Serotonergic regulation of cortical neurovascular coupling and hemodynamics upon awakening from sleep in mice.

Neurovascular coupling (NVC) is the functional hyperemia of the brain responding to local neuronal activity. It is mediated by astrocytes and affected by subcortical ascending pathways in the cortex that convey information, such as sensory stimuli and the animal condition. Here, we investigate the influence of the raphe serotonergic system, a subcortical ascending arousal system in animals, on the modulation of cortical NVC and cerebral blood flow (CBF). Raphe serotonergic neurons were optogenically activated for 30 s, which immediately awakened the mice from non-rapid eye movement sleep. This caused a biphasic cortical hemodynamic change: a transient increase for a few seconds immediately after photostimulation onset, followed by a large progressive decrease during the stimulation period. Serotonergic neuron activation increased intracellular Ca2+ levels in cortical pyramidal neurons and astrocytes, demonstrating its effect on the NVC components. Pharmacological inhibition of cortical neuronal firing activity and astrocyte metabolic activity had small hypovolemic effects on serotonin-induced biphasic CBF changes, while blocking 5-HT1B receptors expressed primarily in cerebral vasculature attenuated the decreasing CBF phase. This suggests that serotonergic neuron activation leading to animal awakening could allow the NVC to exert a hyperemic function during a biphasic CBF response, with a predominant decrease in the cortex.

Contribution of default mode network to game and delayed-response task performance: Power and connectivity analyses of theta oscillation in the monkey.

Neuroimaging studies have demonstrated the presence of a default mode network (DMN) which shows greater activity during rest, and an executive network (EN) which is activated during cognitive tasks. DMN and EN are thought to have competing functions. However, recent studies reported that the two networks show coactivation during some cognitive tasks. To clarify how DMN works and how DMN interacts with EN for cognitive control, we recorded EEG activities in the medial prefrontal (anterior DMN: aDMN), posterior cingulate/precuneus (posterior DMN: pDMN), and lateral prefrontal (EN) areas in the monkey. As cognitive tasks, we employed a monkey-monkey competitive video game (GAME) and a delayed-response (DR) task. We focused on theta oscillation because of its importance in cognitive control. We also examined theta band connectivity among the three network areas using the Granger causality analysis. DMN and EN were found to work cooperatively in both tasks. In all the three network areas, we found GAME-task-related, but no DR-task-related, increase in theta power from the resting level, maybe because of the higher cognitive demand associated with the GAME task performance. The information flow conveyed by the theta oscillation was directed more to aDMN than from aDMN for both tasks. The GAME-task-related increase in theta power in aDMN is supposed to be supported by more information flow conveyed by the theta oscillation from EN and pDMN.

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle.

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Intracellular ATP levels in mouse cortical excitatory neurons varies with sleep-wake states.

Whilst the brain is assumed to exert homeostatic functions to keep the cellular energy status constant under physiological conditions, this has not been experimentally proven. Here, we conducted in vivo optical recordings of intracellular concentration of adenosine 5'-triphosphate (ATP), the major cellular energy metabolite, using a genetically encoded sensor in the mouse brain. We demonstrate that intracellular ATP levels in cortical excitatory neurons fluctuate in a cortex-wide manner depending on the sleep-wake states, correlating with arousal. Interestingly, ATP levels profoundly decreased during rapid eye movement sleep, suggesting a negative energy balance in neurons despite a simultaneous increase in cerebral hemodynamics for energy supply. The reduction in intracellular ATP was also observed in response to local electrical stimulation for neuronal activation, whereas the hemodynamics were simultaneously enhanced. These observations indicate that cerebral energy metabolism may not always meet neuronal energy demands, consequently resulting in physiological fluctuations of intracellular ATP levels in neurons.

Impact of Sleep-Wake-Associated Neuromodulators and Repetitive Low-Frequency Stimulation on Human iPSC-Derived Neurons.

