Parallel On-Chip Analysis of Single Vesicle Neurotransmitter Release

Article pubs.acs.org/ac

Parallel On-Chip Analysis of Single Vesicle Neurotransmitter Release Alexey Yakushenko,† Enno Kaẗ elhön,† and Bernhard Wolfrum*,†,‡ †

Institute of Bioelectronics (PGI-8/ICS-8) and JARAFundamentals of Future Information Technology, Forschungszentrum Jülich, 52425 Jülich, Germany ‡ IV. Institute of Physics, RWTH Aachen University, 52074 Aachen, Germany S Supporting Information *

ABSTRACT: Real-time investigations of neurotransmitter release provide a direct insight on the mechanisms involved in synaptic communication. Carbon fiber microelectrodes are state-of-the-art tools for electrochemical measurements of single vesicle neurotransmitter release. Yet, they lack highthroughput capabilities that are required for collecting robust statistically significant data across multiple samples. Here, we present a chip-based recording system enabling parallel in vitro measurements of individual neurotransmitter release events from cells, cultured directly on planar multielectrode arrays. The applicability of this cell-based platform to pharmacological screening is demonstrated by resolving minute concentrationdependent effects of the dopamine reuptake inhibitor nomifensine on recorded single-vesicle release events from PC12 cells. The experimental results, showing an increased halftime of the recorded events, are complemented by an analytical model for the verification of drug action.

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investigations of only a few cells due to labor-intensive manual handling of each individual probe. This challenge has been addressed by introducing multiprobe CFMs,18−20 which allow a single cell to be mapped for neurotransmitter secretion sites. Yet, because of the finite size of the microelectrode, this technique is not suitable for mapping of neurotransmitter release from individual cells on a network scale. A logical solution to address this challenge is the introduction of a functional substrate that can record from many cells in parallel without the need of manual electrode positioning. A variety of systems employing chip-based electrodes for amperometric detection of neurotransmitter release from single cells has been implemented so far.21−33 In principle, multielectrode arrays (MEAs) can be used for parallel or multiplexed operation of working electrodes to achieve rapid online analysis of neurotransmitter release. Yet, up until now, experimenets are typically performed using patch-clamp amplifiers or potentiostats that are not directed at highly parallel recordings from many individual cells. First steps toward this approach were recently demonstrated by the group of Lindau34 using a complementary metal-oxide-semiconductor (CMOS)-based chip with integrated microelectrodes. However, CMOS technology is generally expensive to process and limits the possibility for flexible and rapid design implementation calling for alternative methods.

eurotransmitters are the chemical messenger molecules in synaptic communication that transduce information between cells. Malfunctioning of this transduction mechanism is related to mental disorders such as depression or schizophrenia.1 Several drugs, including the norepinephrinedopamine reuptake blocker nomifensine, aim at relieving associated symptoms by affecting local neurotransmitter concentrations in the brain. To investigate drug action2 and fundamental effects on synaptic transmission, techniques for real-time observation of neurotransmitter release attracted sound interest in recent years.3−5 First, experimental observations of single exocytotic events were conducted using the patch-clamp technique by recording changes in cellular membrane capacitance upon fusion of secretory vesicles.6 This method can be complemented by a direct electrochemical detection of redox-active neurotransmitters, which is typically performed with carbon-fiber microelectrodes (CFMs) in combination with a patch-clamp amplifier.7 The successful implementation of CFMs to measure neurotransmitter release from large dense core vesicles of different cell types,8,9 as well as from small synaptic vesicles (SSV) of midbrain neurons,10 established the method as a standard for the detection of single vesicle release. Thus, over the last decades, a variety of probebased experiments on neurotransmitter release have led to a fundamental understanding of underlying mechanisms involved in exocytosis.11−14 Additionally, probe-based techniques have been advanced to achieve spatial resolution down to the singlevesicle scale15 and enable scanning for topgraphical and functional imaging.16,17 However, despite being the state-ofthe-art tool for studies of secretory events, CFMs are limited to © XXXX American Chemical Society

Received: February 27, 2013 Accepted: May 6, 2013

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dx.doi.org/10.1021/ac4006183 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

