William Lee and Vladimir Parpura- Wiring neurons with carbon nanotubes

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Frontiers in Neuroengineering www.frontiersin.org May 2009 | Volume 2 | Article 8 | 1

NEUROENGINEERING

GENERAL COMMENTARYpublished: 29 May 2009

doi: 10.3389/neuro.16.008.2009

Wiring neurons with carbon nanotubes

William Lee and Vladimir Parpura*

Department of Neurobiology, Center for Glial Biology in Medicine, Atomic Force Microscopy & Nanotechnology Laboratories, Civitan International Research Center,

Evelyn F. McKnight Brain Institute, University of Alabama, Birmingham, USA

* Correspondence: [email protected]

A commentary on

Carbon nanotubes might improve neuro-

nal performance by favouring electrical

shortcuts.

by Cellot, G., Cilia, E., Cipollone, S.,

Rancic, V., Sucapane, A., Giordani, S.,

Gambazzi, L., Markram, H., Grandolfo, M.,

Scaini, D., Gelain, F., Casalis, L., Prato, M.,

Giugliano, M., and Ballerini, L. (2009). Nat.

Nanotechnol. 4, 126133.

Carbon nanotubes (CNTs) are emerging asnanomaterials with a wide range of biome-

dical applications, including their potentialuse for neuroprosthesis (Bekyarova et al.,2005). Thus, an understanding of howCNTs interact with cellular componentsof the brain is important for engineeringof CNT-based devices. The conductiveproperties and nanoscale features of CNTsmake them better suited as an interface

with neurons for stimulating and recor-ding neural activity than the conventional

bare metal-based electrodes (Keefer et al.,2008). Neurons cultured on CNT substratesshowed enhanced neuronal electrical acti-vity (Lovat et al., 2005; Mazzatenta et al.,2007) suggesting an intimate interaction

between neurons and CNTs. A recent studybyCellot et al. (2009)investigates the natureof CNTneuron interactions and proposesa mechanism in which CNTs can boostneuronal activity by providing a shortcutfor electrical coupling between somatic anddendritic neuronal compartments.

Cellot and colleagues performed whole-

cell patch clamp experiments on hippocam-pal and dorsal root ganglia (DRG) neuronscultured on planar CNT substrates depositedonto glass coverslips. They used a pharmaco-logical approach to block neuronal networkactivity. Cultured neurons were bathed in6-cyano-7-nitroquinoxaline-2,3-dioneand gabazine to inhibit glutamatergic and

gamma-amino-butyric acid-ergic synaptictransmission, respectively. This approachallowed for investigation of electrogenic

properties of patch-clamped neurons sincethe endogenous, as opposed to input/synap-tically driven, electrical excitability of cellsremained intact. To study the contributionof CNTs to neuronal electrical regenerativeproperties, the whole-cell current-clamped

single neurons were stimulated to dischargea train of six spikes/action potentials (APs),at frequencies ranging from 20 to 100 Hz.The post-train changes in neuronal mem-brane potentials were recorded and analy-

zed. The integrated membrane potentialchanges occurring within a 100-ms windowstarting 50 ms after the last AP of the train

were categorized as: (a) after depolarizationpotential (ADP), (b) neutral response (NR),or (c) after hyperpolarization potential(AHP) (Table 1). Interestingly, hippocam-pal neurons grown on CNT-coated glasscoverslips displayed a distribution of post-

train responses biased towards ADP whencompared to the distribution obtained fromneurons cultured on plain glass coverslips(Table 1).

After depolarization potential repre-

sents an electrical excitability based ondendro-somatic electrical coupling ari-sing from summation of dendritic Ca2+

Table 1 | The type of membrane potential events, and their proportion, occurring as a follow-up to

the train of action potentials evoked in hippocampal neurons grown on various substrates.

Post-train event

Substrate AHP NR ADP Source

GLASS

Neurons (n) 26 6 15 Figure 1b

Proportion (%) 55 13 32

CNTS



ITO



ITO+



ITO POOLED

Neurons (n) 30 8 15


RADA16

Neurons (n) 17 13 9 Figure 3e


Listed figures from Cellot et al. (2009)in the utmost right hand column were used as a source for generating

the number of events and their proportion from the whole. It should be noted that such an approach may

lead to some of the minor discrepancies that exist between numbers reported in this table and those found

in the text of the original article. The ITO pooled group represents the combined observations for low and

high conductance substrates, denoted as ITO and ITO+, respectively.

ADP, after depolarization potential; AHP, after hyperpolarization potential; ITO, indium tin oxide; ITO+, high

conductance ITO; NR, neutral response.


