Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three-dimensional motor neuron-Schwann cell coculture model on a microfluidic biochip.

Axonal regeneration and remyelination of peripheral motor neurons (MNs) are critical for restoring neuromuscular motor function after injury or peripheral neuropathy. We examined whether optogenetically mediated light stimulation (OMLS) could enhance the axon outgrowth and myelination of MNs using three-dimensional motor neuron-Schwann cell (MN-SC) coculture on a microfluidic biochip. The biochip was designed to allow SCs to interact with the axons of MNs, while preventing direct contact between SCs and the cell bodies of MNs. Following coculture with SCs on the microfluidic biochip, MNs were transfected with a light-sensitive channelrhodopsin gene. Transfected MNs subjected to repeated light stimulation (20 Hz, 1 hr) produced significantly longer axons than nontransfected MNs. OMLS of MNs greatly increased the number of myelin basic protein (MBP)-expressing SCs, promoting the initiation of myelination of MNs. Ultrastructurally, OMLS of MNs markedly enhanced the thickness of the compact myelin sheath around the MN axons such that the average thickness was closer to that of the theoretical estimates in vivo. Thus, the MN-SC coculture model on a microfluidic biochip augmented by OMLS of MNs is a feasible platform for studying the relationship of neuronal activity with regrowth and remyelination.

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons.

Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.

Primary Motor Neuron Culture to Promote Cellular Viability and Myelination.

A culture system that can recapitulate myelination in vitro will not only help us to better understand the mechanism of myelination and demyelination but also identify possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC co-culture system, MNs survive over 3 weeks and extend long axons. Both viability and axon growth of MNs in the co-culture are markedly enhanced as compared to those of MN monocultures. Co-labeling of myelin basic proteins and neuronal cell microtubules reveals that SCs form myelin sheaths by wrapping around the axons of MNs.

Cubic-phase zirconia nano-island growth using atomic layer deposition and application in low-power charge-trapping nonvolatile-memory devices.

The manipulation of matter at the nanoscale enables the generation of properties in a material that would otherwise be challenging or impossible to realize in the bulk state. Here, we demonstrate growth of zirconia nano-islands using atomic layer deposition on different substrate terminations. Transmission electron microscopy and Raman measurements indicate that the nano-islands consist of nano-crystallites of the cubic-crystalline phase, which results in a higher dielectric constant (κ âˆ¼ 35) than the amorphous phase case (κ âˆ¼ 20). X-ray photoelectron spectroscopy measurements show that a deep quantum well is formed in the Al2O3/ZrO2/Al2O3 system, which is substantially different to that in the bulk state of zirconia and is more favorable for memory application. Finally, a memory device with a ZrO2 nano-island charge-trapping layer is fabricated, and a wide memory window of 4.5 V is obtained at a low programming voltage of 5 V due to the large dielectric constant of the islands in addition to excellent endurance and retention characteristics.

Investigation of the Changes in Electronic Properties of Nickel Oxide (NiOx) Due to UV/Ozone Treatment.

Drastic reduction in nickel oxide (NiOx) film resistivity and ionization potential is observed when subjected to ultraviolet (UV)/ozone (O3) treatment. X-ray photoemission spectroscopy suggests that UV/O3 treatment changes the film stoichiometry by introducing Ni vacancy defects. Oxygen-rich NiOx having Ni vacancy defects behaves as a p-type semiconductor. Therefore, in this work, a simple and effective technique to introduce doping in NiOx is shown. Angle-resolved XPS reveals that the effect of UV/O3 treatment does not only alter the film surface property but also introduces oxygen-rich stoichiometry throughout the depth of the film. Finally, simple metal/interlayer/semiconductor (MIS) contacts are fabricated on p-type Si using NiOx as the interlayer and different metals. Significant barrier height reduction is observed with respect to the control sample following UV/O3 treatment, which is in agreement with the observed reduction in film resistivity. From an energy band diagram point of view, the introduction of the UV/O3 treatment changes the defect state distribution, resulting in a change in the pinning of the Fermi level. Therefore, this work also shows that the Fermi level pinning property of NiOx can be controlled using UV/O3 treatment.

Optogenetic Modulation of Urinary Bladder Contraction for Lower Urinary Tract Dysfunction.

