32nd Annual International Conference of the IEEE EMBS Buenos Aires, Argentina, August 31 - September 4, 2010
A modular 256-channel Micro Electrode Array platform for in vitro and in vivo neural stimulation and recording: BioMEA™ G. Charvet, O. Billoint, S. Gharbi , M. Heuschkel, C. Georges, T. Kauffmann, A. Pellissier, B. Yvert and R. Guillemaud
Abstract— In order to understand the dynamics of large neural networks, where information is widely distributed over thousands of cells, one of today’s challenges is to successfully monitor the simultaneous activity of as many neurons as possible. This is made possible by using the Micro-Electrode Array (MEA) technology allowing neural cell culture and/or tissue slice experimentation in vitro. Thanks to development of microelectronics’ technologies, a novel data acquisition system based on MEA technology has been developed, the BioMEA™. It combines the most advanced MEA biochips with integrated electronics, and a novel user-friendly software interface. To move from prototype (result of the RMNT research project NEUROCOM) to manufactured product, a number of changes have been made. Here, we present a 256-channel MEA data acquisition system with integrated electronics (BioMEA™) allowing simultaneous recording and stimulation of neural networks for in vitro and in vivo applications. This integration is a first step towards an implantable device for BCI (Brain Computer Interface) studies and neural prosthesis.
I. INTRODUCTION
M
icro-electrode array (MEA) technology provides an
elegant way to probe the neural activity distributed over large populations of neurons either in vitro [1] or in vivo [2][3]. MEA also offer the possibility to deliver electrical stimulation to neural networks [4], making them promising technologies to build neural prostheses [5][6]. However, increasing the number of electrodes using conventional electronics is difficult to implement into a compact device. Moreover, high-density devices addressing all channels Manuscript received June 25, 2010. The development of BioMEA™ was supported by The French Ministry of technology (NEUROCOM RMNT Project). Part of the work was also supported by the French National Research Agency (ANR) through Carnot funding. G. Charvet is with the CEA/LETI/CLINATEC, Grenoble, France (e-mail:
[email protected]). O.Billoint is with the CEA/LETI/MINATEC Department DACLE, Grenoble, France (e-mail:
[email protected]). S. Gharbi is with the CEA/LETI/MINATEC Department DTBS, Grenoble, France (e-mail:
[email protected]). B. Yvert is with the CNRS, UMR5228, Bordeaux, France (e-mail:
[email protected]) M. Heuschkel is with Ayanda Biosystems SA, Lausanne, Switzerland (email:
[email protected]). C. Georges is with Bio-Logic SAS, Claix, France (www.bio-logic.info). T. Kauffmann is with Bio-Logic SAS, Claix, France (www.bio-logic.info). A. Pellissier is with Bio-Logic SAS, Claix, France (www.bio-logic.info). R. Guillemaud is with the CEA/LETI/MINATEC Department DTBS, Grenoble, France (e-mail:
[email protected]).
978-1-4244-4124-2/10/$25.00 ©2010 IEEE
independently for simultaneous recording and stimulation are not yet available for electrophysiology studies. To overcome the limitations seen previously, we designed a system with on one hand high-density microelectrode arrays which topology can be adapted to the user needs, and on the other hand integrated electronics (ASIC: Application Specific Integrated Circuit) allowing simultaneous recording and stimulation of neural networks and providing the opportunity to handle a larger number of channels. This paper presents a new data acquisition system named BioMEA™, which can manage 256-channel MEA biochips that are interconnected to four 64-channel ASICs dedicated to the amplification and the multiplexing of the signals and stimulation. The BioMEA™ is the result of a RMNT (Micro and Nano Technology Network) research project NEUROCOM (with MEMSCAP, BioLogic SAS, group ESIEE, CNRS-CNIC University of Bordeaux) where the first prototype was specified and tested by CEA/LETI/MINATEC and CNRS Bordeaux, France. BioMEA™ is both a stimulus generator, and a high sensitivity data acquisition system, which permits 256 electrodes to be stimulated and monitored simultaneously. To move from prototype to manufactured product, a number of changes have been made. In this paper we present the evolutions of the system BioMEA™ and examples of in vitro and also in vivo experimental results. II. THE BIOMEA™ SYSTEM AND EVOLUTIONS The BioMEA™ system comprises an interface to highdensity MEA biochip with 256 electrodes, four dedicated 64channel ASICs (amplification, analog multiplexing, current stimulation) running in parallel, specific acquisition boards, and a user-friendly software. A. Integrated Electronic Interfacing neurons through MEAs using discrete electronics rapidly limits the number of channels, creating the need for highly integrated electronics to achieve sufficient spatial resolution [7]. Therefore a dedicated ASIC named AGNES (Asic for General Neurons Electrical Study) was developed in order to allow simultaneous recording and stimulation on 64 channels [8]. Each channel of this 64-channel CMOS chip (Figure 1) is interfaced with neurons via a microelectrode array and includes a low noise variable gain measurement channel. The preamplifier and the amplifier are based on the same structure which provides a unity DC gain and an AC gain of respectively 75 and 10 in the 0.1Hz to 3kHz bandwidth.
