Low-invasive neural recording in mouse models with diabetes via an ultrasmall needle-electrode.

Diabetes is known to cause a variety of complications, having a high correlation with Alzheimer's disease. Electrophysiological recording using a microscale needle electrode is a promising technology for the study, however, diabetic brain tissue is more difficult to record neuronal activities than normal tissue due to these complications including the development of cerebrovascular disease. Here we show an electrophysiological methodology for diabetic db/db mice (+Leprdb/+Leprdb) using a 4-µm-tip diameter needle-electrode device. The needle electrode minimized the tissue injury when compared to a typical larger metal electrode, as confirmed by bleeding during penetration. The proposed electrode device showed both acute and chronic in vivo recording capabilities for diabetic mice while reducing the glial cells' responses. Because of these device characteristics, the 4-µm-tip diameter needle-electrode will allow electrophysiological studies on diabetes models of not only mice, as proven in this study, but also other animals.

Decoding distributed oscillatory signals driven by memory and perception in the prefrontal cortex.

Sensory perception and memory recall generate different conscious experiences. Although externally and internally driven neural activities signifying the same perceptual content overlap in the sensory cortex, their distribution in the prefrontal cortex (PFC), an area implicated in both perception and memory, remains elusive. Here, we test whether the local spatial configurations and frequencies of neural oscillations driven by perception and memory recall overlap in the macaque PFC using high-density electrocorticography and multivariate pattern analysis. We find that dynamically changing oscillatory signals distributed across the PFC in the delta-, theta-, alpha-, and beta-band ranges carry significant, but mutually different, information predicting the same feature of memory-recalled internal targets and passively perceived external objects. These findings suggest that the frequency-specific distribution of oscillatory neural signals in the PFC serves cortical signatures responsible for distinguishing between different types of cognition driven by external perception and internal memory.

Nanoscale-tipped wire array injections transfer DNA directly into brain cells ex vivo and in vivo.

Genetic modification to restore cell functions in the brain can be performed through the delivery of biomolecules in a minimally invasive manner into live neuronal cells within brain tissues. However, conventional nanoscale needles are too short (lengths of ~10 µm) to target neuronal cells in ~1-mm-thick brain tissues because the neuronal cells are located deep within the tissue. Here, we report the use of nanoscale-tipped wire (NTW) arrays with diameters < 100 nm and wire lengths of ~200 µm to address biomolecule delivery issues. The NTW arrays were manufactured by growth of silicon microwire arrays and nanotip formation. This technique uses pinpoint, multiple-cell DNA injections in deep areas of brain tissues, enabling target cells to be marked by fluorescent protein (FP) expression vectors. This technique has potential for use for electrophysiological recordings and biological transfection into neuronal cells. Herein, simply pressing an NTW array delivers and expresses plasmid DNA in multiple-cultured cells and multiple-neuronal cells within a brain slice with reduced cell damage. Additionally, DNA transfection is demonstrated using brain cells ex vivo and in vivo. Moreover, knockdown of a critical clock gene after injecting a short hairpin RNA (shRNA) and a genome-editing vector demonstrates the potential to genetically alter the function of living brain cells, for example, pacemaker cells of the mammalian circadian rhythms. Overall, our NTW array injection technique enables genetic and functional modification of living cells in deep brain tissue areas, both ex vivo and in vivo.

A floating 5 µm-diameter needle electrode on the tissue for damage-reduced chronic neuronal recording in mice.

Microelectrode technology is essential in electrophysiology and has made contributions to neuroscience as well as to medical applications. However, it is necessary to minimize tissue damage associated with needle-like electrode on the brain tissue and the implantation surgery, which makes stable chronic recording impossible. Here, we report on an approach for using a 5 µm-diameter needle electrode, which enables the following of tissue motions, via a surgical method. The electrode is placed on the brain tissue of a mouse with a dissolvable material, reducing the physical stress to the tissue; this is followed by the implantation of the electrode device in the brain without fixing it to the cranium, achieving a floating electrode architecture on the tissue. The electrode shows stable recording with no significant degradation of the signal-to-noise ratios for 6 months, and minimized tissue damage is confirmed compared to that when using a cranium-fixed electrode with the same needle geometry.

