REVIEW ARTICLE

published: 06 January 2015

doi: 10.3389/fnins.2014.00423

Revealing neuronal function through microelectrode array

recordings
Marie Engelene J. Obien 1*, Kosmas Deligkaris 1,2 , Torsten Bullmann 1 , Douglas J. Bakkum 3 and
Urs Frey 1,2,3
1
RIKEN Quantitative Biology Center, RIKEN, Kobe, Japan
2
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan
3
Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

Edited by: Microelectrode arrays and microprobes have been widely utilized to measure neuronal
Noriko Hiroi, Keio University, Japan activity, both in vitro and in vivo. The key advantage is the capability to record and
Reviewed by: stimulate neurons at multiple sites simultaneously. However, unlike the single-cell or
Ahmed El Hady, Princeton
single-channel resolution of intracellular recording, microelectrodes detect signals from
University/HHMI, USA
Yoonkey Nam, Korea Advanced all possible sources around every sensor. Here, we review the current understanding of
Institute of Science and Technology, microelectrode signals and the techniques for analyzing them. We introduce the ongoing
Korea (South) advancements in microelectrode technology, with focus on achieving higher resolution
*Correspondence: and quality of recordings by means of monolithic integration with on-chip circuitry. We
Marie Engelene J. Obien, Frey
show how recent advanced microelectrode array measurement methods facilitate the
Initiative Research Unit, RIKEN
Quantitative Biology Center, RIKEN, understanding of single neurons as well as network function.
Minatojima-minamimachi 2-2-3
Keywords: microelectrode array, neuronal function, extracellular recording, stimulation, CMOS, multielectrode
Chuo-ku, Kobe, Hyogo 650-0047
array, neuron-electrode interface, multi-scale modeling
Japan
e-mail: [email protected]

INTRODUCTION into a recorded digital form. Typical electrophysiological methods

Studying the function and connectivity of neurons in the brain are shown in Figure 1 and further described below.
involves coordinated, interdisciplinary efforts among scientists At the microscale, patch-clamp can be used to measure cur-
from various fields. Through the years, advancements in genetic rents of single ion channels. The function of single neurons is
markers, immunostaining, optical and electro-optical methods, often explored by direct measurements of the intracellular volt-
electrophysiology, and computational tools have been made age, using patch-clamp or a sharp microelectrode. It is a powerful
to identify neuronal types, explain their molecular machinery, but tedious method and often its use is limited to a few neu-
untangle their wiring, decipher principles of neural coding, and rons per experiment (Wood et al., 2004). Planar patch-clamp
to attribute functional roles to specific regions of the brain. The systems allow rapid in vitro patch-clamping, mostly used for high-
brain is a complex system and its activity spans over multiple throughput ion channel screening of dissociated cells (Dunlop
temporal and spatial scales that require a comprehensive set of et al., 2008). Automated patch-clamp allows for fast in vivo intra-
technologies to address these scales. Innovations in experimental cellular recording and it is feasible to extend the method to
methods to observe and perturb brain activity and in computa- measure several neurons simultaneously (Kodandaramaiah et al.,
tional tools to analyze recorded data are needed to master the 2012). The bulkiness of current micromanipulators and patch-
brain’s complexity and advance our understanding of its func- clamp systems together with the necessity for accurate and precise
tion. Systems biology has allowed to bridge between molecular control have limited simultaneous patch-clamp recordings to a
dynamics and whole cell simulations using multi-scale mod- few—maximum of four and twelve for in vivo (Kodandaramaiah
eling. Applying similar approaches to brain activity will allow et al., 2014) and in vitro (Perin et al., 2011), respectively.
us to gain a more encompassing understanding of it. However, At the macroscale, indirect measurement of large areas of
quantitative data at all these spatial and temporal scales are the brain’s activity is achieved via functional magnetic resonance
a prerequisite. imaging (fMRI), positron emission tomography (PET), and elec-
Electrophysiology has been the preferred means of analyzing troencephalography (EEG). These methods can be used to resolve
brain activity due to the ability to capture a wide range of neu- functional connectivity among brain regions. For example, EEG
ral phenomena, from the spiking activity of individual neurons to detects spontaneous or evoked electrical activity from the scalp
the slower network oscillations of small populations (Llinás, 1988; with low spatial resolution (cm range).
Contreras, 2004; Assad et al., 2014). The electrical nature of neu- In this review, we focus on electrophysiology at the
ronal activity makes it possible to detect signals on electrodes at a mesoscale—extracellular recordings via metal electrodes, open-
distance from the source, but not without caveats. It is necessary gate field-effect transistors (OGFETs) or oxide-semiconductor
to determine the recording capabilities and limits of the device FET (OSFET) integrated into large arrays, so-called microelec-
used and to understand how the neuronal signal is transduced trode arrays (MEAs). This method enables simultaneous and

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 1 | Typical electrophysiological methods. (A) Macroscopic permission from Buzsáki et al. (2012). (B) Mesoscopic and microscopic
recording via electroencephalography (EEG) and mesoscopic recording recording using a tetrode (extracellular) and a glass micropipette
through electrocorticography (ECoG) and implantable electrodes, with the (intracellular), respectively. The fast EAP extracted from the raw tetrode
corresponding representative waveforms recorded in a patient with recordings correlate with the intracellular APs recorded from a pyramidal cell.
drug-resistant epilepsy. The measured signal amplitudes are larger for ECoG (Left) Illustration of cells across cortical layers modified with permission from
and implanted electrodes (local field potential or LFP recording) compared to Buzsáki et al. (2012). (Right) Signals for simultaneous extracellular and
EEG. The waveforms for EEG, ECoG, and implant are modified with intracellular recordings modified with permission from Henze et al. (2000).

long-term recordings of local field potentials (LFPs) and extra- device (needle-type probe, polytrode, neuro dish), the channel
cellular action potentials (EAPs) from a population of neurons at count (multichannel array), the electrode density (HDMEA) or
millisecond time scale. It also allows perturbing neuronal activ- the application (implantable array, in vivo MEA, in vitro MEA)
ity using electrical stimulation. As data obtained from in vivo and more. We would therefore like to briefly explain the termi-
and in vitro experiments are often very similar, the MEA tech- nology used in the context of this review. We generalize the term
nology, concepts, and applications we include here apply to both microelectrodes and MEA to cover both substrate-integrated
and will be helpful for scientists and engineers from either field. planar MEAs and implantable neural probes. We also include
In particular, we explain the interface between the neuron and capacitive-coupled devices, such as multi-transistor arrays in the
the electrode in order to understand how to interpret the record- definition of MEAs. We then distinguish between implantable,
ings. We highlight trends in the development of complementary in vivo MEAs, such as polytrodes and neural probes, and in vitro
metal-oxide-semiconductor (CMOS) based high-density MEAs MEAs that generally include a cell culture dish or some other sort
(HDMEAs). The advantages of HDMEAs include the capability of medium chamber. Further, we classify the different array archi-
to map neuronal activity at sub-cellular resolution, localize single tectures, as will be explained in Section Advances in MEA and
cells, and to constrain full-compartmental neuron models. Probe Devices (Figure 3). Briefly, we distinguish between “fixed
The outline is as follows. Chapter 2 gives an overview of the wiring” arrays, meaning that each transducer in the array has a
MEA technologies, including the comparison between in vivo direct wire to the outside of the array and “multiplexed arrays,”
and in vitro MEA devices from a technical aspect. Chapter 3 in which some sort of switching mechanism is employed within
describes the current understanding on microelectrode record- the array. We use the term “array” to refer to the actual area
ings and introduces the different factors that shape the recorded that encompasses the transducer elements only and we use device
signals. Chapter 4 discusses how to process MEA signals and or MEA to refer to the entire device. With system, we refer to
reviews recent works on using MEAs for neuroscience studies. the MEA and all required components to operate it, such as the
We then conclude in Chapter 5 with perspectives on advanced data acquisition hardware and software. We use the terms “active”
measurements and applications of MEAs for studying neuronal and “passive” to distinguish between devices with active circuit
function. elements, such as transistors, and devices without such elements.

MEA TECHNOLOGY ELECTRODES AND TRANSDUCERS

This chapter reviews the technology involved in MEA develop- There are various techniques for fabricating microelectrodes,
ment. which are reviewed by Li et al. (2003), Park and Shuler (2003),
Huang et al. (2009). Choosing the materials for the insulator, con-
DEVICE TYPES AND TERMINOLOGY ductor, microelectrode, and substrate is crucial, in particular with
Over the years, a wide repertoire of terms has been used to refer respect to biocompatibility. All materials in the MEA that will be
to and distinguish between all the different forms of MEAs, e.g., near to or in contact with cells and tissue need to be tested for tox-
emphasizing the type of transducers used (multi-transistor array, icity in prolonged periods of time (Hassler et al., 2011). It is also
microelectrode array, multielectrode array, micro-nail array, important to consider the biological experiments for which the
capacitive-coupled array, 3D MEA), the type of substrate (active microelectrodes will be used, whether in vivo or in vitro, culture or
array, passive array, silicon array, CMOS array), the shape of the acute preparation. Moreover, deciding the type of MEA to use is

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Obien et al. Revealing neuronal function through MEA recordings

