Sawako Tabuchi

Optogenetic Manipulation of Activity and Temporally Controlled Cell-Specific Ablation Reveal a Role for MCH Neurons in Sleep/Wake Regulation

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.

Orexin Directly Excites Orexin Neurons through Orexin 2 Receptor

Orexin neurons (hypocretin neurons) have a critical role in the regulation of sleep/wakefulness, especially in the maintenance of arousal. Here, we revealed that orexin neurons are directly and indirectly activated by orexin via the orexin 2 receptor (OX2R). Orexin B (1 μm) induced depolarization in orexin neurons, which was still observed in the presence of TTX (1 μm), AP-5 (50 μm), and CNQX (20 μm). In addition, orexin B induced inward currents in the presence of TTX, suggesting a direct activation of orexin neurons. Although orexin B application induced depolarization in orexin neurons of OX1R knock-out mice at comparable levels to wild-type mice, the observation that orexin B failed to depolarize orexin neurons in the OX2R knock-out mice suggested that OX2R was a primary receptor for this response. Moreover, immunoelectron microscopic analyses revealed direct contacts among orexin neurons, which exhibited structural similarities to the glutamatergic synapses. Together, these results suggest that orexin neurons form a positive-feedback circuit through indirect and direct pathways, which results in the preservation of the orexin neuron network at a high activity level and/or for a longer period. Therefore, the activation of orexin neurons through OX2R might have an important role in the maintenance of arousal.

Conditional Ablation of Orexin/Hypocretin Neurons: A New Mouse Model for the Study of Narcolepsy and Orexin System Function

The sleep disorder narcolepsy results from loss of hypothalamic orexin/hypocretin neurons. Although narcolepsy onset is usually postpubertal, current mouse models involve loss of either orexin peptides or orexin neurons from birth. To create a model of orexin/hypocretin deficiency with closer fidelity to human narcolepsy, diphtheria toxin A (DTA) was expressed in orexin neurons under control of the Tet-off system. Upon doxycycline removal from the diet of postpubertal orexin-tTA;TetO DTA mice, orexin neurodegeneration was rapid, with 80% cell loss within 7 d, and resulted in disrupted sleep architecture. Cataplexy, the pathognomic symptom of narcolepsy, occurred by 14 d when ∼5% of the orexin neurons remained. Cataplexy frequency increased for at least 11 weeks after doxycycline. Temporary doxycycline removal followed by reintroduction after several days enabled partial lesion of orexin neurons. DTA-induced orexin neurodegeneration caused a body weight increase without a change in food consumption, mimicking metabolic aspects of human narcolepsy. Because the orexin/hypocretin system has been implicated in the control of metabolism and addiction as well as sleep/wake regulation, orexin-tTA; TetO DTA mice are a novel model in which to study these functions, for pharmacological studies of cataplexy, and to study network reorganization as orexin input is lost.

Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice.

Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. Recent optogenetic studies revealed that the activation or inhibition of orexin neuronal activity affects the probability of sleep/wakefulness transition in the acute phase. To expand our understanding of how orexin neurons maintain wakefulness, we generated new transgenic mice in which orexin neurons expressed archaerhodopsin from Halorubrum strain TP009 (ArchT), a green light-driven neuronal silencer, using the tet-off system (orexin-tTA; TetO ArchT mice). Slice patch clamp recordings of ArchT-expressing orexin neurons demonstrated that long-lasting photic illumination was able to silence the activity of orexin neurons. We further confirmed that green light illumination for 1h in the dark period suppressed orexin neuronal activity in vivo using c-Fos expression. Continuous 1h silencing of orexin neurons in freely moving orexin-tTA; TetO ArchT mice during the night (the active period, 20:00-21:00) significantly increased total time spent in slow-wave sleep (SWS) and decreased total wake time. Additionally, photic inhibition increased sleep/wakefulness state transitions, which is also evident in animals lacking the prepro-orexin gene, orexin neurons, or functional orexin-2 receptors. However, continuous 1h photic illumination produced little effect on sleep/wakefulness states during the day (the inactive period, 12:00-13:00). These results suggest that orexin neuronal activity plays a crucial role in the maintenance of wakefulness especially in the active phase in mice.

Concurrent and robust regulation of feeding behaviors and metabolism by orexin neurons

Orexin neurons in the hypothalamus regulate energy homeostasis by coordinating various physiological responses. Past studies have shown the role of the orexin peptide itself; however, orexin neurons contain not only orexin but also other neurotransmitters such as glutamate and dynorphin. In this study, we examined the physiological role of orexin neurons in feeding behavior and metabolism by pharmacogenetic activation and chronic ablation. We generated novel orexin-Cre mice and utilized Cre-dependent adeno-associated virus vectors to express Gq-coupled modified GPCR, hM3Dq or diphtheria toxin fragment A in orexin neurons. By intraperitoneal injection of clozapine-N oxide in orexin-Cre mice expressing hM3Dq in orexin neurons, we could selectively manipulate the activity of orexin neurons. Pharmacogenetic stimulation of orexin neurons simultaneously increased locomotive activity, food intake, water intake and the respiratory exchange ratio (RER). Elevation of blood glucose levels and RER persisted even after locomotion and feeding behaviors returned to basal levels. Accordantly, 83% ablation of orexin neurons resulted in decreased food and water intake, while 70% ablation had almost no effect on these parameters. Our results indicate that orexin neurons play an integral role in regulation of both feeding behavior and metabolism. This regulation is so robust that greater than 80% of orexin neurons were ablated before significant changes in feeding behavior emerged.