The cross-regional neurons in the brainstem, hypothalamus, and thalamus regulate the central nervous system, including the cerebral cortex, in a sleep-wake cycle-dependent manner. A characteristic brain wave, called slow wave, of about 1 Hz is observed during non-REM sleep, and the sleep homeostasis hypothesis proposes that the synaptic connection of a neural network is weakened during sleep. In the present study, in vitro human induced pluripotent stem cell (iPSC)-derived neurons, we investigated the responses to the neuromodulator known to be involved in sleep-wake regulation. We also determined whether long-term depression (LTD)-like phenomena could be induced by 1 Hz low-frequency stimulation (LFS), which is within the range of the non-REM sleep slow wave. A dose-dependent increase was observed in the number of synchronized burst firings (SBFs) when 0.1-1000 nM of serotonin, acetylcholine, histamine, orexin, or noradrenaline, all with increased extracellular levels during wakefulness, was administered to hiPSC-derived dopaminergic (DA) neurons. The number of SBFs repeatedly increased up to 5 h after 100 nM serotonin administration, inducing a 24-h rhythm cycle. Next, in human iPSC-derived glutamate neurons, 1 Hz LFS was administered four times for 15 min every 90 min. A significant reduction in both the number of firings and SBFs was observed in the 15 min immediately after LFS. Decreased frequency of spontaneous activity and recovery over time were repeatedly observed. Furthermore, we found that LFS attenuates synaptic connections, and particularly attenuates the strong connections in the neuronal network, and does not cause uniform attenuation. These results suggest sleep-wake states can be mimicked by cyclic neuromodulator administration and show that LTD-like phenomena can be induced by LFS in vitro human iPSC-derived neurons. These results could be applied in studies on the mechanism of slow waves during sleep or in an in vitro drug efficacy evaluation depending on sleep-wake state.

Reduction of light source noise from optical intrinsic signals of mouse neocortex by using independent component analysis.

Because the optical intrinsic signal (OIS) of the brain is very weak, noise reduction is essential. Independent component analysis (ICA) is widely used for noise reduction. However, the applicability of ICA to the reduction of light source (LS) noise has not been discussed in detail. In addition, determining the proper number of independent components (ICs) for decomposition is very important to a reasonable classification of the ICs. In this study, we considered the applicability of ICA to LS noise reduction by modeling the impact of LS noise on OIS data. We propose a method for determining the number of ICs that uses the power spectral density of LS noise. To evaluate its usefulness, the method was applied to real OIS data of a mouse's cerebral cortex.

Entrainability of cell cycle oscillator models with exponential growth of cell mass.

Among various aspects of cell cycle, understanding synchronization mechanism of cell cycle is important because of the following reasons. (1)Cycles of cell assembly should synchronize to form an organ. (2) Synchronizing cell cycles are required to experimental analysis of regulatory mechanisms of cell cycles. (3) Cell cycle has a distinct phase relationship with the other biological rhythms such as circadian rhythm. However, forced as well as mutual entrainment mechanisms are not clearly known. In this study, we investigated entrainability of cell cycle models of yeast cell under the periodic forcing to both of the cell mass and molecular dynamics. Dynamics of models under study involve the cell mass growing exponentially. In our result, they are shown to allow only a limited frequency range for being entrained by the periodic forcing. In contrast, models with linear growth are shown to be entrained in a wider frequency range. It is concluded that if the cell mass is included in the cell cycle regulation, its entrainability is sensitive to a shape of growth curve assumed in the model.

Development of distance-selective nerve recruitment for subcortical brain mapping by controlling stimulation waveforms.

During brain surgery, it is important to determine the functional brain area and cortico-cortical pathways so as to keep them intact and preserve patients' quality of life. Cortical and subcortical brain mappings are techniques that deliver direct current stimulation to the brain surface and beneath gray matter to identify the brain area and nerve fibers related to higher-order functions. However, because of the non-selective effect of conventional electrical stimulation methods, it has been difficult to obtain precise spatial distribution of nerve fibers in the subcortical region. We investigated the electrical stimulation of subcortical mapping to evaluate axon-to-electrode distance-selectivity. It was clarified that a conventional rectangular biphasic pulse activates axons non-selectively. We propose double exponential waveforms and show that they can recruit targeted fibers and change the location of a target by manipulating stimulus intensity. These results suggest the usefulness of introducing distance-selective stimulation into subcortical brain mapping.

Reconstruction of fetal vector electrocardiogram from maternal abdominal signals under fetus body rotations.

Fetal electrocardiogram (fECG) and its vector form (fVECG) could provide significant clinical information concerning physiological conditions of a fetus. So far various independent component analysis (ICA)-based methods for extracting fECG from maternal abdominal signals have been proposed. Because full extraction of component waves such as P, Q, R, S, and T, is difficult to be realized under noisy and nonstationary situations, the fVECG is further hard to be reconstructed, where different projections of the fetal heart vector are required. In order to reconstruct fVECG, we proposed a novel method for synthesizing different projections of the heart vector, making good use of the fetus movement. This method consists of ICA, estimation of rotation angles of fetus, and synthesis of projections of the heart vector. Through applications to the synthetic and actual data, our method is shown to precisely estimate rotation angle of the fetus and to successfully reconstruct the fVECG.

