A complete 256-channel reconfigurable system for in vitro neurobiological experiments

Miroslaw Zoladz, Piotr Kmon, Jacek Rauza,

Pawel Grybos, Department of Measurement and Electronics, Faculty of

Electrical Engineering, Automatics, Computer Science and Biomedical Engineering

AGH University of Science and Technology Krakow, Poland

zoladz@agh.edu.pl

Tomasz Kowalczyk, Bartosz Caban Department of Neurobiology,

University of Łódź Łódź, Poland

Abstract— We present a complete reconfigurable measurement system for 256-channel in vitro recordings and electrical stimulation of brain tissue electrophysiological activity. The system is built of: brain tissue life support system, Microelectrode Array (MEA), 4 multichannel integrated electronic circuits for signals conditioning and electrical stimulation, Digitizer and PC Application for measurement, data presentation and storage. The life support system is responsible for keeping brain tissue samples in appropriately saturated artificial cerebrospinal fluid at a very stable temperature. We designed two versions of the ASIC’s that can be easily adopted to the system. These are processed in the CMOS 180nm technology and differ with the main parameters that suits for different types of experiments. The ASIC’s are dedicated to amplification, filtering, and electrical stimulation of the 256 channels while the Digitizer performs simultaneous data acquisition from 256 channels with 14 kS/s sample rate and 12bit resolution. The resulting byte stream is transmitted to PC via USB (Universal Serial Bus). We also show a neurobiological experiment results that confirm the system is able to keep the extracted brain tissue active (posterior hypothalamic slices) and to record local theta field potentials with very small amplitudes from multiple neurons simultaneously.

I. INTRODUCTION

Current technology progress allows to build integrated systems for broad range of experiments. One of these are systems dedicated to experiments where the neuronal activity is recorded [1, 2]. These explorations are run to answer plenty of questions referring to the organization of a human brain or to develop implantable systems for people with disabilities [3]. These type of systems are built of recording electronics combined with recording electrodes. A key requirement of these systems is a need of a multichannel recording in order to effectively analyze observed processes what leads to a need of a minimization of power dissipation and area occupation of a single recording channel. A natural way to fulfill these demands is to use one of the leading submicron processes for fabrication of a multichannel integrated circuits.

This paper describes the detail of a complete 256-channel recording system and its first neurobiological tests as a proof a concept. Section II describes the system architecture. The experimental setup is presented in Section III while the preliminary measurements are described in Section IV. Section V contains a summary.

II. SYSTEM ARCHITECTURE Architecture of the system is presented in Fig. 1. Signals

from brain slices are collected by using 16x16 electrode MEA connected to the Conditioning Board equipped with 4 Conditioning Modules. The Conditioning Module amplifies and filters signals from 64 electrodes. It also reduces number of signals from 64 to 1 by using an analogue multiplexer. Mentioned functionality is realised by a specially designed Application Specific Integrated Circuit (ASIC). The Supply Module provides low noise supply and reference voltages for the Conditioning Module.

Fig. 1. Block diagram of the recording system

The Control Module generates control signals for the analogue multiplexer. Analogue to digital signal conversion is performed by the Digitizer Board which also transmits data

978-1-4799-1471-5/13/$31.00 ©2013 IEEE 250

to the PC. Data acquisition, display and storage is performed by LabVIEW (National Instruments).

