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Objective. Robotic rehabilitation systems have been investigated to assist with motor dysfunction recovery in patients with lower-extremity paralysis caused by central nervous system lesions. These systems are intended to provide appropriate sensory feedback associated with locomotion. Appropriate feedback is thought to cause synchronous neuron firing, resulting in the recovery of function. Approach. In this study, we designed and evaluated an ergometric cycling wheelchair, with a brain–machine interface (BMI), that can force the legs to move by including normal stepping speeds and quick responses. Experiments were conducted in five healthy subjects and one patient with spinal cord injury (SCI), who experienced the complete paralysis of the lower limbs. Event-related desynchronization in the β band (18–28 Hz) was used to detect lower-limb motor images. Main results. An ergometer-based BMI system was able to safely and easily force patients to perform leg movements, at a rate of approximately 1.6 s/step (19 rpm), with an online accuracy rate of 73.1% for the SCI participant. Mean detection time from the cue to pedaling onset was 0.83 ± 0.31 s. Significance. This system can easily and safely maintain a normal walking speed during the experiment and be designed to accommodate the expected delay between the intentional onset and physical movement, to achieve rehabilitation effects for each participant. Similar BMI systems, implemented with rehabilitation systems, may be applicable to a wide range of patients.

Cobalt chloride can create hypoxia-like state in vitro (referred to as chemical hypoxia). Several studies have suggested that chemical hypoxia may cause deleterious effects on myogenesis. The intrinsic underlying mechanisms of myoblast differentiation, however, are not fully understood. Here, we show that cobalt chloride strongly suppresses myoblast differentiation in a dose-dependent manner. The impaired myoblast differentiation is accompanied by downregulation of myogenic regulatory factor myogenin. Under chemical hypoxia, myogenin stability is decreased at mRNA and protein levels. A muscle-specific E3 ubiquitin ligase MAFbx, which can target myogenin protein for proteasomal degradation, is upregulated along with changes in Akt/Foxo and AMPK/Foxo signaling pathways. A proteasome inhibitor completely prevents cobalt chloride-mediated decrease in myogenin protein. These results suggest that cobalt chloride might modulate myogenin expression at post-transcriptional and post-translational levels, resulting in the failure of the myoblasts to differentiate into myotubes.

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Control of the spatial distribution of various cell types is required to construct functional tissues. Here, we report a simple topographical structure changed the spatial cell density. A concave curved boundary was designed, which allowed the spatial descent moving of cells and the change in spatial distributions of co-cultured cells. We utilized the difference in cell motility between myoblast cells (C2C12) and neuronal cells (PC12) to demonstrate the feasibility of spontaneous change in spatial cell density. Without the curved boundaries, high motility cells (C2C12) did not migrate to the adjacent area, which resulted in a slight temporal change (< 15%) in the spatial cell distribution. In contrast, with the curved boundaries, the cell density of the high motility cells in the groove to those cells on the ridge showed an increase exceeding 45%. On the other hand, the temporal change in the spatial cell distribution of low motility cells (PC12) was below 15% with or without the curved boundaries. In addition, as groove width increased, both cells displayed more initially gathering in groove. Importantly, these cell-type dependent results were also maintained under co-culture conditions. Our results suggest that designing topographical interfaces changes spatial cell density without any manipulation and is useful for multi-cellular constructs.

We have demonstrated nanomechanical applications using physicochemical and electrochemical phenomena of inverted-electron beam lithography (I-EBL), which induced in-situ two-dimensional (2-D) processing on wet samples and a living cell after the EB was stoppted in a 100-nm thick SiN membrane. The incident EB generates a virtual electrode and then this induces electrochemical and electrokinetic phenomena around the scanning trajectory. The I-EBL processing has a 120-nm resolution in full-width-at-half-maximum (FWHM) at the deposited line-and-space pattern in 10 mM 3,4-ethylenedioxythiophene (EDOT) diluted water solution. The virtual electrode also causes a electrokinetic local repulsive force with

Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.

We report a fabrication method for flexible and printable thermal sensors based on composites of semicrystalline acrylate polymers and graphite with a high sensitivity of 20 mK and a high-speed response time of less than 100 ms. These devices exhibit large resistance changes near body temperature under physiological conditions with high repeatability (1,800 times). Device performance is largely unaffected by bending to radii below 700 µm, which allows for conformal application to the surface of living tissue. The sensing temperature can be tuned between 25 °C and 50 °C, which covers all relevant physiological temperatures. Furthermore, we demonstrate flexible active-matrix thermal sensors which can resolve spatial temperature gradients over a large area. With this flexible ultrasensitive temperature sensor we succeeded in the in vivo measurement of cyclic temperatures changes of 0.1 °C in a rat lung during breathing, without interference from constant tissue motion. This result conclusively shows that the lung of a warm-blooded animal maintains surprising temperature stability despite the large difference between core temperature and inhaled air temperature.