highly dependent on the type of recorded signals needed, whether Electroporation was shown to facilitate measurement of intracel-
EAPs and/or LFPs or intracellular action potentials (IAPs), single lular activity (Koester et al., 2010; Hai and Spira, 2012).
cell resolution or not. If the MEA is to be used for stimulation, the
charge capacity of electrodes is an important aspect. The electrode ADVANCES IN MEA AND PROBE DEVICES
needs to be able to mediate reactions at the electrode-electrolyte Since the single extracellular microelectrodes used in the middle
interface to allow electron flow in the electrode to transition into of the last century (Weale, 1951; Gesteland et al., 1959), devel-
ion flow in the electrolyte toward stimulating nearby cells (Cogan, opment quickly proceeded to MEAs with multiple transducers
2008). for the purpose of increasing the number of neurons observed
Generally, an important goal of electrode fabrication is to (Thomas et al., 1972; Gross et al., 1977; Pine, 1980; Csicsvari et al.,
achieve low impedance. Low electrode impedance results in 2003) to increase reliability of spike sorting (Gray et al., 1995;
higher signal-to-noise ratio (SNR), with the usual target SNR of Harris et al., 2000) and to allow for source localization (Blanche
5:1 or higher. Uniformity of the electrode impedance across an et al., 2005; Chelaru and Jog, 2005; Frey et al., 2009b; Somogyvári
array of electrodes may also be important to obtain consistent et al., 2012; Delgado Ruz and Schultz, 2014). The advances in
data. lithographic techniques, fueled by the semiconductor industry,
Typically, electrodes are made with metallic conductors such as allowed a gradual increase in performance and reliability of such
gold (Au), titanium nitride (TiN), platinum (Pt), stainless steel, multichannel devices. Passive transducer devices based on elec-
aluminum (Al), and alloys like iridium oxide (IrOx). Since the trodes embedded in glass or silicon substrates with fixed wiring
electrodes used in MEAs are on the micrometer scale, it is a to amplifiers for in vitro and also in vivo applications became
challenge to achieve low electrode impedance with plain conduc- commercially available in the late 90 s and early years of this
tors only. Increasing the effective surface area of electrodes can century. Already early on, silicon-based biosensors for interfac-
be achieved by modification with porous conductive materials ing cells with microelectronics were developed (Bergveld, 1970;
such as Pt-black, Au nanostructures, carbon nanotubes (CNTs), Parce et al., 1989). Active devices, employing FETs were fabricated
and conductive polymers like poly(3,4-ethylenedioxythiophene) and 2D arrays demonstrated (Besl and Fromherz, 2002). Devices
(PEDOT). Emerging materials aside from PEDOT and CNTs using CMOS technology were fabricated in academic facilities
include doped diamond and graphene. By modifying the sur- (DeBusschere and Kovacs, 2001) and industrial foundries, usually
face, the electrode impedance can be decreased drastically and in conjunction with additional processing steps for biocompati-
neuronal recording can be improved (Cui et al., 2001; Franks bility reasons (Berdondini et al., 2002; Eversmann et al., 2003b;
et al., 2005; Ludwig et al., 2006; Keefer et al., 2008; Viswam et al., Franks et al., 2003).
2014). Nam and Wheeler (2011), Kim et al. (2014) for a review of The key advantage of integrating active electronic components
electrode materials and surface modification. on the same substrate as the actual electrodes is the possibil-
Non-metallic electrodes have been mostly used in conjunction ity of a much higher electrode number and density. Due to the
with field-effect transistor (FET) based transducers (Bergveld, possibility of using active switches to time multiplex signals,
1970; Fromherz et al., 1991). An OGFET can, e.g., be obtained integrated circuits make it feasible to transfer data from such
if the fabrication process of a FET is stopped before deposit- high channel counts off chip and to overcome the connectiv-
ing the gate material (Jenkner et al., 2004). Easier to fabricate is ity limitation of passive devices. Additionally, such co-integration
the so-called extended-gate FET (EGFET), in which the FET is allows amplifying the signals with optimal quality, due to minimal
fabricated without modification from a standard CMOS process. parasitic capacitances and resistances (Hierlemann et al., 2011).
Metal and via interconnections are used to extend the gate to the The monolithic co-integration also allows including additional
surface of the chip, where an insulated electrode implements the functionality, e.g., on-chip spike detection, closed-loop capabil-
“extended gate.” Such insulation ensures that no faradaic currents ities, electrical stimulation, electronic chip identification, device
occur. However, as Hierlemann et al., pointed out, devices with calibration, and other type of sensing modalities, such as temper-
metal electrodes also usually connect to a FET directly (Imfeld ature, pH or optical sensing (Baumann et al., 1999; Tokuda et al.,
et al., 2008) or through a filter capacitor (Heer et al., 2006), 2006; Johnson et al., 2013b).
resulting in a largely capacitive recording situation (Hierlemann Figure 2A compares a variety of historical and current devices,
et al., 2011). OGFET, EGFET, and devices that directly connect to illustrate the evolution of MEAs with respect to overall sensing
the electrode to the first FET usually need to include some mea- area and electrode densities. The electrode count is shown with
sures to properly bias the gate or some calibration mechanism, solid lines. The devices are categorized into fixed wiring (Type
which may cause transient currents to flow at the electrode. A&B in Figure 3) and multiplexed arrays (Types C–E in Figure 3).
Whereas for devices with a capacitively coupled front-end stage, Fixed-wiring arrays include devices without any on-chip cir-
controlling the electrode input node is generally not needed. cuitry (Alpha MED Science Co., Ltd.1 ; Multi Channel Systems
Devices with a FET-based transducer, but using a metalized gate GmbH2 ; Thomas et al., 1972; Gross et al., 1977; Pine, 1980;
exposed to the liquid, have also been developed (Jobling et al.,
1981). 1 Alpha MED Science Co., Ltd. MED64: A low-noise and user-friendly multi-
Recently, ultra-small electrodes are being developed to record
electrode array system for in-vitro electrophysiology. Available at: http://www.
intracellular activity, including subthreshold signals, as reviewed med64.com [Accessed December 1, 2014].
in Spira and Hai (2013). This is achieved by 3D structured 2 Multi Channel Systems GmbH. MCS: Innovations in Electrophysiology.
electrodes such as silicon nanowires (Robinson et al., 2013) and Available at: http://www.multichannelsystems.com [Accessed December 1,
Au mushrooms (Hai et al., 2009) penetrating the cell membrane. 2014].

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Obien et al. Revealing neuronal function through MEA recordings

A
Johnson127

3 Ballini14

Montgomery083 Berényi143
Csicsvari033
2

Tokuda065
Olsson053 Eversmann03a6
Lopez143
3

Blanche05C3
DeBusschere014 Fujisawa083
Blanche05B3 3
Kipke033

DeBusschere014

Lopez143

Johnson127
μVrms

Ballini14

Eversmann03a6

FIGURE 2 | Device comparison. MEA comparison with respect to (A) under which these measurements were taken usually differ significantly
electrode density and total sensing area, and (B) parallel recording channel (such as noise bandwidth, in- or exclusion of electrode noise, inclusion of
count and noise level. (A) For devices with a regular sensor pitch, such as ADC quantization noise, etc.). Therefore, this graph only serves as a rough
most in vitro MEA devices, the total area is calculated as number of comparison. The waveforms to illustrate the noise levels are simulated and
electrodes times the pixel area. For all devices, the number of electrode have a spectrum typical for MEA recordings. The simulated spikes are typical
times the inverse of the electrode density matches the total area. The light spikes for acute brain slice measurements recorded with microelectrodes.
gray lines illustrate the number of electrodes. (B) The noise values shown are The recorded amplitudes may vary significantly depending on preparation and
approximated RMS values stated in the respective citations. The conditions sensor characteristics. See Footnotes:3,4,5,6,7 .

Regehr et al., 1989; Nisch et al., 1994; Oka et al., 1999; Litke et al., 3 The area is calculated as the rectangle of the maximum vertical extend times
2004; Segev et al., 2004; Greschner et al., 2014), but also MEAs the maximum horizontal extend, whereas for probes, the horizontal extend is
with on-chip circuitry limited to the surrounding of the array taken as the shaft width.
4 Only a single sub array of 16 electrodes is considered.
(Greve et al., 2007) and arrays that include FETs (Offenhäusser
5 Features a frame rate significantly lower as compared to the other devices.
et al., 1997) and source follower devices directly wired to circuitry 6 Noise values are taken from Lambacher et al. (2010).
outside the array (DeBusschere and Kovacs, 2001). Multiplexed 7 The authors state that with a new acquisition board, the parallel channel
arrays employ some sort of multiplexing within the actual array count could be increased to 1024 at 9300 fps.

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Obien et al. Revealing neuronal function through MEA recordings

(Eversmann et al., 2003a, 2011; Heer et al., 2006; Tokuda et al., The power budget for the entire device, including all circuitry
2006; Aziz et al., 2009; Berdondini et al., 2005, 2009; Frey et al., within the array and surrounding it, is limited by the amount of
2010; Huys et al., 2012; Johnson et al., 2012, 2013a,b; Maccione produced heat that one can tolerate. For the area constraints, one
et al., 2013; Ballini et al., 2014; Bertotti et al., 2014). has to separately consider the area within the array and surround-
For in vivo MEAs, the connectivity limitation is even more ing it. Within the array, the electrode density dictates the available
severe, as connections cannot be wired out on all four sides of area per pixel. Outside the array, the area is limited mostly by
the array, but only on one of the narrow sides. Figure 2A includes the fabrication cost. As a trivial approach to decouple the area
some examples of such devices using fixed wiring (Wise et al., requirement from the noise specifications, one can simply place
1970; Najafi and Wise, 1986; Jones et al., 1992; O’Keefe and Recce, the amplifiers outside the array and directly wire one electrode to
1993; Gray et al., 1995; Bai and Wise, 2001; Csicsvari et al., 2003; one amplifier (Figure 3B). However, this approach still does not
Kipke et al., 2003; Blanche et al., 2005; Olsson and Wise, 2005; allow achieving both a high density and a large electrode count
Fujisawa et al., 2008; Montgomery et al., 2008; Herwik et al., 2009; at the same time. Figure 3 lists these fixed-wiring approaches and
Du et al., 2011; Berényi et al., 2014) and three recent in vivo MEAs typical array architectures using multiplexing within the array to
with multiplexing on the shaft itself (Shahrokhi et al., 2010; Seidl overcome this limitation.
et al., 2011; Lopez et al., 2014). For detailed reviews of in vivo Active switching can be integrated into the array, allowing to
MEAs (see Wise et al., 2008, 2004; Ruther et al., 2010). time multiplex the signals from many electrodes to a few wires
Figure 2B, on the other hand, focuses only on CMOS-based that carry the signals out of the array. We now consider two
devices and illustrates the tradeoff between the number of par- types of time multiplexing, static (Figures 3C,D) and dynamic
allel (or quasi parallel) readout channels and the input referred (Figure 3E) operation (Imfeld et al., 2008). In dynamic mode,
noise of the amplification chain. It illustrates the fundamental each pixel (or electrode) is sampled once within each frame,
fact that a low-noise front-end amplifier requires both area and with typical frame-rates of 2–10 kHz for CMOS-based MEAs
power. Limiting either will inherently increase the noise levels. (Eversmann et al., 2003a; Johnson et al., 2013b) and some devices

Advantages Disadvantages References

A
pads
circuitry Increased
material

B
on On

funconality
Mux
mulplexer

C On
Row Decoder

ount
c
array.

Column Amp/ADC

D m m m Mulple On
2014
m m m switch matrix 2011)
concept

Column Amp/ADC

E On a
Row Decoder

ull Berdond
frame readout Full dout
(APS)

Column Amp/ADC 2013b

Front-end Amplifier Selected Electrode Inactive Electrode Signal Pad Power Supply Pad Switch m Local Memory

FIGURE 3 | Array architectures. This table summarizes and classifies the circuitry for signal conditioning. (C–E) Multiplexed arrays. (C) Signals are
different architectures that are typically used for MEAs. Advantages, multiplexed to the signal pads via column, row addressing in static mode. (D)
disadvantages are stated and representative selected references given. (A,B) More flexible addressing is achieved by adding more routing resources within
Fixed wiring. (A) Electrodes are directly connected to signal pads with no the array in the switch-matrix mode. (E) All electrodes can be sampled at fast
active circuitry. (B) Electrodes are directly connected to on-chip active speeds in full-frame readout implemented in active pixel sensor (APS) MEAs.

