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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 sec, reached its peak 180.2 ± 23.3 sec after the response onset, then gradually returned to the baseline with mean duration of 706.7 ± 113.5 sec. 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.

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.

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.

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.

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

The definitive version is available at www.blackwell-synergy.com, The importance of an appropriate combination of excitability for the higher (neocortex, basal ganglia, and limbic system) and lower (brainstem–spinal cord) motor systems could be necessary for normal behavior during wakefulness and REM sleep. Neurotransmitters such as acetylcholine, the monoamines, GABA, and the orexins regulate the background excitability of the higher and lower motor systems so that an interaction of these systems can be appropriately maintained. Pathophysiological mechanisms of sleep deficiency may be induced, not only by an organic disturbance of brain structures (hardware), but also by dysfunctioning of the neurotransmitter systems (software). From these considerations, this review provides a hypothetical model for "state-dependent interaction of the higher and lower motor systems" for understanding normal behavior and pathophysiological mechanisms of sleep-related disorders such as narcolepsy and the REM-sleep behavioral syndrome.

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.

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.

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 μm to 1.0 mm, 0.20–0.25 μl) 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 μl), a GABAA 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.

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.

Objectives To examine the effect of desmopressin (DDAVP) on bladder contraction and on the neurons that fire in relation to spontaneous bladder contraction (bladder-related neurons) in and around Barrington's nucleus, the micturition center. DDAVP is used for the treatment of nocturnal enuresis because of its antidiuretic action, but the mechanism of this action has not been proved. Methods Urethane-anesthetized Sprague-Dawley male rats (n = 20) were used. DDAVP was infused intravenously or as an intracerebroventricular infusion into the lateral ventricle. Results We encountered three types of bladder-related neurons: those that fired before the start of the contraction (type E1), those that fired synchronous with the bladder contraction (type E2), and those that fired during bladder relaxation (type I). Intravenous infusion caused inhibition in three of five type E1 neurons, excitation in two of five type E2 neurons, and excitation (one neuron) and inhibition (one neuron) of four type I neurons. With intracerebroventricular infusion into the lateral ventricle, two of four type E1 neurons were inhibited, and one of seven type E2 neurons and three of four type I neurons were excited. Bladder contraction was suppressed in 4 of 12 rats by intravenous infusion and in 2 of 8 rats by intracerebroventricular infusion into the lateral ventricle. In all cases, when the bladder contraction was suppressed, an electroencephalogram of larger amplitude and slower frequency appeared. Conclusions DDAVP seems to regulate bladder activity by affecting bladder-related neurons in the micturition center.

Based upon our previous results in cats, we hypothesized that neurons in the central processor systems of the brain generally exhibit state-dependent dynamics alternation of slow fluctuations in spontaneous activity during sleep. To test the validity of this hypothesis across species, we recorded single neuronal activity during sleep from the ventroposterior (VP) thalamic nucleus in unanesthetized, head-restrained rats. Spectral analysis was performed on successive spike-counts of neuronal activity recorded during three stages of the sleep-wakefulness cycle: wakefulness (W, n =6), slow-wave sleep (SWS, n =20), and paradoxical sleep (PS, n =32). We found that firing of VP neurons displayed white-noise-like dynamics over the range of 0.04–1.0 Hz during SWS and 1/ f -noise-like dynamics over the same range during PS. We also demonstrated for the first time that the slow dynamics of neuronal activity during quiet wakefulness (but not drowsiness) are white-noise-like. These results suggest that our hypothesis is true across species. During W and SWS, the brain may be considered as under global inhibition. Conversely, PS may represent a state of global disinhibition in the brain, where neuronal activity exhibits 1/ f -noise-like dynamics. Fluctuations observed in living organisms may be involved in essential processes in generation and function of sleep states.