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Lee and Parpura Wiring neurons with carbon nanotubes


influx triggered by back-propagating APs;pharmacological experiments revealed theADP sensitivity to various voltage-gated

Ca2+

channel blockers (Figure 1, left). Itis then plausible that the CNT-inducedchange of neuronal excitability in the after

math of trains of APs might be through theenhancement of dendro-somatic coupling(Figure 1, right). If so, then reducing thedendritic arborization in the neuron expo-sed to CNTs should result in the absence ofADPs. To assess this possibility, the authorsperformed the experiments using the samestimulation paradigm: a train of APs on

DRG neurons. Unlike those from the hip-pocampus, DRG neurons contain next to

no dendritic arborization. Following sti-mulation, DRG neurons did not exhibit anyADPs. The authors experimentally ruledout the alteration of intrinsic neuronalproperties as a possible cause for the ADPenhancement by CNTs. Here hippocampal

neurons were excited to discharge singleAPs, rather than a train of them. There wasno appreciable difference in the amplitudeor waveform of single APs recorded fromneurons grown on CNT-coated and plainglass coverslips. Altogether, CNTs abilityto enhance neuronal excitability after a

discharge of a train of APs appears to bemediated by an enhanced dendro-somaticelectrical coupling, a phenomenon carriedout by Ca2+ influx via voltage-gated Ca2+channels. It should be noted, however, thatwater-soluble CNTs can reduce depolariza-tion-dependent Ca2+ influx from the extra-cellular space into hippocampal neurons

(Ni et al., 2005). The fact that CNT sub-strates used to grow neurons did not blockADPs, but rather enhanced them, suggests

that the presentation of CNTs (immobi-lized to a planar glass surface or water-soluble form), could dictate the type of

effect exerted onto neurons. Additionally,this finding implies that substrates usedhave an excellent retention of CNTs immo-

bilized to the glass coverslips, so that CNTsare not leaching out, which is consistentwith findings elsewhere (Malarkey et al.,2009).

Carbon nanotube-coatings exhibitelectrical conductivity and a nanoscale sur-face roughness, which is unlike a smoothand non-conductive glass surface. It has

been demonstrated that a CNT substratein a narrow range of conductivity can pro-

mote neuronal growth and neurite out-growth (Malarkey et al., 2009). Althoughnot systematically addressed, it has beenimplicated that surface roughness of CNTscan also affect neurite outgrowth (Sorkin

et al., 2009). To address whether individualsubstrate qualities, conductivity or surfaceroughness, could play a role in mediatingthe observed effect of CNTs on neuronalexcitability, Cellot et al. (2009) used twoadditional substrates to grow hippocam-pal neurons: (a) indium tin oxide (ITO), asubstrate with electrical conductivity and

a surface that lacks nanoscale roughness,and (b) RADA16 peptides, which can self-organize into nanofibers to form a sub-strate that is non-conductive, but displaysa nanoscale surface roughness similar tothat of CNT substrates. Pooled data fromthe examination of cultured neurons grownon ITO substrates showed that these cells

exhibit electrical excitability that closelyresembles that found in neurons grown onglass coverslips, rather than that of neurons

grown on CNT substrates (Table 1). Here,two ITO substrates with different resi-stivity/conductivity, 5 mm (2 S/cm)

and 0.005 mm (2000 S/cm), were used.Hippocampal neurons grew on these ITOsubstrates similar as those cells plated onto

plain glass, albeit neuronal morphologicalfeatures in these conditions were not quan-titatively assessed. Neurons were stimulatedusing the train of APs paradigm. It appearedthat neurons grown on the ITO substratewith higher conductivity (ITO+) displayedmore ADPs than those grown on ITO sub-strate with lower conductivity (2 S/cm);

the latter substrate conductivity is similarto the conductivity of CNT-coating depo-

sited onto glass coverslips (resistivity of11.2 mm, which corresponds to9 S/cmaverage conductivity). This phenomenon,although out of scope of the study, shouldbe re-visited in the future and assessed bysystematically varying conductive proper-

ties of CNTs while keeping their surfaceroughness unchanged. It may turn outthat this could represent a tool for neuro-prosthesis applications when modificationof neuronal excitability would be desira-ble. Nonetheless, the proportion of ADPsrecorded after the last AP from neurons

grown on the ITO substrates were eithersimilar to (ITO+), or lower than (ITO), theproportion recorded from neurons grownon glass coverslips (Table 1). Similarly,neurons cultured on RADA16 peptidesubstrates having a nanofiber meshworksimilar to that formed by CNTs, but non-conductive, displayed a lower proportionof ADPs after a train of APs than neurons

grown on smooth and non-conductiveglass coverslips (Table 1). Additionally, a

FIGURE 1 | Carbon nanotubes enhance excitability of hippocampal

neurons by providing electrical shortcuts. (Left) Dendro-somatic electrical

coupling (yellow bolt) arises from summation of dendritic Ca2+ influx triggered by

back-propagating action potentials (double arrows). (Right) Carbon nanotubes

(CNTs, red lines) can establish intimate contact with neurons causing

enhancement of this electrical coupling.

