The role of orexinergic system in the regulation of cataplexy.

Loss of orexin/hypocretin causes serious sleep disorder; narcolepsy. Cataplexy is the most striking symptom of narcolepsy, characterized by abrupt muscle paralysis induced by emotional stimuli, and has been considered pathological activation of REM sleep atonia system. Clinical treatments for cataplexy/narcolepsy and early pharmacological studies in narcoleptic dogs tell us about the involvement of monoaminergic and cholinergic systems in the control of cataplexy/narcolepsy. Muscle atonia may be induced by activation of REM sleep-atonia generating system in the brainstem. Emotional stimuli may be processed in the limbic systems including the amygdala, nucleus accumbens, and medial prefrontal cortex. It is now considered that orexin/hypocretin prevents cataplexy by modulating the activity of different points of cataplexy-inducing circuit, including monoaminergic/cholinergic systems, muscle atonia-generating systems, and emotion-related systems. This review will describe the recent advances in understanding the neural mechanisms controlling cataplexy, with a focus on the involvement of orexin/hypocretin system, and will discuss future experimental strategies that will lead to further understanding and treatment of this disease.

Increase in Blood Pressure by Local Injection of Ketamine into the Amygdala in Rats.

To confirm that an increase in blood pressure induced by ketamine is mediated through the central nervous system, we examined the effect of ketamine, applied directly to the amygdala, on blood pressure. Six male Sprague-Dawley rats were used in the study. Under head-restrained and unanesthetized condition, 0.2 µL (5 mg/mL) of ketamine was injected in and around the amygdala at a flow rate of 0.2 µL/min through a glass pipette, and the blood pressure was recorded while monitoring the state of the animals by electroencephalogram and electromyogram. After ketamine injection, the injection site was marked by Pontamine Sky Blue infusion. Blood pressure was increased by ketamine injection into the basolateral and central nuclei of the amygdala, endopiriform nucleus and piriform cortex. In a total of 11 responses, an increase in blood pressure started with a mean latency of 193.5 ± 43.0 s, reached its peak 180.2 ± 23.3 s after the response onset, then gradually returned to the baseline with mean duration of 706.7 ± 113.5 s. The mean fluctuation was 17.1 ± 2.5 mmHg. We revealed that blood pressure fluctuations induced by ketamine are associated with the amygdala. Elucidation of the mechanism of ketamine-induced blood pressure increase will lead to understanding of the mechanism of side effects of ketamine, and will contribute to its appropriate use.

[Comparison of Influence of Susceptibility Artifact due to Metallic Embolic Material in MRA Image].

PURPOSE: To identify the influence of susceptibility artifact caused by commonly used trans-catheter embolic devices for vascular lesions in the body on the images of various magnetic resonance angiography (MRA) techniques as an aid to patient screening after endovascular embolization. MATERIALS AND METHODS: We constructed vascular phantoms in which three embolic materials; platinum coil, Inconel coil, and vascular plug, were placed. Each phantom was imaged with three types of MRA techniques as follows: ultra-short echo time magnetic resonance imaging (UTE), three-dimensional fast advanced spin echo (3D-FASE), time-resolved contrast MRA with key hole technique (Key hole). For each embolic material, four reviewers compared the visual capabilities of the vessel lumen and surrounding area of each MRA technique by using a four-point visual scoring system. The quantitative values of susceptibility artifacts generated from each embolic material were compared between each MRA technique. RESULTS: For all MRA techniques, the platinum coil showed the highest visual score (median=four-point) among all the embolic materials (p<0.05). In the platinum coil, the MR signal in the coil was clearly depicted in UTE. For all MRA technique, the quantitative values of the susceptibility artifacts were the lowest in platinum coil among all the embolic materials (p<0.05). CONCLUSION: UTE is less susceptible to susceptibility artifact of embolic materials.

Assessment of the septal area neuronal activity during penile erections in rapid eye movement sleep and waking in the rats.