Micturition is controlled by a network of brainstem neurons involving the Barrington’s nucleus. To depict clearly the brainstem system for micturition control, the present study was designed to record single neuronal activity in the mesopontine tegmentum including the Barrington’s nucleus, and to observe its precise timing in relation to bladder contraction recorded simultaneously. About 1/5 of neurons encountered had firing modulated in relation to bladder contraction. Three types of neurons were distinguished; those which fired only prior to the start of contraction (type E1), those whose firing started shortly prior to and was maintained during contraction (type E2), and those whose firing was strongly suppressed during contraction (type I). Type E2 neurons were most frequently observed in the Barrington’s nucleus and its close vicinity, while the neurons of the other two types were scattered widely in the mesopontine tegmentum. The results show clearly that direct neural signals to induce bladder contraction may arise from the Barrington’s nucleus, and that the nucleus may receive regulatory inputs from wide areas of the mesopontine tegmentum. In addition, the present study clarified that the noradrenergic and cholinergic neurons, which are located in nuclei adjoining the Barrington’s nucleus and function to control sleep/wakefulness, may not be concerned in controlling micturition directly.

In urethane anesthetized rats, the laterodorsal tegmental area was stimulated systematically with a carbon fiber electrode to clarify regions regulating the urinary bladder and/or the external urethral sphincter. Contraction of the former was monitored by bladder pressure and that of the latter by electromyogram. Stimulation of a small area around the ventrolateral edge of the central gray in a plane at the junction of the mesencephalon and pons, where cholinergic neurons in the laterodorsal tegmental nucleus formed the largest mass, induced contraction only of the bladder. Arranged in tandem rostrocaudally with this bladder site, a very small area whose stimulation induced contraction only of the sphincter was found also at the ventrolateral edge of the central gray in a plane slightly caudal to the above. Slightly lateral and caudal to this sphincter site, there were sites the stimulation of which induced contraction of both the bladder and sphincter. It was thus shown physiologically that there were discrete sites in the laterodorsal tegmental area regulating the bladder and sphincter independently.

The precise mechanism by which normal persons wake upon urinary sensation is unclear. The locus caeruleus in the pons is suggested to be involved in the activating systems for arousal. We evaluated the effects of bladder distention on the neural activity in the l. caeruleus under different states of anesthesia.Experiments were performed on 20 male Sprague-Dawley rats. The spontaneous discharge rate of single neurons in and around the l. caeruleus was recorded with the rat under urethane anesthesia administered intraperitoneally, while the electroencephalogram was monitored simultaneously. The changes of the discharge rate of the neurons were observed during saline infusion into the bladder or during tail pinch stimulation. After recording the location of the neurons was confirmed histologically.Of 42 l. coeruleus neurons whose response was examined during deep anesthesia 31 showed an excitatory response to bladder distention, followed by a change in electroencephalogram pattern to a faster and smaller rate, while the remaining 11 showed no response. Of 28 l. coeruleus neurons examined during light anesthesia only 1 neuron was excited and the remaining 27 showed no response. Virtually all of the l. coeruleus neurons unresponsive to bladder distention were responsive to tail pinch stimulation. Of the nonnoradrenergic neurons ventromedial to the l. coeruleus 4 of 7 during deep anesthesia and 5 of 8 during light anesthesia showed an excitatory response to distention.The excitatory response of the l. coeruleus neurons to bladder distention was strongly affected by the state of anesthesia. The response was observed only during deep anesthesia and was accompanied by lightening of the anesthesia. L. coeruleus may be involved in arousal which is mediated by bladder distention.

Under urethane anesthesia, the urinary bladder of male rats was distended by 0.3-0.5 mL of saline. When the animals displayed large amplitude delta waves, the distention caused tonic excitation in 76% (19/25) of the noradrenergic neurons in the locus coeruleus and in 53% (16/30) of the cholinergic neurons in the laterodorsaltegmental nucleus. The response was followed by a shift of electroencephalogram (EEG) pattern to faster waves of smaller amplitude. When EEG amplitude was faster and smaller, the same stimuli caused no response. Neurons in Barrington's nucleus were excited by a small amount of bladder distention independent of EEG patterns. These results suggest that the transition of state, caused by bladder distention, from deep sleep to light sleep is mediated by noradrenergic and cholinergic neurons in the brainstem.