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Obien et al. Revealing neuronal function through MEA recordings

allowing as high as 77 kHz (Bertotti et al., 2014). This mode is same also holds true for the flexibility in the recording band-
similar to image sensors used in cameras. Typically, rectangular width. Some applications may require lower frequency signals
sub-arrays can be chosen as regions of interest and sampled at only, some only spikes in the EAP band, some both bands with
faster rates. From a circuit perspective, the challenge in design- different gain requirements at the same time. The circuits need
ing full-frame readout MEAs lies in the fact that the multiplexing to implement some sort of high-pass filter to block the large 1/f
within the array requires the front-end amplifier to be located noise of the electrode-liquid interface typically observed. MEA
within the pixel itself, as the electrode alone exhibits a high systems can also include stimulation circuitry, covered in the
impedance and therefore cannot drive the multiplexed readout next section, and analog-to-digital conversion (ADC). They need
lines at sufficient speed. Inherently, the available area within the to include an interface to transmit the data and receive com-
pixels is limited in high-density arrays, making it difficult to build mands for controlling the system’s operation. The requirements
very low noise amplifiers. In addition, the electrodes themselves are different for implantable devices, where usually the target
and the activity within the culture medium show wide band noise application is much more defined, but also the power, reliability,
(see Section Noise and SNR), thus requiring a low-pass filter and safety requirements are more stringent. These systems often
within the pixel to prevent noise from being aliased into the sig- implement spike detection or classification and wireless trans-
nal band due to the sampling. Generally full-frame readout arrays mission in the system, either as a monolithic implementation
have a high channel count, and therefore the power budget per or hybrid approach using multiple ICs. They may also be pow-
channel is very limited. ered wirelessly. On the other hand, in vitro MEA systems do not
Alternative approaches to circumvent this issue and to allow require wireless power or data transmission, as they can gener-
for devices in which the circuit itself is not the limiting factor with ally be directly wired to the data-receiving device. In this case,
respect to noise performance have been demonstrated. Arrays often common interface standards are employed, such as USB
operating in static mode (Figures 3C,D) have only switches and (Multi Channel Systems GmbH2 ), Ethernet (Frey et al., 2010),
no amplifiers as active devices within the array. The switches are National Instrument’s DAQ card (Alpha MED Science Co., Ltd.1 ),
used to wire electrodes to front-end amplifiers placed outside of CameraLink (Imfeld et al., 2008), or others. Most of these sys-
the array, where sufficient area for the implementation of low- tems support online storage of the full raw data to hard disks,
noise amplifiers is available. This also decouples the number of sometimes including some form of lossless data compression
electrodes from the number of readout channels, which allows (Sedivy et al., 2007).
budgeting of the available power in more flexible ways. Devices Many of the circuit requirements can be traded against each
that employ a simple column and row based static addressing other, e.g., one can easily lower the noise by increasing the area
are limited in the flexibility of choosing electrodes for parallel or power consumption. The key challenge therefore is to set the
readout. A switch-matrix implementation, which consists of a target specifications for the given application accurately and opti-
large set of routing wires, routing switches, and local memory, mize the systems for it, without overdesigning specific require-
such as SRAM cells within the array, allows the use of complex ments. Further considerations with respect to noise are given
routing paths to rewire a subset of electrodes to the available in Section Noise and SNR. Reviews focusing on circuit related
readout and stimulation channels in a flexible manner. Often, issues can be found here: (Wise et al., 2004, 2008; Harrison, 2008;
such an approach is sufficient to observe biological phenomena Jochum et al., 2009; Gosselin, 2011).
of interest, as typically not all electrodes exhibit activity. However,
experimental protocols tend to get more complex, as one needs STIMULATION
to select the “right” electrodes during the experiment. One of MEAs allow passive observation, and also active influence and
the protocols commonly used for such devices is to first scan the control of neuronal activity. Metal electrodes can deliver electrical
entire array in static mode, i.e., record from each rectangular sub stimuli directly using the microelectrodes, whereas for OGFET-
block for, e.g., a few minutes, run some online or quasi online based devices, typically an extra capacitive stimulation spot is
data processing on the recorded data, and select a more refined used to deliver stimuli (Stett et al., 1997). In addition, monolithic
subset based on the recorded activity and the scientific objective CMOS integration of MEAs opens up the possibility to include
of the experiment. electrical stimulation circuitry directly on-chip, in turn allowing
Apart from the array, CMOS devices also require the design a high degree of flexibility in generating spatiotemporal patterns
of neuronal amplifiers and some sort of data transmitter, either of stimulation, higher spatial resolution for stimulation and direct
of the amplified analog signals or, more typically, of the already on-chip stimulation artifact blanking or suppression.
digitized data. Generally, a neural amplifier needs to have high Already the very first electrophysiological experiments with
input impedance, which is significantly higher than the electrode frogs by Galvani (1791) involved electrical stimulations using
impedance, to ensure signal integrity. The amplifier should be of metal wires connected to various sources, e.g., Leyden jars,
low power to prevent substrate heating that could damage cells Franklin’s magic squares, and even atmospheric electricity dur-
or tissue. For in vitro MEA devices, a variety of target applica- ing lightning. In vivo, electrical stimulation is commonly used
tions have to be considered. Therefore, gain and dynamic range to stimulate nerves for transmitting sensory information to the
requirements can be quite demanding and should be adjustable, brain, such as for cochlear implants (Wilson and Dorman, 2008)
such as to cover applications with maximal amplitudes of a few and retinal implants (Ahuja et al., 2011; Zrenner et al., 2011); to
hundred microvolts in acute slice preparations and, on the other control, e.g., limbs for neurorehabilitation after nervous system
hand, up to 10 mV in measurements from cardiomyocytes. The injury; and to treat disorders, e.g., Parkinson’s disease by deep

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Obien et al. Revealing neuronal function through MEA recordings

brain stimulation using brain pacemakers (Montgomery and methods are used. Jimbo et al. proposed a method to decouple
Gale, 2008). In such applications, the physical distance between the recording amplifiers during stimulation, sample the electrode
the stimulation electrode and target nerves can be rather large, potential during recording and add the stimulation pulse to the
requiring the delivery of high amplitude stimuli. stored electrode potential (Jimbo et al., 2003). This scheme has
Lilly et al. (1955) established charged balanced methods using also been implemented on dedicated ASICs to be used in conjunc-
biphasic brief pulses to limit the damage to the tissue and the tion with MEA devices (Brown et al., 2008; Hottowy et al., 2012;
degradation of the electrodes themselves. Merrill et al. reviewed Tateno and Nishikawa, 2014). Figures 4E,F show stimuli activated
electrical stimulation using electrodes, listing various materials neuronal responses with high spatiotemporal precision. In a study
(Merrill et al., 2005). For in vitro MEAs, effective stimulation pro- to track axonal APs (Bakkum et al., 2013) several ten thousands
tocols were characterized by Wagenaar et al. (2004). The authors of stimuli were required, which was possible without damaging
studied different stimulation parameters (pulse width, amplitude, the electrodes or cells. In this case, voltage-mode stimulation was
pulse shape) that evoke neuronal activity. used, although the stimulation hardware supported both current-
One application of electrical stimulation is the use of it as and voltage-mode (Livi et al., 2010).
a “trigger,” so-called stimulus-triggered averaging (Cheney and Closed-loop experiments, in which neural activity triggers
Fetz, 1985). Electrical stimulation allows delivering trigger pulses electrical stimulation, employing on-chip stimulation circuitry
of high temporal resolution in the order of a few microseconds, have been presented by Hafizovic et al. (2007) and Müller et al.
depending on the stimulation buffer used and the capacitive load (2013). In both cases, the spike detection is performed off-chip on
of the electrode. Stimulation can evoke responses with small dedicated FPGA hardware. The actual decision to stimulate and
temporal jitter, e.g., Bakkum et al. observed a jitter of 160 μs the selection of the stimulation waveform patterns is performed
using passive MEAs (Bakkum et al., 2008). Bakkum et al. used on a personal computer in Hafizovic et al. (2007), whereas in
trigger signals to study the velocity of action potential (AP) prop- Müller et al. (2013) an event engine performing this task is imple-
agation in axons of cultured neurons (Bakkum et al., 2013). mented directly on the FPGA platform, making the latency until
Figure 4A shows how such stimulus-triggered averages revealed stimulation shorter and, importantly, reducing its temporal jitter.
small axonal spikes of different shapes, such as bi- and tri-phasic CMOS-based devices exclusively devoted to stimulation at
types. Figure 4B illustrates the reduction in uncorrelated noise high spatio-temporal resolution of close to 7000 electrode per
with increasing number of averaged repetitions. One potential square millimeter and with variable voltage mode pulses have
issue with delivering electrical stimulation to neuronal cells and been developed as well (Lei et al., 2008, 2011). Circuit considera-
tissue is the occurrence of artifacts in recording channels, due tions for CMOS-based devices for clinical in vivo application are
to the fact that stimulation pulses are typically three to four reviewed (e.g., Ortmanns et al., 2008; Ohta et al., 2009).
orders of magnitude larger than the recorded signals. This cou-
pling between stimulation and recording is difficult to prevent, APPLICATIONS OF IN VITRO CMOS-BASED MEAs
and artifacts are picked up both within the wiring of the array In vitro CMOS MEAs have already been used in a wide vari-
and circuits, but also through the medium of the cell culture or ety of applications, for recording, for electrical stimulation or
tissue. However, as long as the coupling is purely capacitive, arti- for both. Figure 5 lists in vitro CMOS MEAs, their key specifi-
facts usually only prevent recording during the stimulation period cations and preparations for which they have been used so far.
itself. If the amplitude of an artifact is large, which can occur when Some additional in vitro CMOS-based MEAs that are not listed
a recording electrode is near the stimulation electrode, the artifact in Figure 5 can be found here: (Tokuda et al., 2006; Greve et al.,
may saturate the amplification circuits of the recording electrode. 2007; Meyburg et al., 2007; Yegin et al., 2009; Johnson et al., 2012).
This saturation will prevent recording for an extended period In addition, the functionality of some in vivo CMOS MEAs has
of time after the stimulation ended. Figure 4C shows an exam- also been demonstrated using in vitro applications (Aziz et al.,
ple of such a saturated signal from an electrode located 18 μm 2009).
(center-center) away from the stimulation electrode and a signal The two most prominent preparations investigated with
without saturation from an electrode located about a 1 mm away. in vitro CMOS MEAs so far are acute retina preparations from
Figure 4D shows the relationship between the distance from stim- mice (Menzler and Zeck, 2011; Fiscella et al., 2012; Maccione
ulation to recording electrode and the duration of saturation for et al., 2014), rats (Eickenscheidt et al., 2012; Lloyd et al., 2014;
a 11,011-electrode MEA (Frey et al., 2010), without employing Stutzki et al., 2014), rabbits (Zeck et al., 2011; Ballini et al.,
any artifact suppression measures. As long as the amplifiers do 2014; Fiscella et al., 2014), guinea pig (Velychko et al., 2014)
not fully saturate, it is possible to suppress such artifacts in soft- and humans (Reinhardt and Blickhan, 2014); and cultured neu-
ware by subtracting the estimated artifact (based on templates, ronal cells from snails (Eversmann et al., 2003a), rats (Hafizovic
filters or local curve fitting) from the data (Hashimoto et al., et al., 2007; Heer et al., 2007; Gandolfo et al., 2010; Lambacher
2002; Wagenaar and Potter, 2002). To also allow recording from et al., 2010; Bakkum et al., 2013; Ballini et al., 2014) and chicken
electrodes on which saturation would occur, counter measures in (Hafizovic et al., 2007). Additionally, data from acute slices of
hardware have to be employed. One solution is to use a “reset” the cerebellum (Frey et al., 2009a; Obien et al., 2014), cortex
switch that can bring back the saturated amplifier into normal (Ferrea et al., 2012; Medrihan et al., 2014) and olfactory bulb
operation quickly, by resetting the high-pass filter of the front-end (Johnson et al., 2013a) have been shown. Also cultured cardiomy-
amplifier (Heer et al., 2006; Frey et al., 2010). To suppress arti- ocytes were studied (DeBusschere and Kovacs, 2001; Heer et al.,
facts even on the stimulation electrode itself, more sophisticated 2004; Imfeld et al., 2008; Sanchez-Bustamante et al., 2008; Huys