Influence of Inhibitory Serotonergic Inputs to Orexin/Hypocretin Neurons on the Diurnal Rhythm of Sleep and Wakefulness

STUDY OBJECTIVE Serotonergic (5HT) neurons of the dorsal raphe nuclei receive excitatory input from hypothalamic orexin (hypocretin) neurons and reciprocally inhibit orexin neurons through the 5HT1A receptor. However, the physiological significance of this negative feedback circuit for sleep/wakefulness regulation is little understood. DESIGN 5HT1A receptor expression level was specifically and reversibly controlled in the orexin neurons using the Tet-off system. The responsiveness of orexin neurons to 5HT in vitro and the sleep/wakefulness patterns were compared between 5HT1A-overexpressing and control mice. MEASUREMENTS AND RESULTS When the 5HT1A receptor was overexpressed in orexin neurons of Orexin-EGFP; orexin-tTA; TetO Htr1a mice, 5HT-induced inhibition of orexin neurons was prolonged. In the absence of doxycycline, Orexin-tTA; TetO Htr1a mice exhibited severe fragmentation of sleep/wakefulness during the first half of the dark period-the time of maximal activity in nocturnal rodents-without affecting sleep/wakefulness during the light period when sleep time is maximal. However, when the 5HT1A receptor in orexin neurons was reduced to basal expression levels in the presence of doxycycline, sleep/wakefulness patterns in Orexin-tTA; TetO Htr1a mice during the early active period were indistinguishable from those of littermate TetO Htr1a mice. These results strongly suggest that enhancement of inhibitory serotonergic input to orexin neurons caused fragmentation of wakefulness. In contrast, sleep/wakefulness architecture in the light period was unaffected by 5HT1A receptor overexpression in the orexin neurons. CONCLUSION Inhibitory serotonergic input likely functions as negative feedback to orexin neurons in the early dark period and helps stabilize wakefulness bouts, thereby contributing to the diurnal rhythm of sleep and wakefulness.

Progressive Loss of the Orexin Neurons Reveals Dual Effects on Wakefulness.

STUDY OBJECTIVES Narcolepsy is caused by loss of the orexin (also known as hypocretin) neurons. In addition to the orexin peptides, these neurons release additional neurotransmitters, which may produce complex effects on sleep/wake behavior. Currently, it remains unknown whether the orexin neurons promote the initiation as well as the maintenance of wakefulness, and whether the orexin neurons influence initiation or maintenance of sleep. To determine the effects of the orexin neurons on the dynamics of sleep/wake behavior, we analyzed sleep/wake architecture in a novel mouse model of acute orexin neuron loss. METHODS We used survival analysis and other statistical methods to analyze sleep/wake architecture in orexin-tTA ; TetO diphtheria toxin A mice at different stages of orexin neuron degeneration. RESULTS Progressive loss of the orexin neurons dramatically reduced survival of long wake bouts, but it also improved survival of brief wake bouts. In addition, with loss of the orexin neurons, mice were more likely to wake during the first 30 sec of nonrapid eye movement sleep and then less likely to return to sleep during the first 60 sec of wakefulness. CONCLUSIONS These findings help explain the sleepiness and fragmented sleep that are characteristic of narcolepsy. Orexin neuron loss impairs survival of long wake bouts resulting in poor maintenance of wakefulness, but this neuronal loss also fragments sleep by increasing the risk of awakening at the beginning of sleep and then reducing the likelihood of quickly returning to sleep.

Light-induced silencing of neural activity in Rosa26 knock-in mice conditionally expressing the microbial halorhodopsin eNpHR2.0.

Temporally precise inhibition of genetically defined cell populations in intact nervous systems has been enabled by the microbial halorhodopsin NpHR, a fast, light-activated chloride pump. Here, we report the generation of new mouse strains that express eNpHR2-EYFP fusion proteins after Cre- and/or Flp-mediated recombination to silence neural activity in vivo. In these mouse strains, Cre/Flp recombination induced a high-level of eNpHR2-EYFP expression. Slice whole-cell patch clamp experiments confirmed that eNpHR2-EYFP-expressing neurons could be optically hyperpolarized and inhibited from firing action potentials. Thus, these mouse strains offer powerful tools for light-induced silencing of neural activity in genetically defined cell populations.

Orexin 2 receptor positively regulates the activity of orexin neurons

O1-9-3-3 Loss of Goosecoid-like and Dgcr14 in interpeduncular nucleus alters the expression of REM sleep Hiromasa Funato 1,5,6 , Makito Sato 1, Christopher M. Sinton 3, Laurent Gautron 3, S.Clay Williams 1, Joel K. Elmquist 3, Masaru Kuroda 6, Arthur I. Skoultchi 4, Masashi Yanagisawa 1,2,5 1 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, USA 2 Howard Hughes Medical Institute, USA 3 Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, USA 4 Department of Cell Biology, Albert Einstein College of Medicine, New York, USA 5 Center for Behavioral Molecular Genetics, Tsukuba University, Tsukuba, Japan 6 Department of Anatomy, Toho University School of Medicine, Tokyo, Japan