Manipulation techniques of biomolecules have been proposed for biochemical analysis which combine electrokinetic dynamics, such as electrophoresis or electroosmotic flow, with optical manipulation to provide high throughput and high spatial degrees of freedom. However, there are still challenging problems in nanoscale manipulation due to the diffraction limit of optics. We propose here a new manipulation technique for spatiotemporal control of chemical transport in nanofluids using an inverted electron-beam (EB) lithography system for liquid samples. By irradiating a 2.5 keV EB to a liquid sample through a 100-nm-thick SiN membrane, negative charges can be generated within the SiN membrane, and these negative charges can induce a highly focused electric field in the liquid sample. We showed that the EB-induced negative charges could induce fluid flow, which was strong enough to manipulate 240 nm nanoparticles in water, and we verified that the main dynamics of this EB-induced fluid flow was electroosmosis caused by changing the zeta potential of the SiN membrane surface. Moreover, we demonstrated manipulation of a single nanoparticle and concentration patterning of nanoparticles by scanning EB. Considering the shortness of the EB wavelength and Debye length in buffer solutions, we expect that our manipulation technique will be applied to nanomanipulation of biomolecules in biochemical analysis and control.

Proper execution of voluntary movement requires a sensorimotor transformation based on the initial limb state. For example, successfully reaching to a stable target requires the recruitment of different muscle groups depending on limb position at movement initiation. To test whether this transformation could occur at the spinal level, we stimulated the cervical spinal cord of anesthetized monkeys while systematically changing initial posture and examined the modulation of the twitch response induced in the upper limb muscles. In three monkeys, a multichannel microelectrode array was implanted into the C6 segment of the spinal cord and electromyographic electrodes were implanted in 12 limb muscles (five hand, four elbow, and three shoulder muscles). The magnitude and onset latency of the evoked response in each electrode–muscle pair were examined by systematically changing the hand position through nine positions in a horizontal plane with the monkey prone. Among 330 electrode–muscle pairs examined, 61% of pairs exhibited significant modulation of either magnitude or latency of twitch responses across different hand/arm configurations (posture dependency). We found that posture dependency occurred preferentially in the distal rather than proximal muscles and was not affected by the location of the electrode within the stimulated spinal segment. Importantly, this posture dependency was not affected by spinalization at the C2 level. These results suggest that excitability in the cervical spinal cord is affected by initial arm posture through spinal reflex pathways. This posture dependency of spinal motor output could affect voluntary arm movement by adjusting descending motor commands relative to the initial arm posture.

Many of the brain-machine interfaces (BMIs), which detect the motor imagery or the movement states by noninvasive method, use the event related desynchronization (ERD) phenomena. The ERD is known as the intensity change of the specific frequency bands such as mu, beta, theta, and gamma. However, these bands can not be strictly defined because they are slightly different from each user. In this study, we use a 1i-constrained least squares learning to obtain the coefficients of the discriminator which specialize frequency-bands and electrode-positions in each user. This parameter of the discriminator is sparse and it employs only few efficient features for discrimination. Experiment results show that the proposed method can obtain higher performance than the existing method.

The dynamic electromechanical control of spatial structures of biomolecules in aqueous solutions was demonstrated using a nano virtual cathode display. By generating a focused electric field around the biomolecules using an electron beam (EB), the molecules' spatiotemporal responses to the electrical stimuli, such as globule transition of DNA random coils and deformation of planar lipid bilayers and vesicles, were successfully observed. The proposed system may be applied to high resolution and high degree-of-freedom manipulations to measure the mechanical and structural properties of bio-nanomaterials.

Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18–25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.

The goal of this research is to develop a rehabilitation system based on a brain-machine interface (BMI) for paraplegic patients. This requires the ability to detect a patient's desire on the b...

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease-related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well-defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks.

SUMMARY Plasticity of neuronal networks is widely investigated as the basis of learning and stimulus-induced plasticity is one of the remarkable topics. For inducing plasticity and leading a network to a desired state, effective stimuli have to be applied to the network. However, the effect of stimuli on living neuronal networks is difficult to evaluate because of the unsteadiness of the network property. In this paper, we produce a simulated neuronal network and compare the effects of tetanic stimulus at various sites of networks in silico. Using simulation, the tetanic effects can be evaluated irrespective of network transition. We confirmed that the direction of the network plasticity is determined by the tetanized site in the network, and this can be observed only when the proper test-stimulus site is selected. We conclude that this simulation-based approach effectively evaluates the stimulus effect on neuronal networks.

