Kazumi Takahashi

Locus coeruleus neuronal activity during the sleep-waking cycle in mice.

Using extracellular single-unit recordings in nonanesthetized, head-restrained mice, we examined spontaneous and evoked discharges of noradrenaline-containing locus coeruleus (NA-LC) neurons across the sleep-waking cycle. The neurons were all characterized by triphasic broad action potentials. They discharged as either slow (<6 Hz) tonic, single spikes or phasic clusters of spikes specific to wakefulness (W), the discharge rate being highest during active waking and significantly lower during quiet waking. They remained totally silent during both slow-wave sleep (SWS) and paradoxical (or rapid eye movement (REM)) sleep. The phasic unit activity was related to abrupt activation of electromyographic activity occurring either spontaneously or elicited by alerting sensory stimuli. At the transition from waking to sleep, they ceased firing before the onset of cortical synchronization (deactivation), the first sign of electroencephalographic sleep, a significant decrease in firing rate preceding the onset of unit activity of sleep-specific neurons in the basal forebrain (BFB)/preoptic (POA) hypothalamus, as described previously [Takahashi K, Lin JS, Sakai K (2009) Neuroscience 161:269-292]. At the transition from SWS to waking, they fired before the onset of both cortical activation and a significant decrease in activity of sleep-specific neurons. These findings support the previous view that the NA-LC system is involved in both tonic and phasic processes of arousal, and further support our previous proposals that initiation of sleep is caused by decreased activity of waking-promoting neurons (disfacilitation) and that NA-LC neurons play an important role in the sleep/waking switch, that is from waking to sleep and from sleep to waking [Takahashi K, Lin JS, Sakai K (2009) Neuroscience 161:269-292].

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 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.

State-dependent activity of neurons in the perifornical hypothalamic area during sleep and waking

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.

Effects of orexin on the laterodorsal tegmental neurones

Abstract 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.

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.

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.

Firing properties of neurones in the laterodorsal hypothalamic area during sleep and wakefulness

Abstract In undrugged, head‐restrained rats, neuronal activity was recorded in and around the laterodorsal hypothalamic area where orexin neurones are distributed. Among 22 neurones observed across whole sleep–waking states, half (n = 11) were most active during paradoxical sleep and least active during waking. Others were equally more active during paradoxical sleep and waking than during slow‐wave sleep (n = 6), or were most active during waking and least active during paradoxical sleep (n = 3). The majority of neurones started to increase firing activity prior to the transition of sleep–waking states. These results suggest that the area of the hypothalamus containing orexin neurones plays a role in sleep–waking regulation.

Is state-dependent alternation of slow dynamics in central single neurons during sleep present in the rat ventroposterior thalamic nucleus?

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.

2118 Dynamics-transition of central single neuronal activities during sleep

K. Takahashi’, K. Nakamura’, M. Yamamoto’, T. Kodama2, M. Nakao’, N. Katayama’, K. Endo’ Spontaneous single unit activities (n=12) were successfully recorded from the region of the cat’s lateral geniculate nucleus(LGN) during a natural sleep-wakefulness cycle. These activities were categorized into two groups, LGN (n=6) and non-LGN (n=6), by the firing rate and the length of the bursts during slow wave sleep. The LGN group considered as relay neurons had relatively low firing rate (12.7f2.1 spikes/set) and the short bursts (2-7 spikes) during slow wave sleep. On the other hand the non-LGN group considered as perigeniculate or thalamic reticular neurons showed various firing patterns. A power spectral analysis of these activities showed that any neuron in both groups had dynamics-transition between the white spectrum during slow wave sleep and the l/f spectrum during REM sleep. These results support the generality of the phenomenon of the neuronal dynamics-transition observed in the widespread areas of the brain, suggesting an importance of the global modulation of the brain.

2117 Effects of serotonin on fluctuations in single neuronal activities in the region of the lateral geniculate nucleus of a cat

Mamoru Yanagihara’ , Keihachiro Ito 2, Lynda Dauphin2, Robert W. McCarley’ The cholinergic projection from the laterodorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei to the pontomedullary reticular formation may play an important roll in REM sleep. The projection of the PPT/LDT neurons to both sides of the reticular formation (RF) was quantitatively analyzed by a combination of the fluorescence retrograde double labeling with the fluorescence immunohistochemistry for chorine acetyltransferase. Although 2% of PPT neurons project to both sides of the giant cell field of pontine RF (GPRF), 21% of PPT neurons projecting to ipsilateral GPRF have a collateral to the contralateral GPRF. This percentage was reduced by about one half in cases in which the ipsilateral was to the GPRF but the contralateral projection was to a non-giant cellular field. In medullary projection, 0.3-2.4% of PPT neurons project both sides of the medullary RF. In the LDT, 0.8% of neurons project to giant cell field on both sides of pontine RF.