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Journal of Neuroscience Methods 182 (2009) 219–224

Contents lists available at ScienceDirect

Journal of Neuroscience Methods

journa l homepage: www.e lsev ier .com/ locate / jneumeth

ne-to-one neuron–electrode interfacing

lon Greenbauma, Sarit Anavab, Amir Ayalib,∗, Mark Sheina, Moshe David-Pura,shel Ben-Jacobc, Yael Haneina,∗∗

School of Electrical Engineering, Tel-Aviv University, Tel-Aviv, 69978, IsraelDepartment of Zoology, Tel-Aviv University, Tel-Aviv, 69978, IsraelSchool of Physics, Tel-Aviv University, Tel-Aviv, 69978, Israel

r t i c l e i n f o

rticle history:eceived 5 May 2009eceived in revised form 9 June 2009ccepted 10 June 2009

eywords:arbon nanotubesulti electrode arrayseurons

a b s t r a c t

The question of neuronal network development and organization is a principle one, which is closelyrelated to aspects of neuronal and network form-function interactions. In-vitro two-dimensional neu-ronal cultures have proved to be an attractive and successful model for the study of these questions.Research is constraint however by the search for techniques aimed at culturing stable networks, whoseelectrical activity can be reliably and consistently monitored. A simple approach to form small intercon-nected neuronal circuits while achieving one-to-one neuron–electrode interfacing is presented. Locustneurons were cultured on a novel bio-chip consisting of carbon-nanotube multi-electrode-arrays. Thecells self-organized to position themselves in close proximity to the bio-chip electrodes. The organiza-

euronal networkocust

tion of the cells on the electrodes was analyzed using time lapse microscopy, fluorescence imaging andscanning electron microscopy. Electrical recordings from well identified cells is presented and discussed.The unique properties of the bio-chip and the specific neuron–nanotube interactions, together with theuse of relatively large insect ganglion cells, allowed long-term stabilization (as long as 10 days) of prede-fined neural network topology as well as high fidelity electrical recording of individual neuron firing. Thisnovel preparation opens ample opportunity for future investigation into key neurobiological questions

and principles.

. Introduction

One of the most profound open challenges in modern scienceoncerns the emergence of the functioning brain from a collectionf individual neurons. This question translates to how an assem-lage of initially simple elements, namely neurons, develops andrganizes to form a new, complex and highly coordinated systemboth morphologically and functionally): the neural network.

In order to gain insight into the rules governing the develop-ent and mode of operation of neural networks, one needs both

o choose a system that is simple (relative to any in-vivo network),nd to be able to control as many of its variables as possible. In-vitrowo-dimensional cultures of invertebrate neurons offer an attrac-ive preparation for study because of the large size of the cells and

he facility with which they can be cultured in various conditionse.g. extremely low densities; see Rinkevich, 2005; Beadle, 2006or recent reviews). Most importantly, two-dimensional culturesnable easy access for non-invasive optical observations, allowing

∗ Corresponding author. Tel.: +972 3 6409820; fax: +972 3 6409403.∗∗ Corresponding author. Tel.: +972 3 6407698; fax: +972 3 6423508.

E-mail addresses: ayali@post.tau.ac.il (A. Ayali), Hanein@eng.tau.ac.il (Y. Hanein).

165-0270/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.jneumeth.2009.06.012

© 2009 Elsevier B.V. All rights reserved.

a close look at the dynamics of neural growth and network organiza-tion. The experimental results, or the rules discovered in this modelsystem, can then be translated to theoretical predictions and sim-ple model assumptions in order for them to be tested and appliedto more complex systems.

The study of the cybernetics of in-vitro neural networks reacheda turning point with the introduction of the multi-electrode arraytechnology (MEA, also referred to as the Neuro-Chip, e.g. Egertet al., 1998; Streit et al., 2001). The art of Neuro-Chip technologyencompasses interfacing biological neural networks with artificialcomputer-controlled arrays of electrodes. Thus the combination ofin-vitro two-dimensional cultures and MEA enables non-invasivemonitoring of the neural network electrical activity.