Overlapping of extracellularly recorded neural spike waveforms causes the original spike waveforms to become hidden and merged, confounding the real-time detection and sorting of these spikes. Methods proposed for solving this problem include using a multi-trode or placing a restriction on the complexity of overlaps. In this paper, we propose a rapid sequential method for the robust detection and sorting of arbitrarily overlapped spikes recorded with arbitrary types of electrodes. In our method, the probabilities of possible spike trains, including those that are overlapping, are evaluated by sequential Bayesian inference based on probabilistic models of spike-train generation and extracellular voltage recording. To reduce the high computational cost inherent in an exhaustive evaluation, candidates with low probabilities are considered as impossible candidates and are abolished at each sampling time to limit the number of candidates in the next evaluation. In addition, the data from a few subsequent sampling times are considered and used to calculate the “look-ahead probability”, resulting in improved calculation efficiency due to a more rapid elimination of candidates. These sufficiently reduce computational time to enable real-time calculation without impairing performance. We assessed the performance of our method using simulated neural signals and actual neural signals recorded in primary cortical neurons cultured on a multi-electrode array. Our results demonstrated that our computational method could be applied in real-time with a delay of less than 10 ms. The estimation accuracy was higher than that of a conventional spike sorting method, particularly for signals with multiple overlapping spikes.

1/R control is a physiological control method of the total artificial heart (TAH) with which long-term survival was obtained with animal experiments. However, 1/R control occasionally diverged in the undulation pump TAH (UPTAH) animal experiment. To improve the control stability of the 1/R control, appropriate control time constant in relation to characteristics of the baroreflex vascular system was investigated with frequency analysis and numerical simulation. In the frequency analysis, data of five goats in which the UPTAH was implanted were analyzed with first Fourier transform technique to examine the vasomotion frequency. The numerical simulation was carried out repeatedly changing baroreflex parameters and control time constant using the elements-expanded Windkessel model. Results of the frequency analysis showed that the 1/R control tended to diverge when very low frequency band that was an indication of the vasomotion frequency was relative high. In numerical simulation, divergence of the 1/R control could be reproduced and the boundary curves between the divergence and convergence of the 1/R control varied depending on the control time constant. These results suggested that the 1/R control tended to be unstable when the TAH recipient had high reflex speed in the baroreflex vascular system. Therefore, the control time constant should be adjusted appropriately with the individual vasomotion frequency.

We improved adhesiveness of a neuron-like cell, PC12, on a Parylene-C surface by O 2 plasma treatment which changes the surface from hydrophobic to hydrophilic. Neural cell adhesiveness on the plasma-treated Parylene-C was more than twenty times better compared to non-treated Parylene-C and it was close to that on a conventional polystyrene tissue-culture dish.

Tactile motion provides critical information for perception and manipulation of objects in touch. Perceived directions of tactile motion are primarily defined in the environmental coordinate, which means they change drastically with body posture even when the same skin sensors are stimulated. Despite the ecological importance of this perceptual constancy, the sensory processing underlying tactile directional remapping remains poorly understood. The present study psychophysically investigated the mechanisms underlying directional remapping in human tactile motion processing by examining whether finger posture modulates the direction of the tactile motion aftereffect (MAE) induced by inter-finger apparent motions. We introduced conflicts in the adaptation direction between somatotopic and environmental spaces by having participants change their finger posture between adaptation and test phases. In a critical condition, they touched stimulators with crossed index and middle fingers during adaptation but with uncrossed fingers during tests. Since the adaptation effect was incongruent between the somatotopic and environmental spaces, the direction of the MAE reflects the coordinate of tactile motion processing. The results demonstrated that the tactile MAE was induced in accordance with the motion direction determined by the environmental rather than the somatotopic space. In addition, it was found that though the physical adaptation of the test fingers was not changed, the tactile MAE disappeared when the adaptation stimuli were vertically aligned or when subjective motion perception was suppressed during adaptation. We also found that the tactile MAE, measured with our procedure, did not transfer across different hands, which implies that the observed MAEs mainly reflect neural adaptations occurring within sensor-specific, tactile-specific processing. The present findings provide a novel behavioral method to analyze the neural representation for directional remapping of tactile motion within tactile sensory processing in the human brain.