USING microiontophoresis on unanesthetized head-restrained cats, we have found two distinct groups of neurones exhibiting tonic discharge specific to paradoxical sleep (PS) (PS-on neurones) in the mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurones. One group is characterized by a broad action potential, slow conduction velocity and an inhibitory response to a potent cholinergic agonist, carbachol, applied iontophoretically during PS. The other is characterized by a short action potential, fast conduction velocity and an excitatory response to applied carbachol. All PS-on neurones were excited by glutamate. The present findings demonstrate the existence of two types of PS-on neurones and suggest their cholinergic and non-cholinergic neurochemical properties.

In head-restrained rats, spontaneous neuronal activities in the ventroposterior thalamic nuclei were recorded during slow wave sleep (SWS) and paradoxical sleep (PS). The slow dynamics of neuronal activity, represented by a power spectra in the range of 0.04–1.0 Hz, were white noise-like during SWS and 1/f noise-like during PS. The result was similar to that observed previously in other various brain areas in the cat. It supports our hypothesis that the state-dependent alternation of slow dynamics in the activity of the central processor neurons during sleep is common in different species.

Nitric oxide (NO) released from the cholinergic neurons may play important roles in sleep-wake regulation. We reported a possible role of NO, which was mediated through n-methyl-d-aspartic acid (NMDA) receptors in the cholinergic neurons in pons, as a retrograde regulator of norepinephrine (NE) release from the locus coeruleus neurons. In this study, we further investigate the effect of kainate application. There were significant increases in concentration of NO (P < 0.05) for 20 min, in NE (P < 0.05) for 15 min, and in cyclic guanine monophosphate (cGMP; P < 0.05) for 60 min after the initiation of kainate application (5 mmol/L). The difference in the distributions of NMDA and kainate receptors in the dorsolateral pontine tegmental nucleus (LDT) may explain the different firing patterns of LDT neurons.

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.

The basal forebrain including the preoptic area and anterior hypothalamus plays an important role in regulation of slow wave sleep. To examine whether this area is concerned also in a neural mechanism of paradoxical sleep, single neuronal activity in and around the preoptic area along with cortical EEG and neck EMG was recorded in undrugged, 12-h sleep-deprived rats whose head was fixed painlessly. The neurons whose activity was recorded during all states of sleep and wakefulness (n = 98) were classified into following 5 groups according to their firing behavior in relation to sleep-waking states. (1) Neurons most active during paradoxical sleep (n = 26). (2) Those most inactive during paradoxical sleep, some of which were inactive also during slow wave sleep (n = 16). (3) Those specifically active during slow wave sleep (n = 14). (4) Those less active during slow wave sleep than during wakefulness and paradoxical sleep (n = 9). (5) Those whose activity showed no clear correlation with the sleep-waking states (n = 33). About one third of neurons in each of groups (1) to (4) began to increase their sleep-related activity in advance of the shift of sleep/wakefulness state recognized in EEG. These results suggest that the preoptic and anterior hypothalamic areas are involved, at least in rats, in regulation of not only slow wave sleep but also paradoxical sleep.

Summary Recently, we proposed the model consists of a quartet of neuron groups: (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 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 NREM sleep (W-R group). In this chapter, we explain our revised model of sleep–wakefulness regulation in the rat, which includes a neural regulator quartet of NREM sleep, REM sleep, wakefulness, and a coregulator of REM sleep and wakefulness, whose parameters are tuned up so that the model reproduces some of the recent findings concerning the WA neurons. The physiological reality of the model structure is demonstrated by comparing the model neuron activities with the actual neuronal activities across state transition. Among the conceptual and mathematical models, one of the novel features of our model is involvement of the cholinergic W-R neuron group, which is assumed to contribute toward induction of wakefulness as well as REM sleep by selectively mediating autocatalytic activation with the WA or REM neurons. Through activity of this neuron group, the wakefulness–sleep flip-flop and the REM–NREM flip-flop interact with each other in our model. In fact pharmacological or physiological manipulations of REM sleep were shown to affect not only NREM sleep but wakefulness. Our successful modeling suggests that REM sleep is regulated cooperatively/competitively with NREM sleep and wakefulness by well orchestrated interactions among the aforementioned quartet of neural groups distributed in the hypothalamus and the brain stem with the aid of sleep-promoting substances. Finally, further possible updates in the model structure are described considering recent physiological findings.

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.