As current clinical approaches for lower urinary tract (LUT) dysfunction such as pharmacological and electrical stimulation treatments lack target specificity, thus resulting in suboptimal outcomes with various side effects, a better treatment modality with spatial and temporal target-specificity is necessary. In this study, we delivered optogenetic membrane proteins, such as channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to bladder smooth muscle cells (SMCs) of mice using either the Cre-loxp transgenic system or a viral transfection method. The results showed that depolarizing ChR2-SMCs with blue light induced bladder contraction, whereas hyperpolarizing NpHR-SMCs with yellow light suppressed PGE2-induced overactive contraction. We also confirmed that optogenetic contraction of bladder smooth muscles in this study is not neurogenic, but solely myogenic, and that optogenetic light stimulation can modulate the urination in vivo. This study thus demonstrated the utility of optogenetic modulation of smooth muscle as a means to actively control the urinary bladder contraction with spatial and temporal accuracy. These features would increase the efficacy of bladder control in LUT dysfunctions without the side effects of conventional clinical therapies.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc.

Optogenetic Inhibition of the Subthalamic Nucleus Reduces Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the amelioration of levodopa-induced dyskinesia. However, electrical deep brain stimulation cannot specifically activate or inactivate selected types of neurons. OBJECTIVES: We applied optogenetics as an alternative treatment to deep brain stimulation for levodopa-induced dyskinesia, and also to confirm that the mechanism of levodopa-induced dyskinesia amelioration by subthalamic nucleus deep brain stimulation is mediated through neuronal inhibition. METHODS: 6-hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus (AAV) or hSynapsin1-YFP AAV. Two weeks after viral injections, all rats were treated with daily injections of levodopa. Then, the optic fiber was implanted into the ipsilateral subthalamic nucleus. We performed various behavioral tests to evaluate the changes in levodopa-induced dyskinesias after optogenetic expression and illumination in the subthalamic nucleus. RESULTS: The behavioral tests revealed that optical inhibition of the subthalamic nucleus significantly ameliorated levodopa-induced dyskinesia by reducing the duration of the dyskinesias as well as the severity of axial dyskinesia. CONCLUSIONS: These findings will provide a useful foundation for the future development of optogenetic modulation systems that could be considered as an approach to dyskinesia therapy.

Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination.

A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination.

A time-course study of behavioral and electrophysiological characteristics in a mouse model of different stages of Parkinson's disease using 6-hydroxydopamine.

Parkinson's disease (PD) is characterized by abnormal motor symptoms and increased neuronal activity in the subthalamic nucleus (STN) as the disease progresses. We investigated the behavioral and electrophysiological characteristics in a mouse model mimicking the progressive stages of human PD (early, moderate, and advanced) by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB) at three different concentrations (2, 4, and 6 µg/2 µl). Significant changes in motor symptoms were demonstrated between groups in association with relative TH-positive cell loss in the substantia nigra pars compacta (SNc). Moreover, electrophysiologically assessed changes in the mean neuronal firing rate in the STN neurons were comparable to those in the early to advanced stages of human PD. Thus, the mouse model presented herein replicates the unique characteristics of each progressive stage of PD, in both motor and neurophysiological aspects, and therefore can be useful for further investigations of PD pathology.

Characteristics of the neuronal firing patterns in the subthalamic nucleus with graded dopaminergic cell loss in the nigrostriatal pathway.

The aim of this study was to evaluate the neuronal firing changes in the subthalamic nucleus (STN) in a graded mouse model of Parkinson's disease. Unilateral graded dopaminergic cell loss in the substantia nigra pars compacta was achieved by injecting different concentrations of 6-hydroxydopamine (6-OHDA) in the right medial forebrain bundle. Electrophysiological analysis of neuronal firing patterns in the STN revealed an increased firing rate, burst index, and interspike interval coefficient of variation in groups treated with higher 6-OHDA concentrations. The results of this study suggest the detailed pathophysiological characteristics of Parkinson's disease in a mouse model.

An implantable wireless optogenetic stimulation system for peripheral nerve control.

An implantable wireless optogenetic stimulation system with an LED-based optical stimulation cuff electrode was developed for peripheral nerve control. The proposed system consisted of a battery-powered optical cuff electrode, optical stimulation controller, and wireless communication system. The optical cuff electrode had a polydimethylsiloxane (PDMS) structure was designed to illuminate the entire sciatic nerve. The wireless communication system was designed to comply with medical implant communication service (MICS) regulations. To evaluate the proposed system, optogenetic stimulation was performed in optogenetic transgenic mice (Thy1::ChR2). The optical cuff electrode was implanted on the sciatic nerve, and movement was elicited during optical stimulation. The experimental results show that ankle movement can be generated wirelessly using optical stimulation pulse parameters.