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Both can be separately switched to follower configuration. For each channel, an 8-to-1 analog multiplexer (fed by 8 external input signals for the whole ASIC) and a voltage-tocurrent converter allowing uniform current stimulation (+/400µA peak max.) independently of the electrode impedance are used for stimulation. A Sample & Hold circuit allows snapshot style images of the 64 channels (with no time delay between channels as in a sequential reading), and can stand a standard sampling frequency of 13kHz. The circuit’s size is 2.4mm x 11.2mm (0.35µm CMOS process).
The BioMEA™ system is composed of two parts: a MEA holder, and a remote control unit (figure 2).
Fig. 2: The BioMEA™system
Fig. 1: Architecture of ASIC AGNES
B. Electronic system set-up The BioMEA™ system interfaces on 256 electrodes and performs both measurements and stimulations. This system can manage up to four 64-Channels ASICs mounted on a mechanical support allowing electrical interconnections between four ASICs and an interface to a 256-channel array. The complete system includes the ASICs, recording and stimulation electronics boards for the control from a PC, and a dedicated user interface. The BioMEA™ general specifications are in Table I. TABLE I BIOMEA™ SYSTEM : GENERAL SPECIFICATIONS Acquisition Simultaneous on 1 to 256 electrodes 1 and 750 (adjustable for each electrode) Variable gain +/- 2.5 mV (gain 750) Detection range +/- 1.5V (gain 1) 0.1 Hz to 3.5 kHz Bandwith Input Impedance > 1012 ohms Analog output 14 bits resolution 3.3 µV ( measured with MEA 256 electrodes connected RMS noise per channel
average value measured on 2000 channels) over the bandwidth 0.1 Hz to 3.5 kHz
Maximum sampling 74 µs per channel (13 kHz) frequency Current Stimulation Simultaneous on 1 to 64, 128 or 256 electrodes. Number of predefined stimuli patterns : 8 +/- 300 µA (depending on electrode impedance) Current range < 1 µs Time resolution External connector to control independently 8 electrodes
The remote control unit (dimensions: 430 x 312 x 146 mm), based on discrete electronics, includes analog signal adaptation, analog-to-digital converters (ADC 14 bits), digital-to-analog converters (DAC 14 bits), and also a digital interface (microcontroller and FPGA) for ASIC protocol and USB 2.0 data transmission. The MEA holder (dimensions: 280x160x52 mm) is composed of MEA connectivity (for 256 MEA) and four analog integrated circuit AGNES. The physicals characteristics of the MEA holder are listed here: External stimulation (8 channels) Modularity (64-, 128- or 256-electrodes system) due to the specific configuration of MEA holder Compatible with microscopy observation (upright and inverted) Easy and quick MEA/holder connection The system can be also easily adapted to interface various types of MEA biochips or probes, either for in vitro or in vivo studies. C. User friendly interface To move from prototype [9][10] to manufactured product, a number of changes have been made. In particular, BioMEA™ offers a novel user-friendly interface allowing rapid and easy setting of stimulation and acquisition parameters; an adjustable gain of each electrode depending on electrical cell activity measured; and all 256 electrodes can be selected simultaneously for recording and stimulation. Furthermore, all data from the 256 channels can be saved and reloaded with BioMEA™ software (Figure 3) or further analyzed using Spike2 software (Cambridge Electronic Design Limited, UK).
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III. IN VITRO AND IN VIVO VALIDATIONS Scientists mainly use the MEA technology to study in vitro neuronal cell cultures and/or acute tissue brain slices. These cells/tissues are cultured/placed into the MEA biochips just above integrated metallic microelectrodes. It is then possible to monitor electrical activity of the cells/tissue, which is mainly composed of single unit neuronal action potentials or spikes (cell cultures), population spikes and local field potentials (tissue slices). With respect to commercial products, the BioMEA™ system can handle a larger number of electrodes; therefore it will be possible to cover larger neuronal networks (such as mouse spine) with a good spatial resolution. The BioMEA™ has been validated with various biological preparations to show that it can record usual neuronal signals, spikes and LFP with good signal to noise ratio.