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 4 | Stimulation capability of high-resolution CMOS-based MEA. (B) The amount of averaging necessary to detect a spike with a given height
(A) Examples of evoked spikes detected at three sites (columns) along the (0.5–3 σ) with respect to the detection threshold. (C) Left: A raw voltage
same axon. The top row shows individual raw traces, and the other rows trace recorded at an electrode neighboring a stimulation electrode
show traces averaged as indicated. Scale bars, 1 ms horizontal, 10 μV vertical. (Continued)

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 4 | Continued (empty or filled gray boxes) APs detected at a soma located ∼890 μm
saturated for about 4 ms (flat line). Right: A raw voltage trace recorded at away. The line arrow indicates the orthodromic propagation direction.
an electrode located 1.46 mm away from a stimulation electrode did not Scale bar, 20 μm. (F) Voltage traces of somatic APs elicited by biphasic
saturate. (D) The duration of a saturated signal occurring after stimuli is voltage stimuli. Traces in response to eight stimuli are overlaid for each
plotted vs. distance from the stimulation electrode (mean ± s.e.m.; of three stimulation magnitudes (indicated at the top), plotted for all
N = 18 stimulation electrodes from five CMOS-based MEAs). Stimuli effective (black) and four ineffective stimulation sites (gray at the bottom).
consisted of biphasic voltage pulses between 100 and 200 ms duration Stimulation electrode locations are represented as numbered boxes in
per phase and between ± 400 and 800 mV amplitude. (E) Locations of (E). Scale bar, 200 μV. All panels and description adapted with permission
stimulation electrodes that directly evoked (black boxes) or did not evoke from Bakkum et al. (2013).

et al., 2012) and first results from mice organotypic slices were through the formation of dipoles (Niedermeyer and da Silva,
presented (Gong et al., 2014). 2005; Nunez and Srinivasan, 2006). We refer the reader to Einevoll
Certainly, in vitro CMOS-based MEAs, being still an emerg- et al.’s extensive review on the modeling and analysis of LFPs for
ing technology with commercial availability only starting recently, further details (Einevoll et al., 2013). The relationship between
have a high potential for future biomedical research and LFPs and spikes has also been discussed and studied in several
diagnostics (Jones et al., 2011). works (Khazipov et al., 2004; Belitski et al., 2008; Montemurro
et al., 2008; Minlebaev et al., 2011; Kayser et al., 2012; Cingolani,
UNDERSTANDING MEA SIGNALS 2014).
Here, we describe the parameters that contribute to neuronal
signal transduction from the source into digital form. MEA SIGNAL FLOW
We consider the components of the MEA recording and stimu-
WHAT DO MICROELECTRODES DETECT? lation system diagram as shown in Figure 6: (A) the conductive
A microelectrode can detect the changes in the extracellular field extracellular volume where the electric field caused by neu-
caused by the current flows from all ionic processes across the ral signal sources forms; (B) the substrate with the embedded
morphology of the closest neuron and from other nearby cells, microelectrodes; and (C) the hardware connected to the elec-
not only neurons (Buzsáki et al., 2012; Anastassiou et al., 2013). trodes, including amplifiers, filters, digitizer, data transmission,
The effect of the transmembrane currents on the electric field and and stimulator (Stett et al., 2003; Fejtl et al., 2006).
the detected potential on a microelectrode depend on the magni-
tude, sign, and the distance from the recording site (Nunez and Noise and SNR
Srinivasan, 2006), see Section The extracellular space. One crucial aspect of the MEA signal flow is how noise is fed
An AP is a biophysical event that occurs once the neuron’s into the amplification chain and how it affects the SNR of the
transmembrane potential reaches a threshold due to stimuli or recorded data. SNR is the key specification for the amplifier
other inputs (e.g., synapses, gap junctions). On the other hand, we design, regardless of the actual amplification (Jochum et al.,
consider a “spike” to be the signal from a putative AP. For extracel- 2009). It is important to consider where the noise, or interfer-
lular recordings, spikes are commonly identified as voltage signals ence, is injected in the signal chain, as the implications on SNR
that exceed a threshold. During an AP, the initial rapid Na+ ion will differ.
influx creates a sink and results in a large negative spike in the
EAP. Thereafter, the slow K+ efflux produces a source resulting in (a) Biological noise. This is a major source of noise stems from
a small positive spike. In contrast, IAP first shows a positive spike the electrical activity of other cells around the recording elec-
and later a negative volley. EAPs are usually around tens to hun- trode, e.g., APs of distant cells, but also ionic activity, e.g.,
dreds of microvolts in amplitude and <2 ms in duration while subthreshold events in neurites of nearby cells, and synaptic
IAPs are at tens of millivolts and around the same duration as noise due to the stochastic nature of synaptic transmis-
EAPs (Buzsáki et al., 2012). If IAPs can only be detected by direct sion. Several models of biological noise, or sometimes also
access inside the neuron, e.g., patch-clamp, EAPs can be identi- called background noise, have been developed by simulat-
fied when electrodes are placed at the vicinity (∼100 μm) of the ing uncorrelated single-unit spiking activities or examining
spike origin (Henze et al., 2000; Egert et al., 2002), usually at the multi-compartmental neuron models located at distances far
perisomatic area, i.e., around the soma or near the axon initial enough away from the electrodes such that the spikes can-
segment. not be resolved (Eaton and Henriquez, 2005; Martinez et al.,
Aside from measuring single- and multi-unit spiking activity, 2009; Lempka et al., 2011; Jäckel et al., 2012; Camuñas-Mesa
electrodes also sample LFPs. The LFP is assessed by the signal con- and Quian Quiroga, 2013). Although such models replicate
tent in the low-frequency band of the recorded signal (<300 Hz) the average biological noise in experiments, it is possible that
(Belitski et al., 2008; Buzsáki et al., 2012), while EAPs are ana- the cell type, size, and morphology, along with the firing rates
lyzed after filtering the LFP out (300–3000 Hz) (Quian Quiroga, and correlated activity, can affect the shape of the background
2009). Although the contribution of EAPs to LFP is still unclear, a signal. For spike analysis, the LFP is also considered biological
synchrony of APs from many neurons can participate in the gen- noise and filtered out.
eration of LFPs (Buzsáki et al., 2012). The current opinion is that (b) Electrode-electrolyte interface noise. On top of the biological
synchronized synaptic currents in cortical neurons produce LFPs, noise, the liquid-metal interface also adds to noise. At low

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Obien et al. Revealing neuronal function through MEA recordings

Ref Micrograph
μm
2
2001)
(2.4%)
Typ

2
a)

μm Acut (
2

2
(2.9%) a

μm
2

2
(19%)

μm Cultures (Rc):
2

2
(25%)
4096

Tech μm
2
a)

2009a 2014)
2
(7.6%) C

(Sanchez
)
2

μm
2
2012)
2
14.7mm (23%)

2
mm
mW
μm
2

2
(70%)
Oth

μm Cultures
2

2
(11%)

Technolo μm
2

FIGURE 5 | CMOS-based in vitro MEAs. CMOS-based in vitro MEAs, their representative citation for each type of preparation. The specification for each
key specifications and references to biological applications for recording and device are taken from the reference listed on top and may differ for other
stimulation are listed in this table. The application list includes only one versions of the device.

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 6 | MEA stimulation and recording system diagram with neuron, typically an action potential, is transformed by different
the noise sources. The neuron is stimulated by the pulses or parameters across the components of the MEA toward the recorded
waveform generated digitally through the MEA. The response of the signal.

frequencies, such as below 10 Hz, processes at the electrode efficiency factor or PEF (Muller et al., 2012). For in vitro
generate noise with a steep roll-off of 1/f or even 1/f 2 (Hassibi MEAs, area is also of critical importance, as it usually impacts
et al., 2004; Heer, 2005). More relevant for electrophysiol- electrode density and total channel count. The efficient use of
ogy are the frequencies above that, where thermal noise is the the overall area is reflected in the ratio of the actual array area
main contributor (Gesteland et al., 1959; Liu et al., 2007). The divided by the overall chip area (see Figure 5). Quantization
equivalent thermal noise can be calculated as follows: noise is another noise contributor of the hardware. It origi-
 nates from the discretization error made at the ADC part of
  the MEA system. As an approximation for the quantization
vn = 4 · k · T · Re Ze · f ,
noise, typically a value of √1 times the magnitude of the
12
least significant bit (LSB) is used. Typical ADCs applied for
where k is the Boltzmann constant, T is the absolute tem-
 MEA systems have a minimum of 8-bit resolution, with sys-
perature, Re(Ze ) is the real part of the effective electrode
tems that employ off-chip ADCs often using 16-bit or higher
impedance (see Section Neuron-electrode interface), and f
resolution. The transmission of data may also affect the qual-
is the noise bandwidth. Another source of noise is the 50–
ity of the recorded signal, e.g., if a lossy compression has to be
60 Hz hum from power lines. This noise is largely picked
used due to bandwidth constraints.
up between the microelectrode and the connection to the
input of the preamplifier, due to its high impedance at that
The extracellular space
frequency. Hence, minimizing the distance between the elec-
trode and the amplifier is a major design requirement for The analysis of EAPs and LFPs usually assume a homogeneous,
MEA circuits (Harrison, 2008). Proper grounding and shield- resistive extracellular space based on the volume conductor the-
ing of the MEA setup can minimize interference. ory, i.e., Kirchhoff ’s current law or charge conservation and
(c) Device noise. Finally, the device or the system that amplifies Ohm’s law (Nunez and Srinivasan, 2006). The difference in wave-
and digitizes the signals further adds to noise. Usually, the forms of a signal recorded at different locations in the tissue is
front-end amplifier is the most important factor to consider. mainly due to how each neuronal source linearly sums up, with
A general design objective for such amplifiers is to ensure source contributions weighted inversely proportional to their dis-
that the signal acquisition system does not limit the system tance (Nunez and Srinivasan, 2006). Under the assumption of a
performance with regard to noise. As discussed above, this is purely homogeneous, isotropic, and ohmic extracellular medium,
a design tradeoff in which also power and circuit area may Maxwell’s equations of electromagnetism can be rewritten with
play a role. For example, if the maximal allowed contribu- appropriate Laplace boundary conditions, such that for a single
tion to noise from the circuitry is set to 10%, the amplifier point current source the following equation holds true for the
noise needs to be 45% or less as compared to the noise of potential at an electrode, Ve (Klee and Rall, 1977; Nunez and
the electrode. A commonly used figure of merit that captures Srinivasan, 2006; Anastassiou et al., 2013):
the tradeoff between noise and amplifiers’ supply current is I
the noise efficiency factor (NEF) proposed in Steyaert and Ve = ,
4π σ r
Sansen (1987). This figure has also been adapted to capture
the different supply voltages used to allow a better compar- where I is the point current, σ is the conductivity of the medium,
ison with respect to power consumption, coined the power and r is the distance between the point source and the recording