Fig. 2. Conditioning board

III. CONDITIONING ASIC’S We designed two versions of multichannel integrated circuits to cover broad range of neurobiological experiments. These can be easily and fast mounted on the final system to satisfy different requirements of explorations. Both ASICs are processed in the CMOS 180nm technology and consists of 64 channels. The photo of chips mounted on the PCB is presented in the Fig. 3. The Fig. 3 a) shows the NEURO_A chip that occupies 2.5mm x 5mm and consists of 64 recording channels while the Fig. 3 b) presents the NRSRF64 chip that occupies 5mm x 5mm and consist of 64 channels dedicated to both recording and stimulation. The simplified single channel schemes of NEURO_A and NRSRF64 chips are presented in Fig. 4. The single recording channel of the NEURO_A chip consists of three stages: the preamplifier, the band pass filter and the front-end amplifier. The cut-off frequencies of the band-pass filter are formed thanks to the subtraction of the two output signals of the low pass filters. The consecutive stages are AC coupled what results in no dc offset propagation. The user is capable to control main chip parameters by off chip inputs, i.e. the lower cut-off frequency can be control by the VG input that is common for all 64 channels while the band pass filter is controlled by the three digital lines. The detailed analysis of the particular blocks of the recording channel can be found in [4, 5]. The recording channel of the NRSRF64 chip is built of the two stages only: the preamplifier and the front-end amplifier because the amplification and a band bass filtration are realized in the first stage. The chip is also equipped with the 64 stages responsible for electrical stimulation of the tissue. The main system parameters, i.e. voltage gain, lower and upper cut-off frequency, upper cut-off frequency, voltage offsets, and stimulation parameters are controlled by the on chip digital registers. Therefore, the user is capable to set independent

parameters of the recording and stimulating channels to meet broad range of the neurobiological experiments.

Fig. 3. Photography of multichannel ASICs for neurobiological experiments: a) NEURO_A b) NRSRF64.

Fig. 4. Simplified scheme of the single conditioning channel of the ASICs for neurobiological experiments. a) NEURO_A b) NRSRF64

251

Each channel can generate current pulses with a preset value, polarity and duration. The current value can be controlled together for all channels using four 8-bit DACs or separately for each channel with six current buffers placed at the output of the channel. To minimize the switching time from stimulation to recording phase, the consecutive stages are DC coupled. Additionally, recording channel is equipped with DC voltage cancellation circuit. The proposed in NRSRF64 chip architecture allows to obtain higher system functionality while increasing the uniformity of main system parameters [6]. The main parameters of the NEURO_A and NRSRF64 chips are presented in the Table I.

TABLE I SUMMARY OF BASIC PARAMETERS OF ASICS

NEURO_A NRSRF64 Low cut-off frequency [Hz] 1 – 60 0.06 – 100 High cut-off frequency [kHz] 3.5 - 15 4.7/12

Minimum amplitude of recognisable spikes (µV) 150 50

Input referred noise of the system [µV]RMS (recording channel bandwidth setting)

18 (60 Hz – 6.5 kHz)

8 (100 Hz – 12 kHz)

Gain [V/V] 140 140/1100 Power dissipation [µW] 220 25 Area occupation of the recording channel [mm2] 0.095 0.13

Current stimulation with 8-bit resolution in six ranges Not available ± (0.5; 2; 8; 32;

128; 512) µA

IV. EXPERIMENTAL SETUP The experimental setup sketch is presented in the Fig. 5.

Experiments were performed on the brain tissues extracted from the anesthetized rats [7]. The brain was removed and placed in oxygenated, cold (3–5◦C) artificial cerebrospinal fluid. Transverse slices (500μm) were made from both hippocampi using a tissue slicer (Stoelting Co.). Slices were

preincubated in oxygenated ACSF at the temperature of 20◦C for 1 h after the dissection. Next, slices were transferred into the MEA and are delicately fixed in position with nylon mesh. To keep the brain slices alive animate, one has to provide them with artificial cerebrospinal fluid saturated appropriately (with a proper ionic composition and nutrient content). Additionally, there is a strong demand for keeping the fluid temperature at 35°C with a high precision, i.e. ±0.25°C. The fluid is delivered to the chamber via a valve (fluid flow speed control) and preheated (fluid oversaturation elimination). The bottom of the chamber which contains the MEA is made of glass while the chamber sides and flange are made of silicone. The liquid excess is removed by suction from the chamber thanks to the tube placed opposite to the liquid inlet side. Required temperature is maintained by heating the MEA glass substrate. The substrate is warmed up by the heating elements by the brass block improving temperature stabilization and homogenous heat distribution. The correct interpretation of the measured signals requires establishing their origins, i.e. the places on the tissue from which they were collected. For this purpose the camera with suitable optics is mounted up on the chamber. The tissue is illuminated from below through a glass rod placed in a hollow hole in the brass block. Dedicated connectors combine the MEA printed circuit board (PCB) with the main board PCB equipped with integrated circuit modules. Fig. 5 shows the photo of the experimental stand.