To understand the central mechanism of penile erections during rapid eye movement (REM) sleep and waking, single units were recorded from the septal area in un-anesthetized head-restrained rats simultaneous with erections. Erectile events were assessed by pressure in the bulb of the corpus spongiosum of the penis and bulbospongiosus-muscle activity. Of 143 recorded neurons, 36% showed increased activity (E-type) and 24% decreased activity (I-type) during different phases of erection in REM sleep, while 10% were E-type and 35% were I-type during erections in waking. Most E-type neurons were recorded from the dorsal and intermediate part of lateral septum, whereas I-type neurons were from the medial septum. The findings illustrate the extensive network of various types of neurons in the septal area that fire in concert in relation to erection during REM sleep and waking. This study provides a unique prospective of the septal area for perpetuation of erectile circuitry during sleep.

Sleep as a biological problem: an overview of frontiers in sleep research.

Sleep is a physiological process not only for the rest of the body but also for several brain functions such as mood, memory, and consciousness. Nevertheless, the nature and functions of sleep remain largely unknown due to its extremely complicated nature and lack of optimized technology for the experiments. Here we review the recent progress in the biology of the mammalian sleep, which covers a wide range of research areas: the basic knowledge about sleep, the physiology of cerebral cortex in sleeping animals, the detailed morphological features of thalamocortical networks, the mechanisms underlying fluctuating activity of autonomic nervous systems during rapid eye movement sleep, the cutting-edge technology of tissue clearing for visualization of the whole brain, the ketogenesis-mediated homeostatic regulation of sleep, and the forward genetic approach for identification of novel genes involved in sleep. We hope this multifaceted review will be helpful for researchers who are interested in the biology of sleep.

Chronic alterations in monoaminergic cells in the locus coeruleus in orexin neuron-ablated narcoleptic mice.

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.

[Regulation of sleep and wakefulness through the monoaminergic and cholinergic systems].

Sleep and wakefulness are regulated in the brainstem and hypothalamus. Classical brain dissecting or stimulating studies have proposed the concept of an ascending reticular activating system, presently known as the wakefulness center, located in the caudal midbrain/rostral pontine (mesopontine) areas, comprising the serotonergic, noradrenergic and cholinergic neural populations. These neural groups, in association with the histaminergic and orexinergic neurons in the hypothalamus, activate the cerebral the cortex through the thalamus or basal forebrain. This activating (waking) system is controlled by the slow wave sleep (SWS) generating system in the preoptic area, which receives inhibitory signals from the waking center. The mesopontine area is also involved in the regulation of rapid eye movement (REM) sleep. Reciprocal interactions between the cholinergic/glutamatergic excitatory systems and the aminergic/GABAergic inhibitory systems are crucial for the regulation of REM sleep. In the REM activating system, mutual excitatory interactions between cholinergic and glutamatergic neurons serve to maintain the state of REM sleep. The REM activating system in the mesopontine area receives GABAergic inhibitory signals from several neural groups in the periaqueductal gray and the medulla. Thus, sleep and wakefulness are controlled by the interplay of various neural populations located in several areas in the central nervous system.

Acupuncture of the sacral vertebrae suppresses bladder activity and bladder activity-related neurons in the brainstem micturition center.

Acupuncture of the sacral vertebrae has therapeutic effects in patients with overactive bladders. The mechanism of these effects, however, remains unclear. The present study, using urethane-anesthetized rats, investigated the effects of acupuncture stimulation of the sacral vertebrae on bladder activity and bladder activity-related neurons in and around Barrington's nucleus. In 95 of 147 trials (64.6%), acupuncture stimulation of the sacral vertebrae for 1 min suppressed bladder contraction for 27-2347s. Acupuncture-induced suppression of bladder contraction was blocked by intraperitoneal injection of bicuculline (Bic). Acupuncture stimulation strongly affected bladder activity-related neurons, including those which fired only prior to the start of contraction (Type E1), those whose firing was maintained during contraction (Type E2), and those whose firing was strongly suppressed during contraction (Type I). All Type E1 neurons and most (93.8%) Type E2 neurons decreased firing when bladder activity was suppressed by acupuncture stimulation. Four of 14 (28.6%) Type I neurons exhibited an excitatory response while 3 of 14 (21.4%) exhibited an inhibitory response. These findings suggest that acupuncture stimulation of the sacral vertebrae suppresses bladder contraction and changes the firing properties of bladder activity-related neurons in and around Barrington's nucleus, and that these changes are mediated by GABAergic systems.

