Ion conducting polymer microelectrodes for interfacing with neural networks

Journal of Neuroscience Methods 160 (2007) 16–25

Ion conducting polymer microelectrodes for interfacing with neural networks Tobias Nyberg ∗ , Akiyoshi Shimada 1 , Keiichi Torimitsu 2 NTT Basic Research Laboratories, NTT Corporation, Atsugi, Kanagawa 243-0198, Japan Received 6 April 2006; received in revised form 3 August 2006; accepted 9 August 2006

Abstract We have examined the stimulation and recording properties of conjugated polymer microelectrode arrays as interfaces with neural networks of dissociated cortical cells. In particular the stimulation properties were investigated as a means of supplying a neural network with information. The stimulation efficiency at low stimulation voltages was evaluated and referenced to bare indium tin oxide (ITO) electrodes. The polymer electrodes were electrochemically polymerized from a blend of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT-PSS) and ethylenedioxythiophene (EDOT) onto ITO microelectrodes. Dissociated cortical cells were then plated on the electrodes and cultivated to form neural networks. Polymer electrode stimulation evoked a much greater response from the network than stimulation from ITO electrodes. Neural interfaces using polymer electrodes could be maintained for several months. © 2006 Elsevier B.V. All rights reserved. Keywords: Electrical stimulation; Neural network; Microelectrode array; MEA; Conducting polymer; PEDOT-PSS

1. Introduction Microelectrode arrays (MEAs) can be used to study different properties of neural networks such as network formation, network dynamics and memory functions (Droge et al., 1986; Meister et al., 1994; Kamioka et al., 1996; Jimbo et al., 1999; Beggs and Plenz, 2004). As our main interest is to clarify how neural networks process and store sensory input it is useful to stimulate the neuronal network and observe the response. For retinal explants this can be elegantly solved with light stimuli (Meister et al., 1994). For dissociated cultures without any normal sensory input a direct method for supplying the network with an input is to use electrodes to excite the network electrically (Jimbo et al., 1999; Wagenaar et al., 2004). Electrical interfacing with microelectrode arrays is also of interest for tentative neuronal prosthesis such as retinal interfaces (Zrenner et al., 1999) and interfaces with the peripheral nervous system (Nyberg et al., 2002; Stieglitz et al., 2002). All the above applications ∗

Corresponding author. E-mail addresses: [email protected] (T. Nyberg), [email protected] (A. Shimada), [email protected] (K. Torimitsu). 1 Tel.: +81 46 240 3610; fax: +81 46 270 2364. 2 Tel.: +81 46 240 3360; fax: +81 46 270 2364. 0165-0270/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2006.08.008

place a size restriction on the electrode in terms of obtaining an electrode density that gives the interface sufficient resolution to record selectively from and pass information to discrete neuronal populations. Ultimately the interface would allow for a one to one relationship between electrodes and neurons, which would imply a very high-density interface of small sized electrodes. Although modern photolithography makes it easy to fabricate high-density arrays of electrodes, the impedance of the electrodes increases as they are scaled down. The role of the electrode is essentially to act as a transducer of ionic current to electronic current for recording purposes and vice versa during stimulation. A lower impedance electrode simply allows a larger current to pass for a set voltage than a higher impedance electrode. A low impedance electrode thus disperses less of the energy of the signal to be recorded or the stimulus to be outputted than a higher impedance electrode. For recording purposes, one can compensate for a high impedance electrode to some degree by increasing the sensitivity of the amplifier. For stimulation purposes the applied voltage to the electrode can be increased, within certain limits, to overcome the problem of using a high impedance electrode and output a sufficiently large current to stimulate the neural tissue. The practical limit of the potential that can be applied is defined as the maximum potential that does not lead to electrochemical reactions that will either damage the electrode or have a detrimental effect on the neural tissue.

