Interfacing graphene with peripheral neurons: influence of neurite outgrowth and NGF axonal transport

Graphene displays properties that make it appealing for neuroregenerative medicine, yet the potential of large-scale highly-crystalline graphene as a conductive peripheral neural interface has been scarcely investigated. In particular, pristine graphene offers enhanced electrical properties that can be advantageous for nervous system regeneration applications. In this work, we investigate graphene potential as peripheral nerve interface. First, we perform an unprecedented analysis aimed at revealing how the typical polymeric coatings for neural cultures distribute on graphene at the nanometric scale. Second, we examine the impact of graphene on the culture of two established cellular models for peripheral nervous system: PC12 cell line and primary embryonic rat dorsal root ganglion (DRG) neurons, showing a better and faster axonal elongation using graphene. We then observe that the axon elongation in the first days of culture correlates to an altered nerve growth factor (NGF) axonal transport, with a reduced number of retrogradely moving NGF vesicles in favor of stalled vesicles. We thus hypothesize that the axon elongation observed in the first days of culture could be mediated by this pool of NGF vesicles locally retained in the medial/distal parts of axons. Furthermore, we investigate electrophysiological properties and cytoskeletal structure of peripheral neurons. We observe a reduced neural excitability and altered membrane potential together with a reduced inter-microtubular distance on graphene and correlate these electrophysiological and structural reorganizations of axon physiology to the observed vesicle stalling. Finally, the potential of another 2D material as neural interface, tungsten disulfide, is explored

The impact of carbon based materials on hippocampal cells: from neurons to networks.

Tissue engineering and regenerative medicine require the constant development of synthetic materials to manufacture scaffolds thatbetter integrate into the target tissues (O\u2019Brien, 2011; Ku et al, 2013; Harrison et al, 2014). In this framework, newly synthesized nanomaterials made of pure carbon, in particular Carbon Nanotubes (Ijima, 1991) and Graphene (Novoselov et al, 2004) applications to biology received particular attention due to their outstanding physicochemical properties (Hirsch, 2010). Our team has performed pioneer works during the last decade, about the interactions of neural cells with carbon nanotubes (Lovat et al, 2005; Mazzatenta et al, 2007; Cellot et al, 2009; Cellot et al, 2011; Fabbro et al, 2012; Bosi et al, 2015), and with graphene (Fabbro et al, 2015; Rauti et al, 2016) or, more in general, with synthetic substrates (Cellot et al, 2016). The major aim of my work has been to use traditional and novel physiology tools to investigate further these \u201cneuro-hybrid systems\u201d, and to understand how far Carbon Nanotubes and Graphene can be pushed in neuroscience applications. With this aim, in the first part of my PhD I further elucidated the behavior of newly formed synapses in primary dissociated neurons when interfaced to bi-dimensional substrates of Multi-walled Carbon Nanotubes. I then addressed the homeostasis of invitro neural networks interfaced to pure graphene and I characterized for the first time the changes induced by this material in neurons. As last step, I set up a more complex biological in-vitro model, consisting of lesioned organotypic Entorhinal-Hippocampal cultures (Perederiy and Westbrook, 2013) and we described the regenerative features of Carbon Nanotubes in this lesion model. During my PhD I was also involved in two side projects: in the first one, in collaboration with Sebastian Reinhartz and Matthew Diamond (SISSA), we refine the possible approaches of the optogenetic technique, by manipulating neuronal responses with different light waveforms (Reinhartz et al, MS in preparation, in the appendix). In the second one, in collaboration with the group of Manus Biggs, from the National University of Galway, Ireland, we tested the biocompatibility and addressed the neural behavior of primary neural cells interfaced with Indium Tin Oxide (ITO) substrates with different roughness, thickness and conducting profiles (Vallejo-Giraldo et al, 2017)

Optogenetic Brain Interfaces

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 multimodal control over neural function and genetic targeting of specific cell-types. 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.United States. Defense Advanced Research Projects Agency (Space and Naval Warfare Systems Center, Pacific Grant/Contract No. N66001-12-C-4025)University of Wisconsin--Madison (Research growth initiative; grant 101X254)University of Wisconsin--Madison (Research growth initiative; grant 101X172)University of Wisconsin--Madison (Research growth initiative; grant 101X213)National Science Foundation (U.S.) (MRSEC DMR-0819762)National Science Foundation (U.S.) (NSF CAREER CBET-1253890)National Institutes of Health (U.S.) (NIH/NIBIB R00 Award (4R00EB008738)National Institutes of Health (U.S.) (NIH Director’s New Innovator award (1-DP2-OD002989))Okawa Foundation (Research Grant Award)National Institutes of Health (U.S.) (NIH Director’s New Innovator Award (1DP2OD007265))National Science Foundation (U.S.) (NSF CAREER Award (1056008)Alfred P. Sloan Foundation (Fellowship)Human Frontier Science Program (Strasbourg, France) (Grant No. 1351/12)Israeli Centers of Research Excellence (I-CORE grant, program 51/11)MINERVA Foundation (Germany