The abundance, morphology, and functional properties of mitochondria become altered in response to denervation. To gain insight into the regulation of this process, mitochondrial enzyme activities and gene expression involved in mitochondrial biogenesis and dynamics in mouse gastrocnemius muscle was investigated. Sciatic nerve transactions were performed on mice, and then gastrocnemius muscles were isolated at days 5 and 30 after surgery. Muscle weight was decreased significantly by 15% and 62% at days 5 and 30 after surgery, respectively. The activity of citrate synthase, a marker of oxidative enzyme, was reduced significantly by 31% and 53% at days 5 and 30, respectively. Enzyme histochemical analysis revealed that subsarcolemmal mitochondria were largely lost than intermyofibrillar mitochondria at day 5, and this trend was further progressed at day 30 after surgery. Expression levels of peroxisome proliferator-activated receptor, γ coactivator 1 (PGC-1)α, estrogen-related receptor α (ERRα), and mitofusin 2 were down-regulated throughout the experimental period, whereas those of PGC-1β, PRC, nuclear respiratory factor (NRF)-1, NRF-2, TFAM, and Lon protease were down-regulated at day 30 after surgery. These results suggest that PGC-1α, ERRα, and mitofusin 2 may be important factors in the process of denervation-induced mitochondrial adaptation. In addition, other PGC-1 family of transcriptional coactivators and DNA binding transcription factors may also contribute to mitochondrial adaptation after early response to denervation.

We address an estimation method of isometric muscle tension of fingers, as fundamental research for a neural signal-based prosthesis of fingers. We utilize needle electromyogram (EMG) signals, which have approximately equivalent information to peripheral neural signals.The estimating algorithm comprised two convolution operations. The first convolution is between normal distribution and a spike array, which is detected by needle EMG signals. The convolution estimates the probability density of spike-invoking time in the muscle. In this convolution, we hypothesize that each motor unit in a muscle activates spikes independently based on a same probability density function. The second convolution is between the result of the previous convolution and isometric twitch, viz., the impulse response of the motor unit. The result of the calculation is the sum of all estimated tensions of whole muscle fibers, i.e., muscle tension.We confirmed that there is good correlation between the estimated tension of the muscle and the actual tension, with >0.9 correlation coefficients at 59%, and >0.8 at 89% of all trials.

Neural interface devices that will allow signals from the human nervous system to control external equipment are extremely important for the next generation of prosthetic systems. A novel multichannel regeneration-type nerve electrode designed to record from and stimulate peripheral nerves has been developed to allow the control of artificial hands and to generate artificial sensations. In this study a novel flexible regeneration microelectrode based on the nerve regeneration principle was designed and fabricated using MEMS technologies. The electrode, which was fabricated on a 25-µm-thick parylene C substrate, has multiple fluidic channels. Each fluidic channel was 100µm wide×30µm high×1500µm long and featured multiple electrodes inside them as recording and stimulating sites. They also served as guidance channels for the regenerating axons. © 2009 Wiley Periodicals, Inc. Electron Comm Jpn, 92(4): 29–34, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10059

We are developing a brain-machine interface (BMI) called “RatCar," a small vehicle controlled by the neural signals of a rat's brain. An unconfined adult rat with a set of bundled neural electrodes in the brain rides on the vehicle. Each bundle consists of four tungsten wires isolated with parylene polymer. These bundles were implanted in the primary motor and premotor cortices in both hemispheres of the brain. In this paper, methods and results for estimating locomotion speed and directional changes are described. Neural signals were recorded as the rat moved in a straight line and as it changed direction in a curve. Spike-like waveforms were then detected and classified into several clusters to calculate a firing rate for each neuron. The actual locomotion velocity and directional changes of the rat were recorded concurrently. Finally, the locomotion states were correlated with the neural firing rates using a simple linear model. As a result, the abstract estimation of the locomotion velocity and directional changes were achieved.

The present study introduces an approach to detect and classify extracellulary recorded action potentials of neurons, usually termed spike sorting. Our approach is based on template matching, which is an optimal filter under Gaussian noise; however, this approach is usually expensive in computational time. To speed up the filter, it is important to curtail the matching process when the distance between template and waveform exceeds some threshold. We approach this aspect of the problem using the frame of similarity detection algorithms (SSDA) and Davies-Bouldin validation indices (DBVI). Windowing pair of the filter was selected in DBVI-based order and a signal which has a rapidly increasing error was discarded to reduce the computational time. DBVI is a function of the ratio of the sum of within-cluster scatter to between-cluster separation; thus, using this order we can expect to separate a signal and a noise in fewer window point calculation than full point matching. This matching process performed well, with a shortter computational time and fewer incorrect classifications than other ordering methods such as time-based or amplitude-based order. © 2009 Wiley Periodicals, Inc. Electron Comm Jpn, 92(11): 57–63, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecj.10066