To clarify a relation between contractile condition of the urinary bladder and vigilance condition, bladder pressure was monitored simultaneously with cortical electroencephalogram (EEG) in urethane-anesthetized rats. Slow waves of large amplitude and those of lower amplitude appeared in EEG alternately. The bladder showed no contractile activity in the former EEG condition, while it contracted intermittently or continuously to urinate in the latter EEG condition. The relative power of EEG delta waves was significantly different in these two conditions. These results suggest that activity of the micturition system changes according to vigilance conditions, which may serve to prevent enuresis during sleep.

The effects of electrical stimulation to the septum on penile erections in rats were examined to clarify the mechanisms for regulation of erectile responses during different states of vigilance. Penile responses were assessed by changes in pressure in the corpus spongiosum of penis (CSP) and electromyography (EMG) of the bulbospongiosus (BS) muscle. In anesthetized and un-anesthetized rats, stimulation in and around the septum induced three erectile patterns; 1) a Normal type response, which was indistinguishable from a spontaneous erection, characterized by a slow increase in CSP pressure with sharp CSP pressure peaks associated with BS muscle bursts, 2) Mixed type response, in which high frequency CSP pressure peaks were followed by a Normal type response, and 3) a Prolonged type response, evoked only in the anesthetized rat, consisting of a single sharp CSP peak followed by a slow increase in CSP pressure and a return to baseline with multiple subsequent events repeated for up to 960 s. In addition, a Micturition type response was also observed involving high frequency CSP pressure oscillations similar to the pressure pattern seen during spontaneous micturition. We found that erections were induced after stimulation to the lateral septum (LS), but not from the medial septum (MS). In anesthetized rats, a few responses were also obtained following stimulation of the horizontal limb of diagonal band (HDB). In un-anesthetized rats, responses were also induced from the HDB and the ventral limb of diagonal band (VDB) and the adjoining areas. The effective sites for eliciting erection during rapid eye movement (REM) sleep were located in the dorsal and intermediate parts of the LS, whereas the ventral part of the LS was the most effective site for eliciting erections during wakefulness. These results suggest a functional role for penile erection in the septum, and further suggest that subdivisions of the LS may have different roles in the regulation of penile erection during wakefulness and REM sleep.

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 of450s and duration ranged from 32s to42 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, p0.001). 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.

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.

Nitric oxide released from the cholinergic neurons in the pons may play important roles in sleep-wake regulation. However, there are few reports demonstrating the mechanisms of nitric oxide release in the cholinergic neurons in the pons. The present study investigated the effects of drug delivery of N-methyl-D-aspartic acid on nitric oxide and the neurotransmitters released in the laterodorsal tegmental nucleus (LDT), one of the major cholinergic cell groups in the pons, in rats by in vivo microdialysis with a view to clarifying nitric oxide functions in the cholinergic system. The application of N-methyl-D-aspartic acid (1 mM) into the LDT induced a significant increase in NO(2)and NO(3) for 40 min (P

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 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.

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.

Neurons containing orexins are located in the perifornical hypothalamic area and are considered to have a role in sleep-wake regulation. To examine how this area is involved in the regulation of sleep and wakefulness, we recorded neuronal activity in undrugged, head-restrained rats across sleep-waking cycles. Recordings were made in the perifornical hypothalamic area where orexin-immunoreactive neurons are distributed (PFH), and in the area dorsal to the PFH, including the zona incerta and subincertal nucleus (collectively referred to as ZI). The 40 neurons recorded from in the PFH were divided into five groups: (1) neurons most active during paradoxical sleep (PS, n=14, 35%), (2) neurons active during both waking (W) and PS (n=12, 30%), (3) neurons most active during W (n=7, 18%), (4) neurons most active during slow-wave sleep (SWS, n=3, 7.5%), and (5) neurons whose activity had no correlation with sleep-waking states (n=4, 10%). Of 30 neurons recorded from in the ZI, the corresponding numbers were 13 (43%), seven (23%), six (20%), three (10%), and one (3.3%). In both areas, neuronal activity fluctuated more during PS than during W. Waking-specific neurons (group 3) in the PFH generated action potentials with longer durations than those produced by other types of neurons. About half of the neurons in the PFH that were classified in groups 1, 2, and 3 increased their firing rate after the transition from one state to another, while higher percentages of neurons of groups 1 and 2 in the ZI than those in the PFH increased their firing rate prior to the state shift from SWS to PS. In these ZI neurons, however, the firing rate varied considerably at the state shift. These results suggest that the PFH and ZI are involved in the regulation of PS or W, especially the regulation of phasic events during PS or the maintenance of W. The ZI appears to be more closely involved than the PFH in the induction of PS or some phasic phenomena associated with PS.