Role of the lateral preoptic area and the bed nucleus of stria terminalis in the regulation of penile erection.

To elucidate the role of the preoptic area (POA) in the regulation of penile erection, we examined the effects of electrical stimulation in and around the POA on penile erection in rats, which was assessed by changes in pressure in the corpus spongiosum of the penis (CSP) and electromyography (EMG) of the bulbospongiosus (BS) muscle. In unanesthetized and anesthetized rats, four types of responses were induced by stimulation in and around the POA; (1) normal type responses, which were similar to spontaneously occurring erections, characterized by slow increase in CSP pressure and sharp peaks concurrent with BS muscle bursting; (2) muscular type responses, which included sharp CSP pressure peaks (muscular component) with almost no vascular component; (3) mixed type responses, which included a sequence of high-frequency CSP peaks followed by low-frequency CSP peaks; and (4) micturition type responses, which had higher-frequency and lower-amplitude CSP peaks than other responses which were identical to those of normal micturition. In unanesthetized condition, erections were evoked by stimulation of the lateral preoptic area (LPOA), medial preoptic area (MPOA), bed nucleus of the stria terminalis (BST), paraventricular nucleus (PVN), reuniens thalamic nucleus (Re) and lateral septum (LS). Lower-intensity stimulation evoked erections from the LPOA, BST, PVN and RE, but not the MPOA. In anesthetized condition, stronger stimuli were required and effective sites were restricted to the LPOA, MPOA and BST. These findings suggest that the lateral and medial subdivisions of the preoptic area play different roles in mediating penile erection.

Penile erection and micturition events triggered by electrical stimulation of the mesopontine tegmental area.

The cholinergic neurons in the laterodorsal tegmental nucleus (LDT) play a crucial role in the regulation of rapid eye movement (REM) sleep. Because penile erection occurs during REM sleep, the involvement of the LDT in penile erection was examined in unanesthetized head-restrained rats. To detect penile erection, corpus spongiosum of the penis (CSP) pressure was measured through a telemetric device with simultaneous bulbospongiosum (BS) muscle EMG recording through stainless wires. Electrical stimulation in and around the LDT induced the following three CSP pressure patterns: 1) a full erection pattern indistinguishable from the nonevoked or spontaneous erection, characterized by a slow increase in CSP pressure with additional sharp CSP peaks associated with BS muscle bursts, 2) a muscular pattern characterized by sharp CSP pressure peaks but in the absence of a vascular component, i.e., without an increase in baseline CSP pressure, and 3) a mixed-type response characterized by high-frequency CSP pressure peaks followed by a full erection response. Full erections were evoked in and around the LDT, including more medially and ventrally. The sites for inducing mixed-type events were intermingled with the sites that triggered full erections in the anterior half of the LDT, whereas they were separated in the posterior half. The sites for muscular responses were lateral to the sites for full erections. Finally, a CSP pressure response identical to micturition was evoked in and around the Barrington's nucleus and in the dorsal raphe nucleus. These results suggest that the LDT and surrounding region are involved in the regulation of penile erection. Moreover, different anatomical areas in the mesopontine tegmentum may have specific roles in the regulation of penile erection and micturition.

Acupuncture stimulation to the sacral segment affects state of vigilance in rats.

The effects of acupuncture stimulation to the sacral segment on electroencephalograms (EEGs) and activity of locus coeruleus (LC) neurons were examined in urethane-anesthetized rats. In 71 of 112 trials, when EEGs displayed small amplitude and high frequency, stimulation to the sacral segment-induced large amplitude and slow EEGs with a latency of <450s and duration ranged from 32s to >42 min. Stimulus-induced EEGs comprised significant increases in delta power and significant decreases in theta and beta powers. After intraperitoneal administration of bicuculline, stimulation to the sacral segment failed to induce changes in EEG pattern. Firing rate of noradrenergic LC neurons decreased significantly from 2.9+/-1.5 to 1.1+/-0.8 Hz (n=11, p<0.001). Decreased neuronal activity exhibited close relationships with increased EEG amplitude. These results suggest that acupuncture stimulation to the sacral segment changes the state of animals from light anesthesia to deep anesthesia, and that this change is mediated by GABAergic systems suppressing the activity of noradrenergic LC neurons.