V. PRELIMINARY RESULTS Transverse posterior hypothalamic area (PHa) preparations

has been used in order to test the experimental setup. Recently, we have shown that PHa slices perfused with 75 μM cholinergic agonist carbachol (CCH) generated specific LFP pattern – theta rhythm [8]. Fig. 7. shows theta activity recorded from PHa slice after 75 μM CCH perfusion. Fig. 8. (left) shows the PHa slice on the matrix of electrodes with a

Figure 5. Sketch of experimental setup for multichannel in vitro multichannel neural activity recording.

252

16x16 crisscross of electrode positions on top. The broken line indicates the location of the supramammilary region (SuM) of the PHa (left). The right panel of Fig. 8. shows the amplitude level matrix of 256 channels MEA recorded from

presented PHa slice, based on selected time period. The generation of well-synchronized theta rhythm in the present study confirms that the brain tissue is in good condition and hence it confirms that the experimental stand is able to provide suitable conditions for neurons to remain active and to generate physiological pattern of oscillations. Moreover,

recordings of different local field potential activity acquired from several different electrodes confirm that the system is able to conduct simultaneous multichannel recordings.

VI. SUMMARY A measurement system has been presented for in vitro

recording of exctracellular neural signals from 256 electrodes from acute brain tissue The critical elements of the system are: - properly selected MEA (electrodes shape, diameter and spacing), - complex 64-channel ASIC’s with AC coupling circuit, high impedance amplifier and band-pass filter in each channel with a large tuning range, - acquisition system capable of digitisation and transmission of the 256 signals with 14kS/s sampling rate and 12bit resolution, - tissue life support system equipped with temperature control and artificial cerebrospinal fluid supply, - proper Optical camera for tissue sample inspection i.e. signal origins determination.

ACKNOWLEDGMENT This research and development project was supported by

National Science Centre in the years 2010-2013 (N N505 559 139), and by the Polish Ministry of Science and Higher Education (2013), and National Science Centre Poland in the years 2011-2014 (2011/01/B/N24/00373).

REFERENCES [1] Ahuja A. K. et al. (2008), “An In Vitro Model of a Retinal Prosthesis”,

2008, IEEE Transactions on Biomedical Engineering, 55 (6), 1744 –1753.

[2] Hochberg L. R., et al. (2012), “Reach and grasp by people with tetraplegia using a neutrally controlled robotic arm”, Nature, vol. 485, pp. 372-377

[3] Litke A. et al. (2004), "What does the eye tell the brain?: Development of system for the large-scale recording of retinal output activity" IEEE Transactions on Nuclear Science, 51, 1434-1440.

[4] P. Grybos, et al. (2011), “64 channel neural recording amplifier with tunable bandwidth in 180 nm CMOS technology”, Metrology and Measurements, vol. XVIII (4), 631-644.

[5] Gryboś P. (2002), "Low Noise Multichannel Integrated Circuits in CMOS Technology for Physics and Biology Applications." Monography 117, AGH Uczelniane Wydawnictwa Naukowo-Dydaktyczne, Kraków 2002, available at: www.kmet.agh.edu.pl/www/asics

[6] P. Kmon, P. Gryboś, “Energy Efficient Low-Noise Multichannel Neural Amplifier in Submicron CMOS Process”, IEEE Transactions on Circuits and Systems—I: Regular Papers, July 2013

[7] Kowalczyk, T., Bocian, R., Caban, B. and Konopacki, J. (2013), Atropine-sensitive theta rhythm in the posterior hypothalamic area: In vivo and in vitro studies. Hippocampus. [Epub ahead of print] doi: 10.1002/hipo.22167

Fig. 7. Theta activity recorded from posterior hypothalamic area slice.

Fig. 8. PHa slice on MEA with a 16x16 crisscross of electrode positions on top (left). Right panel shows the amplitude matrix of 256 channels MEANote that the position of the SuM is indicated with black broken line.

253