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For platinum electrodes, the −0.55 to +0.94 V range versus Ag/AgCl has been considered a safe potential window as regards avoiding gas formation at the electrodes (Rose and Robblee, 1990). Methods used for reducing the impedance of microelectrodes often involve increasing the area in contact with the electrolyte by creating a rough electrode surface. Conducting polymers have been utilized to increase the surface roughness and improve the performance of planar electrodes (Cui et al., 2001; Cui and Martin, 2003). Another approach is to make the electrode material highly permeable to the electrolyte. Here the electrolyte will be in contact with the bulk material of the electrode and consequently the interface area of the electrode will be much larger than that of an impermeable electrode (Ghosh and Inganas, 1999, 2000; Nyberg et al., 2002; Kim et al., 2004). The stimulating and recording performance of the electrode can consequently be enhanced by increasing the height of the electrode, within certain limits, while still retaining a small lateral surface area. In effect, this type of electrode makes use of three dimensions for its electrical properties. For any high-resolution neural interface with a high electrode density the use of a three-dimensional electrode appears to be a very attractive approach for enhancing the electrical properties of the interface. 2. Materials and methods 2.1. Microelectrode arrays The MEAs consisted of a square array of 8 × 8 ITO electrodes photolithographically defined in a 0.5 ␮m thick photoresist layer. The array pitch was 150 ␮m and the sides of the ITO square electrode were 50 ␮m, as shown in Fig. 1.

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2.2. Electrochemical methods The polymer electrode growth, impedance measurements, current voltage scans and pulse measurements were undertaken with an Autolab PGSTAT30 equipped with a multiplexer module SCNR16 (Ecochemie, The Netherlands). PEDOT-PSS electrodes were prepared from a mixture of PEDOT-PSS (H.C. Starck, Japan) and the monomer EDOT (which was a gift from Bayer, Japan). Ten microliters of EDOT was dissolved in 10 ml of PEDOT-PSS by stirring and sonification. The electrodes were fabricated by passing a charge of 12.5 ␮C per electrode (0.5 C/cm2 ) in the potentiostatic mode at a potential of 0.8–0.9 V as referenced to an Ag/AgCl electrode. Unless otherwise stated, the polymer electrodes were deposited on 32 of the ITO electrodes following the configuration in Fig. 1. Freshly made electrodes were characterized within hours of fabrication in 0.15 M NaCl. Impedance spectroscopy was undertaken for each electrode at 51 frequencies logarithmically distributed between 1 Hz and 100 kHz. Pulse tests were performed for each electrode by outputting voltage controlled biphasic pulses with lengths of 100 ␮s and 1 ms per phase. 2.3. Topographic characterization Atomic force microscopy was performed in water or air using a Digital Instruments Nanoscope IV (Veeco Metrology Group, USA) in tapping mode. Optical profiling was undertaken with a VK-9510 violet laser color 3D profile microscope (Keyence, Japan). 2.4. Cell culture The MEAs were coated with a blend of laminin (20 ␮g/ml; Sigma, Japan) and poly-d-lysine (50 mg/ml, M.W. 70,000–150,000; Sigma) in Dulbecco’s Modified Eagle Medium (Gibco, Japan) at 37 ◦ C overnight. Dissociated cortical cells from Wistar rat embryos (embryonic day 18) were cultivated in 1–1.5 ml of cell medium. The protocol described by Kamioka et al. (1996) was followed for the dissociation procedure and medium composition. In addition 2.5 mg/ml insulin, 50 U/ml penicillin and 50 ␮g/ml streptomycin (Sigma) were added to the medium. The cultures were kept at 37 ◦ C in 10% CO2 and one-half of the medium was changed every 3–4 days. 2.5. Neuronal recording and stimulation

Fig. 1. MEA with 64 electrodes divided into groups of polymer (lower left corner) and ITO electrodes, before cell plating. Stimulus tests were performed by scanning the stimulus site at 1 s intervals, starting at the upper left corner and ending at the lower right corner. The site was shifted to the next column to the right, shifting down a row after the eighth column was reached and then restarting from the first column of the next row.