Noradrenergic projection originating in the locus coeruleus, serotonergic projection from the dorsal raphe nucleus, and cholinergic projection from neurons gathering in the laterodorsal tegmental nucleus and scattering in the pedunculopontine tegmental nucleus constitute three diffuse projection systems arising from the brainstem and innervating wide areas of the brain. They may function as controllers of sleep and wakefulness. We have investigated functional roles of the projections by recording neuronal activity in these brainstem nuclei, and by observing effects of stimulation of the brainstem nuclei. The projection from the locus coeruleus is an arousal system, since the noradrenergic neurons are active specifically during waking, and activation of the noradrenergic projection excites upper brain structures. Functions of the serotonergic projection are still mysterious, since its action on upper brain is inhibitory in spite of waking-specific activity of the neurons. A group of cholinergic neurons constitute a system to induce and maintain paradoxical sleep. The cholinergic projection may have another role, i.e. to induce a rapid, transient elevation of vigilance level by their phasic response to novel, unfamiliar stimuli.

In undrugged, head-restrained rats, neuronal activity was recorded in and around the laterodor-sal hypothalamic area where orexin neurones are distributed. Among 22 neurones observedacross whole sleep–waking states, half ( n =11) were most active during paradoxical sleep andleast active during waking. Others were equally more active during paradoxical sleep and wakingthan during slow-wave sleep ( n =6), or were most active during waking and least active duringparadoxical sleep ( n =3). The majority of neurones started to increase firing activity prior to thetransition of sleep–waking states. These results suggest that the area of the hypothalamus con-taining orexin neurones plays a role in sleep–waking regulation. Key words hypothalamus, neuronal activity, orexin, rapid eye movement sleep, wakefulness. INTRODUCTION In the laterodorsal hypothalamic area, which largelyoverlaps with the feeding centre, 1 neurones contain-ing recently identified peptides, orexins (orexin A andB), are distributed.

Orexin, a hypothalamic neuropeptide, has been revealed to be involved in sleep regulation. To elucidate functions of orexin in brainstem sleep regulation mechanism, we examined the effects of orexin applied from micropipettes with air pressure on neurones in and around the laterodorsal tegmental nucleus (LDT). In five of seven cholinergic neurones and six of nine non-cholinergic neurones orexin induced long-lasting excitation. These results suggest that hypothalamic orexin neurones may affect the LDT neurones directly, and thereby participate in control of sleep.

Cholinergic neurons in the laterodorsal tegmental nucleus (LDT) have important roles in the regulation of sleep or waking in adult animals. In neonatal animals, sleep is largely occupied by paradoxical sleep. To investigate the relation between the cholinergic neurons in the LDT and the development of neonatal sleep, we dissected the LDT of rat by micropunch method at postnatal day 1–45 and measured the activity of choline acetyltransferase (ChAT). Either specific or total activity of ChAT was weak in the first week, increased strikingly in the second week and then moderately thereafter. The time course of the increase in ChAT activity correlates well to that of the decrease in the amount of paradoxical sleep or body twitches after birth.