Three-micrometer-diameter needle electrode with an amplifier for extracellular in vivo recordings.

Microscale needle-electrode devices offer neuronal signal recording capability in brain tissue; however, using needles of smaller geometry to minimize tissue damage causes degradation of electrical properties, including high electrical impedance and low signal-to-noise ratio (SNR) recording. We overcome these limitations using a device assembly technique that uses a single needle-topped amplifier package, called STACK, within a device of ∼1 × 1 mm2 Based on silicon (Si) growth technology, a <3-µm-tip-diameter, 400-µm-length needle electrode was fabricated on a Si block as the module. The high electrical impedance characteristics of the needle electrode were improved by stacking it on the other module of the amplifier. The STACK device exhibited a voltage gain of >0.98 (-0.175 dB), enabling recording of the local field potential and action potentials from the mouse brain in vivo with an improved SNR of 6.2. Additionally, the device allowed us to use a Bluetooth module to demonstrate wireless recording of these neuronal signals; the chronic experiment was also conducted using STACK-implanted mice.

Donut-Shaped Stretchable Kirigami: Enabling Electronics to Integrate with the Deformable Muscle.

Electronic devices used to record biological signals are important in neuroscience, brain-machine interfaces, and medical applications. Placing electronic devices below the skin surface and recording the muscle offers accurate and robust electromyography (EMG) recordings. The device stretchability and flexibility must be similar to the tissues to achieve an intimate integration of the electronic device with the biological tissues. However, conventional elastomer-based EMG electrodes have a Young's modulus that is ≈20 times higher than that of muscle. In addition, these stretchable devices also have an issue of displacement on the tissue surface, thereby causing some challenges during accurate and robust EMG signal recordings. In general, devices with kirigami design solve the issue of the high Young's modulus of conventional EMG devices. In this study, donut-shaped kirigami bioprobes are proposed to reduce the device displacement on the muscle surface. The fabricated devices are tested on an expanding balloon and they show no significant device (microelectrode) displacement. As the package, the fabricated device is embedded in a dissolvable material-based scaffold for easy-to-use stretchable kirigami device in an animal experiment. Finally, the EMG signal recording capability and stability using the fabricated kirigami device is confirmed in in vivo experiments without significant device displacements.

A Magnetically Assembled High-Aspect-Ratio Needle Electrode for Recording Neuronal Activity.

Microelectrode devices, which enable the detection of neuronal signals in brain tissues, have made significant contributions in the field of neuroscience and the brain-machine interfaces. To further develop such microelectrode devices, the following requirements must be met: i) a fine needle's diameter (<30 µm) to reduce damage to tissues; ii) a long needle (e.g., ≈1 mm for rodents and ≈2 mm for macaques); and iii) multiple electrodes to achieve high spatial recording (<100 µm in pitch). In order to meet these requirements, this study herein reports an assembly technique for high-aspect-ratio microneedles, which employs a magnet. The assembly is demonstrated, in which nickel wires of length 750 µm and diameter 25 µm are produced on a silicon substrate. The impedance magnitude of the assembled needle-like electrode measured at 1 kHz is 5.6 kΩ, exhibiting output and input signal amplitudes of 96.7% at 1 kHz. To confirm the recording capability of the fabricated device, neuronal signal recordings are performed using mouse cerebra in vivo. The packaged single microneedle electrode penetrates the barrel field in the primary somatosensory cortex of the mouse and enables the detection of evoked neuronal activity of both local field potentials and action potentials.

Locally induced neuronal synchrony precisely propagates to specific cortical areas without rhythm distortion.

