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.

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.

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.

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.

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.

Inhibitory effect of cervical trachea and chest wall vibrations on cough reflex sensitivity and perception of urge-to-cough in healthy male never-smokers.

BACKGROUND: Non-pharmacological options for symptomatic management of cough are desired. Although chest wall mechanical vibration is known to ameliorate cough reflex sensitivity, the effect of mechanical vibrations on perceptions of urge-to-cough has not been studied. Therefore, we investigated the effect of mechanical vibration of cervical trachea, chest wall and femoral muscle on cough reflex sensitivity, perceptions of urge-to-cough as well as dyspnea. METHODS: Twenty-four healthy male never-smokers were investigated for cough reflex sensitivity, perceptions of the urge-to-cough and dyspnea with or without mechanical vibration. Cough reflex sensitivity and urge-to-cough were evaluated by the inhalation of citric acid. The perception of dyspnea was evaluated by Borg scores during applications of external inspiratory resistive loads. Mechanical vibration was applied by placing a vibrating tuning fork on the skin surface of cervical trachea, chest wall and femoral muscle. RESULTS: Cervical trachea vibration significantly increased cough reflex threshold, as expressed by the lowest concentration of citric acid that elicited five or more coughs (C5), and urge-to-cough threshold, as expressed by the lowest concentration of citric acid that elicited urge-to-cough (Cu), but did not significantly affect dypnea sensation during inspiratory resistive loading. On the other hand, the chest wall vibration not only significantly increased C5 and Cu but also significantly ameliorated the load-response curve of dyspnea sensation. CONCLUSIONS: Both cervical and trachea vibrations significantly inhibited cough reflex sensitivity and perception of urge-to-cough. These vibration techniques might be options for symptomatic cough management.

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.

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.

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.

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.

Robustness of the blind source separation with reference against uncertainties of the reference signals.

The fetal electrocardiogram (ECG) could provide clinical information concerning physiological conditions of the fetus. In order to extract fetal ECG, we proposed the novel algorithm, the blind source separation with reference (BSSR), which successfully extracts a complete waveform of QRS complex and avoids uncertainty in the order of the extracted signals. In the BSSR, the reference signal is supposed to be generated from the ultrasonic Doppler signal. Thus generated reference is expected to suffer from uncertainties in waveform and occurrence timing. Based on simulations, the BSSR is shown to have robustness against the uncertainties of reference signals. In addition, it is shown how the robustness depends on the order of power of correlation function between the reference and extracted signals, which composes a performance function of the BSSR.

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.