The mesopontine tegmentum, which contains both cholinergic and non-cholinergic neurons, plays a crucial role in behavioral state control. Using microiontophoresis in unanesthetized cats, we have examined the effect of cholinergic and monoaminergic drugs on two putative cholinergic neurons located mostly in the laterodorsal tegmental nucleus and X area (or the cholinergic part of the nucleus tegmenti pedunculopontinus, pars compacta): one (type I-S) exhibiting slow tonic discharge during both waking and paradoxical sleep, and the other (PGO-on) displaying single spike activity during waking and burst discharges in association with ponto-geniculo-occipital (PGO) waves during paradoxical sleep. We found that: (i) application of carbachol, a potent cholinergic agonist, inhibited single spike activity in both PGO-on and type I-S neurons, but had no effect on the burst activity of PGO-on neurons during paradoxical sleep; the inhibition was associated with either blockade or increased latency of antidromic responses, suggesting membrane hyperpolarization; (ii) application of glutamate, norepinephrine, epinephrine, or histamine resulted in increased tonic discharge in both PGO-on and type I-S neurons; this was state-independent and resulted in a change in the firing mode of PGO-on neurons from phasic to tonic; (iii) application of serotonin had only a weak state-dependent inhibitory effect on a few type I-S neurons; and (iv) application of dopamine had no effect on either type of neuron. The present findings suggest that cholinergic, glutamatergic and monoaminergic (especially noradrenergic, adrenergic and histaminergic) inputs have the capacity to strongly modulate the cholinergic neurons, altering both their rate and mode of discharge, such as to shape their state specific activity, and thereby contribute greatly to their role in behavioral state control.

The relation between unit activity in the laterodorsal tegmental (LDT) area and the state of the urinary bladder was examined in urethane-anesthetized rats. Neurons in the LDT area can be classified into two populations: broad-spike (possibly cholinergic) and brief-spike (non-cholinergic). When the rats showed cortical electroencephalographic activity with large amplitude lower frequency, indicative of deep anesthesia, more than 40% of the broad-spike neurons was excited and about 10% was inhibited by infusion of saline into the bladder. The response was followed by decrease in amplitude and slight increase in frequency of the cortical activity, i.e., lightening of anesthesia. During light anesthesia, excitation was observed only in less than 10% of the units, while 17% was inhibited. In the brief-spike neurons, a similar proportion (about 20%) was excited and less than 10% was inhibited by the distention during either state of anesthesia. About 10% of the broad-spike neurons in the LDT area and 30% of the brief-spike neurons examined were discharged prior to the bladder contraction. Such neurons of the brief-spike category were encountered frequently outside of the central gray; lateral, caudal and ventral to the main mass of cholinergic neurons in the LDT area. These results suggest the possible involvement of the broad-spike (cholinergic) neurons in the elevation of vigilance level caused by bladder distention. The brief-spike (non-cholinergic) neurons firing with relation to bladder contraction may be part of the micturition reflex center.

From indirect evidence we have proposed that cholinergic versus non-cholinergic neurons in the laterodorsal tegmental nucleus can be distinguished with the duration of their extracellularly recorded action potentials, “broad” spikes for the former, “brief” for the latter. To test this assumption more directly, we labelled single neurons recorded extracellularly in and around the laterodorsal tegmental nucleus with biocytin or neurobiotin, and processed the sections with reduced nicotinamide adenine dinucleotide phosphate-diaphorase, a proven marker for cholinergic neurons in the laterodorsal tegmental nucleus. Biocytin or neurobiotin which was deposited at the site of recording was incorporated into single neurons. Among 171 trials (91 for broad-spike and 80 for brief-spike neurons), marking was successful in 68 cases (29 for broad-spike and 39 for brief-spike neurons). Almost all (21/22) of the broad-spike neurons located within the laterodorsal tegmental nucleus were positive for reduced nicotinamide adenine dinucleotide phosphate-diaphorase staining, i.e. they were cholinergic, while all of the brief-spike neurons in and outside of the laterodorsal tegmental nucleus lacked the diaphorase activity, and were thus non-cholinergic. The present study shows that, after extracellular labelling of single neurons by biocytin or neurobiotin, cholinergic neurons in the laterodorsal tegmental nucleus are confidently distinguished from non-cholinergic ones in the corresponding area with their spike shapes. It is also shown that the cholinergic neurons distinguished by this criterion are characterized by their tonic firing at slightly lower rate and larger cell size than the brief-spike non-cholinergic ones.

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. © 2013 Tsujino et al.