Suppression of anodal break excitation by electrical stimulation with down-staircase waveform for distance-selective nerve recruitment.

Electrical nerve stimulation using extracellular electrodes is widely performed in clinical medicine as well as basic medical science. It has been reported that selective recruitment of nerve fibers on the basis of the distance between the electrode and the axon is possible without moving the electrode and only by modifying the waveform of electrical stimulation. However, computer simulations have not reproduced the complete nature of the distance-selectivity of the stimulus owing to the difficulty in numerical analysis. In this paper, we propose a minor modification to the myelinated axon model to overcome this difficulty. We confirm that this modification improves the numerical stability of the simulation and enables us to obtain the spatio-temporal dynamics of axons, including the electrode-to-axon distance-dependency. In addition, we propose a novel stimulation method using a down-staircase waveform for distance-selective nerve recruitment. Simulations confirm that the method works well. We show the spatial distribution of axons activated by the down-staircase stimulation, which would be helpful to determine the stimulation parameters for distance-selective nerve recruitment.

Parameter exploration of staircase-shape extracellular stimulation for targeted stimulation of myelinated axon.

Spatio-temporal dynamics of a mathematical model of myelinated axon in response to staircase-shape extracellular electrical stimulation, which was developed for selective nerve stimulation, is investigated by the computer simulation. It is shown that the response is classified into four types: subthreshold response, cathodic excitation, anodal block and anodal break excitation. Based on the simulation results, simple diagrams representing the response characteristics of the axon are constructed as functions of stimulation parameters and distance between the axon and electrode. The diagram would be useful for determining simulation parameters for dynamic targeted stimulation of myelinated axon.

Separation of multiunit signals by independent component analysis in complex-valued time-frequency domain.

Multiunit recording with a multi-electrode in the brain has been widely used in neuroscience studies. After the data recording, neuronal spikes should be sorted according to spike waveforms. For the spike sorting, independent component analysis (ICA) has recently been used because ICA potentially solves the problem to separate even overlapped multiple neuronal spikes into the single. However, we found that multiunit signals are recorded in each electrode channel with channel-specific delay. This situation does not satisfy the instantaneous mixture condition prerequisite for most of ICA algorithms. Actually, this delayed mixture situation was shown to degrade the performance of an ordinary ICA. In this study, in order to overcome this problem, complex-valued processing in the time-frequency domain is applied to multiunit signals by the wavelet transform. In the space spanned by the wavelet coefficients, the condition of instantaneous mixture is almost fulfilled. By application to a synthetic multiunit signal, the ICA algorithm extended to complex-valued signals makes much improvement in spike sorting performance so that even overlapped multiple spikes are successfully separated. Taken together, the complex-valued method could be a powerful tool for spike sorting.

Enhancement of synchronization between hippocampal and amygdala theta waves associated with pontine wave density.

Theta waves in the amygdala are known to be synchronized with theta waves in the hippocampus. Synchronization between amygdala and hippocampal theta waves is considered important for neuronal communication between these regions during the memory-retrieval process. These theta waves are also observed during rapid eye movement (REM) sleep. However, few studies have examined the mechanisms and functions of theta waves during REM sleep. This study examined correlations between the dynamics of hippocampal and amygdala theta waves and pontine (P) waves in the subcoeruleus region, which activates many brain areas including the hippocampus and amygdala, during REM sleep in rats. We confirmed that the frequency of hippocampal theta waves increased in association with P wave density, as shown in our previous study. The frequency of amygdala theta waves also increased with in associated with P wave density. In addition, we confirmed synchronization between hippocampal and amygdala theta waves during REM sleep in terms of the cross-correlation function and found that this synchronization was enhanced in association with increased P wave density. We further studied theta wave synchronization associated with P wave density by lesioning the pontine subcoeruleus region. This lesion not only decreased hippocampal and amygdala theta frequency, but also degraded theta wave synchronization. These results indicate that P waves enhance synchronization between regional theta waves. Because hippocampal and amygdala theta waves and P waves are known to be involved in learning and memory processes, these results may help clarify these functions during REM sleep.

Multi-neuron action potentials recorded with tetrode are not instantaneous mixtures of single neuronal action potentials.

