Multimodal Functional Analysis Platform: 2. Development of Si Opto-Electro Multifunctional Neural Probe with Multiple Optical Waveguides and Embedded Optical Fiber for Optogenetics.

We have developed a Si opt-electro multifunctional neural probe with multiple waveguides and embedded optical fiber for highly accurate optical stimulation. The Si opt-electro multifunctional neural probe had 16 recording sites, three optical waveguides, and metal cover for suppressing light leakage. The other opt-electro multifunctional neural probe had an optical fiber in the trench of the probe shank, which leads to fewer damages to tissues. We evaluated the electrochemical properties of the recording sites and confirmed that the neural probe had suitable characteristics for neural recording. We also demonstrated the optical stimulation to the neurons expressing ChR2 using our probe. As a result, we succeeded in multisite optical stimulation and observed that no light leakage from the optical waveguides because of the metal cover. From in vivo experiments, we successfully recorded optically modulated local field potential using the fabricated Si neural probe with optical waveguides. Moreover, we applied current source density analysis to the recorded LFPs. As a result, we confirmed that the light-induced membrane current sinks in the locally stimulated area. The Si opto-electro multifunctional neural probe is one of the most versatile tools for optogenetics.

Multimodal Functional Analysis Platform: 3. Spherical Treadmill System for Small Animals.

In the application of advanced neuroscience techniques including optogenetics to small awake animals, it is often necessary to restrict the animal's movements. A spherical treadmill is a beneficial option that enables virtual locomotion of body- or head-restrained small animals. Besides, it has a wide application range, including virtual reality experiments. This chapter describes the fundamentals of a spherical treadmill for researchers who want to start experiments with it. First, we describe the physical aspect of a spherical treadmill based on the simple mechanical analysis. Next, we explain the basics of data logging and preprocessing for behavioral analysis. We also provide simple computer programs that work for the purpose.

Multimodal Functional Analysis Platform: 1. Ultrathin Fluorescence Endoscope Imaging System Enables Flexible Functional Brain Imaging.

To elucidate the expression mechanisms of brain functions, we have developed an ultrathin fluorescence endoscope imaging system (U-FEIS) that can image cells in the brain at any depth while minimizing the invasion. The endoscope part of U-FEIS consists of a GRIN lens and a 10,000-pixel image fiber with a diameter of 450 µm. The specialized microscope of U-FEIS is within 30 cm square and includes lenses and optical filters optimized for the endoscope. Using U-FEIS, we successfully visualized neurons expressing GFP with single-cell resolution and recorded the multineuronal activities in vitro and in vivo. U-FEIS can also perform imaging and optical stimulation simultaneously. Therefore, U-FEIS should be a powerful optical tool in neuroscience research.

Multimodal Functional Analysis Platform: 4. Optogenetics-Induced Oscillatory Activation to Explore Neural Circuits.

To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research.

Thyroid hormone insufficiency alters the expression of psychiatric disorder-related molecules in the hypothyroid mouse brain during the early postnatal period.

The functional role of thyroid hormone (TH) in the cortex and hippocampus of mouse during neuronal development was investigated in this study. TH insufficiency showed a decrease in the expression of parvalbumin (PV) in the cortex and hippocampus of pups at postnatal day (PD) 14, while treatment with thyroxine from PD 0 to PD 14 ameliorated the PV loss. On the other hand, treatment with antithyroid agents in adulthood did not result in a decrease in the expression of PV in these areas. These results indicate the existence of a critical period of TH action during the early postnatal period. A decrease in MeCP2-positive neuronal nuclei was also observed in the cortical layers II-IV of the cerebral cortex. The brains were then stained with CUX1, a marker for cortical layers II-IV. In comparison with normal mice, CUX1 signals were decreased in the somatosensory cortex of the hypothyroid mice, and the total thickness of cortical layers II-IV of the mice was lower than that of normal mice. These results suggest that TH insufficiency during the perinatal period strongly and broadly affects neuronal development.

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.

Resting-state functional connectivity analysis of the mouse brain using intrinsic optical signal imaging of cerebral blood volume dynamics.