Neural multisite recordings were obtained with a purpose built recording/stimulation system (Jimbo et al., 2003) at a sampling rate of 40 kHz. The preamplifier input impedance was 1 G at 1 kHz. Of the 64 available electrodes, we used 63 to record neural activity; 1 amplifier channel was used to record a trigger signal. One stimulation trial was performed by sequentially scanning the stimulus site through all 64 electrodes with a 1 s interval using one biphasic pulse of 100 ␮s/phase with a leading negative phase. This trial was repeated 10 times for each stimulation voltage, with a 5 s interval between trials. The series of amplitudes we used ranged from 0.1 to 0.5 V with

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an increment of 0.1 V. Stimulation trials were carried out on cultures that exhibited synchronized bursting after 2 weeks in culture and for at least 60 electrodes. 2.6. Immunostaining The cultures were first briefly rinsed with phosphate buffer saline (PBS) prior to fixation in a 4% paraformaldehyde solution for 15 min, followed by three 5–10 min washes in PBS. The cultures were then incubated at 37 ◦ C for 30–60 min with primary antibodies against neurofilament (mouse anti-neurofilament m, 145 kDa, monoclonal antibody, MAB1621; Chemicon, USA) diluted with PBS (1:50). This was followed by a brief rinse in PBS and then three washes in PBS of 5 min each. The cultures were then incubated with secondary antibodies conjugated with Alexa 488 (Alexa Fluor 488 chicken anti-mouse IgG; Molecular Probes, USA), used at a PBS dilution of 1:400 (2.5 ␮g/ml), for 30 min at room temperature followed by three brief washes. The samples were mounted with cover slips and imaged using a Nikon Eclipse TE300 microscope (Nikon, Japan) equipped with a Canon EOS D60 digital camera (Canon, Japan). 2.7. Analysis We analyzed the data on a personal computer using purpose written algorithms implemented in a MATLAB 7.0 environment (The MathWorks Inc., USA). Neuronal spikes were detected with an algorithm that searched for the signature of a local minimum point at least 20 ␮V below the average of the two nearest local maxima. The spikes had to occur in a maximum time window of 3.2 ms or they were discarded. The flank times, defined as the times between the minimum and maximum values of the waveform, had to be at least 100 ␮s.

Fig. 2. Bode plot of polymer and ITO electrodes averaged over eight electrodes for each type of electrode. Impedance (circles) and phase (solid line) of polymer electrodes and impedance (squares) and phase (dashed line) of ITO electrodes. The full length of the error bars represents two standard deviations.

The difference between the impedances of the ITO and polymer electrodes did not affect the signal shape of the neural activity to any greater degree, but we noticed that some recording channels that were influenced by hum noise when recording from an ITO electrode suffered less if the ITO electrode was substituted for a polymer electrode. Voltage controlled pulse measurements revealed that the peak current output of the polymer electrode was proportional to the applied potential, as shown in Fig. 3. The same figure makes it clear that the polymer electrodes could sustain a higher current output for a longer time than the ITO electrodes showing

3. Results 3.1. Polymer electrodes The polymer electrodes grown at 0.8 V exhibited a rounded geometry with a height of around 2 ␮m in the dried state, as estimated from AFM and optical profiling measurements (data not shown). We estimate the height in the swollen state to be considerably greater but, as the vertical range of the AFM was limited to around 5 ␮m, and the optical profiling only could be performed in air, it was difficult to obtain the height in solution. Nevertheless, the swollen state seems to be several micrometers. Increasing the growth potential to 0.9 V gave a more square geometry with a larger lateral area and less height in the dried state. The electrical parameters of the 0.9 V electrodes were similar to the electrodes grown at 0.8 V. Unless otherwise mentioned we will henceforth use ‘polymer electrode’ to refer to electrodes grown at 0.8 V. The impedance of the polymer electrodes was markedly lower than the impedance of the ITO electrodes for low and medium frequencies, as shown in Fig. 2. The 1 kHz magnitude of the impedance was around 5 k for the polymer electrodes whereas the value for the ITO electrodes was around 270 k.

Fig. 3. Average current amplitude induced by voltage pulses for polymer and ITO electrodes, the average is over eight electrodes per type. The pulses were biphasic with a leading negative phase. A 100 ␮s long negative potential followed by a 100 ␮s long positive potential was applied (short pulse) at 1 ms. This was repeated at 2 ms but with each phase having a length of 1 ms (long pulse). The pulse shape is shown in the inset. Black lines denote increasing current from the polymer electrodes for voltage pulses with amplitudes of 0.1–0.5 V, increasing with intervals of 0.1 V. The red line indicates the current from the ITO electrodes subjected to voltage pulses of 0.5 V amplitude.

