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.