Enhanced efficacy of human brain-derived neural stem cells by transplantation of cell aggregates in a rat model of Parkinson's disease.

OBJECTIVE: Neural tissue transplantation has been a promising strategy for the treatment of Parkinson's disease (PD). However, transplantation has the disadvantages of low-cell survival and/or development of dyskinesia. Transplantation of cell aggregates has the potential to overcome these problems, because the cells can extend their axons into the host brain and establish synaptic connections with host neurons. In this present study, aggregates of human brain-derived neural stem cells (HB-NSC) were transplanted into a PD animal model and compared to previous report on transplantation of single-cell suspensions. METHODS: Rats received an injection of 6-OHDA into the right medial forebrain bundle to generate the PD model and followed by injections of PBS only, or HB-NSC aggregates in PBS into the ipsilateral striatum. Behavioral tests, multitracer (2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) and [(18)F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([(18)F]-FP-CIT) microPET scans, as well as immunohistochemical (IHC) and immunofluorescent (IF) staining were conducted to evaluate the results. RESULTS: The stepping test showed significant improvement of contralateral forelimb control in the HB-NSC group from 6-10 weeks compared to the control group (p<0.05). [(18)F]-FP-CIT microPET at 10 weeks posttransplantation demonstrated a significant increase in uptake in the HB-NSC group compared to pretransplantation (p<0.05). In IHC and IF staining, tyrosine hydroxylase and human ß2 microglobulin (a human cell marker) positive cells were visualized at the transplant site. CONCLUSION: These results suggest that the HB-NSC aggregates can survive in the striatum and exert therapeutic effects in a PD model by secreting dopamine.

Optogenetic inactivation of the subthalamic nucleus improves forelimb akinesia in a rat model of Parkinson disease.

BACKGROUND: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the therapeutic effects in patients with Parkinson disease (PD) but cannot specifically activate or inactivate different types of neurons. Recently, a new technology based on optogenetics has been developed to modulate the activity of specific neurons. However, the therapeutic effects of optical inactivation in the subthalamic nucleus (STN) have not been fully investigated. OBJECTIVE: To perform various behavioral tests to evaluate changes in motor functions in a PD rat model after optogene expression and, unlike previous studies, to assess the therapeutic effects of direct optogenetic inactivation in the STN. METHODS: 6-Hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus or an equivalent volume of phosphate-buffered saline. Three weeks after injection of adeno-associated virus or phosphate-buffered saline, the optic fiber was implanted into the ipsilateral STN. A stepping test, a cylinder test, and an apomorphine-induced rotation test were performed in 3 sequential steps: during light-off state, during light stimulation, and again during light-off state. RESULTS: Stepping tests revealed that optical inhibition of the STN significantly improved 6-hydroxydopamine-induced forelimb akinesia. PD motor signs, as assessed by cylinder and apomorphine tests, were not affected by optical inhibition. Immunofluorescence revealed that halorhodopsin was highly expressed and colocalized with vesicular glutamate transporter 2 in the STN. CONCLUSION: Optogenetic inhibition in the STN may be effective in improving contralateral forelimb akinesia but not in changing forelimb preference or reducing dopaminergic receptor supersensitivity. These findings are useful as a basis for future studies on optogenetics in PD.

Feedback control of electrode offset voltage during functional electrical stimulation.

Control of the electrode offset voltage is an important issue related to the processes of functional electrical stimulation because excess charge accumulation over time damages both the tissue and the electrodes. This paper proposes a new feedback control scheme to regulate the electrode offset voltage to a predetermined reference value. The electrode offset voltage was continuously monitored using a sample-and-hold (S/H) circuit during stimulation and non-stimulation periods. The stimulation current was subsequently adjusted using a proportional-integral (PI) controller to minimise the error between the reference value and the electrode offset voltage. During the stimulation period, the electrode offset voltage was maintained through the S/H circuit, and the PI controller did not affect the amplitude of the stimulation current. In contrast, during the non-stimulation period, the electrode offset voltage was sampled through the S/H circuit and rapidly regulated through the PI controller. The experimental results obtained using a nerve cuff electrode showed that the electrode offset voltage was successfully controlled in terms of the performance specifications, such as the steady- and transient-state responses and the constraint of the controller output. Therefore, the proposed control scheme can potentially be used in various nerve stimulation devices and applications requiring control of the electrode offset voltage.