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Cell Culture. In vitro experiments were performed on a catecholamine-containing rat pheochromocytoma cell line (PC12),37 kindly provided by Agnes Dreier and Prof. Joachim Weis from Uniklinik Aachen, Germany. A stock PC12 culture was grown in Petri dishes using Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS and 1% penicillin-streptomycin mixture (Pen/Strep, penicillin 100 units/mL, streptomycin 100 μg/mL), kept in the incubator at 37 °C and 5% CO2 and was passaged every 3−4 days. Before cell plating, the chips were sterilized by UV light for 30 min. Afterward, the chips were incubated for 30 min with 100 μL of 50 μg/mL solution of poly-L-lysine (PLL) to enhance cell adhesion. Cells were trypsinated from an 80% confluent cell culture in a Petri dish and collected into a 15 mL Falcon tube. After centrifugation, the cell pellet was resuspended in 1 mL of supplemented DMEM medium. Subsequently, 80 μL of supplemented DMEM medium were added inside the ring of the chip. A volume of 20 μL of the cell suspension were pipetted into the reservoir for a final concentration of approximately 285 000 cells/cm2, and the cells were allowed to sediment and adhere in the incubator at 37 °C for 2 h. Cell Fixation for SEM. PC12 cells 2 h after seeding on MEAs were washed thoroughly twice in prewarmed physiological buffer (in millimolar, HEPES 10, glucose 5, MgCl2 1.2, CaCl2 2, KCl 5, and NaCl 150). Afterward, the buffer was exchanged to a prewarmed 3.2% gluteraldehyde solution in HEPES buffer, and the cells were incubated for 2 h at 37 °C. Subsequently, the cells were washed twice at room temperature in HEPES buffer and deionized water. Then a series of 10 min incubations in rising concentrations of ethanol in water solutions (10%, 30%, 50%, 70%, and 90%) was applied. Afterward, three 5-min incubations in 95% ethanol and again in 100% ethanol were performed. As a last step, the samples were dried using carbon dioxide critical point drying. Amplifier System and Characterization. The amplification system for the in vitro recordings of the neurotransmitter release consisted of two parts: a main amplifier equipped with a 16 bit analog-to-digital converter (ADC) (NI USB-6255, National Instruments, Austin, TX) and a novel current preamplifying head-stage developed in our institute, PicoAmp64. The main amplifier along with the ADC allows simultaneous data acquisition from 64 channels at 10 kHz sampling rate (∼200 μs temporal resolution). It also provides postamplification gain between 1 and 100 at a maximal input voltage of ±10 V. The preamplifying head-stage (see S1 in the Supporting Information) delivers output signal amplification of 50 mV/pA using low noise operational amplifiers (OPA129, Texas Instruments, Dallas, TX), which corresponds to ±200 pA current range and 6.1 fA current bit resolution with a 16 bit ADC. The amplification can be modified substituting the 5 GΩ feedback resistance of the current-to-voltage converter. An analog low-pass filter is integrated to attenuate frequencies above a cutoff frequency of 1 kHz (−6 dB). A MEA can be inserted directly into the preamplifier box, minimizing the capacitance of the feedlines and reducing the noise in the amperometric recordings. Each electrode of the 64channel MEA can be individually biased to a maximum potential of ±1.25 V vs the reference electrode. The platform uses planar platinum MEAs fabricated as described above, with electrode diameters between 3 and 12 μm and noise characteristics directly related to the electrode area (see S2 in

Here, we report on the development and implementation of a platform for parallel recordings of vesicular neurotransmitter release in real time. The system operates with MEA chips comprising 64 simultaneously addressable microelectrodes with a minimum electrode diameter of 3 μm. It allows electrochemical measurements in the subpicoampere regime with automated analysis of single vesicle release characteristics. In this work, we demonstrate the potential of the system by distinguishing concentration-dependent effects of the dopamine reuptake blocker nomifensine on the vesicular neurotransmitter release in vitro. We observe minute but statistically significant drug-induced changes by exploiting the acquired large data sets. The results are interpreted by an analytical model that takes into account geometric aspects of the cell-substrate interface. Overall, because of the high number of recording sites, large bandwidth, and the ability to record single-vesicle signals, our system provides all the toolkits needed for electrochemical measurements of neurotransmitter release in drug-screening applications.