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Obien et al. Revealing neuronal function through MEA recordings

electrode. Since the membrane currents are distributed over the can accurately describe the interface at subcellular resolution.
cable-like morphology of a neuron, a line source approximation Detailed characterizations of the electrode model for various
(LSA) of current sources was also proposed (Holt, 1997; Gold materials have been carried out, see Section Electrodes and
et al., 2006; Einevoll et al., 2007). Transducers. Other studies on similar neuron-electrode equiv-
The presence of numerous cell bodies, dendritic structures, alent circuits were conducted by Ingebrandt et al. (2005), Joye
axonal bundles, blood vessels, and white matter in brain tissue et al. (2008), Thakore et al. (2012). These models assume that a
raises questions as to whether the brain can really be consid- tight seal between the neuron and electrode is needed to mea-
ered as purely ohmic. Moreover, the frequency spectra observed sure EAPs from isolated neurons. In the in vivo situation, such
in LFP and EEG (Pritchard, 1992; Freeman et al., 2003; Bédard close contacts usually do not exist and models usually focus less
et al., 2006a; Buzsaki, 2006; Bédard and Destexhe, 2009; Miller on the electrode properties themselves, but more on the electric
et al., 2009; Milstein et al., 2009) led to uncertainties regarding field generated by current sources in a conductive volume (Lind
the role of extracellular space in frequency dependent filtering. et al., 1991; Moffitt and McIntyre, 2005; Gold et al., 2006). For
Pettersen and Einevoll (2008) clarified that in a purely resistive HDMEAs, such volume conductor models match measurements
and homogeneous extracellular medium, amplitude variability for, e.g., the idealized case of point source in saline (Obien et al.,
and low-pass filtering of EAPs occur due to the spatial sepa- 2013), but also for complex neuronal morphologies in acute brain
ration of correlated current sources and sinks during a spike. slices (Frey et al., 2009a). In cell cultures, it has been observed
Similarly, Lindén et al. (2010) found that an intrinsic dendritic that EAPs are also detected by electrodes that do not have a tight
low-pass filtering affects the LFP, not the extracellular space. seal with the isolated neuron, even by electrodes that are relatively
Other interesting studies described how low-pass filtering effects distant from the neuronal source (Bakkum et al., 2013). Thus,
can be achieved in a medium of radially decaying conductivity we generalize the neuron-electrode model in Figure 7B, which
(exponential) around the source (Bédard et al., 2004, 2006b). applies to tissue slices and dissociated cell cultures.
Already in 1968, Robinson (1968) suggested that inhomo- One important assumption for this generalization is that we
geneities, such as the presence of glial cells in brain tissue, can can treat the MEA surface as an insulator allowing us to sepa-
considerably impact the extracellular recording of spiking activ- rate the neuron-electrode interface problem into two parts: (i)
ity. He also argued that since the resistance of the paths around the “fluid”-side and (ii) “metal”-side. We are able to do this separa-
glial cells are lower (for signals at 1 kHz) than the paths through tion because the high input impedance of MEA amplifiers largely
them (due to the membranes), the extracellular signals would prevents any effect of the metal electrode on the potential at the
flow between the cells, not through them. Thus, the structures “fluid”-side of the interface. This is valid, as long as the impedance
in the tissue can cause directional differences in the conduc- on the “metal”-side seen by the electrode is much larger as com-
tion of signals (Rice et al., 1993; Okada et al., 1994). Similar pared to the tissue or fluid impedance at all frequencies of interest.
results were achieved by Nelson et al. (2013) across fiber and The generalized interface model can then be interpreted such that
cell obstructions. Various studies explored different properties an electrode detects the average voltage present at the record-
of brain tissue conduction, such as anisotropy (Nicholson and ing site, as claimed by Robinson (1968), Nunez and Srinivasan
Freeman, 1975; Logothetis et al., 2007); anisotropy and inhomo- (2006), Nelson et al. (2008). The detected voltage is then shaped
geneity (Ranck, 1963a,b; Hoeltzell and Dykes, 1979; Goto et al., by the electrical characteristics of the interface. It should be noted
2010); and capacitive property (Gabriel et al., 1996a,b; Bédard that the model, as shown here, is adapted for the recording sit-
et al., 2004; Bédard and Destexhe, 2009). Whole brain analysis uation, focusing on the understanding of the neuronal signals
of the electrical tissue properties at the microscale may be use- as recorded by MEAs. Similar models have also been developed
ful for modeling and analyzing EAPs and LFPs from different and used for the application of electrical stimulation using micro-
groups of neurons in different brain areas. Using the four-point electrodes or capacitive stimulation spots, as discussed in Section
probes method (Kelvin sensing, with separate pairs of current- Stimulation.
carrying and voltage-sensing electrodes) is advisable for measur-
ing the electrical impedance of brain tissue, since it minimizes the “Fluid”-side: voltage at the electrode by volume conduction. For
influence of the impedance of the current carrying electrodes. simple geometries of the “fluid”-side, assuming that the MEA sur-
face is an insulating infinite plane and the fluid a homogenous,
Neuron-electrode interface isotropic medium, we can apply the method of images to the
Using an equivalent circuit model, the interface between neurons point-source equation given in Section The extracellular space,
and microelectrodes in vivo has been described and character- such that the potential Ve at any given electrode e can be solved
ized by Robinson (1968). Later, this concept has been adapted for using the following equation (Obien et al., 2013):
substrate integrated MEA devices, e.g., to compare metal micro- 1  In
electrodes with OGFET devices in simulations (Grattarola and Ve = .
2π σ rn
Martinoia, 1993). This representation of the neuron-electrode
interface was then coined the point-contact model (Weis and In represents the nth point current source and rn represents
Fromherz, 1997) and is shown in Figure 7A. It is a standard the distance between the point source and the recording elec-
model of the electrical characteristics of the interface, which has trode, with n = 1. . . N, where N is the number of individual
also been extended to an area-contact model (Buitenweg et al., point sources. For electrodes larger than an ideal point electrode,
2003; Fromherz, 2003) to consider the spatial distributions that Ve can be solved at multiple locations of the surface area of

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 7 | MEA neuron-electrode interface. (A) The classic point or area interface separating the problem into two parts. Upper—“Fluid”-side: The
contact model derived from Fromherz (2003). The cell membrane is potential at the electrode sites can be solved using the volume conductor
represented with an equivalent model based on the Hodgkin-Huxley model theory. The MEA surface is assumed to be an insulator such that the
of the squid axon (Hodgkin and Huxley, 1952). CM represents the method of images can be applied on Coulomb’s law to solve the potential at
capacitance across the neuronal membrane, i.e., the lipid bilayer. The any point on the MEA surface. The neuron-electrode distance influences the
voltage-gated ion channels (K for potassium and Na for sodium) are signal amplitude measured at the electrodes. High spatial resolution allows
represented by non-linear conductances, gK and gNa , and the leak is shown for recording at several locations of a single neuron, with large negative
as a linear conductance, gL . The reversal potentials that drive the flow of spikes located at the perisomatic area and positive spikes at the dendritic
ions are represented by EK , ENa , and EL . The ion flow is shown by IK , INa , IL , area, i.e., return current. Lower—“Metal”-side: The voltage measured at the
and IC . The other elements are described in the text. Vrec is the recorded electrode is shaped by the electrical parameters of the electrode-electrolyte
voltage signal. Typical IAP and EAP recordings are shown. The location of interface, represented by Ze ’ as the effective electrode impedance and Za ’
the scissors indicates where the “cut” can be made to separate the as the effective input impedance. This model is derived from Robinson
neuron-electrode interface into two parts. (B) Generalized neuron-electrode (1968), Nelson et al. (2008), Hierlemann et al. (2011).

the microelectrode and then averaged. The larger the electrode model, the input to the circuit is a low impedance voltage source
area, the larger the averaging effect (Grimnes and Martinsen, with the value corresponding to the potential resulting from the
2008). Anisotropy can also be incorporated in this model easily currents in the volume conductor discussed above. This voltage
(Nicholson and Freeman, 1975). However, more complex geome- (Ve ) is connected to the effective electrode impedance Ze , con-
tries of, e.g., the MEA device (such as in vivo neural probes) or sisting of Rspread , Rm , Re , Ce . Rspread is the spreading resistance,
an inhomogeneous medium generally require a finite element which is the resistance a current sees, that spreads from the micro-
method to solve for the electric field and the potential at the electrode into the electrolyte. Its value is mostly dependent on
electrode. the electrode geometry and the electrolyte conductivity. Re and
The orientation and distance between the neuronal source and Ce are the resistance and capacitance, respectively, of a simplified
the measuring electrode affect the amplitude and shape of the model of the electric double layer that forms at the electrode-
signals detected, as discussed in Section The extracellular space. electrolyte interface. This is a reduction of the more complex
The spread and decay of the signal over the MEA surface plane is model, consisting of a constant-phase-angle impedance, a charge-
highly correlated with the distance of the signal source from the transfer resistance, and a Warburg impedance. Rm is an additional
surface. This makes it possible to estimate the distance between resistance representing the metallic part of the microelectrode.
a current source and the MEA electrodes by measuring the volt- The effective amplifier input impedance, Za , is connected in
ages at high spatial resolution using an HDMEA (Obien et al., series to Ze , which includes the actual input impedance of the
2013). The same concept can be applied to estimate the neuron- amplifier Za and the shunting paths to ground outside the ampli-
electrode distance given a good model of the membrane currents fier (Rs and Cs ). Input amplifiers are designed to have a high
of the neuron being recorded (Somogyvári et al., 2005, 2012; Frey Za (above 10 M at 1 kHz) to limit the influence of Za on the
et al., 2009b; Delgado Ruz and Schultz, 2014). measured voltage (Robinson, 1968). The shunt resistance (Rs )
is usually negligible, but the shunt capacitance (Cs ) reduces Za ,
“Metal”-side: signal transformation by the electrode-electrolyte especially at higher frequencies (Robinson, 1968; Nelson et al.,
interface. The “metal”-part of the model is an equivalent circuit 2008). Cs is the combination of all capacitances from connec-
of the microelectrode modified from Robinson (1968), Franks tors and wires from the bath to the amplifier, and the capacitance
et al. (2005), Nelson et al. (2008), Hierlemann et al. (2011). In this from metal of the electrode (through the insulation) to the bath

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Obien et al. Revealing neuronal function through MEA recordings