Requirement of tryptophan hydroxylase during development for maturation of sensorimotor gating.

Deficits in sensorimotor gating, a function to focus on the most salient stimulus, could lead to a breakdown of cognitive integrity, and could reflect the "flooding" by sensory overload and cognitive fragmentation seen in schizophrenia. Sensorimotor gating emerges at infancy, and matures during childhood. The mechanisms that underlie its development are largely unclear. Here, we screened the mouse genome, and found that tryptophan hydroxylase (TPH) is implicated in the maturation of sensorimotor gating. TPH, an enzyme involved in the biosynthesis of serotonin, proved to be required only during the weaning period for maturation of sensorimotor gating, but was dispensable for its emergence. Proper serotonin levels during development underlie the mature functional architecture for sensorimotor gating via appropriate actin polymerization. Thus, maintaining proper serotonin levels during childhood may be important for mature sensorimotor gating in adulthood.

A quartet neural system model orchestrating sleep and wakefulness mechanisms.

Physiological knowledge of the neural mechanisms regulating sleep and wakefulness has been advanced by the recent findings concerning sleep/wakefulness-related preoptic/anterior hypothalamic and perifornical (orexin-containing)/posterior hypothalamic neurons. In this paper, we propose a mathematical model of the mechanisms orchestrating a quartet neural system of sleep and wakefulness composed of the following: 1) sleep-active preoptic/anterior hypothalamic neurons (N-R group); 2) wake-active hypothalamic and brain stem neurons exhibiting the highest rate of discharge during wakefulness and the lowest rate of discharge during paradoxical or rapid eye movement (REM) sleep (WA group); 3) brain stem neurons exhibiting the highest rate of discharge during REM sleep (REM group); and 4) basal forebrain, hypothalamic, and brain stem neurons exhibiting a higher rate of discharge during both wakefulness and REM sleep than during nonrapid eye movement (NREM) sleep (W-R group). The WA neurons have mutual inhibitory couplings with the REM and N-R neurons. The W-R neurons have mutual excitatory couplings with the WA and REM neurons. The REM neurons receive unidirectional inhibition from the N-R neurons. In addition, the N-R neurons are activated by two types of sleep-promoting substances (SPS), which play different roles in the homeostatic regulation of sleep and wakefulness. The model well reproduces the actual sleep and wakefulness patterns of rats in addition to the sleep-related neuronal activities across state transitions. In addition, human sleep-wakefulness rhythms can be simulated by manipulating only a few model parameters: inhibitions from the N-R neurons to the REM and WA neurons are enhanced, and circadian regulation of the N-R and WA neurons is exaggerated. Our model could provide a novel framework for the quantitative understanding of the mechanisms regulating sleep and wakefulness.

Suppressive effect of acupuncture stimulation to the sacral segment on the state of vigilance and the brainstem cholinergic neurons.

The effects of acupuncture stimulation to the sacral segment on the electroencephalogram (EEG) and activity of the cholinergic neurons in the laterodorsal tegmental nucleus (LDT) were examined in urethane-anesthetized rats. When EEG was small amplitude and higher frequency, the stimulation to the sacral segment induced large amplitude and slow EEG with latencies ranged from 45 sec to 12 min, and durations from 48 sec to 56 min. The stimulus induced EEG is composed of significant increase in delta power and significant decrease in theta and beta powers. Firing rate of the cholinergic LDT neurons significantly decreased from 2.9+/-1.5 Hz to 1.1+/-0.8 Hz after the stimulus (n=12, p<0.05). The decrease of neuronal activity always preceded to the start of large and slow EEG, while the increase of the activity always preceded to the change of EEG from large slow wave to small faster wave. These results suggest that the acupuncture stimulation to the sacral segment changes the state of the animals from light anesthesia to deep anesthesia, and that the change is mediated by the suppression of the cholinergic neurons in the LDT.

