Neural micro-electrode arrays that are transparent over a broad wavelength spectrum from ultraviolet to infrared could allow for simultaneous electrophysiology and optical imaging, as well as optogenetic modulation of the underlying brain tissue. The long-term biocompatibility and reliability of neural micro-electrodes also require their mechanical flexibility and compliance with soft tissues. Here we present a graphene-based, carbon-layered electrode array (CLEAR) device, which can be implanted on the brain surface in rodents for high-resolution neurophysiological recording. We characterize optical transparency of the device at >90% transmission over the ultraviolet to infrared spectrum and demonstrate its utility through optical interface experiments that use this broad spectrum transparency. These include optogenetic activation of focal cortical areas directly beneath electrodes, in vivo imaging of the cortical vasculature via fluorescence microscopy and 3D optical coherence tomography. This study demonstrates an array of interfacing abilities of the CLEAR device and its utility for neural applications.

Optogenetics is a technique for controlling subpopulations of neurons in the intact brain using light. This technique has the potential to enhance basic systems neuroscience research and to inform the mechanisms and treatment of brain injury and disease. Before launching large-scale primate studies, the method needs to be further characterized and adapted for use in the primate brain. We assessed the safety and efficiency of two viral vector systems (lentivirus and adeno-associated virus), two human promoters (human synapsin (hSyn) and human thymocyte-1 (hThy-1)) and three excitatory and inhibitory mammalian codon-optimized opsins (channelrhodopsin-2, enhanced Natronomonas pharaonis halorhodopsin and the step-function opsin), which we characterized electrophysiologically, histologically and behaviorally in rhesus monkeys (Macaca mulatta). We also introduced a new device for measuring in vivo fluorescence over time, allowing minimally invasive assessment of construct expression in the intact brain. We present a set of optogenetic tools designed for optogenetic experiments in the non-human primate brain.

Electrical stimulation using implantable electrodes is widely used to treat various neuronal disorders such as Parkinson's disease and epilepsy and is a widely used research tool in neuroscience studies. However, to date, devices that help better understand the mechanisms of electrical stimulation in neural tissues have been limited to opaque neural electrodes. Imaging spatiotemporal neural responses to electrical stimulation with minimal artifact could allow for various studies that are impossible with existing opaque electrodes. Here, we demonstrate electrical brain stimulation and simultaneous optical monitoring of the underlying neural tissues using carbon-based, fully transparent graphene electrodes implanted in GCaMP6f mice. Fluorescence imaging of neural activity for varying electrical stimulation parameters was conducted with minimal image artifact through transparent graphene electrodes. In addition, full-field imaging of electrical stimulation verified more efficient neural activation with cathode leading stimulation compared to anode leading stimulation. We have characterized the charge density limitation of capacitive four-layer graphene electrodes as 116.07-174.10 μC/cm based on electrochemical impedance spectroscopy, cyclic voltammetry, failure bench testing, and in vivo testing. This study demonstrates the transparent ability of graphene neural electrodes and provides a method to further increase understanding and potentially improve therapeutic electrical stimulation in the central and peripheral nervous systems.

Transparent graphene-based neural electrode arrays provide unique opportunities for simultaneous investigation of electrophysiology, various neural imaging modalities, and optogenetics. Graphene electrodes have previously demonstrated greater broad-wavelength transmittance (∼90%) than other transparent materials such as indium tin oxide (∼80%) and ultrathin metals (∼60%). This protocol describes how to fabricate and implant a graphene-based microelectrocorticography (μECoG) electrode array and subsequently use this alongside electrophysiology, fluorescence microscopy, optical coherence tomography (OCT), and optogenetics. Further applications, such as transparent penetrating electrode arrays, multi-electrode electroretinography, and electromyography, are also viable with this technology. The procedures described herein, from the material characterization methods to the optogenetic experiments, can be completed within 3-4 weeks by an experienced graduate student. These protocols should help to expand the boundaries of neurophysiological experimentation, enabling analytical methods that were previously unachievable using opaque metal-based electrode arrays.

Objective There is great interest in designing implantable neural electrode arrays that maximize function while minimizing tissue effects and damage. Although it has been shown that substrate geometry plays a key role in the tissue response to intracortically implanted, penetrating neural interfaces, there has been minimal investigation into the effect of substrate footprint on the tissue response to surface electrode arrays. This study investigates the effect of micro-electrocorticography device geometry on the longitudinal tissue response. Approach The meningeal tissue response to two micro-electrocorticography devices with differing geometries was evaluated. The first device had each electrode site and trace individually insulated, with open regions in between, while the second device had a solid substrate, in which all sixteen electrode sites were embedded in a continuous insulating sheet. These devices were implanted bilaterally in rats, beneath cranial windows, through which the meningeal tissue response was monitored for one month after implantation. Electrode site impedance spectra were also monitored during the implantation period. Main Results It was observed that collagenous scar tissue formed around both types of devices. However, the distribution of the tissue growth was different between the two array designs. The mesh devices experienced thick tissue growth between the device and the cranial window, and minimal tissue growth between the device and the brain, while the solid device showed the opposite effect, with thick tissue forming between the brain and the electrode sites. Significance This data suggests that an open architecture device would be more ideal for neural recording applications, in which a low impedance path from the brain to the electrode sites is critical for maximum recording quality.