electrical coupling

+ CNTs


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Lee and Parpura Wiring neurons with carbon nanotubes


comparison of input resistance and capaci-tance measurements from neurons culturedon glass, CNT, ITO, and RADA16 peptidesdid not show any statistically significantdifference between these different sub-strates which indicates that these intrinsicproperties of neurons were unaltered. Thus,

either the conductivity or the nanotextu-ring per se might not be responsible forthe shift in the distribution of responsestowards ADPs recorded in neurons grownon CNTs (Table 1). Such an effect might bespecific to a substrate with the combinationof electrical, chemical, and structural pro-perties of CNTs.

As alluded to earlier, one possible expla-nation to the observation of ADP frequencyshift is that the CNTs with their size andtubular shape as well as conductive cha-racteristics could represent a shortcut for

electrical coupling between two (somaticand dendritic) neuronal compartments.The authors substantiated this provocative

hypothesis by theoretical modeling using atwo-compartment spiking neuron model.They were, indeed, able to reproduce CNT-mediated ADP and its sensitivity to Ca2+channel blockers. They furthermore usedmodeling strategy to predict the effect ofthis CNT-mediated modulation of neuro-nal excitability on the neuronal networks.

The prediction made based on computersimulations was then observed experimen-

tally using electrophysiological recordingfrom hippocampal neurons. Combined,these results indicate the existence of electri-cal coupling between CNTs and neurons.Such CNTneuron interfaces should then

be evident under electron microcopy (EM);the ultrastructural investigation usingtransmission EM showed tight contactsbetween CNTs and neuronal membranes.These hybrid areas should be of great inte-rest for further investigations. One of theissues to address would be whether CNTscould induce Ca2+ hot spots via cluste-

ring of voltage-gated Ca2+ channels and/ortheir increased expression at the plasmamembrane. Such action would represent

an additional regulatory point for CNTmodulation of the neuronal excitability.This would be especially interesting forneuroengineering, since it has been demon-strated that relatively low conductivity(0.430.9 S/cm) woven polyester substratesenhanced the cell adhesion of endothelial

cells and the level of interleukin-6 mRNAin monocytes/macrophages (Jakubiec et al.,1998). Similar relatively low conductivityCNT substrates (0.3 S/cm) caused neuro-nal morphological changes (Malarkey et al.,2009). Whether variations in conductivityof CNTs, while keeping their surface rough-ness relatively constant, can also correlate to

changes in neuronal protein expression andexcitability, and be harnessed for neuroen-gineering will need to be tested in carefullydesigned studies.

Taken together, Cellot et al. (2009)

suggest that CNTs can establish intimatecontacts with neurons resulting in theenhancement of neuronal electrical excita-

bility. A proposed mechanism of action isthat CNTs provide additional dendro-soma-tic electrical coupling pathways in parallelto the normal coupling route occurringthrough the neuronal cytosol/plasma mem-brane (Figure 1). While this model is thesimplest explanation for neuronal enhance-ment by CNTs, the neuronal morphologi-

cal changes induced by CNT substrates thatcould alter the passive neuronal membrane

properties, as well as modifications of thelevel and spatio-temporal subcellular cha-racteristics of protein expression, cannotbe ruled out. The results from this studyoffer further support for the potential useof CNT-based electrical interfaces and scaf-

folds for guided regeneration of lost neu-rons at the site of brain injury.

ACKNOWLEDGMENTS

We thank Randy F. Stout Jr for his com-ments on a previous version of this com-mentary. The authors work is supported

by a grant from the National Institute ofMental Health (MH 069791 to VP). Wededicate this work to Glenn I. Hatton, whose

energy and creativity inspired new views ofastrocyte-neuronal interactions.

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Received: 29 April 2009; published online: 29 May 2009.

Citation: Lee W and Parpura V (2009) Wiring neu-

rons with carbon nanotubes. Front. Neuroeng. 2:8. doi:

10.3389/neuro.16.008.2009

Copyright: 2009 Lee and Parpura. This is an open-

access publication subject to an exclusive license agreement

between the authors and the Frontiers Research Foundation,

which permits unrestricted use, distribution, and reproduc-

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source are credited.

William Lee and Vladimir Parpura- Wiring neurons with carbon nanotubes

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