Although the MEA is a powerful tool for recording and stimu-lating large populations of neurons simultaneously it has limitedresolution and rather short-term capabilities on the single neuronlevel. This is mainly due to the fact that cells tend to migrate andin many culturing conditions also form dense clusters (Trenkner

and Sidman, 1977; Maar et al., 1997; Segev et al., 2003). To over-come this challenge several elaborate micro-fabrication approacheswere devised to stabilize cells to specific electrodes after the cellshave been manually brought to the desired location (Regehr etal., 1989; Maher et al., 1999; Zeck and Fromherz, 2001; Merz and

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20 A. Greenbaum et al. / Journal of Ne

romherz, 2005). It is also important to mention here the greatody of work dedicated to pattern cells using chemical modifica-ion of the MEA surface. Methods such as direct lift-off process, softithography using polydimethylsiloxane (PDMS) stamps and sev-ral related approaches have been successfully applied to achieveetwork patterning (Branch et al., 1998; Chang et al., 2003; Schollt al., 2000; Kam et al., 2001; Sorribas et al., 2002). However, dueo strong neuronal motility, these methods fail to achieve stabilitynd long-term electrode–cell coupling.

An ideal method would facilitate the positioning as well as thetabilization of individual cells at close proximity to recording elec-rodes to allow the study of well-defined networks by high fidelity

onitoring of the electrical activity and connectivity of each neuronn the network for long periods of time.

We have recently developed a unique carbon-nanotube (CNT)ased MEA in which the CNT electrodes are used to position andtabilize the cells and the network by way of strong mechanicalnteractions between the neurons and the CNTs (Anava et al., 2009;orkin et al., 2009). In the presented technique cells are not manipu-ated to reach the electrode site, rather the interaction between theells and the electrodes facilitate the cell positioning and the conse-uence stabilization. The highly-conductive CNTs also function asecording and stimulation sites, thus forming an optimized inter-ace with the neurons to achieve long-term electrical recordings. Inhe present work, we demonstrate the power of this approach byhe use of specially designed CNT substrates to pattern predefinedmall size networks of locust frontal ganglion neurons and recordheir electrical activity.

. Methods

.1. Fabrication of CNT MEA

CNTs were synthesized by a computer controlled chemicalapor deposition (CVD) system on p-type silicon substrates with a00 nm thermal oxide layer. The entire fabrication process has beenescribed in detail in a previous publication (Gabay et al., 2007).riefly, underlying TiN lines are used as conducting tracks. These

ines are passivated with sputtered Si3N4 which is later exposed athe regions of the active electrode using a reactive ion etch step. Ahin nickel layer is e-beam evaporated at the openings. The processs concluded with a CNT thermal CVD growth procedure utilizinghe nickel as a catalyst material. Test samples with isolated CNTslands (with various diameters) were also fabricated in a similarashion and were used to test the interaction between the cells andhe CNT surface.

.2. Chemical vapor deposition (CVD)

The lithography steps described above were followed by a CNTrowth process. The CVD process consists of an initial purge step inydrogen followed by a temperature ramping stage to the growthemperature. Once the temperature of the furnace had reached00 ◦C, a constant volumetric flow of 20 sccm (standard cubic cen-imeter per minute) of ethylene gas was added. The duration of thistep was 10 min.

.3. Neuronal cultures

The procedures for dissection and dissociation of locust frontalanglion neurons for primary cell culture have been previously

escribed in detail (Shefi et al., 2002a,b; Shefi et al., 2005; Fuchst al., 2007). Briefly, following dissection, neurons were dispersedy enzymatic treatment and mechanical dissociation and plated onither a culture dish that was pre-coated with Concanvalin A (ConA,.5 mg/ml, Sigma–aldrich, St. Louis, MO, USA) or on prefabricated

nce Methods 182 (2009) 219–224

CNT substrates. Plated neurons are fully differentiated adult neu-rons that have lost their original dendrites and axon, leaving onlythe soma. Culture media (L-15, Sigma) was enriched with 5% locusthemolymph. Throughout, the cells were maintained in darkness,under controlled temperature and humidity.

2.4. Light microscopy and culture monitoring

Cultures were maintained for as long as 10DIV in a special envi-ronmental chamber. The environmental chamber was placed on amicroscope stage (Nikon TS100) and images were acquired usinga CCD camera (Nikon DS-Fi1) controlled by a control unit (NikonDS-L2) to take time lapse images every 5–30 min. The pictures weretaken in magnification of 5×, 10×, 20× or 40×. Since locust culturesare sensitive to light, a programmed controller was used to controlthe illumination in order to minimize exposure of the culture tolight.