Transparent carbon nanotubes promote the outgrowth of enthorino-dentate projections in lesioned organ slice cultures

The increasing engineering of carbon-based nanomaterials as components of neuro-regenerative interfaces is motivated by their dimensional compatibility with subcellular compartments of excitable cells, such as axons and synapses. In neuroscience applications, carbon nanotubes (CNTs) have been used to improve electronic device performance by exploiting their physical properties. Besides, when manufactured to interface neuronal networks formation in vitro, CNT carpets have shown their unique ability to potentiate synaptic networks formation and function. Due to the low optical transparency of CNTs films, further developments of these materials in neural prosthesis fabrication or in implementing interfacing devices to be paired with in vivo imaging or in vitro optogenetic approaches are currently limited. In the present work, we exploit a new method to fabricate CNTs by growing them on a fused silica surface, which results in a transparent CNT-based substrate (tCNTs). We show that tCNTs favour dissociated primary neurons network formation and function, an effect comparable to the one observed for their dark counterparts. We further adopt tCNTs to support the growth of intact or lesioned Entorhinal-Hippocampal Complex organotypic cultures (EHCs). Through immunocytochemistry and electrophysiological field potential recordings, we show here that tCNTs platforms are suitable substrates for the growth of EHCs and we unmask their ability to significantly increase the signal synchronization and fibre sprouting between the cortex and the hippocampus with respect to Controls. tCNTs transparency and ability to enhance recovery of lesioned brain cultures, make them optimal candidates to implement implantable devices in regenerative medicine and tissue engineering. This article is protected by copyright. All rights reserved

Carbon-based transparent microelectrodes for optical investigation and electrophysiology

The goal of this work was to develop carbon based transparent electrodes for advancement of microelectrode array (MEA) technology by allowing the possibility of combining optical methods with classical electrophysiology. Recent years have seen a surge of interest in novel methods such as optogenetics and calcium imaging with the focus on understanding the complex neuronal networks. The conventional microelectrode materials obstruct the optical access, which is from the substrate side with an inverted microscope, and this limitation is overcome by using carbon materials. This work was focused on three main materials - carbon nanostructures, graphene and graphene/PEDOT:PSS (poly(3,4- ethylenedioxythiophene)). The transparency often comes at the cost of high electrochemical impedance. This challenge was tackled by using a novel combination of chemical vapour deposited (CVD) graphene and PEDOT:PSS. Carbon nanostructures were grown at 550 °C by CVD with acetylene as the carbon source. The morphology was studied by scanning electron microscope (SEM) and the presence of nanostructures mixed with amorphous carbon confirmed by Raman spectroscopy. The semitransparent nature was revealed by UV-Vis measurements. The electrochemical impedance was in the acceptable range for electrophysiological recordings. The functionality of the carbon nanostructure microelectrodes was confirmed by recording electrogenic signals from cardiomyocytes where the optical inspection of the cells through the semitransparent microelectrodes was possible. The mechanical robustness and biocompatibility was revealed by studying the electrode-cell ultrastructure. Graphene was grown by CVD with methane as the carbon source and integrated in the MEA fabrication process. The largely single layer graphene was investigated with SEM and Raman spectroscopy. The excellent transparency over the entire microelectrode was revealed by optical transmittance measurements. The graphene microelectrodes displayed high electrochemical impedance which led to high noise during electrophysiology. The functionality of the transparent graphene mircoelectrodes was checked with cardiomyocytes where high amplitude signals were detected similar to recording with standard electrodes, however, the smaller amplitude signals went unrecorded owing to the high noise. Graphene/PEDOT:PSS microelectrodes were fabricated by electrodeposition of the conducting polymer PEDOT:PSS on graphene microelectrodes. Optical microscopy revealed that PEDOT:PSS followed the graphene surface and the continuous coverage of the latter by the former reduced to sparse coverage with decreasing amount of PEDOT:PSS. Raman spectroscopy, especially in the case of lower PEDOT:PSS amounts, revealed the presence of PEDOT:PSS on regions which appeared transparent optically. This information was crucial in understanding the electrodeposition mechanism. The electrochemical impedance was found to be comparable with the commercially available TiN microelectrodes and the applicability was tested with cardiomyocytes. Optical imaging was possible through the transparent microelectrodes. An optimum balance between the optical transparency and electrochemical impedance was obtained which allows flexibility in producing microelectrodes for a wide range of applications. This work presents a comprehensive view on carbon based transparent microelectrodes for novel applications employing combinations of electro- and opto-physiology. The electrodes fabricated in this work are expected to go a long way in assistance with decoding the complex biological systems and provide insights on the single cell level

Roadmap on semiconductor-cell biointerfaces.

This roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world