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MATERIALS AND METHODS Microfabrication of Microelectrode Arrays (MEAs). The MEAs were fabricated in the clean-room using standard microfabrication technologies. Briefly, borosilicate wafers (Schott AG, Mainz, Germany) of 100 mm diameter and 500 μm thickness were used as a substrate. First, a double layer resist (LOR 3b, Microchem, Newton, MA and AZ nLOF 2070, MicroChemicals, Ulm, Germany) was spin-coated onto the wafer and the electrode and feedline geometries were patterned using optical photolithography (MA-6, SUESS MicroTec AG, Garching, Germany). Then, a stack of metal layers (10 nm Ti/ 150 nm Pt/10 nm Ti) was deposited by electron-beam deposition. The titanium layers served to facilitate adhesion to the substrate and the following passivation layer. Electrodes and feedlines were defined by lift-off and were then passivated with a stack of five alternating SiO2 (200 nm) and Si3N4 (100 nm) layers (ONONO, 800 nm overall) by plasma enhanced chemical vapor deposition (PECVD) (Sentech Instruments GmbH, Berlin, Germany). ONONO was chosen to improve the passivation of the feedlines from the electrolyte solution by decreasing stress and lowering the number of pinholes.35 A second lithography was performed to remove the passivation layer at the contact pads and microelectrode positions. The passivation layer was etched down to the platinum layer by reactive ion etch (RIE) (Oxford Instruments, Abingdon, U.K.) using subsequently CHF3/CF4 and Ar/O2 gas mixtures to etch the Ti adhesion layer as well as the remaining resist and possibly formed Teflon-like residues.36 The processed wafers were diced into individual chips (24 chips/wafer), and the yield of fully functional chips was in the range of 95%. Approximately 20 chips were used during this study. In order to culture cells directly on the chip, a few additional encapsulation steps followed. First, the chip was flip-chipped on the printed circuit board carrier with a low-temperature solder paste (ASNBIN/500, Amtech, Branford, CT). The contact surface between the chip and the carrier was isolated with medical epoxy (302-3M, Epoxy Technology, Billerica, MA). Glass rings were glued onto the carrier with polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning GmbH, Wiesbaden, Germany) and cured to define a 100 μL reservoir for the cell medium. The final dimensions of the encapsulated chip were 24 mm × 24 mm, and the effective working area formed by the microelectrode array constitutes 1.6 mm × 1.6 mm. B



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Figure 1. Parallel recordings of neurotransmitter release from PC12 cells on a MEA. (a) 64-Channel MEA. Insets: (i) Live-dead staining of PC12 cells on MEA, 2 h after plating. Calcein (green) indicates live and ethidium homodimer (red) indicates dead cells (ii) zoom on a single electrode, scale bars 200, 200, and 50 μm. (b) Simultaneous amperometric recordings of spontaneous exocytotic events from 10 electrodes. Each trace represents one electrode, scale bar 50 pA. (c) Histogram of the detected catecholamine amount (Npeaks = 3099) showing two normally distributed subgroups of vesicles centered at 3.72 ± 0.05 and 5.28 ± 0.45 zmol1/3, respectively. (d) SEM images of FIB cuts of a PC12 cell fixed on chip 2 h after plating. Subfigure i demonstrates the top view, ii−iv cross sections. White arrows indicate the cells, yellow arrows electrodes, scale bars (i−iv) 10, 5, 2.5, and 1 μm.