This study investigated the correlation of firing pattern of the cholinergic neurons in the brainstem and the fluctuation of blood pressure during paradoxical sleep (PS) in unanesthesized, head-restrained rats. Of 13 cholinergic neurons recorded from the laterodorsal tegmental nucleus, five showed phasic firing, which was in close correlation with the fluctuation of the blood pressure during PS. The increase in firing was observed 1–3s prior to the increase of blood pressure. The results suggest that a population of cholinergic neurons in the brainstem are closely involved in the fluctuation of blood pressure during PS.

Although cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei have been shown to have a pivotal role in neural mechanisms of paradoxical sleep, their function during wakefulness is less understood. To examine the latter, we have recorded from "broad-spike neurons", which were distinguished by their long spike duration, in the laterodorsal tegmental nucleus of undrugged, head-restrained rats, and examined their response properties to sensory stimuli such as light touch to the tail, air puff to the face, 2 kHz pure tone and flashes of light. Broad-spike neurons from the locus coeruleus and dorsal raphe nucleus were studied for comparison; these neurons have been demonstrated to be noradrenergic and serotonergic, respectively. The broad-spike neurons in the laterodorsal tegmental nucleus have also been suggested to be cholinergic. There were two kinds of responses: (1) a simple increase or decrease in firing, reflecting an elevated level of vigilance; and (2) a phasic response composed of a single spike or brief, high frequency burst, usually diminishing or disappearing upon repetition of the stimulus. When two or more types of stimuli were effective in a neuron, they evoked responses of the same quality. Most of the dorsal raphe neurons displayed only the simple increase of firing, whereas the locus coeruleus neurons gave a phasic response with rather weak attenuation upon repetition. Compared with these, the laterodorsal tegmental neurons were heterogeneous: about one-quarter showing only a simple change of firing (half increasing, half decreasing); and two-thirds displaying phasic responses. The latter response of many neurons attenuated strongly upon repetition. The laterodorsal tegmental neurons were classified into several groups according to their spontaneous firing behavior during sleep and wakefulness, but every neuron in a group did not show the same type of response. For example, some of the neurons which were most active during paradoxical sleep and essentially silent during wakefulness decreased or stopped firing upon sensory stimulation, while others in this group had strong phasic responses. These results suggest that putative cholinergic neurons in the laterodorsal tegmental nucleus have heterogenous properties not only with respect to their spontaneous activity during sleep and wakefulness but also with respect to their response to sensory stimulation. Some of these neurons may function to induce a global attentive state in response to a novel stimulus.

The laterodorsal tegmental nucleus (LDT) is the largest aggregation in the brainstem of cholinergic neurons whose axons reach the thalamus as part of a diffuse projection to the forebrain. We measured the regional blood flow in the thalamus by means of laser Doppler flowmetry, and examined whether the blood flow was regulated by the ascending cholinergic nerve fibers originating in the LDT. Experiments were performed on urethane-anesthetized rats whose upper cervical spinal cord was transected to avoid response of systemic blood pressure following LDT stimulation. The ascending cholinergic nerve fibers were excited by electrical or chemical stimulation applied to the LDT. The regional thalamic blood flow increased in response to repetitive electrical stimulation and chemical stimulation with L-glutamate to the LDT. The response, starting several seconds after the onset of electrical stimulation and lasting as long as 1 min, was reduced by i.v. scopolamine, a cholinergic muscarinic receptor antagonist. The results indicate that regional blood flow in the thalamus is increased by excitation of the ascending cholinergic nerve fibers originating in the LDT mainly through the cholinergic muscarinic receptors.

Using head-restrained unanesthetized rats, we examined the effects of ionophoretically applied prostaglandin (PG) D2 and PGE2 on the activity of neurons in the preoptic/anterior hypothalamic areas that were classified according to the activity change across sleep-wakefulness cycles. About 24% (84/345) of the neurons had a higher firing rate during slow wave sleep than during wakefulness (S-neurons), and about 14% (47/345) were more active during wakefulness than during slow wave sleep (W-neurons). PGD2 had an excitatory effect on about one-third of the S-neurons examined, while a similar percentage of W-neurons were excited by PGE2. These results suggest that PGD2 and PGE2, influencing sleep-related neurons in the preoptic/anterior hypothalamic areas, are key substances underlying the regulation of sleep and wakefulness.