T. Nyberg et al. / Journal of Neuroscience Methods 160 (2007) 16–25

that the polymer electrodes have a larger charge storage capacity. The main charging of the ITO electrodes was completed within 100 ␮s. The faradic current, which involves electrochemical changes to the solution, made a negligible contribution to the total current. For the 0.5 V pulses, it was less than 40 nA as evaluated from the residual current tail of long pulses on

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the polymer electrodes. With the ITO electrodes a large capacitive current can be seen when the polarity is switched, the peak amplitude for the positive phase at the switch was double that of the negative phase. A similar but less prominent phenomenon can be seen for the polymer electrodes, and is especially visible with 1 ms pulses, as shown in Fig. 3. The peak current amplitude

Fig. 4. Screenshot of activity from an MEA. Each window represents a 1.5 s long recording from an electrode, the full height of each window corresponds to 200 ␮V. The lower left corner beneath the dashed line shows the recorded data of square polymer electrodes. The window in the lower right corner shows the trigger signal. (a) A burst of activity from a culture at 5 DIV, most activity is seen on the ITO electrodes. Two ITO electrodes are heavily influenced by hum noise. (b) A burst of activity from the same culture at 8 DIV. Activity is more evenly dispersed on both the ITO and polymer electrodes.

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for the polymer electrodes was found to decrease to around 25% of its original value after 2 months under culture conditions. 3.2. Spontaneous activity and biocompatibility Spontaneous activity could be detected by both ITO and polymer electrodes after 5 days in vitro. As the networks matured, the bursting frequency increased, as noticed by others (Kamioka et al., 1996). During the stimulation trial we observed spontaneous activity at all the working channels. We noticed that the activity of three out of four young cultures grown on substrates with ITO and polymer electrodes appeared more intense on the ITO part, as seen in Fig. 4a. However, this difference levelled out with time, as seen in Fig. 4b. When we inspected the network morphology of a 22 DIV sample we saw two distinct types of neuron behaviour on the polymer electrodes, as shown in Fig. 5. The neurons followed the contours of the round polymer electrodes. For the square type 0.9 V electrodes, the neural processes could also be seen traversing the polymer surface showing that the material exhibited the biocompatibility needed for neurons to grow on the electrodes. 3.3. Evoked activity The stimulation efficacy protocol was carried out under normal conditions without blocking agents; the stimulated activity was thus concealed by a background of spontaneous activity, as seen in Fig. 6a. However, after averaging the responses for 10 sequences, the stimulation-induced activity becomes clearly visible, as seen in Fig. 6b. After stimulation, a stimulus artefact reducing circuit (Jimbo et al., 2003) blanked out the recording for 1.2–4.25 ms depending on the channel. To avoid any differences in our results caused by the different time lags we have omitted, from our calculations, any data obtained during the first 4.25 ms after stimulation.

Fig. 5. Epifluorescence image taken of neurons stained with antibodies for neurofilament with a culture age of 22 DIV. The round polymer electrodes on the left were fabricated at 0.8 V and the square electrodes on the right at 0.9 V. The red background is caused by fluorescence from the insulating layer of the photoresist. Many neurons seemed to follow the circumference of the round electrodes whereas the square electrodes were more freely traversed by neuronal processes.

Fig. 6. Total stimulated and spontaneous activity recorded from all the electrodes on a substrate with polymer and ITO electrodes. The data are divided into 10 ms bins. The ticks indicate when stimulation was applied. Stimulation by a polymer electrode is marked with a star. Activity is presented as the average of the recorded spikes per electrode. (a) One trial of 0.2 V stimulations and (b) average of 10 trials of 0.2 V stimulations. The inset shows a magnification of the 16–16.25 s interval. Each bar represents a bin of 10 ms.