Propagation of oscillatory spike firing activity at specific frequencies plays an important role in distributed cortical networks. However, there is limited evidence for how such frequency-specific signals are induced or how the signal spectra of the propagating signals are modulated during across-layer (radial) and inter-areal (tangential) neuronal interactions. To directly evaluate the direction specificity of spectral changes in a spiking cortical network, we selectively photostimulated infragranular excitatory neurons in the rat primary visual cortex (V1) at a supra-threshold level with various frequencies, and recorded local field potentials (LFPs) at the infragranular stimulation site, the cortical surface site immediately above the stimulation site in V1, and cortical surface sites outside V1. We found a significant reduction of LFP powers during radial propagation, especially at high-frequency stimulation conditions. Moreover, low-gamma-band dominant rhythms were transiently induced during radial propagation. Contrastingly, inter-areal LFP propagation, directed to specific cortical sites, accompanied no significant signal reduction nor gamma-band power induction. We propose an anisotropic mechanism for signal processing in the spiking cortical network, in which the neuronal rhythms are locally induced/modulated along the radial direction, and then propagate without distortion via intrinsic horizontal connections for spatiotemporally precise, inter-areal communication.

Fiber bundle endomicroscopy with multi-illumination for three-dimensional reflectance image reconstruction.

Bundled fiber optics allow in vivo imaging at deep sites in a body. The intrinsic optical contrast detects detailed structures in blood vessels and organs. We developed a bundled-fiber-coupled endomicroscope, enabling stereoscopic three-dimensional (3-D) reflectance imaging with a multipositional illumination scheme. Two illumination sites were attached to obtain reflectance images with left and right illumination. Depth was estimated by the horizontal disparity between the two images under alternative illuminations and was calibrated by the targets with known depths. This depth reconstruction was applied to an animal model to obtain the 3-D structure of blood vessels of the cerebral cortex (Cereb cortex) and preputial gland (Pre gla). The 3-D endomicroscope could be instrumental to microlevel reflectance imaging, improving the precision in subjective depth perception, spatial orientation, and identification of anatomical structures.

Heterogeneous Redistribution of Facial Subcategory Information Within and Outside the Face-Selective Domain in Primate Inferior Temporal Cortex.

The inferior temporal cortex (ITC) contains neurons selective to multiple levels of visual categories. However, the mechanisms by which these neurons collectively construct hierarchical category percepts remain unclear. By comparing decoding accuracy with simultaneously acquired electrocorticogram (ECoG), local field potentials (LFPs), and multi-unit activity in the macaque ITC, we show that low-frequency LFPs/ECoG in the early evoked visual response phase contain sufficient coarse category (e.g., face) information, which is homogeneous and enhanced by spatial summation of up to several millimeters. Late-induced high-frequency LFPs additionally carry spike-coupled finer category (e.g., species, view, and identity of the face) information, which is heterogeneous and reduced by spatial summation. Face-encoding neural activity forms a cluster in similar cortical locations regardless of whether it is defined by early evoked low-frequency signals or late-induced high-gamma signals. By contrast, facial subcategory-encoding activity is distributed, not confined to the face cluster, and dynamically increases its heterogeneity from the early evoked to late-induced phases. These findings support a view that, in contrast to the homogeneous and static coarse category-encoding neural cluster, finer category-encoding clusters are heterogeneously distributed even outside their parent category cluster and dynamically increase heterogeneity along with the local cortical processing in the ITC.

Ultrastretchable Kirigami Bioprobes.