filter out hard-to-analyze small amplitude peaks and noise bursts. Peak start and end points were defined as one sample before the current rises above zero and the last point before the current descends again below zero. The peak half-time was determined as the full width at half the maximal amplitude (FWHM). Average numbers of neurotransmitter molecules per peak N were calculated by integrating the peaks to obtain the transferred charge Q using Faraday’s law: Q = nNe, where n is the number of released electrons in the oxidation reaction (n = 2 for catecholamines), and e is the electron charge (1.6 × 10−19 C). Overlapping peaks were discarded from the analysis. The data was analyzed using self-programmed Matlab (The MathWorks Inc., Natick, MA) and Python (Python Software Foundation) routines. Statistical Analysis. Statistical analysis was performed on pooled mean values from each electrode according to the procedure suggested and verified by Colliver et al.39 for neurotransmitter release recordings with CFMs. In brief, single neurotransmitter release events at one electrode were pooled across different traces of the same experiment and averaged. Only electrodes with 10 events or more were considered. These averaged values from single electrodes were considered as single independent experiments. Mean pooled values from single electrodes were pooled across the same chip as well as different chips measured under the same conditions. Only the pooled mean values were used for statistical tests during pharmacological screening experiments. Simulations. In computer aided simulations, we model the current response of an electrode channel as a function of the electrode distance from the point of neurotransmitter release. In short, we hereby proceed in three steps: First, we find a filter

the Supporting Information). The low-noise characteristics allow currents in the sub-picoampere regime to be read out simultaneously at all electrodes. Prior to cell culture experiments, selected MEAs were analyzed with electrochemical impedance spectroscopy in order to determine their capacitance in cell culture medium (VSP300, Biologic). Values in the range of 8−12 pF were constantly obtained for well-functioning 12 μm Pt microelectrodes. Also, cyclic voltammetry with dopamine-containing solutions was used to verify the functionalty of electrodes and obtain voltammograms characteristic for dopamine (data not shown). As an indirect measure to monitor the correct electrode performance, amperometric root-mean-square (rms) noise values were evaluated. Thus, functionality of each microelectrode was checked between different cell culture experiments. Electrodes with deviating values were not used due to suspicion of pinhole formation and deterioration of the passivation layer. The cell experiments were usually performed in a twoelectrode setup (64 working microelectrodes and a chlorinated silver pellet reference electrode). A counter electrode can be omitted as the measured currents are in the picoamp range, i.e., too low to significantly affect the potential at the reference electrode. The MEA chips were reused many times (∼15 cell cultures) and showed only slight deterioration in the noise properties over time. Data Analysis. The analysis of exocytotic events recorded by amperometry was performed according to the guidelines recommended by Mosharov and Sulzer.38 Shortly, all traces were postprocessed with a Kaiser window smoothing (m = 11, beta = 14). The peak amplitude threshold was set at 5 pA to C



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that models the properties of our analog measurement circuitry. Second, we use this result to estimate the kinetics of vesicular neurotransmitter release from experimentally determined data. Third, we derive a function that describes the reaction probability of a single neurotransmitter molecule in dependence of the time since its release for the two cases of immediate and negligible cellular reuptake. This function is then convoluted with the vesicular release kinetics and filtered with the amplifier response to describe the full characteristics of the signal. In order to compare calculated peaks with experimentally determined data, we first need to find a model for the response of the analog circuitry that was used for the data acquisition in our lab. This response is mainly characterized by the system’s filter set that uses a simple resistor-capacitor circuit. It operates as a one-pole low-pass filter at a cutoff frequency of 1 kHz and exhibits a gain-magnitude frequency response of about −3 dB at the cutoff frequency and a decline of −6 dB per octave in the stop band domain. Since the filter is the largest contributor to the device response characteristics, we limit our model of the circuitry to the filter, which can be ideally described via a firstorder Butterworth filter operating at 1 kHz. We can now estimate the temporal neurotransmitter release kinetics from experimental data if we use two additional approximations: First, neurotransmitters react instantly upon collisions with the electrode, and second, the sharpest peaks that we can find in the experimental data correspond to release events featuring a negligible cell−electrode distance as seen on some SEM images of the FIB cuts (Figure 1d and S3 in the Supporting Information). Accordingly, these peaks provide the release kinetics of the cell filtered with the analog circuitry of the measurement setup, since diffusive processes can be neglected over these indistinguishable cell-chip gaps. We then estimate a parameter-dependent function (a simple triangular peak was used in this work) that describes the rate of neurotransmitter release as a function of time. Afterward, we convolute this function with the above found amplifier impulse response and fit the obtained curve to the sharpest recorded peaks by varying the function’s parameters. Hereby, we find that a peak width of 1.25 ms matches the release kinetics in the experimental data (see S4 in the Supporting Information). In the next step, we find a function that describes the diffusive transport of the neurotransmitters from the cell to the electrode surface. We base this calculation on the simple model of an infinite flat electrode that is arranged in parallel to an infinite flat cell. Hereby, two different cases are considered: a fully absorbing and a fully reflecting cell surface. Both can be calculated via the following formula that provides the absorption probability P(n, z, a) of a one-dimensional random walker in between two absorbing boundaries as a function of the number of steps n that are performed since its release.40,41 Its initial position in the number step length dx from the lower boundary is given by the value z and the distance h between the two boundaries in the number of steps is given by a.

