatory input to the Hcrt cells. In vitro electrophysiological studies showed that bath application of TRH caused concentration-dependent membrane depolarization, decreased input resistance, and increased firing rate of identified Hcrt neurons. In the presence of tetrodotoxin, TRH induced inward currents that were associated with a decrease in frequency, but not amplitude, of miniature postsynaptic currents (PSCs). Ion substitution experiments suggested that the TRH-induced inward current was mediated in part by Ca2+ influx. Although TRH did not significantly alter either the frequency or amplitude of spontaneous excitatory PSCs, TRH (100 nm) increased the frequency of spontaneous inhibitory PSCs by twofold without affecting the amplitude of these events, indicating increased presynaptic GABA release onto Hcrt neurons. In contrast, TRH significantly reduced the frequency, but not amplitude, of miniature excitatory PSCs without affecting miniature inhibitory PSC frequency or amplitude, indicating that TRH also reduces the probability of glutamate release onto Hcrt neurons. When injected into the LHA, TRH increased locomotor activity in wild-type mice but not in orexin/ataxin-3 mice in which the Hcrt neurons degenerate postnatally. Together, these results are consistent with the hypothesis that TRH modulates behavioral arousal, in part, through the Hcrt system.

Ectopic overexpression of orexin alters sleep/wakefulness states and muscle tone regulation during REM sleep in mice.

Orexins (also called hypocretins), which are neuropeptides exclusively expressed by a population of neurons specifically localized in the lateral hypothalamic area, are critically implicated in the regulation of sleep/wake states. Orexin deficiency results in narcoleptic phenotype in rodents, dogs, and humans, suggesting that orexins are important for maintaining consolidated wakefulness states. However, the physiological effect of constitutive increased orexinergic transmission tone, which might be important for understanding the effects of orexin agonists that are promising candidates for therapeutic agents of narcolepsy, has not been fully characterized. We report here the sleep/wakefulness abnormalities in transgenic mice that exhibit widespread overexpression of a rat prepro-orexin transgene driven by a β-actin/cytomegalovirus hybrid promoter (CAG/orexin transgenic mice). CAG/orexin mice exhibit sleep abnormalities with fragmentation of non-rapid eye movement (REM) sleep episode and a reduction in REM sleep. Non-REM sleep was frequently disturbed by short episodes of wakefulness. EEG/EMG studies also reveal incomplete REM sleep atonia with abnormal myoclonic activity during this sleep stage. These results suggest that endogenous orexinergic activity should be appropriately regulated for normal maintenance of sleep states. Orexinergic transmission should be activated during wakefulness, while it should be inactivated or decreased during sleep state to maintain appropriate vigilance states.

Critical Role of Neuropeptides B/W Receptor 1 Signaling in Social Behavior and Fear Memory

Neuropeptide B/W receptor 1 (NPBWR1) is a G-protein coupled receptor, which was initially reported as an orphan receptor, and whose ligands were identified by this and other groups in 2002 and 2003. To examine the physiological roles of NPBWR1, we examined phenotype of Npbwr1 −/− mice. When presented with an intruder mouse, Npbwr1 −/− mice showed impulsive contact with the strange mice, produced more intense approaches toward them, and had longer contact and chasing time along with greater and sustained elevation of heart rate and blood pressure compared to wild type mice. Npbwr1 −/− mice also showed increased autonomic and neuroendocrine responses to physical stress, suggesting that impairment of NPBWR1 leads to stress vulnerability. We also observed that these mice show abnormality in the contextual fear conditioning test. These data suggest that NPBWR1 plays a critical role in limbic system function and stress responses. Histological and electrophysiological studies showed that NPBWR1 acts as an inhibitory regulator on a subpopulation of GABAergic neurons in the lateral division of the CeA and terminates stress responses. These findings suggest important roles of NPBWR1 in regulating amygdala function during physical and social stress.

