Tomomi Tsunematsu

Tomomi Tsunematsu; Thomas S. Kilduff; Edward S. Boyden; Satoru Takahashi; Makoto Tominaga; Akihiro Yamanaka

Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. To help determine how these neurons promote wakefulness, we generated transgenic mice in which orexin neurons expressed halorhodopsin (orexin/Halo mice), an orange light-activated neuronal silencer. Slice patch-clamp recordings of orexin neurons that expressed halorhodopsin demonstrated that orange light photic illumination immediately hyperpolarized membrane potential and inhibited orexin neuron discharge in proportion to illumination intensity. Acute silencing of orexin neurons in vivo during the day (the inactive period) induced synchronization of the electroencephalogram and a reduction in amplitude of the electromyogram that is characteristic of slow-wave sleep (SWS). In contrast, orexin neuron photoinhibition was ineffective during the night (active period). Acute photoinhibition of orexin neurons during the day in orexin/Halo mice also reduced discharge of neurons in an orexin terminal field, the dorsal raphe (DR) nucleus. However, serotonergic DR neurons exhibited normal discharge rates in mice lacking orexin neurons. Thus, although usually highly dependent on orexin neuronal activity, serotonergic DR neuronal activity can be regulated appropriately in the chronic absence of orexin input. Together, these results demonstrate that acute inhibition of orexin neurons results in time-of-day-dependent induction of SWS and in reduced firing rate of neurons in an efferent projection site thought to be involved in arousal state regulation. The results presented here advance our understanding of the role of orexin neurons in the regulation of sleep/wakefulness and may be relevant to the mechanisms that underlie symptom progression in narcolepsy.

Tomomi Tsunematsu; Takafumi Ueno; Sawako Tabuchi; Ayumu Inutsuka; Kenji F. Tanaka; Hidetoshi Hasuwa; Thomas S. Kilduff; Akira Terao; Akihiro Yamanaka

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.

Akihiro Yamanaka; Sawako Tabuchi; Tomomi Tsunematsu; Yugo Fukazawa; Makoto Tominaga

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.

Sawako Tabuchi; Tomomi Tsunematsu; Sarah Wurts Black; Makoto Tominaga; Megumi Maruyama; Kazuyo Takagi; Yasuhiko Minokoshi; Takeshi Sakurai; Thomas S. Kilduff; Akihiro Yamanaka

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.

Tomomi Tsunematsu; Sawako Tabuchi; Kenji F. Tanaka; Edward S. Boyden; Makoto Tominaga; Akihiro Yamanaka

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.

Ayumu Inutsuka; Azusa Inui; Sawako Tabuchi; Tomomi Tsunematsu; Michael Lazarus; Akihiro Yamanaka

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.

Tomomi Tsunematsu; Fu Ly; Akihiro Yamanaka; Kanako Ichiki; Tanoue A; Takeshi Sakurai; van den Pol An

Water homeostasis is a critical challenge to survival for land mammals. Mice display increased locomotor activity when dehydrated, a behavior that improves the likelihood of locating new sources of water and simultaneously places additional demands on compromised hydration levels. The neurophysiology underlying this well known behavior has not been previously elucidated. We report that the anti-diuretic hormone arginine-vasopressin (AVP) is involved in this response. AVP and oxytocin directly induced depolarization and an inward current in orexin/hypocretin neurons. AVP-induced activation of orexin neurons was inhibited by a V1a receptor (V1aR)-selective antagonist and was not observed in V1aR knock-out mice, suggesting an involvement of V1aR. Subsequently activation of phospholipase Cβ triggers an increase in intracellular calcium by both calcium influx through nonselective cation channels and calcium release from calcium stores in orexin neurons. Intracerebroventricular injection of AVP or water deprivation increased locomotor activity in wild-type mice, but not in transgenic mice lacking orexin neurons. V1aR knock-out mice were less active than wild-type mice. These results suggest that the activation of orexin neurons by AVP or oxytocin has an important role in the regulation of spontaneous locomotor activity in mice. This system appears to play a key role in water deprivation-induced hyperlocomotor activity, a response to dehydration that increases the chance of locating water in nature.

Robert Scharf; Tomomi Tsunematsu; Niall McAlinden; Martin D. Dawson; Shuzo Sakata; Keith Mathieson

Controlling neural circuits is a powerful approach to uncover a causal link between neural activity and behaviour. Optogenetics has been widely adopted by the neuroscience community as it offers cell-type-specific perturbation with millisecond precision. However, these studies require light delivery in complex patterns with cellular-scale resolution, while covering a large volume of tissue at depth in vivo. Here we describe a novel high-density silicon-based microscale light-emitting diode (μLED) array, consisting of up to ninety-six 25 μm-diameter μLEDs emitting at a wavelength of 450 nm with a peak irradiance of 400 mW/mm2. A width of 100 μm, tapering to a 1 μm point, and a 40 μm thickness help minimise tissue damage during insertion. Thermal properties permit a set of optogenetic operating regimes, with ~0.5 °C average temperature increase. We demonstrate depth-dependent activation of mouse neocortical neurons in vivo, offering an inexpensive novel tool for the precise manipulation of neural activity.

Tomomi Tsunematsu; Kenji F. Tanaka; Akihiro Yamanaka; Amane Koizumi

Melanopsin (OPN4) is a photosensitive G-protein-coupled photopigment and its ectopic expression enables control of neural activity induced by blue light. Here we report that we successfully expressed OPN4 in hypothalamic orexin/hypocretin neurons of double-transgenic mice (orexin-tTA; Bitet-O human OPN4 [hOPN4]/mCherry mice). In the double-transgenic mice, hypothalamic orexin neurons selectively expressed hOPN4 as well as mCherry as a reporter. We conducted slice patch-clamp recordings on hOPN4/mCherry-expressing orexin neurons, which showed long-lasting activation initiated by blue light even after the light was switched off. Optical fiber-guided blue light stimulation in the hypothalamus successfully initiated the electroencephalography pattern that reflects long-lasting wakefulness in the mice in vivo. Taken together, the results indicate that ectopic expression of hOPN4 in orexin neurons enables long-lasting activation of orexin neurons by blue light to control sleep/wakefulness of the mice.

Tomomi Tsunematsu; Akihiro Yamanaka

Orexin, also called hypocretin, is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Orexin exhibits its physiological effects after binding two G-protein-coupled receptors, orexin 1 receptor and orexin 2 receptor. Impairment of the orexin signal, either by deletion of the prepro-orexin or orexin 2 receptor gene or by the ablation of orexin neurons, results in a sleep disorder similar to narcolepsy, suggesting that the orexin system plays an important role in the regulation of sleep/wakefulness. In addition, previous studies have suggested that orexin is involved in energy and fluid homeostasis, emotion regulation, stress responsiveness, and reward. However, growing evidence also suggests that orexin affects the function of peripheral tissues via direct activation of orexin receptors or through activation of autonomic nervous or endocrine systems. In this review, we discuss the physiological roles of orexin not only in the central nervous system but also in the peripheral tissues.