2.5. Immunohistochemistry

The procedure was preformed following (Loesel et al., 2006):cultures (7DIV) were fixed in 4% paraformaldehyde (freshly pre-pared from powder) in phosphate buffered saline (PBS) for 20 min,were rinsed twice in PBS and incubated for 30 min in permeabi-lization solution (5% Goat serum, 1 mg/ml BSA, 0.3% Triton). Thecultures were rinsed again twice in PBS incubated with primaryantibody, anti-HRP serum in blocking solution (PBS containing1 mg/ml BSA and 5% normal goat serum), at a dilution of 1:10,000 at4 ◦C over night. The next day, after rinsing in PBS, cultures were incu-bated with secondary antibody, Cy2-conjugated goat anti-rabbit(Jackson Immunoresearch, West Grove, PA) used at a dilution of1:200 in blocking solution for 4 h at room temperature. After rinsingtwice with PBS the cultures were mounted under glass cover slipsusing Fluorescent mounting medium (S3023, DakoCytomation). Alllabeled preparations were analyzed and photographed using a ZeissLSM 510 confocal microscope (Carl Zeiss, Jena, Germany).

2.6. Neurons labeling

Cell cultures were fixed in 4% paraformaldehyde (freshly pre-pared from powder) in phosphate buffered saline (PBS) for 20 minand were rinsed twice in PBS and incubated 2 h with DiO (V-22889,Molecular Probes) according to the manufacturer’s protocol. Thecultures were then rinsed several times in PBS and mounted influorescent mounting medium (S3023, DakoCytomation).

2.7. Scanning electron microscopy

Scanning electron microscopy observations were preformed asfollows: samples were fixed for 30 min in a PBS (79382, Fluka)solution with 2.5% glutaraldehyde (49629, Fluka) and 4% sucrose.The fixed samples were then dehydrated by rinsing for 5 min inincreasing concentrations of ethanol (25%, 50% and 75%), keepingthe sample covered with each of the ethanol solutions, followedby 10 min rinses with 96% and 100% ethanol solutions. Finally, thedehydrated samples were critical point dried using a Balzers Unioncritical-point drier and chrome coated (6 nm layer) (Emiteck K575X,SEM coating unit). The samples were examined using a JEOL 6700Fscanning electron microscope.

2.8. Extracellular recording

Extra-cellular recording were conducted utilizing low noisepre-amplifiers board (MEA-1060, amplifier, gain 1200× with aband-pass filter 200 Hz–5 KHz, Multi Channel Systems). The sig-nals collected from the microelectrodes were sampled at a 10 KHz

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ampling rate and stored on a personal computer equipped with128-channel, 12-bits data acquisition board (MC-Card, Multi

hannel Systems) and a MC-Rack data acquisition software (Multihannel Systems, version 3.2.20).

. Results

We first explored the manner by which locust neuronal net-orks develop on CNT substrates, and examined how theseetworks differ from networks cultured on control substrates cov-red uniformly with an adhesive layer (ConA). Cells from 10 locust

ig. 1. A comparison between locust neuronal networks plated on ConA substrate and CNetwork and same field of view as in (A) after 7DIV. A cell that retains its position is markeame network as in (C) after 7DIV. Four examples of cells that retain their position are mmage showing locust neurons (stained with neuron-specific anti-HRP immunostaining)30 �m diameter).

ig. 2. Time lapse observation of cell migration toward CNT islands. Culture is 7DIV. Tim80 min (F) 410 min. Scale bar, 25 �m. Arrows indicate specific features discussed in the t

nce Methods 182 (2009) 219–224 221

ganglia were plated on each substrate type and maintained in theincubator.

As illustrated in Fig. 1A, the neurons cultured on the ConA sam-ples developed processes and initially formed elaborate networks.However, once interconnections have been formed, cells tended tomigrate toward one another leading to the appearance of neuronal

clusters (Fig. 1B). Only after 10DIV the networks reached stabilityand were characterized by several dense clusters connected by thickbundles of neural processes. These results are in accordance withour previously reported results (Shefi et al., 2002a,b; Fuchs et al.,2007).

T substrate. (A) A neuronal network over ConA substrate after 5DIV. (B) The samed by a white asterisk. (C) A neuronal network over CNT substrate after 5DIV. (D) Thearked by a black asterisks. Scale bar, 60 �m in (A)–(D). (E) A confocal fluorescenceon CNT MEA. The single cells are positioned exactly on the round CNT electrodes

e between frames, relative to image (A): (B) 150 min (C) 210 min (D) 220 min (E)ext.

2 uroscience Methods 182 (2009) 219–224

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Fig. 3. Interactions between Locust cells and CNTs (A) A three-dimensional render-ing of a fluorescence confocal image of a neuron wrapped around a CNT island. Neuralcells were fluorescently labeled with the lipophilic trace DiO. Scale bar, 40 �m. (B)A HRSEM image of the cell-CNT interface. Scale bar, 1 �m. Arrows indicate tautneuronal processes.

