Carbon nanotubes: artificial nanomaterials to engineer single neurons and neuronal networks

Abstract

In the past decade, nanotechnology applications to the nervous system have often involved the study and the use of novel nanomaterials to improve the diagnosis and therapy of neurological diseases. In the field of nanomedicine, carbon nanotubes are evaluated as promising materials for diverse therapeutic and diagnostic applications. Besides, carbon nanotubes are increasingly employed in basic neuroscience approaches, and they have been used in the design of neuronal interfaces or in that of scaffolds promoting neuronal growth in vitro. Ultimately, carbon nanotubes are thought to hold the potential for the development of innovative neurological implants. In this framework, it is particularly relevant to document the impact of interfacing such materials with nerve cells. Carbon nanotubes were shown, when modified with biologically active compounds or functionalized in order to alter their charge, to affect neurite outgrowth and branching. Notably, purified carbon nanotubes used as scaffolds can promote the formation of nanotube-neuron hybrid networks, able per se to affect neuron integrative abilities, network connectivity, and synaptic plasticity. We focus this review on our work over several years directed to investigate the ability of carbon nanotube platforms in providing a new tool for nongenetic manipulations of neuronal performance and network signaling.

Figure 1

Carbon nanotubes as scaffolds for neuronal growth. (a) Examples of chemical modifications on carbon nanotubes. Reprinted with permission from ref (17). Copyright 2011 The Royal Society of Chemistry. (b) MWCNT and glass growth supports used as the control condition in most studies are characterized by different roughness. AFM images (A,C) and three-dimensional plot profiles (B,D) of MWCNT and glass substrates, respectively. Reprinted with permission from ref (27). Copyright 2011 The Society for Neuroscience.

Figure 2

Neurons in close contact with CNT scaffolds show improved network activity and single-cell integrative abilities. (a) Exemplificative voltage-clamp recordings of postsynaptic currents (PSCs) recorded from hippocampal neurons cultured on glass or on CNT substrates; PSC frequency is significantly increased in CNT cultures. Reprinted from ref (28). Copyright 2005 American Chemical Society. (b) Top, scanning electron microscopy images showing SWCNTs substrate features (A) and the close and intimate contacts between CNTs and the neuronal membrane of cultured hippocampal neurons (subsequent micrographs in B–F). Reprinted with permission from ref (29). Copyright 2007 The Society for Neuroscience. Scale bar (in E): A, 1 μm; B, 200 μm; C, 25 μm; D, 10 μm; E, 2 μm; F, 450 nm. Bottom, transmission electron microscopy micrographs from planar sections of hippocampal cultures grown on CNTs, showing a single nanotube (highlighted in the box) “pinching” the neuronal membrane (bottom part of the image). Reprinted with permission from ref (30). Copyright 2009 Nature Publishing group. (c) Hippocampal neurons cultured on control glass (CTRL) or on CNT substrate (CNT) were forced to fire a train of six action potentials (by injection of current steps, i; top) in order to assess the presence of an additional afterhyperpolarization or afterdepolarization at the end of the train (gray shadow). CNT scaffolds significantly increased the fraction of neurons showing an afterdepolarization. Reprinted with permission from ref (30). Copyright 2009 Nature Publishing group. (d) The “electrotonic” hypothesis has been formulated in order to explain the increased ability to generate an afterdepolarization in neurons cultured on a CNTs layer. The hypothesis assumes that intracellular compartments are electrically exposed to CNTs, which could act as electrical shortcuts between distal cellular compartments. Reprinted with permission from ref (30). Copyright 2009 Nature Publishing group.

Figure 3

(a) Left, bright-field image of the experimental setting employed for paired patch-clamp recordings from hippocampal neurons cultured on glass or on CNTs scaffolds. The presynaptic neuron is forced to fire one or more action potentials, and the evoked postsynaptic currents (PSCs) are recorded from the postsynaptic neuron. Scale bar is 15 μm. Right, cultures interfaced to CNTs show a strong increase in the probability of finding connected cell pairs. (b) Left, confocal images from immunocytochemistry experiments highlighting the presynaptic component (vescicular GABA transporter,VGAT, clusters, in green) and the postsynaptic component (GABA receptor γ₂ subunit clusters, in red) in control (A) and CNT cultures (B), whose colocalization is the morphological evidence of the presence of synapses. A1–3 and B1–3 show the VGAT and GABA receptor γ₂ subunit signals separately (A1, A2, and B1, B2) or merged (A3, B3) in control and CNT cultures, respectively. Scale bars: A and B, 10 μm; A1–3 and B1–3, 2 μm. Cultures on CNTs show a marked increase in the number of colocalized clusters, i.e., synapses (right). (c) In paired recordings on control neurons, a train of action potentials elicited in the presynaptic neuron usually evokes depressing PSCs in the postsynaptic neuron, while in cells cultured on CNT scaffolds PSCs are usually not depressing. Reprinted with permission from ref (27). Copyright 2011 The Society for Neuroscience.

Figure 4

CNT scaffolds support the growth of spinal explants and remotely boost synaptic connectivity. (a) Scanning electron microscopy micrograph of a peripheral neuronal fiber of a spinal explant grown on a CNT layer, with numerous and very tight contacts between CNTs and the neuronal membrane (red arrows). Scale bar is 500 nm. (b) Left, schematic representation of the experimental setting: the dorsal root ganglion was electrically stimulated, while the evoked postsynaptic currents (ePSCs) were recorded from homolateral ventral interneurons. Middle, superimposed ePSCs recorded from interneurons from explants cultured on glass or on CNTs at the resting membrane potential (−56 mV), at the reversal potential for inhibitory currents (−40 mV, red; the excitatory component of the evoked response), and at the reversal potential for excitatory currents (0 mV, blue; the inhibitory component of the evoked response). The amplitude of both the excitatory and the inhibitory components was strongly increased in explants interfaced to CNT scaffolds (right). (c) Transmission electron microscopy micrographs from sagittal sections of spinal explants grown on CNTs, showing healthy tissue (mitochondria are highlighted by arrowheads and neuronal microtubule sections by boxes) and close contacts between CNTs and the bottom part of the explant (arrows). Scale bars: top left, 1 μm; top right, 500 nm; bottom left, 500 nm; bottom right, 200 nm. Reprinted from ref (10). Copyright 2012 American Chemical Society.