Hypotensive effect of endothelin-1 via endothelin-B-receptor pathway on pulmonary circulation is enhanced in rats with pulmonary hypertension

The pharmacological characterization of endothelin-1 (ET-1) in the pulmonary circulation in pulmonary hypertension (PH) is not known precisely. We investigated the effect of intravenous injection of ET-1 (1000 pmol/kg) on right ventricular systolic pressure (RVSP) (which is equal to systolic pulmonary arterial pressure) in rats with monocrotaline-induced PH. ET-1 decreased RVSP in PH rats; however, ET-1 did not alter RVSP in control rats, suggesting that ET-1 causes dilatation of the pulmonary artery in PH rats. Under pretreatment with the endothelin-A- (ET(A)) receptor antagonist BMS 193884, ET-1 decreased RVSP in PH rats more than in control rats, suggesting that pulmonary vasodilator action of ET-I mediated via the ET(B)-receptor pathway is augmented in PH rats. Under pretreatment with the ET(A/B)-receptor antagonist SB 209670, the effect of ET-1 in lowering pulmonary arterial pressure was abolished in both groups of rats. These results suggest that the hypotensive effect of ET-1 on pulmonary circulation mediated via the ET(B)-receptor pathway is enhanced in PH rats compared with control normal rats. It is considered that the blockade of only the ET(A)-receptor pathway is preferable to the blockade of both the ET(A)- and ET(B)-receptor pathways in the treatment of PH.

Afferent Control of the Hypocretin/Orexin Neurons

Since the discovery of the hypocretins (Hcrt) [1] or orexins [2] and the subsequent link to narcolepsy in animals [3, 4] and humans [5, 6] a decade ago, it has become apparent that the Hcrt system plays a central role in a number of physiological and behavioral functions. The well-established clinical symptomatology of narcolepsy including excessive daytime sleepiness and cataplexy gave rise to the hypothesis that the Hcrt system was important for the maintenance of wakefulness [7]. Although subsequent research has suggested that Hcrt activity may be related to wakefulness associated with motivated behaviors rather than wakefulness per se [8], other studies have demonstrated that the Hcrt system has a role in other functions such as energy metabolism, reward, and addiction [9, 10].

Critical role of neuropeptides B/W receptor 1 signaling in social behavior and fear memory

Neuropeptide B/W receptor 1 (NPBWR1) is a G-protein coupled receptor, which was initially reported as an orphan receptor, and whose ligands were identified by this and other groups in 2002 and 2003. To examine the physiological roles of NPBWR1, we examined phenotype of Npbwr1 mice. When presented with an intruder mouse, Npbwr1 mice showed impulsive contact with the strange mice, produced more intense approaches toward them, and had longer contact and chasing time along with greater and sustained elevation of heart rate and blood pressure compared to wild type mice. Npbwr1 mice also showed increased autonomic and neuroendocrine responses to physical stress, suggesting that impairment of NPBWR1 leads to stress vulnerability. We also observed that these mice show abnormality in the contextual fear conditioning test. These data suggest that NPBWR1 plays a critical role in limbic system function and stress responses. Histological and electrophysiological studies showed that NPBWR1 acts as an inhibitory regulator on a subpopulation of GABAergic neurons in the lateral division of the CeA and terminates stress responses. These findings suggest important roles of NPBWR1 in regulating amygdala function during physical and social stress. Citation: Nagata-Kuroiwa R, Furutani N, Hara J, Hondo M, Ishii M, et al. (2011) Critical Role of Neuropeptides B/W Receptor 1 Signaling in Social Behavior and Fear Memory. PLoS ONE 6(2): e16972. doi:10.1371/journal.pone.0016972 Editor: Xiaoxi Zhuang, University of Chicago, United States of America Received August 12, 2010; Accepted January 10, 2011; Published February 24, 2011 Copyright: 2011 Nagata-Kuroiwa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Grants-in-Aid for Scientific Research on Priority Areas ‘‘System study on higher-order brain functions’’, the 21st Century COE Program for University of Tsukuba and Kanazawa University from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, ERATO from the Japan Science and Technology Agency, Takeda Science Foundation and Uehara Memorial Foundation. M.Y. is an investigator at the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tsakurai@med.kanazawa-u.ac.jp