Postnatal development of cholinergic neurons in the mesopontine tegmentum revealed by histochemistry.

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT) play a role in the regulation of several kinds of behavior. Some of them, such as locomotion, motor inhibition or sleep, show dramatic changes at a certain period of postnatal development. To understand the neural substrate for the development of these physiological functions, we studied the development of cholinergic neurons in the LDT and PPT of postnatal and adult rats using histochemical staining of NADPH-diaphorase (NADPH-d) and immunohistochemical staining of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). At postnatal day 1 (P1), ChAT- and VAChT-stained cells localized more dorsally than those of NADPH-d-stained cells, and at P7 their distributions became similar to those of NADPH-d-stained cells. The number of NADPH-d-stained cells increased rapidly after birth, reaching the adult level by P7. In contrast, the number of ChAT- and VAChT-stained cells and the intensity of their staining decreased from P1 to P3 and then increased through P21. The volume of the LDT increased during the second postnatal week. These findings indicate that cholinergic neurons in the LDT develop their cholinergic properties during the second postnatal week and mature functionally thereafter. We discuss these results in light of the several physiological functions regulated by the cholinergic neurons in the mesopontine tegmentum.

Subthalamic neurons coordinate basal ganglia function through differential neural pathways.

The subthalamic nucleus (STN) is a key component of basal ganglia circuitry that mediates a variety of motor functions. The STN neurons send glutamatergic projections to the output structures of basal ganglia, including the substantia nigra pars reticulata (SNr) and the entopeduncular nucleus, and also innervate the globus pallidus (GP). However, the mechanism by which the STN regulates motor functions in the neural circuitry is not fully understood. Here we performed conditional ablation of the STN neurons by using immunotoxin-mediated cell targeting. We then analyzed dopamine (DA)-mediated motor behavior and firing activity of the SNr and GP neurons. Ablation of the STN neurons increased spontaneous movement and reduced hyperactivity in response to DA stimulation. Ablation of these neurons modulated the pattern and rate of spontaneous firing of the SNr neurons, although it did not substantially affect spontaneous firing of the GP neurons. The ablation attenuated DA-induced suppression of the firing rate of the SNr neurons and inhibited DA-induced elevation of the rate of the GP neurons. In addition, pharmacological blockade of GP activation in response to DA stimulation inhibited the suppression of SNr activity and the resultant motor activation. These results suggest that the STN neurons suppress spontaneous behavior through their direct projection to the output neurons and that, in response to DA, they contribute to expression of behavior by acting on the output neurons mainly through the GP-mediated pathways. We conclude that the STN coordinates motor behavior through differential neural pathways depending on the state of DA transmission.

Orexinergic projections to the cat midbrain mediate alternation of emotional behavioural states from locomotion to cataplexy.

Orexinergic neurones in the perifornical lateral hypothalamus project to structures of the midbrain, including the substantia nigra and the mesopontine tegmentum. These areas contain the mesencephalic locomotor region (MLR), and the pedunculopontine and laterodorsal tegmental nuclei (PPN/LDT), which regulate atonia during rapid eye movement (REM) sleep. Deficiencies of the orexinergic system result in narcolepsy, suggesting that these projections are concerned with switching between locomotor movements and muscular atonia. The present study characterizes the role of these orexinergic projections to the midbrain. In decerebrate cats, injecting orexin-A (60 microm to 1.0 mm, 0.20-0.25 microl) into the MLR reduced the intensity of the electrical stimulation required to induce locomotion on a treadmill (4 cats) or even elicit locomotor movements without electrical stimulation (2 cats). On the other hand, when orexin was injected into either the PPN (8 cats) or the substantia nigra pars reticulata (SNr, 4 cats), an increased stimulus intensity at the PPN was required to induce muscle atonia. The effects of orexin on the PPN and the SNr were reversed by subsequently injecting bicuculline (5 mm, 0.20-0.25 microl), a GABA(A) receptor antagonist, into the PPN. These findings indicate that excitatory orexinergic drive could maintain a higher level of locomotor activity by increasing the excitability of neurones in the MLR, while enhancing GABAergic effects on presumably cholinergic PPN neurones, to suppress muscle atonia. We conclude that orexinergic projections from the hypothalamus to the midbrain play an important role in regulating motor behaviour and controlling postural muscle tone and locomotor movements when awake and during sleep. Furthermore, as the excitability is attenuated in the absence of orexin, signals to the midbrain may induce locomotor behaviour when the orexinergic system functions normally but elicit atonia or narcolepsy when the orexinergic function is disturbed.