An ultrastretchable film device is developed that can follow the shape of spherical and large deformable biological samples such as heart and brain tissues. Although the film is composed of biocompatible parylene for the device substrate and metal layers of platinum (Pt)/titanium (Ti), which are unstretchable materials, the film shows a high stretchability by patterning slits as a "Kirigami" design. A Pt/Ti-microelectrode array embedded in 11 µm thick parylene film with 5 × 91 slits exhibits a film strain of ≈250% at 9 mN strain-force (0.08 MPa in stress) with a Young's modulus of 23 kPa, while the 3 × 91-slit film shows a Young's modulus of 3.6 kPa. The maximum strains of these devices are ≈470% and ≈840%, respectively. It is demonstrated that the Kirigami-based microelectrode device can simultaneously record in vivo electrocorticogram signals from the visual and barrel cortices of a mouse by stretching the film and tuning the electrode gap. Moreover, wrapping the Kirigami device around a beating mouse's heart, which shows large and rapid changes in the volume and the surface area, can record the in vivo epicardial electrocardiogram signals. Such a small Young's modulus for a stretchable device reduces the device's strain-force, minimizing the device-induced stress to soft biological tissues.

Associative-memory representations emerge as shared spatial patterns of theta activity spanning the primate temporal cortex.

Highly localized neuronal spikes in primate temporal cortex can encode associative memory; however, whether memory formation involves area-wide reorganization of ensemble activity, which often accompanies rhythmicity, or just local microcircuit-level plasticity, remains elusive. Using high-density electrocorticography, we capture local-field potentials spanning the monkey temporal lobes, and show that the visual pair-association (PA) memory is encoded in spatial patterns of theta activity in areas TE, 36, and, partially, in the parahippocampal cortex, but not in the entorhinal cortex. The theta patterns elicited by learned paired associates are distinct between pairs, but similar within pairs. This pattern similarity, emerging through novel PA learning, allows a machine-learning decoder trained on theta patterns elicited by a particular visual item to correctly predict the identity of those elicited by its paired associate. Our results suggest that the formation and sharing of widespread cortical theta patterns via learning-induced reorganization are involved in the mechanisms of associative memory representation.

Nanoscale-Tipped High-Aspect-Ratio Vertical Microneedle Electrodes for Intracellular Recordings.

Intracellular recording nanoscale electrode devices provide the advantages of a high spatial resolution and high sensitivity. However, the length of nanowire/nanotube-based nanoelectrodes is currently limited to <10 µm long due to fabrication issues for high-aspect-ratio nanoelectrodes. The concept reported here can address the technological limitations by fabricating >100 µm long nanoscale-tipped electrodes, which show intracellular recording capability.

Dissolvable base scaffolds allow tissue penetration of high-aspect-ratio flexible microneedles.

Microscale needle technology is important in electrophysiological studies, drug/chemical delivery systems, optogenetic applications, and so on. In this study, dissolvable needle-base scaffold realizes penetration of high-aspect-ratio flexible microneedles (e.g., <5 µm diameter and >500 µm length) into biological tissues. This methodology, which is applicable to numerous high-aspect-ratio flexible microneedles, should reduce the invasiveness and provide safer tissue penetrations than conventional approaches.

Super multi-channel recording systems with UWB wireless transmitter for BMI.

In order to realize a low-invasive and high accuracy Brain-Machine Interface (BMI) system for clinical applications, a super multi-channel recording system was developed in which 4096 channels of Electrocorticogram (ECoG) signal can be amplified and transmitted to outside the body by using an Ultra Wide Band (UWB) wireless system. Also, a high density, flexible electrode array made by using a Parylene-C substrate was developed that is composed of units of 32-ch recording arrays. We have succeeded in an evaluation test of UWB wireless transmitting using a body phantom system.

Characteristics of temporal fluctuation of the vertical ground reaction force during quiet stance in Parkinson's disease.

Stance instability is seen in late stage Parkinson's disease (PD). Stabilometer-based center-of-pressure (COP) evaluation is an easy, routine method for measuring postural control ability. Most of the stabilometer- and force plate-based studies on upright postural control have discussed horizontal COP component control. Previous studies on vertical component control have been few, and no fractal analysis-based study on the component has been reported. We aimed to show the influence of neurological changes and aging on the vertical component and the difference in fluctuation pattern behavior in healthy young and elderly subjects as well as Parkinsonian patients. Detrended fluctuation analysis was used to study characteristics of fluctuation of vertical ground reaction force. In the three groups, all scaling exponents (α, α1, α2), which are time-correlated data of vertical ground reaction force, had a value >0 and <0.5 (0<α<0.5). Additionally, α and α2 were significantly different between PD and the other groups. Significant differences were observed between PD and the other groups regarding RMS, maximum peak value, and coefficient of variance. We demonstrated statistically significant differences in vertical ground reaction force between Parkinsonian patients and the other groups, suggesting that a neurological influence with PD may be markedly reflected in the vertical ground reaction force.