Fig. 3: BioMEA™ user interface
D. MEA Technology The BioMEA™ has been designed modularly, allowing the use of MEA biochips including 64, 128 or 256 recording/stimulation electrodes on the same system. A wide choice of MEA biochips configurations manufactured by Ayanda Biosystems is currently available (figure 4). It includes MEA biochips based on planar electrodes in a single- or multi-well format (up to 9 wells) and 3D tipshaped electrode that are well suited for experimentation using tissue slices [11].
A. In vitro validations As an example, whole dissociated wistar rat hippocampal neurons cell cultures (P1) have been used using MEA biochips. Typical spontaneous activity could be recorded from dissociated hippocampal neurons with a 256-electrodes MEA biochip (Electrode diameter: 46µm and Electrode Spacing: 200µm) coated with PEI 1% prior to cell culture. Typical single unit action potentials with amplitude of up to approximately 100µVpp and a noise level around 4µVrms at 14DIV are displayed in Figure 5.
Fig 5: Examples of typical spontaneous activity recorded from dissociated hippocampal neurons. Top: 20 recording traces out of 256 from dissociated rat hippocampal neurons (P1) at 14 DIV coated with PEI 1% prior to cell culture. Bottom: enlarged signal of single unit action potentials.
B. In vivo Validation The BioMEA™ system was initially designed for in vitro studies. However, as it was said previously, the interface with the MEA biochip can be easily adapted to various connectors. Therefore it became possible to adapt the BioMEA™ to in vivo studies with wired connections to small animals. Experimental procedures and animal care were carried out in accordance with the European Community Council Directive of 24 November 1986 (86/609/EEC). Fig. 4: Ayanda MEA biochips with 64 or 256 Microelectrodes
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First tests were performed at CEA/LETI/Clinatec®, France under Prof. A.L. Benabid’s scientific direction within the framework of a Brain Computer Interface (BCI) project in Clinatec®. The first goal was to record brain activity of a freely moving rat (electro corticography, ECoG). Screws were fixed in the skull of the animal and used as electrodes. They were connected by wire through a free-rotating connector to an ASIC AGNES. Brain signals were acquired with a reduced noise and an interesting bandwidth from 0.1Hz up to 3kHz for BCI studies (Figure 6). Other in vivo validations were performed on rats at GIN (Grenoble Institut de Neurosciences) [12] for investigations on epilepsy, using recording and stimulation capabilities of the system.
for research applications in neurosciences and was successfully validated through typical in vitro and in vivo investigations on various biological preparations. Owing to these good obtained results, an industrial transfer was organised with BioLogic, France with the objective of commercialisation of an instrument for in vitro applications. The BioMEA™ offers unprecedented capabilities to address applications such as evaluation of neural plasticity, functional screening and toxicology/safety pharmacology. The BioMEA™ product is distributed by Bio-Logic SAS (www.bio-logic.info) and Ayanda Biosystems SA (www.ayanda-biosys.com). Further developments on electronics miniaturization towards implantable devices with improved security are also in progress through the project BCI in CEA/LETI/ Clinatec®. ACKNOWLEDGMENT The authors wish also to thank C. Moulin (CEA), M. Trevisiol (CEA), S. Joucla (CNRS), P. Meyrand (CNRSLNR), F. Goy (BioLogic), J.P. .Rostaing (CEA): partners of the Neurocom Project. The authors wish also to thanks Prof. H. Markram, M. Giugliano and L. Gambazzi from EPFLBMI, Switzerland, for their involvement in in vitro validations and Prof. A.L Benabid (CEA), C. Moro (CEA), C. Mestais (CEA), F. Sauter (CEA), A. Depaulis (GIN), O. David (GIN), S. Saillet (GIN) for their involvement in the in vivo validations. REFERENCES
Fig. 6: In vivo recording with wired connection on a rat with implanted electrodes (a) In-vivo BioMEA™ set up; (b) Electrocorticogram Signal.
IV. TOWARDS IMPLANTABLE DEVICE The achievement of the AGNES ASIC development through the Neurocom project was the first step towards integration of an electrophysiological monitoring system. The current hardware is based on large electronics boards for control and wired connection as well as on large ASIC carriers that can be miniaturized easily. This further miniaturization is an important step towards development of implantable devices for in vivo investigations or therapeutic functionalities (prostheses). In order to achieve novel in vivo tools, CEA-LETI is working on a new low-power ASIC suited for electrical monitoring that will include ADC and a simpler control. Therefore, it will become possible to develop a first generation of recording implants, by adding a low-power micro-controller for system management and RF link. Security of such implants is also an important issue, especially as it will be developed for usage on humans. This work is currently in progress in the project BCI in the context of Clinatec® lead by Prof. A.L. Benabid. V. CONCLUSION An innovative system, the BioMEA™, has been developed in the context of the Neurocom project. It provides new functionalities of recording and stimulation on large MEA
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