OBJECTIVE: Resting-state functional connectivity (rsFC) of the human brain is closely related with neurological and psychiatric disorders. Mice are widely used to investigate the physiological mechanisms of such disorders, because of the applicability of invasive experimental techniques. Thus, studies on rsFC of the mouse brain are essential to link physiological mechanisms with these disorders in humans. In this study, we investigated the applicability of intrinsic optical signal imaging of cerebral blood volume (IOSI-CBV) for rsFC analysis of the mouse brain. APPROACH: Transcranial IOSI-CBV images were collected from the brains of un-anesthetized wild-type mice with a cooled-CCD camera. The time traces of all pixels were averaged to create a global signal (GS). Marginal and partial correlation analyses were performed to estimate the rsFC based on CBV signals both with and without GS removal. The consistency of the results were confirmed by comparing them with to the rsFCs data reported in the previous studies. MAIN RESULTS: We confirmed that GS correlated with heart rate fluctuation in the FC frequency band. The marginal correlation coefficient of CBV with GS removal was consistent with measurements using conventional optical imaging methods relying on oxygenated hemoglobin concentration and cerebral blood flow. SIGNIFICANCE: These results suggest the applicability and usefulness of the transcranial IOSI-CBV method to estimate rsFC of the mouse brain.

Some adverse actions of chlorothalonil at sublethal levels in rat thymic lymphocytes: Its relation to Zn2.

Chlorothalonil, a polychlorinated aromatic fungicide, is considered non-toxic to small mammals. However, chlorothalonil inactivates sulfhydryl enzymes and depletes cellular glutathione. Chlorothalonil increases intracellular Zn2+ concentration ([Zn2+]i) in mammalian cells possibly because intracellular Zn2+ is released via zinc-thiol/disulfide interchange. The effects of chlorothalonil at sublethal concentrations on the cellular content of nonprotein thiols ([NPT]i) and [Zn2+]i were examined using flow cytometry in rat thymocytes. Low concentrations (0.3-1 µM) of chlorothalonil increased, but high concentrations (3-10 µM) decreased [NPT]i. These effects of chlorothalonil were partly attenuated by an intracellular Zn2+ chelator. Chlorothalonil at 0.3-10 µM increased [Zn2+]i in a concentration-dependent manner, which was largely dependent on the release of intracellular Zn2+. Both the decrease in [NPT]i and increase in [Zn2+]i increase the vulnerability of cells to oxidative stress. Chlorothalonil at 1-10 µM potentiated the cytotoxicity of H2O2 (300 µM). It was also the case for 10 µM pentachloronitrobenzene, but not 10 µM pentachlorophenol. In conclusion, chlorothalonil at low (sublethal) micromolar concentrations is cytotoxic to mammalian cells under oxidative stress.

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 novel extraction method of fetal electrocardiogram from the composite abdominal signal.

In contrast to the ultrasonic measurement of fetal heart motion, the fetal electrocardiogram (ECG) provides clinically significant information concerning the electrophysiological state of a fetus. In this paper, a novel method for extracting the fetal ECG from abdominal composite signals is proposed. This method consists of the cancellation of the mother's ECG and blind source separation with the reference signal (BSSR). The cancellation of the mother's ECG component was performed by subtracting the linear combination of mutually orthogonal projections of the heart vector. The BSSR is a fixed-point algorithm, the Lagrange function of which includes the higher order cross-correlation between the extracted signal and the reference signal as the cost term rather than a constraint. This realizes the convexity of the Lagrange function in a simple form, which guarantees the convergence of the algorithm. By practical application, the proposed method has been shown to be able to extract the P and T waves in addition to the R wave. The reliability and accuracy of the proposed method was confirmed by comparing the extracted signals with the directly recorded ECG at the second stage of labor. The gestational age-dependency of the physiological parameters of the extracted fetal ECG also coincided well with that of the magnetocardiogram, which proves the clinical applicability of the proposed method.

Tri-n-butyltin-induced blockade of store-operated calcium influx in rat thymocytes.