Multiunit recording with multi-site electrodes in the brain has been widely used in neuroscience studies. After the data recording, neuronal spikes should be sorted according to the pattern of spike waveforms. For the spike sorting, independent component analysis (ICA) has recently been used because ICA has potential for resolving the problem to separate the overlapped multiple neuronal spikes. However the performance of spike sorting by using ICA has not been examined in detail. In this study, we quantitatively evaluate the performance of ICA-based spike sorting method by using simulated multiunit signals. The simulated multiunit signal is constructed by compositing real extracellular action potentials recorded from guinea-pig brain. It is found that the spike sorting by using ICA hardly avoids significant false positive and negative errors due to the cross-talk noise contamination on the separated signals. The cross-talk occurs when the multiunit signal of each recording channel have significant time difference; this situation does not satisfy the assumption of instantaneous source mixture for the major ICA algorithms. Since the channel delay problem is hardly resolved, an ICA algorithm which does not require the instantaneous source mixing assumption would be appropriate for use of spike sorting.

The 3D position estimation of neurons in the hippocampus based on the multi-site multi-unit recordings with silicon tetrodes.

The mechanical strain of the neural tissue induced by the implant of neuronal electrode is one of the important factors responsible for the quality and performance of extracellular recording of neuronal activities in the brain because the mechanical strain could kill or inactivate the neurons. In order to evaluate the effect of the implant of neural electrode, we propose a method to estimate the three-dimensional distribution of electrophysiologically active neurons near the electrode based on the multi-site multi-unit recording data. The spatial distribution of the active neurons emerges the region in the neural tissue that could be killed or inactivated by the implant of the electrode. The proposed method will be useful for the in situ assessment of the neural electrode implanted in the brain.

Phase-locking of spontaneous and tone-elicited pontine waves to hippocampal theta waves during REM sleep in rats.

Temporal relationships between hippocampal theta waves and pontine waves (P waves) during rapid-eye-movement (REM) sleep were investigated in rats. P waves were phase-locked to the positive theta peak. The phase relationships of P waves elicited by a tone stimulus (P(E) waves) to hippocampal theta waves were also analyzed to qualitatively clarify the mechanism of phase-locking between these two phenomena. P(E) waves occurred at the positive theta peak, as seen for spontaneous P waves. This phase preference of P(E) waves could be understood as that of the response probability to tone stimulus. These data suggest that the P-wave generator receives inputs that mimic theta waves. As hippocampal theta waves and P waves are known to be involved in learning and memory processes during REM sleep, the present studies could help to clarify these functions.

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.

Instantaneous acceleration and amplification of hippocampal theta wave coincident with phasic pontine activities during REM sleep.

Rapid eye movement (REM) sleep is characterized by hippocampal theta waves and phasic spike-like waves originating from the pons, termed ponto-geniculo-occipital (PGO) waves in cats and pontine (P) waves in rats. While the theta wave and PGO/P wave have been suggested to participate in higher-order brain functions, their generation mechanisms and roles in brain functions have been studied independently. Therefore, the present study investigated instantaneous aspects of the relationship between theta waves and PGO/P waves in both cats and rats. Theta wave was instantaneously accelerated several hundred milliseconds before the negative peak of the PGO/P wave in both animals, and was also amplified just before PGO/P wave occurrence. Considering the integrated knowledge provided by studies of both animals, these results suggest that PGO/P wave-related activities in the pons are delivered to the theta wave generator. The activations of the theta wave coincident with PGO/P wave might facilitate cooperative contribution to higher-order brain functions in REM sleep.

Sleep EEG dynamics in rat barrel cortex associated with sensory deprivation.

Sleep is involved in the development and maintenance of neural networks. We investigated how somatosensory deprivation affects EEG dynamics of adult rats during sleep, which might be a result of changes in neural organization. Rats' whiskers were clipped unilaterally daily and the resulting changes in the balance of EEG spectral powers between the intact and sensory deprived barrel cortices were recorded for a month. Both delta and theta band spectral powers in the deprived cortex initially decreased in terms of their ratio to the intact cortex. Subsequently, the ratio was restored to control levels. This non-monotonic change in EEG activity might reflect the re-organization process of the cortical circuit.

Theta wave amplitude and frequency are differentially correlated with pontine waves and rapid eye movements during REM sleep in rats.

The present study examined the correlations between the dynamics of hippocampal theta waves and pontine waves (P waves) and rapid eye movements (REMs) densities during REM sleep. Theta wave peak frequency and theta amplitude were estimated as the parameters of theta wave dynamics in each 3s segment. The peak frequency and theta amplitude were positively correlated with P wave and REMs densities, however their detailed correlation properties were distinct from each other. Dependency of peak frequency on P wave/REMs density did not change significantly from that on REMs/P wave density. On the other hand, dependency of the theta amplitude on P wave/REMs density significantly increased with an increased REMs/P wave density. Because hippocampal theta waves and P waves are involved in learning and memory functions during REM sleep, the correlation between theta parameters and P wave density might help to clarify these functions.