Gait phase detection from sciatic nerve recordings in functional electrical stimulation systems for foot drop correction.

Cutaneous afferent activities recorded by a nerve cuff electrode have been used to detect the stance phase in a functional electrical stimulation system for foot drop correction. However, the implantation procedure was difficult, as the cuff electrode had to be located on the distal branches of a multi-fascicular nerve to exclude muscle afferent and efferent activities. This paper proposes a new gait phase detection scheme that can be applied to a proximal nerve root that includes cutaneous afferent fibers as well as muscle afferent and efferent fibers. To test the feasibility of this scheme, electroneurogram (ENG) signals were measured from the rat sciatic nerve during treadmill walking at several speeds, and the signal properties of the sciatic nerve were analyzed for a comparison with kinematic data from the ankle joint. On the basis of these experiments, a wavelet packet transform was tested to define a feature vector from the sciatic ENG signals according to the gait phases. We also propose a Gaussian mixture model (GMM) classifier and investigate whether it could be used successfully to discriminate feature vectors into the stance and swing phases. In spite of no significant differences in the rectified bin-integrated values between the stance and swing phases, the sciatic ENG signals could be reliably classified using the proposed wavelet packet transform and GMM classification methods.

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson's disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [(18)F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

Improvement of wireless power transmission efficiency of implantable subcutaneous devices by closed magnetic circuit mechanism.

Induction coils were fabricated based on flexible printed circuit board for inductive transcutaneous power transmission. The coil had closed magnetic circuit (CMC) structure consisting of inner and outer magnetic core. The power transmission efficiency of the fabricated device was measured in the air and in vivo condition. It was confirmed that the CMC coil had higher transmission efficiency than typical air-core coil. The power transmission efficiency during a misalignment between primary coil and implanted secondary coil was also evaluated. The decrease of mutual inductance between the two coils caused by the misalignment led to a low efficiency of the inductive link. Therefore, it is important to properly align the primary coil and implanted secondary coil for effective power transmission. To align the coils, a feedback coil was proposed. This was integrated on the backside of the primary coil and enabled the detection of a misalignment of the primary and secondary coils. As a result of using the feedback coil, the primary and secondary coils could be aligned without knowledge of the position of the implanted secondary coil.

Improvement of signal-to-interference ratio and signal-to-noise ratio in nerve cuff electrode systems.

Cuff electrodes are effective for chronic electroneurogram (ENG) recording while minimizing nerve damage. However, the ENG signals are usually contaminated by electromyogram (EMG) activity from the surrounding muscles, stimulus artifacts produced by the electrical stimulation and noise generated in the first stage of the neural signal amplifier. This paper proposed a new cuff electrode to reduce the interference from EMG signals and stimulus artifacts. As a result, when an additional middle electrode was placed at the center of the cuff electrode, a significant improvement in the signal-to-interference ratio was achieved at 11% for the EMG signals and 12% for the stimulus artifacts when compared to a conventional tripolar cuff. Furthermore, a new low-noise amplifier was proposed to improve the signal-to-noise ratio. The circuit was designed based on a noise analysis to minimize the noise, and the results show that the total noise of the amplifier was below 1 µV for a cuff impedance of 1 kΩ and a frequency bandwidth of 300 to 5000 Hz.

Spontaneous synchronized burst firing of subthalamic nucleus neurons in rat brain slices measured on multi-electrode arrays.

The current study presents an organotypic rat midbrain slice culture that served as a consistent and informative framework, where the STN neurons and their interconnectivity were closely examined with respect to electrophysiological and pharmacological properties. From multi-electrode array recordings, it was found that the majority of STN neurons spontaneously fired in bursts rather than tonically under control conditions, and the neural activity between pairs of burst-firing STN neurons was tightly correlated. This spontaneous synchronized burst firing was also affected by a glutamate receptor antagonist, yet unaffected by a GABA receptor antagonist. Moreover, even when the STN was isolated from all its known external inputs, spontaneous synchronized burst firing was still observed under control conditions and consistently switched to tonic firing following the application of a glutamate receptor antagonist. Therefore, the results indicated the existence of glutamatergic projections to the STN in the slice preparation, and these excitatory synaptic connections appeared to originate from axon collaterals within the STN rather than other basal ganglia nuclei. It could be concluded that the STN neurons and their interconnectivity are essential requirements in the rat brain slice preparation to produce spontaneous synchronized burst firing.