(Robinson, 1968). The ratio of Ze (mostly Ce ) and Za is of recording site geometry are not crucial. This is because LFPs only
importance, so if the electrode impedance is low enough, the vary in a spatial scale much larger than the size of electrodes used
influence of shunt capacitance to the signal is small (Robinson, for extracellular recordings, e.g., by a few hundred micrometers
1968; Nelson et al., 2008). HDMEAs require small electrodes to (Katzner et al., 2009) or even by 1 mm (Destexhe et al., 1999). In
achieve a high resolution, and therefore also the Ce is usually addition, LFPs are of lower temporal frequency, making electrode
small. However, monolithic integration allows keeping Cs small noise a more important factor as in that range, it is dominated by
too. For example, Cs is estimated to be below 0.5 pF for the 1/f 2 noise, which makes larger electrodes more favorable.
HDMEA presented in Frey et al. (2010), whereas passive MEA can It is therefore important to choose optimal electrode sizes
have a significantly larger parasitic capacitance, depending on the depending on the targeted application. In addition, a high density
thickness of the insulation and the track width [e.g., James et al. of electrodes will inherently limit the electrode size.
measured values of 60–100 pF (James et al., 2004) and Nisch et al.
estimated it to be below 15 pF (Nisch et al., 1994)]. For measure- PRACTICAL APPLICATION OF MICROELECTRODE
ments requiring a high accuracy despite having a device with a RECORDINGS
large Cs , capacitance compensation circuits can be used, as those Here, we provide a brief overview on how to extract relevant
commonly used in patch-clamp amplifiers and, e.g., also used for information from distorted, convoluted, and noisy recorded sig-
highly accurate tissue impedance measurements (Logothetis et al., nals. We then review relevant applications of MEAs for the study
2007). of single neurons and networks using various techniques and
preparations.
Effect of electrode size and density
Sizes of published microelectrodes range from 5 to 50 μm in MEA SIGNAL PROCESSING AND SPIKE SORTING
diameter (Kim et al., 2014). Larger electrodes have a higher pos- MEA signal processing usually includes (1) filtering the raw data
sibility of getting physically near the neurons and of picking up traces, (2) spike detection, and (3) spike sorting.
higher amplitude spikes (Camuñas-Mesa and Quian Quiroga, First, the raw signal is processed to separate the fast APs from
2013), e.g., studies by Moxon (1999), Paik et al. (2003), Ward LFP and noise by applying a band-pass filter (Quian Quiroga,
et al. (2009), Andersen et al. (2010) claim that larger record- 2007), with a typical narrow band of 300–3000 Hz. Filtering
ing electrodes can record from more neurons simultaneously. methods aim to attain higher SNR and lower false positive rates.
However, large electrodes (>50 μm diameter) can average out The filtering process can add phase distortions and therefore alter
a neuron’s spatially localized peak signal amplitude with nearby the shape of the detected EAP. One can avoid such phase dis-
smaller amplitude signals. This reduces the peak signals, which tortions by using non-causal filters when future inputs are also
can result in a lower SNR. Electrode size also affects the elec- used for computation. In hardware implementations and online
trode impedance Ze , which in turn determines electrode noise filters, causal filters are typically used though, as non-causal filters
(see Section Noise and SNR). With that, there are three effects would require the usage of a data buffer (Quian Quiroga, 2009).
for which SNR improves with larger electrodes (reduced electrode Depending on the scientific goal, good practice is to record data
noise, reduced attenuation due to large Ze /Za ratio, and increased with wide-band filters (e.g., 1–7000 Hz) and negligible phase dis-
chance to “being at the right spot”), and one effect for which SNR tortion, then apply the narrower band filters only for the purpose
gets worse with larger electrodes (increased signal averaging). of the extraction of spike timing information, for which undis-
As discussed above, for EAP recording in the 300–3000 Hz torted spike shapes are not needed. One can then still use the spike
frequency band, electrode noise is mostly thermal and compa- timing information generated by the spike sorter to re-extract the
rably small, especially if some sort of electrode coating is used undistorted spike shapes from the original data.
and the electrode size is >5 μm in diameter. Without consider- Once the signal is filtered, the spikes are detected. Amplitude
ing electrode noise, Camuñas-Mesa et al. studied via simulation thresholding is commonly used, although other spike detec-
the optimal electrode size for an in vivo situation, considering tion methods have been implemented, e.g., two-point procedure
neuronal background activity. For their simulation parameters, (Borghi et al., 2007; Maccione et al., 2009) and template-matching
they found 40 μm to be the optimum (Camuñas-Mesa and Quian (Kim and McNames, 2007). The threshold is usually set as a mul-
Quiroga, 2013). For HDMEAs, the situation is a bit different. tiple (5 times) of the baseline noise level, calculated as the root
Most importantly, there is no need to enlarge the electrode to be mean square (RMS) of the signals with a mean value of zero. In
close to the location with the largest signal, as there will always the presence of many spikes, the threshold can be estimated using
be another electrode “at the right spot”. Secondly, the effective a measure based on the median, which is less sensitive to outliers
input capacitance can be significantly smaller as compared to pas- and therefore more robust with regard to spike frequency (Quian
sive devices, due to a small Cs , which in turn allows for a smaller Quiroga et al., 2004).
Ce . As a result, small electrodes are much more preferable in this After spike detection, spike shapes are grouped according
situation, with only electrode noise being the limiting factor. to their spike shape, which is referred to as spike sorting.
LFP and EAP recordings from neurons located distant to the Several feature extraction techniques have been used, e.g., prin-
electrodes feature lower spatial frequencies and therefore allow cipal component analysis or PCA (Quian Quiroga, 2007) and
for larger electrodes without signal degradation than recordings wavelet transform (Mallat, 1989). In the ideal case, distinct neu-
from neurons within close proximity. Especially for LFPs, Nelson rons will have spikes whose features belong to well-separated
and Pouget (2010) discussed that the electrode impedance and clusters, and each neuron will only be part of one cluster. In

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Obien et al. Revealing neuronal function through MEA recordings

practice, spike sorting often requires user supervision in order Kreuz (2013) for further details and methods on quantifying
to manually evaluate the performance of the procedure and cor- synchronization.
rect for errors, e.g., to merge nearby clusters or remove outliers.
For a detailed explanation of the spike sorting steps, the reader MEAs and neuronal cultures
is referred to other review articles (Lewicki, 1998; Einevoll et al., Since Pine reported the first MEA recordings from dissociated
2012a). Available spike sorting packages and frameworks include neuronal cultures in 1980 (Pine, 1980), the method has been
Wave_Clus (Quian Quiroga et al., 2004), NeuroQuest (Kwon expanded for pharmacological tests, diagnostics, and investi-
et al., 2012), SigMate (Mahmud et al., 2012), UltraMegaSort gation of neuronal growth and connectivity. Combination of
(Hill et al., 2011), EToS (Takekawa et al., 2010, 2012), and immunostaining, fluorescence microscopy, and MEA recording
QSpike tools (Mahmud et al., 2014), among others. HDMEAs can allows the identification of neuronal types and synapses, e.g.,
improve spike sorting performance since with high-resolution GABAergic and glutamatergic, and the analysis of neuronal elec-
spatial information, one can more efficiently separate individual trical activity in long-term cultures. Using this technique, Ito et al.
neurons (Gray et al., 1995; Jäckel et al., 2011; Franke et al., 2012). (2013) observed a correlation between synapse densities and elec-
A number of concerns have been raised regarding the effec- trical activity of cultured rat cortical networks (Figures 8A,B).
tiveness of spike sorting. In fact, it is difficult to validate spike The initial increase in glutamatergic and also GABAergic synapses
sorting algorithms and it is important to test them based on was accompanied with increasing electric activity, which reached
realistic simulated data (Einevoll et al., 2012b). For in vivo experi- a plateau after 28 days in culture when the synapses reached their
ments, or in acute recordings where the electrodes can move with final density.
respect to the neurons, drift may occur and alter the recorded More complex neuronal culture analyses can be done using
signal. Another issue is the amplitude variability of APs from a HDMEAs such as burst pattern tracking (Gandolfo et al., 2010)
single neuron that can lead to clustering errors, either intrinsi- and functional connectivity estimation (Maccione et al., 2012).
cally or due to bursts (McCormick et al., 1985; Henze et al., 2000; By plating low-density cultures, it is feasible to not only optically
Delescluse and Pouzat, 2006; Stratton et al., 2012), such that one visualize the network of stained neurons, but also to estimate the
cluster may contain the large amplitude spikes and the second one functional connections and to obtain detailed functional maps
the smaller amplitude ones (Van Dijck et al., 2012). at cellular resolution (Maccione et al., 2012), see Figures 8C,D.
Maccione et al. processed and analyzed the HDMEA signals by
USING MEAs FOR NEUROSCIENCE STUDIES ad hoc developed spatio-temporal filtering and by applying a
MEA recordings have been employed to understand neuronal cross-correlation based method.
communication, information encoding, propagation, and pro-
cessing in neuronal cultures as well as in brain slices and retina MEAs and brain slices
explants (Taketani and Baudry, 2006). Recent works start to take A brain slice is a 3D environment of neurons that can be placed
full advantage of the unique abilities of HDMEAs. on MEAs to monitor electrical activity. Cutting the brain into
very thin slices has allowed access to neurons deep in the brain
Bursts for imaging, i.e., mapping the anatomy. The same method can
Bursts and burst rates of APs in a neuron or across a network be used for recording the activity of neurons that are otherwise
of neurons is a common feature extracted from data in MEA difficult to reach and identify in vivo. This requires a setup to
applications. Bursts have several meanings and functions in neu- keep the neurons viable, i.e., by perfusion with artificial cere-
roscience, e.g., synchronization, information carrier, and motor brospinal fluid (ACSF) with continuous carbogen (95% oxygen
pattern generation. Single neurons can exhibit bursting, or burst and 5% carbon dioxide) gassing. The neurons and network
firing, when APs fire at a high frequency for a period of time, fol- structure in slices are physiologically and biochemically more
lowed by a quiet period. Bursts can be triggered by the network similar to the in vivo situation. It is possible to observe LFPs
activity (environment) or can be intrinsic to the neuron (pheno- and oscillations inherent in different states of the brain. Such
type of the cell). There are many algorithms to detect the presence recordings have been done for different brain areas, e.g., hip-
of bursts from single neurons (see Samengo et al., 2013; Bakkum pocampus, suprachiasmatic nucleus, etc. For instance, MEAs have
et al., 2014 for some methods). been employed to investigate the disruption of normal network
Besides single neuron bursting, population-wide synchronous waves and oscillations in the brain caused by the absence of cer-
activities are also of interest. For example, repetition of activa- tain ion channels in neurons. In one particular case, Simeone
tion patterns (Abeles and Gerstein, 1988; Sun et al., 2010) can et al. studied the effect of the delayed rectifier potassium chan-
be considered as memory traces, replayed by the appearance of nel α-subunit Kv1.1 to the oscillations in the hippocampus shown
a similar stimulus or due to internal processes that occur, e.g., in Figures 9A–C (Simeone et al., 2013). By reducing or elimi-
during sleep (O’Neill et al., 2008; Abel et al., 2013). Bakkum nating the expression of Kv1.1 in the axons of the hippocam-
et al. (2014) investigated parameters for and compared the per- pal tri-synaptic pathway, the authors were able to observe an
formance of various burst detectors on population-wide bursts. increase in occurrence of fast ripples (80–200 Hz bandwidth, 50%
An inter-spike interval (ISI) based network burst detector was longer duration) and high frequency oscillations associated with
able to identify small and large bursts better than other tech- epilepsy, as shown in Figure 9C. Similar applications have been
niques in cultured networks. Rate-based detectors detected larger done using HDMEAs. Medrihan et al. (2014) showed that the
bursts only, while prematurely identifying the end of bursts. See absence of synapsin II (Syn II), a protein related to epilepsy,

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Obien et al. Revealing neuronal function through MEA recordings

Purkinje cells (PCs) in acute cerebellar slices was demonstrated

using HDMEAs (Frey et al., 2009a). One important factor is to
ensure tissue adhesion on the MEA surface. Adhesion can be
achieved by cellulose nitrate coating (Egert et al., 2002), but also
by a slice anchor typically used for patch-clamp recordings. EAPs
were observed along the PC layer and, after spike sorting, the EAP
footprint of a single PC was analyzed. The negative spikes were
recorded around the perisomatic area of the neuron, while posi-
tive spikes were obtained along the molecular layer corresponding
to the dendrites of the PC. A comparison of the high spatiotempo-
ral resolution recording with simulations of a full-compartmental
model based on the stereotypical morphology of a PC was done.
Figure 10 shows both measured and simulated EAP data from
PCs at high resolution. Although the planar geometry of PC is
advantageous, similar results might be obtained from neurons in
other brain areas.
Aside from acute preparations, MEAs have been used to ana-
lyze the brain function using organotypic slice cultures. For
example, Ito et al. studied the functional connectivity in hip-
pocampal and cortical organotypic cultures (Ito et al., 2014).
They analyzed the network activity at different frequency ranges
using the wavelet transform of the cross-correlogram.

