IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING, VOL. 54, NO. 2, FEBRUARY 2007 331 Communications Multisite Recording of Extracellular Potentials Produced by Microchannel-Confined Neurons In-Vitro E. Claverol-Tinturé*, J. Cabestany, and X. Rosell Abstract—Towards establishing electrical interfaces with patterned in vitro neurons, we have previously described the fabrication of hybrid elastomer-glass devices polymer-on-multielectrode array technology and obtained single-electrode recordings of extracellular potentials from confined neurons (Claverol-Tinturé et al., 2005). Here, we demonstrate the feasibility of spatially localized multisite recordings from individual mi- crochannel-guided neurites extending from microwell-confined somas with good signal-to-noise ratios (20 dB) and spike magnitudes of up to 300 . Single-cell current source density (scCSD) analysis of the spatio-temporal patterns of membrane currents along individual processes is illustrated. Index Terms—Cell patterning, current source density analysis, Helix aspersa, microfluidics, multichannel electrophysiology, neural interfaces, neuronal culture, PDMS, snail neurons. I. INTRODUCTION Multisite recording of neuronal activity with substrate integrated mi- croelectrode arrays (MEAs) [2]–[4] is a promising alternative to the use of less scalable conventional electrophysiology [5]–[7] and pho- tobleaching-prone voltage-sensitive dyes [8], [9]. In particular, deeper understanding of the complex relationship between cell morphology, active channel distribution and the associated spatio-temporal patterns of neuronal activity [10]–[13] could be obtained by the achievement of multisite MEA recordings from unambiguously identifiable compart- ments of neurons cultured in isolation. Yet, the intrinsic anatomical plasticity of unconstrained developing cells results in continued changes in the relative position of the cellular compartments with respect to recording sites and, as a consequence, in unpredictable and time-changing signal-to-noise ratios. To address this issue and ensure long-term proximity between electrodes and neurons, efforts are focusing on the development of cell patterning techniques such as microcontact printing of adhesion promoting molecules [14], [15] and confinement by 3-D microstructures, e.g., fabricated by photo-thermal etching of wells and trenches in agarose layers [16], [17]. Recently, soft-lithography has emerged as a low-cost low-complexity technology [18] suitable for fabrication of polymeric microstructures of use in cell guiding. With this approach, patterning of mass neuronal cultures has been achieved [19]–[21] and, in conjunc- tion with substrate integrated electrodes, single-site recordings from individual oocytes [22] and myocytes [23] have also been realized. Manuscript received September 21, 2005; revised April 23, 2006. This work was supported in part by the Spanish Ministry for Science and Education through the Ramón y Cajal programme. Asterisk indicates corresponding author. *E. Claverol-Tinturé is with the Department of Electronics, Technical Uni- versity of Catalonia, Barcelona 08028, Spain. He is also with the Centre for Re- search in Biomedical Engineering (CREBEC), Barcelona 08028, Spain (e-mail: ectmail@eel.upc.edu). J. Cabestany is with the Department of Electronics, Technical University of Catalonia, Barcelona 08028, Spain. X. Rosell is with the Department of Electronics, Technical University of Cat- alonia, Barcelona 08028, Spain. He is also with the Centre for Research in Biomedical Engineering (CREBEC), Barcelona 08028, Spain. Digital Object Identifier 10.1109/TBME.2006.880903 Fig. 1. Sketch of the multielectrode device, consisting of a sandwich structure assembled by overlaying a polymer film (60–80 thick) including microw- ells and microchannels on a planar multielectrode array [1]. The array was fab- ricated by photolithographically defining a set of parallel ITO electrodes on a glass substrate. Neurons were individually selected after tissue dissociation and positioned within microwells in contact with the poly-L-lysine treated glass sub- strate. As cells sproute neurites into the microchannels, the substrate embedded electrodes record extracellular potentials generated by active neurites. We have previously reported on hybrid polymer-MEA devices, fabricated with a combination of soft-lithography and classic pho- tolithography technologies, and demonstrated single-channel extracel- lular recordings from patterned neurites of isolated neurons [1]. Here we describe multi-point recordings from individual channel-confined neurites in vitro and illustrate the application of this technique to single-cell current source density analysis (scCSD). II. METHODS A. Device Fabrication Fig. 1 shows a sketch of the device. A glass substrate with embedded parallel Indium-Tin-Oxide (ITO) electrodes was sandwiched with a PDMS film containing microwells connected by microchannels. Fabrication procedures have been described previously [1]. Briefly, ITO was sputtered on glass substrates to a resistivity of 80 ohm/sq or purchased with similar resistivity (PGO, Germany). The commercial substrates had a barrier layer between glass and ITO. A set of 18 parallel electrodes, 40 in width, were defined by standard photolithography techniques. The substrates were sonicated in ace- tone, isopropanol, neutral detergent and milli-Q grade water before assembly with elastomeric overlays. The overlays consisted of PDMS films (60–80 thick) with microcavities (40 in diameter) and microchannels (cross section fabricated by microhole punching and soft-lithography from SU-8 masters [1] and laid on substrates with only coarse alignment. The sealed-well, as opposite to open-well, configuration was established by spreading silicone grease (Bayer, Germany) over the microwells to electrically insulate them. The substrate was treated with poly-L-lysine (0.1 mg/ml) for 24 hr prior to overlaying of the PDMS films in order to promote cell adhesion and growth. 0018-9294/$25.00 © 2007 IEEE