An evoked burst of activity lasted for less than an average of 300 ms, as shown in the inset of Fig. 6b. Some bursts had a time span during which there was slight residual activity that extended beyond this time frame. To compare the stimulation efficiency we summed the activity within a time window of 25 ms following the stimulus. The 25 ms time frame gives the response of the early phase activity, characterized by the precise and reproducible activity of the network (Jimbo et al., 2000). Stimulation trials performed at 36 DIV for three samples revealed the efficiency of stimulating with polymer electrodes, as shown in Fig. 7a. The minimum stimulus voltage of 0.1 V generated a weak response for samples 1 and 3, while the third sample exhibited a clear response. For stimulus strengths above 0.1 V the polymer electrodes evoked a response that was clearly stronger than that of the ITO electrodes. Whereas the activity

T. Nyberg et al. / Journal of Neuroscience Methods 160 (2007) 16–25

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polymer and ITO electrodes at a stimulus of 0.1 V were almost the same. Increasing the stimulation voltage to 0.2 V greatly increased the response from the polymer electrodes while the response from the ITO electrodes decreased. If we look at the total response we can see that the 0.2 V polymer stimulations were clearly effective in exciting the network of all three samples. The relative differences between the different samples were also clearly reduced at this voltage compared with the 0.1 V stimuli. In contrast to the early phase it can also be seen that samples 1 and 2 exhibited peak activity for stimulus voltages smaller than the maximum voltage. For the ITO electrodes the response to stimulation increased as the stimulation voltage increased in the 0.2–0.5 V range. The polymer electrodes from only one sample exhibited a monotonously increasing response with stimulation voltage. In general the 300 ms response showed a relatively larger standard deviation than the early phase response. We can expect the stimulation trials to interact with the dynamics of the network and affect consecutive trials. To investigate this we summed the response for each trial and averaged

Fig. 7. Evoked activity at 36 DIV from sample 1 (blue bars polymer electrodes), sample 2 (green bars polymer electrodes) and sample 3 (grey bars polymer electrodes). The evoked activity from ITO electrodes is shown as white bars in front of the respective sample’s polymer bar. The recorded activity from all electrodes due to stimulation by either polymer or ITO electrodes was averaged over ten 10 stimulation trials to yield the mean recorded response per electrode on the MEA. The full length of the error bars represents two standard deviations. (a) Average activity recorded by one electrode within 25 ms of the stimulus and (b) average activity recorded by one electrode within 300 ms of the stimulus.

evoked from samples 1 and 3 steadily increased with stimulus voltage, that of sample 2 levelled out after 0.3 V. For sample specific ranges the activity was roughly proportional to the stimulus strength, as can be seen in Fig. 7a. To investigate the total response of the network containing a greater amount of synapse mediated activity than the early phase, we extended the summation time frame to a window of 300 ms following the stimulus. Increasing the time frame to more than 300 ms would include some longer bursts but would also lead to an incorporation of more unwanted spontaneous activity in the result. The total evoked response for polymer stimulation was still much greater than for ITO stimulation, even if the relative difference was decreased compared with the early phase response, as shown in Fig. 7b. For sample 1, the responses of the

Fig. 8. Relative intensities for samples 1–3 (black, red and green lines) at 36 DIV. The data were averaged over stimulus voltages of 0.3–0.5 V for all electrodes. The full length of the error bars represents two standard deviations. (a) Early phase response and (b) total response.

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the results with the sum of the responses from all the trials. This result was then summed and averaged for all the stimulus voltages resulting in a relative intensity of activity for each trial. Depending on when the trial was performed in the series, the resulting relative intensity then revealed the efficiency of this particular position, within the series, in evoking a response. The trend was a slightly larger response for the early phase and total response of the first trial in each series compared with the nine consecutive trials, as seen in Fig. 8a and b. The behaviour of samples 1 and 3 was followed for an extended time of 56 and 49 DIV, as shown in Fig. 9 (data not shown for sample 3). For the polymer electrodes, the maximum early phase response was acquired at 36–42 DIV, as shown in Fig. 9a. The ITO response also reached its maximum value around 36–42 DIV. As seen in Fig. 9b, even though there was an increase in the evoked activity for the last measurement, a similar increase was seen for sample 3. In both cases the trials were terminated the week after the last presented measurements, as there was almost no activity. During the intervening time, the