MEAs and retina

The planar arrangement of retinal ganglion cell (RGC) bodies
and axons is highly compatible with MEA recordings from retina
explants. Responses of RGCs can be recorded using different types
of light stimulations (Segev et al., 2004; Wässle, 2004; Jones et al.,
2011). This allowed the identification of cell types of popula-
tions of RGCs and the mapping of their receptive fields (Meister
et al., 1994; Chichilnisky, 2001), in different regions of the retina.
FIGURE 8 | Neuronal culture studies using MEAs. (A,B) Combination of Fiscella et al. (2012) established a methodology applied to mice
MEAs with immunostaining and microscopy to analyze the relationship
retina that uses light stimulation and HDMEAs to identify, select,
between the development of synapses and electrical activity of neurons,
adapted with permission from Ito et al. (2013). (A) Plot showing the number and record from defined populations of RGCs. After spike sort-
of synapses along the neuronal dendrites in a long-term primary culture. ing the HDMEA recordings, the EAP footprints of detected RGCs
The glutamatergic (red) and GABAergic (green) synapses along the were obtained, as shown in Figures 11A,B. Each detected RGC is
dendrites of neurons were obtained by immunostaining from cultures at assigned to one of the four types of ON–OFF direction-selective
7–35 days in vitro (DIV). The number of synapses at the dendrites
continuously increased for 3 weeks and saturated afterwards. The same is
RGCs, depending on the occurrence of the response to different
true for synapses at the soma (not shown), which saturated after 30 DIV. light stimulation patterns (see Figures 11C–E).
(B) Plotted data from MEA recordings of a long-term culture. A similar Another study on retina (macaque) using HDMEAs revealed
pattern is observed from the firing rate and synchronized burst rate the identification of the type, location, and strength of the func-
measured by a MED64 MEA device from 7 to 35 DIV. Both the firing and
tional input of each cone photoreceptor to each RGC (Field
burst rates increased until 30 DIV, which eventually saturated afterwards.
(C,D) Application of HDMEAs to analyze the functional connectivity of
et al., 2010). Populations of midget, parasol, and small bistratified
neurons in vitro, adapted with permission from Maccione et al. (2012). RGCs were recorded simultaneously in the presence of white noise
Fluorescent images of stained neurons on an HDMEA are shown with “visual” stimulation. The spatial receptive field and response time
arrows indicating the functional connectivity (from white—weak to of RGCs were detected by computing the spike-triggered average
red—strong) obtained by analyzing spike trains using cross-correlation.
of the stimuli. Afterwards, the detected clusters of cells obtained
by PCA were further stimulated with 10-fold smaller pixels (5 ×
5 μm2 ) to reveal finer details of the receptive fields. The method
decreases tonic inhibition in mouse hippocampal slices, thus was able to map putative cones accumulated across the receptive
increasing synchronized bursts (see Figures 9D,E). THIP (4,5,6,7- field of RGCs, which were verified by overlaying a microscopy
tetrahydroisoxazolo[5,4-c]pyridin-3-ol; gaboxadol), a selective image of cones labeled with peanut agglutinin (see Figure 11F).
agonist of δ subunit-containing GABAA receptors, restores tonic The authors were able to quantify the strength of connectiv-
inhibition. ity between different RGC types and different types of cones
Depth recording of EAPs from neurons up to 100 μm dis- (sensitive to red, green, or blue). These exhibit the capability
tance from the MEA surface was also shown (Egert et al., 2002; of HDMEAs, combined with advanced stimulation and analy-
Frey et al., 2009b). Subcellular resolution recording from single sis techniques, to resolve the functional connectivity of neurons

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 9 | Waves in acute hippocampal slices revealed by MEAs. (A–C) CA3sp (100 μV), WT CA3sr (200 μV), KO CA1sp (20 μV), and WT CA1sr
Studying the effect of the delayed rectifier potassium channel α-subunit Kv1.1 (200 μV). CA, cornus ammonis; DG, dentate gyrus. (D,E) Studying the effect
to sharp waves in in vitro hippocampal slices using MEAs, modified with of deleting synapsin II (Syn II) to the tonic inhibition in mouse hippocampal
permission from Simeone et al. (2013). (A) Image of a Kcna1-null (knock-out slices using HDMEAs, adapted with permission from Medrihan et al. (2014).
of the gene encoding Kv1.1) hippocampal slice on an MEA. Black squares (D) Mean firing rate computed from each electrode from WT and Syn II
correspond to the electrodes. The regions of the hippocampus are also knock-out hippocampal slices before and after THIP treatment. THIP:
indicated. (B) The sharp waves in wild-type (WT) and Kcna1-null hippocampi (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; gaboxadol), a selective agonist
are initiated in CA3 that spread with similar time-courses. (C) Representative of δ subunit-containing GABAA receptors. (E) Raster plots showing highly
sharp waves from WT and Kcna1-null hippocampi recorded at the location of synchronized bursts, x-axis corresponds to time, y-axis corresponds to pixels
red boxes in (A). The sharp waves are longer (with ripples) in Kcna1-null (electrode). THIP reduced the high frequency bursts in Syn II knock-out
compared to WT. Scale bars: horizontal, 50 ms; vertical, 50 μV except for WT hippocampus. Scale bar: 1 min.

in the retina at single-cell resolution. There are also other recent to map the flow of APs. The axons do not necessarily need a
works on population coding in the retina using MEA recordings tight contact on the electrodes, since the potential due to the APs
(Marre et al., 2012; Tkačik et al., 2014). was also detected from other surrounding electrodes, with lower
amplitude compared to the electrode nearest the axon.
MEAs and axonal signal tracking
Taking advantage of the spatiotemporal resolution and high sig- NEURONAL MODELING AND HDMEA RECORDINGS
nal quality of HDMEAs, tracking the propagation of APs between Computational modeling is useful to interpret the dynamics
cells can be performed. Bakkum et al. (2013) achieved this in and processing of neurons and networks. MEA recordings are
dissociated neuronal cultures (see Figures 12A–C). Axonal sig- commonly analyzed to model neuronal networks (Taketani and
nals are difficult to identify using conventional methods: thin Baudry, 2006; Kreuz, 2013; Samengo et al., 2013). Here, we
axons are difficult to patch and extracellular signal amplitudes are focus on the use of HDMEA data to analyze and model single
rather low compared to those from the soma. A major accom- neurons.
plishment of this work is the capability to electrically image
the propagation of APs along axons, across the topology of the Localization of neurons
whole neuronal network. By using HDMEAs that can record and Neuronal circuits are arranged with high spatial precision and
dynamically stimulate at defined locations, with little artifact to specificity and therefore, spatial information is an important
the signals, it was possible to quantify the direction, velocity, and factor in deciphering neuronal activity. Microscopy, fluores-
extent of axonal AP propagation. The stimulation and record- cent markers, and transgenic animals have enabled researchers
ing techniques are shown in Figures 12B,C. This is a suitable to localize and classify neurons in a high-throughput man-
platform to study the role of axons in neuronal computation in ner. Together with dynamic multineuron Ca-imaging using
the future. spinning-disk confocal microscopy with two-photon excitation,
Axonal conduction was also measured by Zeck et al. (2011) spatial and functional information can be obtained simulta-
from rabbit retina using HDMEAs. The authors were able to mea- neously. However, the temporal resolution of MEA recordings
sure the velocity of axonal AP caused by stimuli and discovered can capture neuronal responses better than these imaging tech-
that similar RGC types respond with the same latency and con- nologies (Delgado Ruz and Schultz, 2014) and the optical tools
duct with similar velocity (see Figure 12D). Except for the area described above may not be applicable to all experiments, e.g.,
where axons are myelinated, axonal signals were detected from due to the unavailability of the transgenic animals, the duration of
all stimulated RGCs. This work also shows that when axons are the experiment, optical access such as in in vivo experiments with
very near or flat on the electrode array surface, it is possible freely moving animals, etc. Therefore, localization of neurons in

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 10 | High-resolution mapping of spontaneous Purkinje cell the soma at 0.4 ms to the proximal dendrites at 0.6 ms and covers the
activity using HDMEAs. (A–E) HDMEA recordings from an acute slice dendritic area, while the soma repolarizes. Frequency band: 180 Hz–3.5 kHz.
preparation of the caudal half of the cerebellar vermis. (A) Activity map of the (F–H) Matching simulated and measured EAP footprints. (F) Comparison of
detectable spike activity in the recording area. Small dots correspond to the the recorded average single-unit spikes (black traces) and the spikes
electrodes used for recording (∼30% of the available electrodes). Events calculated from a compartment-model simulation of a PC (green traces).
exceeding a threshold of ±36 μV were used to calculate the color-coded Scale bar: vertical is 100 μV, horizontal is 1.9 ms. (G) Illustration of the
event rate. Scale bar: 0.3 mm. (B) Close-up of a region with high activity position and orientation of the simulated PC, with the center of the soma
delimited in (A). All units identified by spike sorting are marked, i.e., the located [blue diamond in (F)] 40 μm above the chip surface. (H) Simulated
somatic region is blue and the dendritic region is red. Scale bar: 0.1 mm. (C) potential on the chip surface along a line parallel to the soma-dendrite axis
Schematic of the basic cellular structures in the cerebellar slice (Gray, 1918). [dashed blue line in (F,G)] during the spike evolution at 0.1 ms intervals. The
Scale bar: 0.1 mm. ML, molecular layer; PCL, Purkinje cell layer; GL, granular black and white dots on the potential line of maximal amplitude (bold blue
layer; CF, climbing fiber; MF, mossy fiber; PF, parallel fiber; PC, Purkinje cell; line) represent the HDMEA spatial resolution (18 μm pitch). Significant spatial
GgC, Golgi cell; SC, stellate cell; BC, basket cell. (D) Footprint of a PC undersampling of the potential distribution curve can be observed by
selected from the region shown in (B). Scale bar: vertical is 200 μV, horizontal reducing the lateral spatial resolution by 50% (black dots only, pitch 36 μm),
is 1.9 ms. (E) Current source density (CSD) analysis for the cell shown in (D) especially for the largest negative peak. All panels and descriptions adapted
at several points in time (green: sink; yellow: source). The sink moves from with permission from Frey et al. (2009a).