Pupper boundary(n , z , a) =

a−1 v=1

a

sin

π v (a − z ) πv sin a a

(3)

(4)

while D represents the diffusion coefficient (0.6 × 10−9 m2/s for dopamine42). In order to calculate the first case of a reflecting cell surface, we make use of the inherent symmetry of the system. Since a reflected pathway equals a pathway in the same system mirrored at the cell surface, we define the release point of the random walker to be centrally in between two absorbing boundaries. Hence, we set the initial position z to a/2, while a/ 2 represents the cell−chip distance h in steps of the random walker. Then we calculate P(n, a/2, a) and transform it to the reaction probability density Preflecting(t, h). In the second case of a fully absorbing cell, we release the random walker in close proximity to the cell surface, which is here the lower boundary, by setting z to the average radius (75 nm) of a vesicle43 and a to h/dx. Then, we calculate Pupper boundary(n, z/dx, a), before we transform Pupper boundary into the desired result Pabsorbing(t, h). In both cases, we use a spatial step width of 25 nm for our calculation. In the last step, we can now calculate the expected shape of the current peaks as a function of the cell−chip distance for the two reuptake characteristics of the cell. Herefore, Preflecting(t, h) and Pabsorbing(t, h) are convolved with the estimated cellular release kinetics and then filtered with the emulated filter of the measurement setup. The simulated reaction probabilities for the case of a reflecting boundary and three different cell− electrode distances can be found in Figure 3b.

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RESULTS AND DISCUSSION On-Chip Measurement of Single Vesicle Release. We cultured a catecholamine-releasing PC12 (rat pheochromocytoma) cell line44 directly on-chip and recorded single vesicle neurotransmitter release events amperometrically. Typically, there were more than 95% of living cells during the measurementsas shown using live-dead staining (Figure 1a). Both spontaneous and chemically stimulated exocytotic events were recorded using the Pt microelectrodes biased to +0.6 V vs an Ag/AgCl pellet reference electrode. Given a random spatial distribution of the cells, the number of microelectrodes with active cells as well as the frequency of the detected events differed significantly between experiments (Figure 1b). The recorded current traces were subjected to automated analysis and specific characteristics of single neurotransmitter release events, such as the number of detected molecules (catecholamine amount), peak amplitude, and half-time per vesicle were extracted according to the guidelines of Mosharov and Sulzer.38 We pooled the data from multiple experiments to produce a histogram of the cubic root of the catecholamine amount. This value, which is proportional to vesicular radius, had a mean value of 4.84 ± 0.03 zmol1/3 or 89300 ± 1 700 molecules (npeaks= 3099, error is SEM). Conforming to previously reported data,45 a double-hump shaped distribution

with

∑ cosn− 1

v=1

dx 2 = 2D × dt

(1)

1 a

a−1

∑ cosn− 1 πv

Temporal and spatial step width of the random walk, dt and dx, are linked via the following formula that can be derived from the general solution of the one-dimensional diffusion equation

P(n , z , a) = Plower boundary(n , z , a) + Pupper boundary(n , z , a)