cell cultures had degenerated so that little or no activity could be detected after the last trial. For sample 1, the cell layer had clearly delaminated from the substrate and was not in contact with the electrodes. We were able to record from and stimulate a culture with polymer electrodes for up to 4 months whereupon the culture had to be terminated so that we could undertake equipment maintenance. At this time only sparse low amplitude activity could be detected on 20 of the electrodes. The total response of the polymer electrodes for sample 1 decreased with time while the total response for the ITO electrodes clearly peaked at 42 DIV, as seen in Fig. 9c and d. For sample 3, the responses of both the polymer and ITO electrodes peaked at 36 DIV. Plotting the early phase and total response of each polymer electrode of sample 1 revealed that the decreased response over time was caused by a decrease common to all electrodes (data not shown for sample 3), as shown in Fig. 10a and c. The early phase activity from the polymer electrodes was very homogenous with most electrodes displaying similar capabilities to excite the network, as seen in Fig. 10a. In contrast the early phase activity of

Fig. 9. Time series of early phase and total evoked activity for sample 1 at different stimulation voltages. The data correspond to the average response for any electrode over 10 trials (see Fig. 6). (a) Early phase, stimulation by polymer electrodes, (b) early phase, stimulation by ITO electrodes, (c) total activity, stimulation by polymer electrodes and (d) total activity, stimulation by ITO electrodes.

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the ITO electrodes was more dispersed, with some electrodes showing a large response, as shown in Fig. 10b. However, as with the polymer electrodes, the total activity resulting from ITO electrode stimulation decreased with time, as seen in Fig. 10d. In Fig. 10a, some electrodes exhibit maximum activity for a low stimulus voltage. However, the average activity over all the electrodes increased monotonously with increasing stimulus strength, as seen in Fig. 9. The characteristics of sample 3 were similar to those of sample 1 (data not shown). 4. Discussion The polymer electrodes described in this work make use of the PEDOT-PSS water dispersion of swelled polymer particles to provide the counterions for electropolymerization. Previous studies of polymer electrodes fabricated by using PEDOT-PSS and polypyrrole have shown that these electrodes are fast with a high charge delivery capacity and act as supercapacitors (Ghosh and Inganas, 1999, 2000; Nyberg et al., 2002). This is due to the swelled nature of the polymer matrix in an aqueous environment, providing high ionic conductivity and a large contact area between the electroactive material and the electrolyte. In the current work we chose to use EDOT as the monomer for

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electropolymerization as it is more stable than pyrrole in an aqueous environment (Yamato et al., 1995). The electrical performance of the electrode is affected by the degradation of the polymer material as well as the interface between the polymer and ITO. We believe these factors to be jointly responsible for the deterioration in electrode performance over time. Nevertheless, we found the performance of the electrodes to be adequate for more than 1 month, which is reasonable for a large number of experiments on dissociated cells. A concern when introducing a new cell culture material is that it should be biocompatible. The stimulation response from an electrode will not only depend on the strength of the pulse it can deliver but also on the local network morphology and the distance to the neurons (Rattay, 1998). For instance, if the electrodes were to repel neurons, effectively increasing the distance between electrode and neuron, the efficacy of the stimulation would be decreased. As neurons were able to grow on the polymer material, namely the square polymer electrodes shown in Fig. 5, the material itself does not seem to invoke any adverse reaction from the neurons. Instead we attribute the growth pattern near the round 0.8 V electrodes to the topography of the electrode as noticed by others for topographically patterned guidance structures (Hirono et al., 1988; Britland et al., 1996;

Fig. 10. Time series of evoked activity per stimulating electrode for sample 1. For each trial day the responses from the stimulation voltages of 0.1–0.5 V are displayed. Data correspond to the average response over 10 trials, as measured by all the electrodes, caused by the stimulation from the respective electrode. (a) Early phase, stimulation by polymer electrodes, (b) early phase, stimulation by ITO electrodes, (c) total activity, stimulation by polymer electrodes and (d) total activity, stimulation by ITO electrodes.

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Fig. 10. (Continued ).