Tri-n-butyltin (TBT), one of environmental pollutants, disturbs intracellular Ca(2+) homeostasis by increasing intracellular Ca(2+) concentration ([Ca(2+)]i). Effect of TBT on oscillatory change in [Ca(2+)]i (Ca(2+) oscillation) of rat thymocytes was examined using a laser microscope with fluo-3-AM in order to further elucidate the TBT toxicity related to intracellular Ca(2+). The Ca(2+) oscillation was completely attenuated by 300nM TBT. Since store-operated Ca(2+) channels are involved in the generation of Ca(2+) oscillation, the action of TBT on an increase in [Ca(2+)]i by Ca(2+) influx through store-operated Ca(2+) channels was examined. The increase in [Ca(2+)]i by the store-operated Ca(2+) influx was not affected by 3nM TBT. However, TBT at 10nM or more significantly reduced the increase in [Ca(2+)]i. It is likely that TBT attenuates the Ca(2+) oscillation by reducing the Ca(2+) influx through store-operated Ca(2+) channels.

Evaluation of multidrug cancer chronotherapy based on cell cycle model under influences of circadian clock.

The intracellular circadian clock mechanisms are known to affect various substantial cellular machinery such as cell cycle progression, inflammatory response, apoptosis, and DNA repair. Cancer growth in various tissues is still under circadian control, which may be at least partly underlain by the survived connections between the intracellular machinery and the clock. Considering such findings, chronotherapy has been applied to cancer treatments, in which anti-cancer drugs are administered in scheduled circadian times so as to resolve the trade-off between damages against the normal and cancer cells. However, any effective administration strategy has not yet been established especially in a quantitative sense. In this study, we develop an automaton model of cell division cycle interacting with circadian clock and suffering from a probability of cell death. A cancer cell is modeled by shortening/ lengthening the cell division interval and a transition to motility state under starving condition. Population proliferating dynamics in 3D space are simulated under the diffusion of nutrient factor and the anti-cancer drugs from a vessel. The simulation results show that the drug administration schedule could differentiate the damages against proliferation of normal and cancer cells. This implies the existence of optimal timing for the drug administration, which could provide an efficient strategy of chronotherapeutic treatment of cancer.

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.

A phase dynamics model of human circadian rhythms.

Nonphotic entrainment of an overt sleep-wake rhythm and a circadian pacemaker-driving temperature/melatonin rhythm suggests existence of feedback mechanisms in the human circadian system. In this study, the authors constructed a phase dynamics model that consisted of two oscillators driving temperature/melatonin and sleep-wake rhythms, and an additional oscillator generating an overt sleep-wake rhythm. The feedback mechanism was implemented by modifying couplings between the constituent oscillators according to the history of correlations between them. The model successfully simulated the behavior of human circadian rhythms in response to forced rest-activity schedules under free-run situations: the sleep-wake rhythm is reentrained with the circadian pacemaker after release from the schedule, there is a critical period for the schedule to fully entrain the sleep-wake rhythm, and the forced rest-activity schedule can entrain the circadian pacemaker with the aid of exercise. The behavior of human circadian rhythms was reproduced with variations in only a few model parameters. Because conventional models are unable to reproduce the experimental results concerned here, it was suggested that the feedback mechanisms included in this model underlie nonphotic entrainment of human circadian rhythms.

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.

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.

Phase-locking of spontaneous and elicited ponto-geniculo-occipital waves is associated with acceleration of hippocampal theta waves during rapid eye movement sleep in cats.

We investigated the temporal relationship between hippocampal theta waves and ponto-geniculo-occipital waves (PGO) during rapid eye movement sleep (REM sleep) in cats. In addition, we analyzed the relationship between hippocampal theta waves and PGO as elicited by tone stimulus (PGO(E)) in order to quantitively characterize the PGO wave generator mechanism. The results showed that a spontaneous PGO tended to be phase-locked to the theta wave, which was more clearly observed in the single PGO than in the cluster. However, cluster PGO(E) tended to be phase-locked as well as single PGO(E). It was therefore suggested that the generator of PGO is activated in relation to the hippocampal theta wave. An acceleration of the theta wave associated with PGO occurrence was found, and was more markedly observed than with the cluster PGO. Although the magnitude of it was less than in the spontaneous case, an acceleration around the PGO(E) was also observed. These results suggest that the generators of theta and PGO receive some common activations, especially when a cluster PGO is generated. The interaction between PGO and hippocampal theta waves is expected to be involved in the possible functions of REM sleep.

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.