22 A. Greenbaum et al. / Journal of Ne

In contrast, neurons cultured on the CNT samples were veryuick to form stable networks, with most of the cells located eithern close proximity and direct contact to a CNT island or directlyn the CNT surface (Fig. 1C and D). The data in Fig. 1C and D clearlyeveals the relative stability of the networks formed on the CNT pat-erned surfaces, in particular in comparison with cultures on ConAoated surfaces. Cultures remained stable for as long as 10DIV evenfter connectivity between cells has been formed. Apparently thedhesion to the CNTs is strong enough to compete with the forcesxerted by the neuronal processes. Fig. 1E exemplifies the neu-ons’ selectivity to the CNT islands, where single neurons positionedhemselves almost exclusively on the CNT surfaces. The stability ofhe locust neuronal networks over CNT substrates and the selec-ivity of the locust neurons to the CNT islands are in accordanceith our previous findings with mammalian neurons (Gabay et

l., 2005; Sorkin et al., 2006). Mammalian neuronal patterns onNT electrodes (Gabay et al., 2005; Sorkin et al., 2006) are con-picuously stable and can maintain their shape for as long as threeonths in appropriate culturing conditions. The advantage of sta-

ilizing invertebrate neuronal cultures using CNT islands comparedith the more common chemical patterning schemes is discussed

t length in Sorkin et al., 2006.Since cell positioning in our preparation is not forced but rather

riven by inherent cellular properties, it was important to explorehe process of cell stabilization on the CNT islands. This waschieved by time lapse and HRSEM imaging. Fig. 2 shows lighticroscope images of an individual cell cultured on CNTs, taken at

ix different time points during development of its neuronal pro-esses. Initially (at 7DIV) the cell was located between two CNTslands (CNT1 and CNT2, see Fig. 2A). The cell processes were dis-inctly bound to the two neighboring CNT islands (Fig. 2C). Whenne of the processes disconnected from CNT2, the cell migratedoward CNT1, to which the cell was still connected (Fig. 2E). Notehat the shape of the cell body in Fig. 2 changes from round (Fig. 2A)o irregular (Fig. 2C), as the processes bound to the two CNT islands.

hen the upper link is broken, the soma returns to its originalound shape (Fig. 2F). A plausible simple explanation to the abovebservation may be the effects of tension forces which developedn the cell processes (Anava et al., 2009; Sorkin et al., 2009).

As highlighted above the cell-CNT interaction is a major factornabling the stability of the networks. Fig. 3A is a confocal image oflocust neuron associated to a CNT island. The processes of the cellxtend from its soma and encircle the CNT island. Further details ofhe interaction between the neuron and the CNTs are demonstratedn the HRSEM image in Fig. 3B. The cell and its processes are onhe right-hand side of the image and on the left-hand side are theNTs. The apparent tautness of the processes (marked with arrows)estifies to the existence of tension forces in the dendrites.

Non-neuronal cells may also contribute to organization of neu-ons on the CNT islands (data not shown). The mechanism of neuronigration as a result of interactions with non-neuronal cells (e.g.

lia) was described by others (Distasi et al., 2002).We have thus far demonstrated the effectiveness of CNT islands

s a method for creating stable networks. Two additional advan-ages of CNTs, are their good electrical conductivity, and theirxcellent electrochemical specific capacitance (Gabay et al., 2007).hus, high density CNT coating is a most suitable interface mate-ial for neuronal applications. By integrating the CNT growth withicrofabrication processes, fully functional MEA devices were real-

zed. Electrode arrays with electrode diameters of 80 �m were usedo record electrical activity from the locust frontal ganglion (FG)

eurons (Fig. 4).

After 5DIV spontaneous electrical activity was measured (dataot shown). To enhance the electrical activity and to validate thatecorded signals are biological in nature, the network was chemi-ally excited with the muscarinic agonist Pilocarpine (PILO 10−4 M)

Fig. 4. A network of single cells over CNT electrodes 6DIV. The black circles arethe CNT islands (80 �m diameter); Examples of single cells over CNT electrodes aremarked with a white arrow. Scale bar, 80 �m.

A. Greenbaum et al. / Journal of Neuroscience Methods 182 (2009) 219–224 223

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ig. 5. CNT electrode recordings of electrical activity form a single cell, in the preseB) The probability density function of the inter-spike intervals. (C) A single recordine). (D) Light microscope image of the recording site in (A–C). The depicted cell is

Priel and Albuquerque, 2002; Rand et al., 2008). Fig. 5 illustrateshe electrical activity recorded after the introduction of PILO. Theeuronal activity was characterized by bursting events (Fig. 5A). Aursting event is a short time window (2–3 s) of extensive electri-al activity, followed by a long period of time (10–40 s) in which nolectrical activity is recorded. The inter spike intervals (ISI) distribu-ion (Fig. 5B) clearly shows two time scales, a time scale of hundredsf milliseconds, which corresponds to the time intervals within theurst, and a time scale of few seconds which correspond to the time

ntervals between bursting events. Fig. 5C shows an action potentialecorded from a single cell, and averaged activity of the cell (dashedine). Voltage traces were averaged over more than 200 spikes. Theocust neurons large soma size allowed us to confirm the existencef only one neuron cell at the recording site (Fig. 5D).