Nyberg et al., 2002). It should be noted that we used a resist thickness of only 0.5 ␮m and a polymer height of several micrometers. We are currently using a thicker resist layer to reduce the amount of polymer protruding from the surface. In this case, when the polymer electrode can be grown level with the substrate surface, the influence of the topography should be reduced and the effect of the polymer material on the growing network should be more apparent. However, the topography of the electrode could also be used to influence the growth of the neural tissue and this could be of interest in applications where directed nerve growth is desired (Chang et al., 2001; Nyberg et al., 2002). The main cause of the relatively large response to stimulation from polymer compared to ITO stimulation is undoubtedly connected with the larger current output. As the current is increased, neurons with a higher threshold to excitation may be recruited, leading to an increase in the number of neurons that drive the initial excitation of the burst (Wagenaar et al., 2004). It is interesting to note the way in which the early phase response differs from the total response. For all samples the early response increased with increasing stimulus voltage even though sample 2 levelled out with the 0.4 and 0.5 V stimulus. In the same manner, only the total response for sample 3 increased for all increasing stimulus strengths. Obviously there is an upper limit to the amount of activity the network can present. The limit will depend on such aspects of the network layout as the wiring and the cell density. This means we cannot expect exactly the same response from

each network, instead the response will bear the signature of the network. The signature will also be dependent on the experimental layout. The slightly larger total response for the first trial in each series shows that the network was affected by the longer rest of 2 min between voltage series, compared with the 5 s rest between trials. The intrinsic properties of the cells (Soleng et al., 2003) and synaptic efficiency may both be affected by the prolonged stimulation and yield a lesser response. The same phenomena may also be behind some of the reduction in the evoked total activity of samples 1 and 2 for the high stimulus strength trials, as these trials came at a late stage during the experiment. We note that the average early phase activity, which includes less synaptic activity, always exhibited the maximum evoked activity for the greatest stimulation strength, as shown in Fig. 9. The decrease in the evoked activity of the polymer electrode, after more than 1 month of culturing (Figs. 9 and 10), is probably influenced by the deterioration of the polymer electrode, which reduces the stimulation current of the electrodes. However, the similar reduction in the evoked activity from the ITO electrodes, which we expect to be more stable, suggests that the decrease can to some extent be attributed to the changing properties of an aging neural network. A comparison of the individual ITO electrode responses with the polymer electrode response seen in Fig. 10 shows the uniform efficiency of the latter. Using a simple model, we estimated

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that it is very probable that the action potential initiates around the electrode edges due to the potential difference there. The homogenous response from the polymer electrodes in Fig. 10a and c is therefore due to a local excitation of the network spreading outwards from the electrode. On a millimetre scale, the most distant polymer electrodes were ∼1.4 mm apart, the network architecture allows for a number of different initiation sites for a burst. From this point of view, the network architecture around one electrode is similar to that around any other electrode. On the other hand, the inhomogeneous response from the ITO electrodes seen in Fig. 10b and d, probably results from inefficient stimulation owing to the low current output from these electrodes. In this case it is harder to use stimulation to probe the layout of the network. These early results show that polymer electrodes can be used for more than 1 month to interface successfully with neural networks. As these are our first results for interfacing polymer electrodes with neural networks we believe that there is much room left for the development and optimisation of the polymer electrodes. 5. Conclusions Polymer electrodes can be used at low stimulating potentials for the efficient stimulation of neuronal tissue for more than 1 month and interfacing can be maintained for several months. Polymer electrodes can also be tuned through a range of current densities, within a small voltage range, to allow for varying degrees of stimulation. The stimuli from polymer electrodes were vastly more efficient than with ITO electrodes. It is also important to consider that the material is satisfactorily biocompatible to be used as an electrode in this experiment. The shape of the polymer electrode can be used to influence the morphology of the neuronal network. This may benefit applications where the directed growth of neurons is of interest. Acknowledgement T. Nyberg’s work was supported in part by the Japan Society for the Promotion of Science. References Beggs JM, Plenz D. Neuronal avalanches are diverse and precise activity patterns that are stable for many hours in cortical slice cultures. J Neurosci 2004;24:5216–29. Britland S, Perridge C, Denyer M, Morgan H, Curtis A, Wilkinson C. Morphogenetic guidance cues can interact synergistically and hierarchically in steering nerve cell growth. Exp Biol Online 1996;1:2.

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