Intrasulcal electrocorticography in macaque monkeys with minimally invasive neurosurgical protocols.

Electrocorticography (ECoG), multichannel brain-surface recording and stimulation with probe electrode arrays, has become a potent methodology not only for clinical neurosurgery but also for basic neuroscience using animal models. The highly evolved primate's brain has deep cerebral sulci, and both gyral and intrasulcal cortical regions have been implicated in important functional processes. However, direct experimental access is typically limited to gyral regions, since placing probes into sulci is difficult without damaging the surrounding tissues. Here we describe a novel methodology for intrasulcal ECoG in macaque monkeys. We designed and fabricated ultra-thin flexible probes for macaques with micro-electro-mechanical systems technology. We developed minimally invasive operative protocols to implant the probes by introducing cutting-edge devices for human neurosurgery. To evaluate the feasibility of intrasulcal ECoG, we conducted electrophysiological recording and stimulation experiments. First, we inserted parts of the Parylene-C-based probe into the superior temporal sulcus to compare visually evoked ECoG responses from the ventral bank of the sulcus with those from the surface of the inferior temporal cortex. Analyses of power spectral density and signal-to-noise ratio revealed that the quality of the ECoG signal was comparable inside and outside of the sulcus. Histological examination revealed no obvious physical damage in the implanted areas. Second, we placed a modified silicone ECoG probe into the central sulcus and also on the surface of the precentral gyrus for stimulation. Thresholds for muscle twitching were significantly lower during intrasulcal stimulation compared to gyral stimulation. These results demonstrate the feasibility of intrasulcal ECoG in macaques. The novel methodology proposed here opens up a new frontier in neuroscience research, enabling the direct measurement and manipulation of electrical activity in the whole brain.

Simultaneous recording of ECoG and intracortical neuronal activity using a flexible multichannel electrode-mesh in visual cortex.

Electrocorticogram (ECoG) is a well-balanced methodology for stably mapping brain surface local field potentials (LFPs) over a wide cortical region with high signal fidelity and minimal invasiveness to the brain tissue. To directly compare surface ECoG signals with intracortical neuronal activity immediately underneath, we fabricated a flexible multichannel electrode array with mesh-form structure using micro-electro-mechanical systems. A Parylene-C-based "electrode-mesh" for rats contained a 6×6 gold electrode array with 1-mm interval. Specifically, the probe had 800×800 µm(2) fenestrae in interelectrode spaces, through which simultaneous penetration of microelectrode was capable. This electrode-mesh was placed acutely or chronically on the dural/pial surface of the visual cortex of Long-Evans rats for up to 2 weeks. We obtained reliable trial-wise profiles of visually evoked ECoG signals through individual eye stimulation. Visually evoked ECoG signals from the electrode-mesh exhibited as well or larger signal amplitudes as intracortical LFPs and less across-trial variability than conventional silver-ball ECoG. Ocular selectivity of ECoG responses was correlated with that of intracortical spike/LFP activities. Moreover, single-trial ECoG signals carried sufficient information for predicting the stimulated eye with a correct performance approaching 90%, and the decoding was significantly generalized across sessions over 6 hours. Electrode impedance or signal quality did not obviously deteriorate for 2 weeks following implantation. These findings open up a methodology to directly explore ECoG signals with reference to intracortical neuronal sources and would provide a key to developing minimally invasive next-generation brain-machine interfaces.