. Discussion

The challenges of the study of form-function interactions andheir role in neuronal network development and organization arewo fold. On the one hand there is a need to generate stable topolo-ies and even controlled and predefined network wiring diagrams.n the other, there is the challenge of high-resolution and high-delity electrical recording. Neuronal networks cultured on CNTubstrates offer an appropriate and promising answer to these dif-erent challenges.

We presented a model system of locust ganglion neurons cul-ured on specially designed substrates composed of CNT islands.ased on our observations, we believe that mechanical forces canxplain most of the details of the specific neuron-CNT interactions.e have recently described process entanglement as a neuronal

nchorage mechanism (Sorkin et al., 2009) and suggested a regu-ative role for neurite mechanical tension in network developmentAnava et al., 2009). Our current results re-confirm these reports

s we demonstrate straightening of the neuronal segments follow-ng attachment to the CNT islands. This is the result of mechanicalension that is created along the cell’s processes and pulls the cell’soma toward the end of the process; the site of attachment to theNT in most cases.

ilocarpine (40 �mol). (A) A raster plot demonstrating three typical busting events.ion potential and the averaged activity of the single cell over 200 samples (dashedll from the cell culture shown in Fig. 5 at 6DIV. Scale bar, 80 �m.

The effect of tension forces on the position of neurons is alsoreported in previous studies, in which single cells were physicallyconfined by specially constructed cages or wells (Claverol-Tintureet al., 2007; Maher et al., 1999). In these studies, tension competeswith the devices used for cells confinement. The tension gener-ated along maturing processes tends to pull the somata towardsthe edge of the cage, and the cells migrate in the direction of thelargest process (Maher et al., 1999). In other cases tension forcesalong processes were reported to mechanically pull neuron cellsout of microwells (Claverol-Tinture et al., 2007), or of cages (Maheret al., 1999). Hence our novel preparation is unique in harnessingthe mechanical forces to work toward keeping cells in place andstabilizing the network.

We demonstrated that a large stable network of single self orga-nized cells is formed in our low density cultures (around 500cells). Each single cell inhabits a CNT electrode thus, a one-to-oneneuron electrode interface is created, which provides high selectiv-ity electrical recordings. The network stabilization and electricalrecordings from single cells, as presented here, provide a proofof concept. We believe that the relatively low signals recordedfrom locust cells, as presented here, can be further improved. Thisimprovement can be obtained by reducing the electrode size andincreasing the electrode sealing by forcing cells to reside on topof the electrodes (i.e. by embedding the CNT islands in wells).The relatively low signals can be readily understood as the con-sequence of an incomplete sealing of the recording electrodes. It iswell known that partial exposure of the electrode to the electrolytesolution reduces the recorded signal amplitude. In the extreme case,of a completely exposed electrode, or highly conducting mediumthe signal will be undetected (Regehr et al., 1989). Thus, largerelectrodes are in fact less advantageous and smaller electrodes (pro-vided the noise level is within the noise level of the amplificationsystem) are preferred. When using high cell densities and in partic-

ular when performing MEA recordings from a tissue, significantlyhigher signals can be obtained (Shoval et al., 2009). In these casesthe culture or the tissue may contribute to better electrode sealing.

We have been unable yet to neither record spontaneous activ-ity nor signal propagation in our locust cultures. Activity was so

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ar recorded from single cells and only after chemical stimulation.t is likely that culturing conditions are of utmost importance andarameters such as cell density and the degree of connectivity areritical to maintain spontaneous activity and signal propagation.aturity of the culture is also critical to ensure sufficient synap-

ic development. Indeed, the ability to explore these and relatedffects is one of the strength of the proposed scheme and will allows and others to explore the link between the single cell and themall network realms. These issues are beyond the scope of theurrent publication and will be the focus of our future work. Weelieve that by using such preparations it will be possible to care-ully monitor the activity of the cells as they turn from individual,solated entities, into wired circuits. This in turn will allow thexploration of fundamental questions related to network develop-ent and activity. Questions such as: when and how spontaneous

ctivity is initiated, when do chemical and electrical synapses begino appear, and more.

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