Input of orexin/hypocretin neurons revealed by a genetically encoded tracer in mice.

The finding of orexin/hypocretin deficiency in narcolepsy patients suggests that this hypothalamic neuropeptide plays a crucial role in regulating sleep/wakefulness states. However, very little is known about the synaptic input of orexin/hypocretin-producing neurons (orexin neurons). We applied a transgenic method to map upstream neuronal populations that have synaptic connections to orexin neurons and revealed that orexin neurons receive input from several brain areas. These include the amygdala, basal forebrain cholinergic neurons, GABAergic neurons in the preoptic area, and serotonergic neurons in the median/paramedian raphe nuclei. Monoamine-containing groups that are innervated by orexin neurons do not receive reciprocal connections, while cholinergic neurons in the basal forebrain have reciprocal connections, which might be important for consolidating wakefulness. Electrophysiological study showed that carbachol excites almost one-third of orexin neurons and inhibits a small population of orexin neurons. These neuroanatomical findings provide important insights into the neural pathways that regulate sleep/wakefulness states.

The orexinergic synaptic innervation of serotonin- and orexin 1-receptor-containing neurons in the dorsal raphe nucleus.

Orexin/hypocretin has been well demonstrated to excite the serotonergic neurons in the dorsal raphe nucleus (DRN). We studied the morphological relationships between orexin-containing axon terminals and serotonin- as well as orexin-receptor-containing neurons in the dorsal raphe nucleus. Using immunohistochemical techniques at the light microscopic level, orexin A (OXA)-like immunoreactive neuronal fibers in the DRN were found to make close contact with serotonergic neurons, while some of the serotonergic neurons also expressed the orexin 1 receptor (OX1R). At the electron microscopic level, double-immunostaining experiments showed that the orexin A-like immunoreactive fibers were present mostly as axon terminals that made synapses on the serotonin- and orexin 1-receptor-containing neurons. While only axodendritic synapses between orexin A-containing axon terminals and serotonergic neurons were detected, the synapses made by orexin A-containing axon terminals on the orexin 1-receptor-containing neurons were both axodendritic and axosomatic. The present study suggests that excitation effect of orexin A on dorsal raphe serotonergic neurons is via synaptic communication through orexin 1 receptor.

State-dependent effects of orexins on the serotonergic dorsal raphe neurons in the rat.

The serotonergic dorsal raphe (DR) neurons play an important role in sleep-wakefulness regulation. Orexinergic neurons in the lateral hypothalamus densely project to the brainstem sites including the DR. To test the effects of orexins on the serotonergic DR neurons, we applied orexin A (0.1 mM) by pressure to these neurons in unanesthetized and urethane anesthetized rats. Orexin A caused excitation in 10 of 15 neurons under unanesthetized condition. The excitation was characterized by slow onset (0-18 s), long lasting duration (15-150 s) and state-dependency. Orexin A applied during REM sleep or slow wave sleep induced significant excitation while during wakefulness, the similar amount of orexin A did not increase the firing rate any more. In the anesthetized animals, orexin A induced excitation in four of eight neurons. The excitation had slow onset and was long lasting. These results suggest that orexinergic neurons exert excitatory influence on the serotonergic DR neurons to maintain tonic activity of them, thereby participating in regulation of sleep-wakefulness cycles and other functions.