Abstract-The brain is a large network of interconnected neurons where each cell functions as a nonlinear processing element. Unraveling the mysteries of information processing in the complex networks of the brain requires versatile neurostimulation and imaging techniques. Optogenetics is a new stimulation method which allows the activity of neurons to be modulated by light. For this purpose, the cell-types of interest are genetically targeted to produce light-sensitive proteins. Once these proteins are expressed, neural activity can be controlled by exposing the cells to light of appropriate wavelengths. Optogenetics provides a unique combination of features, including multi-modal control over neural function and genetic targeting of specific celltypes. Together, these versatile features combine to a powerful experimental approach, suitable for the study of the circuitry of psychiatric and neurological disorders.The advent of optogenetics was followed by extensive research aimed to produce new lines of light-sensitive proteins and to develop new technologies: for example, to control the distribution of light inside the brain tissue or to combine optogenetics with other modalities including electrophysiology, electrocorticography, nonlinear microscopy, and functional magnetic resonance imaging.In this paper, the authors review some of the recent advances in the field of optogenetics and related technologies and provide their vision for the future of the field.

Objective Spatial localization of neural activity from within the brain with electrocorticography (ECoG) and electroencephalography (EEG) remains a challenge in clinical and research settings, and while microfabricated ECoG (micro-ECoG) array technology continues to improve, complimentary methods to simultaneously modulate cortical activity while recording are needed. Approach We developed a neural interface utilizing optogenetics, cranial windowing, and micro-ECoG arrays fabricated on a transparent polymer. This approach enabled us to directly modulate neural activity at known locations around micro-ECoG arrays in mice expressing Channelrhodopsin-2 (ChR2). We applied photostimuli varying in time, space and frequency to the cortical surface, and we targeted multiple depths within the cortex using an optical fiber while recording micro-ECoG signals. Main Results Negative potentials of up to 1.5 mV were evoked by photostimuli applied to the entire cortical window, while focally applied photostimuli evoked spatially localized micro-ECoG potentials. Two simultaneously applied focal stimuli could be separated, depending on the distance between them. Photostimuli applied within the cortex with an optical fiber evoked more complex micro-ECoG potentials with multiple positive and negative peaks whose relative amplitudes depended on the depth of the fiber. Significance Optogenetic ECoG has potential applications in the study of epilepsy, cortical dynamics, and neuroprostheses.

Predicting the distribution of light inside any turbid media, such as biological tissue, requires detailed information about the optical properties of the medium, including the absorption and scattering coefficients and the anisotropy factor. Particularly, in biophotonic applications where photons directly interact with the tissue, this information translates to system design optimization, precision in light delivery, and minimization of unintended consequences, such as phototoxicity or photobleaching. In recent years, optogenetics has opened up a new area in deep brain stimulation with light and the method is widely adapted by researchers for the study of the brain circuitries and the dynamics of neurological disorders. A key factor for a successful optogenetic stimulation is delivering an adequate amount of light to the targeted brain objects. The adequate amount of light needed to stimulate each brain object is identified by the tissue optical properties as well as the type of opsin expressed in the tissue, wavelength of the light, and the physical dimensions of the targeted area. Therefore, to implement a precise light delivery system for optogenetics, detailed information about the optical properties of the brain tissue and a mathematical model that incorporates all determining factors is needed to find a good estimation of light distribution in the brain. In general, three measurements are required to obtain the optical properties of any tissue, namely diffuse transmitted light, diffuse reflected light, and transmitted ballistic beam. In this report, these parameters were measured in vitro using intact rat brain slices of 500 μm thickness via a two-integrating spheres optical setup. Then, an inverse adding doubling method was used to extract the optical properties of the tissue from the collected data. These experiments were repeated to cover the whole brain tissue with high spatial resolution for the three different cuts (transverse, sagittal, and coronal) and three different wavelengths (405, 532, and 635 nm) in the visible range of the spectrum. A three-dimensional atlas of the rat brain optical properties was constructed based on the experimental measurements. This database was linked to a Monte Carlo toolbox to simulate light distribution in the tissue for different light source configurations.