Plower boundary(n , z , a) =

1 a

πv πv πvz sin sin a a a (2) D



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Figure 2. Effect of dopamine transporter blocker nomifensine on the reuptake of released dopamine. (a) Schematics of on-chip dopamine (orange circles) release from a PC12 cell, its oxidation at the electrode into dopamine o-quinone (red circles), and reuptake into the cell via dopamine transporter (DAT, light blue). (b) Inhibition of dopamine reuptake with a DAT blocker nomifensine (green diamonds). (c) Exemplary peaks of a standard control PC12 cell (blue) and a cell treated with 10 μM nomifensine (red). (d) Nomifensine dose response, N indicates the number of electrodes used to attain the mean value, only electrodes with 10 events or more were analyzed. (e) Histogram of peak half-times for control (blue, mean = 1.40 ± 0.07 ms, Nelectrodes = 42) and 10 μM nomifensine (red, mean = 2.12 ± 0.26 ms, Nelectrodes = 37), errors are SEM.

by a varying number of events detected from each electrode was eliminated. This approach significantly reduced the standard deviation of the data (see S5 in the Supporting Information), at the same time, however, strongly decreased the total number of data points. The unique power of the platform presented in our work is its parallel measurement capabilities that allow carrying out 64 independent experiments on a single chip simultaneously. Combined with the described statistical method for reducing variance in the data, while still keeping the number of total events significantly higher than in previous works, our system can be utilized for discriminating, otherwise hardly detectable, effects of pharmaceutical substances in vitro. Effect of DAT Inhibitor Nomifensine. As a proof-ofprinciple we have studied the inhibiting action of the DAT blocker nomifensine,46 frequently used for in vitro studies of reuptake mechanisms.47,48 Its working mechanism is schematically shown in Figure 2a,b. In this simple picture, which ignores the limited reuptake kinetics of dopamine, one could assume that the peak’s half-time is affected by the drug. Nomifensinetreated cells should show longer peaks compared to the control owing to an extended dwelling time and subsequent oxidation of dopamine molecules in the cell-electrode gap caused by the blocked DAT (Figure 2c). In our in vitro pharmacological screening experiments, the PC12 cells (passages 17−23) cultured on chip were exposed to a range of nomifensine concentrations up to 50 μM and incubated for 10−20 min prior to measurements. Spontaneous as well as stimulated (1 M KCl solution injections diluted to on-chip end bulk concentration of 25 mM) neurotransmitter release was recorded for approximately 10 min for each chip. The resulting averaged pooled means of the peaks’ half-times (Figure 2d) show a steady increase with the drug dose as the number of blocked DATs rises. The differences between concentrations are statistically significant (one-way ANOVA, P = 0.0136, Ntotal= 167 electrodes, 8628 events). Yet, in pairwise comparison only

was obtained revealing two normally distributed subgroups of LDCVs (Figure 1c). In our experiments, the inherent vesicular size distribution was further influenced by the distance from the release site to the electrode and the direction of the flux of released neurotransmitters. To investigate the magnitude of these distances, scanning electron microscope (SEM) images were taken of fixed cells on the chip that had been dissected using focused ion beam (FIB) cutting. The images from 15 sites indicate a wide distribution of the cell-to-electrode distance (Figure 1d and S3 in the Supporting Information), namely, from virtually no gap to several micrometers. Moreover, single electrodes were observed to have varying numbers of cells within their vicinity. The thickness of the passivation layer (800 nm) might have an affect on the distribution of cell−electrode distances but does not interfere with the ability to detect release events. A further reduction of the passivation thickness could reduce the average cell-to-electrode distance. However, according to our experience, very thin PECVD layers compromise the noise levels due to an increase of pinholes forming in the silicon oxide/nitride layers. Taking into account this broad cell-to-electrode distance distribution together with the aforementioned significant neurotransmitter release variance across different electrodes, a highly scattered data set was usually obtained. That is, every electrode could still be considered an independent experiment. However, when pooling all events and averaging, some electrodes would be underrepresented and some overrepresented, due to a strongly varying number of events per electrode, biasing the overall means. Therefore, an adapted method for reducing the variance in the data, proposed by Colliver et al.,39 was used for pharmacological data analysis. Essentially, we pooled and averaged the values from each individual electrode during one experiment (further referred to as pooled mean), producing one data point per every active electrode. Thus, the bias caused E