MEA recordings has been of interest for in vivo and acute slice Somogyvári et al. (2012) proposed spike CSD (sCSD) to esti-
in vitro experiments too. mate the CSD after optimizing for the best locations of the sources
Based on the volume conductor theory several current source from the recording electrodes that recreates the spike data (see
density (CSD) methods have been proposed to solve for the cur- Figure 13A). The method has been used to analyze recordings
rent sources and sinks from LFP and EAP data (Nicholson and from a 16-electrode probe in vivo. Although sCSD has been used
Freeman, 1975; Mitzdorf, 1985; Plenz and Aertsen, 1993; Okada to solve for the CSD at the optimized locations of the sources,
et al., 1994; Pettersen et al., 2010; Łȩski et al., 2011). A volume it assumes that the number of electrodes is equal to the number
CSD approach for measurements using a 3D MEA has also been of sources to solve for. The over-simplification of the number of
done (Riera et al., 2014). These methods approximate the location current sources in sCSD results in errors, especially when the ori-
of the sources prior to solving the CSD and may not be suitable entation of the neuron being analyzed is at an angle with respect
for localizing single neurons. Different methods to localize sin- to the measuring electrodes.
gle neurons depend on the source models used, e.g., monopole On the other hand, Delgado Ruz and Schultz (2014) intro-
source type models such as exponential decay and inverse power duced a neuronal-based model for localization, utilizing known
law models (Blanche et al., 2005; Chelaru and Jog, 2005; Kubo current distributions and morphological traits. The method was
et al., 2008), dipole models (Blanche et al., 2005; Mechler and tested in simulations and in vivo recordings using high-density
Victor, 2012), line source models (Somogyvári et al., 2005, 2012), probes. The authors showed that different morphologies and
and simplified line model fitted to the perisomatic area of a ion channel distributions of neurons elicit different localiza-
full-compartmental neuron model (Delgado Ruz and Schultz, tion accuracies (see Figures 13B–D). This method, however,
2014). assumes that the experimenter knows the type (morphology and

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 11 | Identification of retinal ganglion cell receptive fields using recording block of the HDMEA. The yellow square indicates the location of
HDMEAs. (A–E) Characterization and analysis of HDMEA recordings from the light stimulus, with the gray squares indicating the center of the stimulus
defined populations of mouse retinal ganglion cells (RGCs), adapted with at four positions. Middle panel: Raster plots corresponding to four stimulation
permission from Fiscella et al. (2012). (A) Each trace shows the average (thick locations indicated in the left panel. Each dot corresponds to a single EAP.
black lines) of the 959 superimposed EAPs (gray lines). The electrode Each raster plot shows the response to five repetitions of the same stimulus.
locations are indicated in (B). The propagation speed of the spike was The firing rate of the RGC (averaged from five responses) is indicated below.
calculated to be 0.7 m/s. (B) Footprint of an RGC over an area of 0.025 mm2 . Right panel top: Polar plot showing the responses of the RGC to motion of a
The highest peak-to-peak amplitude is shown by the thick dark waveform. bar in 8 directions at 45◦ radial intervals. Right panel bottom: Inter-spike
(C–E) Physiological response of RGCs. Left panel: RGC footprint on a (Continued)

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 11 | Continued responses to white noise stimuli. Top middle panel: Receptive field
interval distribution showing the time intervals between consecutive radius vs. the first principal component of the response time course.
spikes. (C) Blue = ON RGC. (D) Red = OFF RGC. (E) Green = The clusters reveal different RGC types. Surrounding panels: Identified
ON-OFF RGC. (F) Classification of RGC types and receptive fields at RGC types highlighted at the top middle panel. The RGCs are
single cone resolution, adapted with permission from Field et al. (2010). stimulated with fine-grained white noise to reveal single cone receptive
The RGCs were recorded simultaneously and classified using the fields. Scale bars: 50 μm.

FIGURE 12 | Imaging axonal signal propagation using HDMEAs. (A–C) method. The red crosshair represents the stimulated electrode. The colored
Axonal propagation of a cultured neuron on an HDMEA, adapted with dots represent the latencies of detected APs with respect to the largest
permission from Bakkum et al. (2013). (A) Live image of a neuron at 21 DIV voltage signal indicated by the arrow. Scale bar, 40 μm. (D) Axonal
transfected with red fluorescent protein (RFP). The axon is highlighted. propagation of an RGC from rabbit retina, adapted with permission from Zeck
(B) Illustration of the distributed stimulation method. The crosshair et al. (2011). Consecutive electrical images of the EAP propagation allow for
represents the location of the “somatic” AP observed while stimulating the calculation of axonal conduction velocity. (a) Image of a somatic AP (blue
different electrodes represented by colored dots (color represent the median spot in the first window) propagating along the proximal axon. (b) Image of a
latency until AP detection, where light gray corresponds to electrodes that biphasic spike recorded from an axon. (c) Plot indicating the distance traveled
did not evoke an AP). The small dots represent the location of the HDMEA of the AP in time. Open symbols represent data calculated from recordings at
electrodes. Scale bar, 40 μm. (C) Illustration of the single-site stimulation 16.4 kHz; closed symbols are recordings at 8.2 kHz.

current distributions) of neurons being measured for localiza- across the neuron’s morphology, measured by multielectrodes,
tion and that the dynamics of neurons of the same type are can then be used to constrain compartmental models (Gold et al.,
stereotypical. 2007). Frey et al. (2009a) used this approach to model a full-
compartmental Purkinje neuron using HDMEA recordings, see
Constraining compartmental models Figure 10. This shows that using high-density EAP recordings, it
Aside from localization of neurons, it has also been demon- is possible to model the ion channel dynamics during neuronal
strated that with known morphology, it is possible to estimate function.
the ion channel density from extracellular recordings. Gold et al.
(2006, 2007) simulated realistic extracellular signals based on OUTLOOK
adjusting the ion channel distributions in full-compartmental We have shown the current status of MEA research in terms of
models (see Figure 14). With such a method, the EAP waveforms technology, the understanding of signal transduction, and the

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 13 | Localization of single neurons. (A) Spike current source normalized CSD distributions [I(d)] with different d-values. Localization is
density (sCSD) method by Somogyvári et al. (2012), figure modified with done by solving for dopt . The optimum d (dopt ) is chosen as the value where
permission. The experimental setup is shown on the left, where the neuron I(d) is the most spike-like, i.e., similar to the normalized amplitude of the EAP
is oriented at a distance d parallel to the in vivo MEA. The highest amplitude during the whole duration of the spike. Thus, the EAP and sCSD color maps
comes from the current sources at the soma of the neuron (sink) and is are similar. (B–D) Localization of simulated neurons using simplified line
detected by multiple electrodes. The forward solution at d is given by the T(d) model by Delgado Ruz and Schultz (2014), figures adapted with permission.
matrix, which transforms the CSD on the neuron to the EAP detected by the (B) The simulated neurons are CA1 pyramidal, L2/3 pyramidal, double
MEA. The EAPs are shown in the voltage traces per electrode, where one bouquet or DB (not shown), NPY interneurons, and PV interneuron.
spike is plotted as a color map, indicating the spatial EAP pattern in time. The Localization depends on the location of the sodium trough, which
sCSD obtained from the EAP signals by inverse solution T−1 (dopt ) is shown corresponds to the moment when currents are concentrated near the soma.
on the right. The EAP spatio-temporal map is transformed into a series of (Continued)

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Obien et al. Revealing neuronal function through MEA recordings

FIGURE 13 | Continued neuron-electrode distances under 35 μm and increased thereafter,

As shown by the color map embedded on the neuron morphologies, especially along the Z axis. (D) The localization errors were not
the sodium trough (red) is displaced from the soma for NPY due to similar for all simulated neurons. The differences in morphology and
the contribution of the dendritic arbor and axon, leading to higher electrophysiology cause the errors, although the maximum EAP
localization error along the Y axis shown in (D). (C) Localization (location of sodium trough) is more or less confined to the
results for CA1, where the errors along X–Z axes remained low for perisomatic area.

FIGURE 14 | Ion channel density estimation. Adapted from Gold et al. intracellular recording (dotted line). (D) First column: The details of the
(2006). (A) The extracellular action potentials (EAPs) solved in a grid intracellular signal simulation for each compartment. White solid lines in
from the multicompartmental model of a CA1 pyramidal neuron. The (A) indicate the locations of the compartments. Second column: The
dotted black line indicates the tip of the electrode used to measure the simulated membrane currents in the same compartments as the first
EAPs. (B) Enlarged image of the EAP at the electrode tip. Location is column. The net membrane current across the soma and proximal
indicated by the white dotted line in (A). Solid line in the plot dendrites best estimates the EAP waveform. Third column: Membrane
corresponds to the simulated EAP, which is superimposed with the current components in terms of Na+ , K+ , and mixed-ion capacitive
recorded EAP shown as dotted line. (C) Comparison of the simulated current. Last column: Conductivity densities of the A, C, D, K, and M
intracellular signal (solid line) at the proximal apical trunk to the type K+ currents. For further details, see Gold et al. (2006).

application to neuroscience studies. After years of MEA devel- A promising route is the combination of MEAs with other
opment, what is next? One path is to continuously improve the modalities. Aside from electrical recording and stimulation,
devices, i.e., better SNR, higher spatial resolution, more paral- brain activity mapping and manipulation at cellular resolution
lel readouts, scalability, portability, and increased ease-of-use. have also been done using optical methods, e.g., fluorescent
Additionally, device flexibility and biocompatibility are targets calcium indicators, genetic markers, optogenetics, two-photon
for long-term in vivo recording and stimulation. Another microscopy, etc. Similar to extracellular recordings, the pres-
approach is to enhance MEA signal pre-processing for experi- ence of many molecules and compartments in the brain with
menters to easily extract meaningful information from record- different optical properties render optical recording and analysis
ings in real time. This is crucial for applications where fast, challenging. It is of interest to pinpoint the advantages and
online analysis is required, e.g., closed-loop experiments and constraints of both electrophysiological and optical methods
brain machine interfaces (BMIs) combined with stimulation to determine how they can complement each other. Another
therapies. example is the use of optogenetics to manipulate the activity of

Frontiers in Neuroscience | Systems Biology January 2015 | Volume 8 | Article 423 | 22

Obien et al. Revealing neuronal function through MEA recordings

specific cellular subpopulations. By using MEAs to measure the Assad, J. A., Berdondini, L., Cancedda, L., De Angelis, F., Diaspro, A., Dipalo, M.,
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ACKNOWLEDGMENTS 5696–5709. doi: 10.1523/JNEUROSCI.0009-08.2008
This work was supported by the Japan Society for Promotion of Berdondini, L., van der Wal, P. D., Guenat, O., de Rooij, N. F., Koudelka-Hep, M.,
Science (JSPS) under Grants-in-Aid for Young Scientists (B) (No. Seitz, P., et al. (2005). High-density electrode array for imaging in vitro elec-
trophysiological activity. Biosens. Bioelectron. 21, 167–174. doi: 10.1016/j.bios.
25730183) and Swiss National Science Foundation Ambizione 2004.08.011
Grant PZ00P3_132245. Berdondini, L., Imfeld, K., Maccione, A., Tedesco, M., Neukom, S., Koudelka-Hep,
M., et al. (2009). Active pixel sensor array for high spatio-temporal resolution
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