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Figure 3. Modeling of the neurotransmitter release. (a) Reaction probability distribution of a single neurotransmitter molecule as a function of time since its release at the cell surface (blue), convolution of this distribution with the cellular release kinetics (red), and simulated signal after filtering. Inset: Estimated normalized kinetics of the cellular neurotransmitter release. (b) Calculated signals for different cell−electrode distances without reuptake back into the cell. (c) Half-times of the simulated signals as a function of the cell−electrode distance for the cases of full and no reuptake. Lines are 2nd polynomial fits (R2 = 0.999). (d,e) Double histograms of the recalculated distance vs catecholamine amount for control (d) (Npeaks = 2343) and cells treated with 10 μM nomifensine (e) (Npeaks = 1254), colorbar is relative frequency in %.

the 10 μM result (2.12 ± 0.26 ms) is significantly different from the control (1.40 ± 0.07 ms), with the mean half-time being 0.72 ms or 51% longer for the drug-affected cells (Tukey test, P = 0.0069). Additionally, there is a decline in half-times after 10 μM, which can be attributed to the drug toxicity as previously observed by Cui et al.49 The histograms of pooled peaks for the control vs 10 μM nomifensine (Figure 2e) illustrate the minute changes in the release pattern in vitro that are difficult to distinguish without applying a solid statistical analysis. Overall, the results shown here were combined from 8628 events on 167 electrodes. Being equivalent to highly time-consuming measurements from 167 single cells using CFMs, it emphasizes the effectiveness of the presented technique. In vivo investigations on the effect of nomifensine usually measure bulk concentrations of dopamine upon perfusion with the drug. In a previous study, the increase in different brain regions of rats has been reported to range from 87% to 5633%.50 However, these experiments cannot be directly compared to our single-cell measurements, as we measure the effect of the DAT inhibitor on the half-time of each single vesicle release event separately and not the bulk concentrations. In the case of cells growing on the chip, the neurotransmitter molecules have the possibility to diffuse away into the culture medium immediately after being released, whereas in the intact brain they are restricted to the narrow interstitial space and accumulate rapidly to high concentrations when the reuptake is blocked. Modeling the Drug Effect. Because of the lack of truly comparable single vesicle data on the effect of nomifensine in the literature and also for a better understanding of the expected effect, we resorted to computer modeling to assess the validity of our results. For that purpose we used computeraided simulations to model the current response at a single

electrode as a function of the electrode distance from the point of neurotransmitter release. The model, described in detail in the Materials and Methods section, is comprised of three steps: first, we modeled the filtering properties of our analog measurement circuitry; second, we estimated the neurotransmitter release kinetics of a LDCV from experimentally determined data; third, we derived a relation that describes the probability of the oxidation of a single neurotransmitter molecule at the electrode as a function of time since its release. This probability was calculated for the extreme cases of immediate (absorbing boundary) and negligible (reflecting boundary) cellular reuptake. At last, we convolved the oxidation probability with the release kinetics and applied the modeled filter (Figure 3a,b and S4 in the Supporting Information). The results obtained using the analytical model show a similar trend as observed in the experimental data. Figure 3c demonstrates the divergence of the simulated half-time values of the control and drug-affected cell as a function of the cell− electrode distance ranging from undetectable differences, when the cell is located directly on the electrode, to 3 ms at a distance of 2.5 μm. This aspect signifies that the effect of blocked reuptake is easier to distinguish at larger time scales when measuring from cells located further away. Furthermore, as the half-time values differ with the cell−electrode distance, they can be used to approximate a distance from the neurotransmitter release site to the electrode. We fitted a function to find the relation between the half-times and distances for the case of simulated immediate cellular reuptake. We applied this relation to convert the experimental half-time values into distances. On the basis of these calculated distance values and the detected amount of neurotransmitters, we constructed double histograms for control and nomifensine-treated cells from the experimental data (Figure 3d,e). Although in reality both F



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confirmed the trend observed in the experimental data and demonstrated that the effect should become more apparent on longer time-scales but vanishes for vesicles that are released very close to the recording site. We believe that the presented easy-to-use system has a high potential for analyzing chemical release from cells on a network scale. Furthermore, it can help to elucidate complex pharmacological effects requiring ample statistics for definitive results. Moreover, we envision a further improvement in terms of high-throughput via integration of MEAs into standard 96well plates. Such an improved cell-based assay would allow screening of up to 96 chemicals on a single cell passage, further reducing the deviations that might arise from the use of different passages of the same cell line.

populations lie in between the extreme cases of reflecting and absorbing boundaries, this visualization clearly shows their underlying distinctive nature. For the control group, only large vesicles are detected at longer distances, but when the DAT blocker is applied many smaller vesicles are being detected from release sites lying further away, as the released content is not being rapidly reuptaken by the cell. Moreover, because of exactly the same reason, many smaller vesicles, which were undetectable in the control experiments, just surpass the 5 pA current detection threshold after the drug application and appear on the histogram with the catecholamine amount of approximately 3 zmol1/3. Overall, the most frequent cell− electrode distance as can be deduced from the histograms lies somewhere between 0.5 to 1.0 μm. If we take into account only these distances, the expected difference in the half-times of the detected release events, as provided by our model, would be approximately 0.05 to 0.36 ms. This difference is actually significantly smaller than what we measure experimentally. Additionally, one has to keep in mind that the model for the control group is based on a perfectly absorbing boundary. In reality, the reuptake is further limited by the kinetics of dopamine uptake due to transporter density and turnover rate. In fact, the latter is expected to lie in the range of one turnover per second,51 and therefore, irrelevant on the time scales of our experiment. As a consequence, we can only consider a one-time capture event of an individual molecule for each transporter. Thus, the difference in half-time, as predicted by our model, has to be seen as an upper boundary and cannot explain the large difference observed in the experimental data in terms of peak broadening due to uptake blocking. However, the experimental result is obtained by averaging events that are detected from all distances. The shifted weight caused by the additional detection of vesicles released from distances several micrometers away could be responsible for the observed longer half-times of 0.72 ms, assuming that nomifensine does not directly affect vesicle release or cell-substrate interaction.

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ASSOCIATED CONTENT

S Supporting Information *

Electronic diagram of a single unit of the preamplifying headstage; noise characteristics of the sensor; additional SEM images of the FIB cuts of PC12 cells on the chip; modeled cellular release in the case of a negligible cell−electrode distance and comparison to the shortest experimentally obtained peaks; and comparison of the pooled mean data statistics to mean pooled data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-2461-61-3285. Fax: +49-2461-61-8733. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Francesca Santoro and Bastian Haberkorn for help with the cell culture, Elke Brauweiler-Reuters for SEM images, Marko Banzet for clean-room assistance, Norbert Wolters for building electronic equipment, and Dieter Lomparski for software development. We additionally thank Björn Falkenburger, Christian Wolfrum, Marc Spehr, and Andreas Offenhäusser for helpful discussions. We acknowledge funding by the Helmholtz Young Investigator program.

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CONCLUSIONS We have developed a cell-based platform that enables parallel high-throughput measurements of single vesicle neurotransmitter release in real time. A distinctive advantage of the approach is the in vitro assessment of single vesicle release characteristics from cells directly cultured on a functional substrate. The availability of an array of recording sites and the ability to measure from all of them simultaneously increases the throughput manifold compared to existing methods. In turn, through the increased throughput, large data sets are generated in a short time providing the statistics required for the investigation of subtle pharmacological effects of chemical substances on cells. The efficiency of our system for high-throughput screening was demonstrated on a catecholamine-releasing PC12 cell line, using the DAT blocker nomifensine, which inhibits the reuptake of dopamine into the cell. We have produced a large data set, markedly exceeding the number of data points previously reported in the literature, and applied a statistical approach that reduces the variance between the experiments. From a series of nomifensine concentrations, a statistically significant difference between control and nomifensine-treated cells was seen in the detected peak half-times. The experimental data was supported by a computational model. Exocytotic events with (control) and without reuptake (nomifensine) were modeled for verification of the drug effect. The model

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