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We designed a miniaturized and thin polydimethylsiloxane (PDMS) microchannel device compatible with commercial microelectrode array (MEA) chips. It was optimized for selective axonal ablation by laser microdissection (LMD) to investigate the electrophysiological and morphological responses to a focal injury in distinct network compartments over 45 days in vitro (45 DIV). Low-density cortical or hippocampal networks (

OBJECTIVE In light of recent progress in mapping neural function to behavior, we briefly and selectively review past and present endeavors to reveal and reconstruct nervous system function in Caenorhabditis elegans through simulation. APPROACH Rather than presenting an all-encompassing review on the mathematical modeling of C. elegans, this contribution collects snapshots of pathfinding key works and emerging technologies that recent single- and multi-center simulation initiatives are building on. We thereby point out a few general limitations and problems that these undertakings are faced with and discuss how these may be addressed and overcome. MAIN RESULTS Lessons learned from past and current computational approaches to deciphering and reconstructing information flow in the C. elegans nervous system corroborate the need of refining neural response models and linking them to intra- and extra-environmental interactions to better reflect and understand the actual biological, biochemical and biophysical events that lead to behavior. Together with single-center research efforts, the Si elegans and OpenWorm projects aim at providing the required, in some cases complementary tools for different hardware architectures to support advancement into this direction. SIGNIFICANCE Despite its seeming simplicity, the nervous system of the hermaphroditic nematode C. elegans with just 302 neurons gives rise to a rich behavioral repertoire. Besides controlling vital functions (feeding, defecation, reproduction), it encodes different stimuli-induced as well as autonomous locomotion modalities (crawling, swimming and jumping). For this dichotomy between system simplicity and behavioral complexity, C. elegans has challenged neurobiologists and computational scientists alike. Understanding the underlying mechanisms that lead to a context-modulated functionality of individual neurons would not only advance our knowledge on nervous system function and its failure in pathological states, but have directly exploitable benefits for robotics and the engineering of brain-mimetic computational architectures that are orthogonal to current von-Neumann-type machines.

Using C. elegans as a model organism, we present on an optimization strategy for reducing the spatial needs and power consumption in an optical connectome implementation. By means of a cluster analysis algorithm1, the interconnectivity of 279 neurons can be subdivided into 3 groups. This clustering reveals 2 independent neural populations, whose members interconnect only within their cluster-community and through a relay group of inter-cluster connections. Using this strategy, the expected spatial needs could be cut down by one fourth, thereby reducing the required light intensities by the same amount. A follow-up sub-partitioning of the individual clusters led to an additional power saving of up to 7%.

With exactly 302 neurons and about 8000 connections, the hermaphrodite of the soil-dwelling ringworm Caenorhabditis elegans features one of the simplest nervous systems in nature. The Si elegans project will provide a reverse-engineerable model of this nematode by emulating its nervous system and embodying it in a virtual world. The hardware will consist of 302 individual FPGAs, each carrying a neuron-specific neural response model. The FPGA neurons will be interconnected by an electro-optical connectome to distribute the signal at the axonal output or gap-junction pin of an FPGA neuron onto the respective synaptic input or gap-junction pins of postsynaptic FPGA neurons. This technology will replicate the known connectome of the nematode to allow for a biomimetic parallel information flow between neurons. This chapter focuses on the comparison of different electro-optical connectome concepts and on the required implementation steps with their advantages and disadvantages being explained.

We present an optical system that combines IR (1064 nm) holographic optical tweezers with a sub-nanosecond-pulsed UV (355 nm) laser microdissector for the optical manipulation of single neurons and entire networks both on transparent and non-transparent substrates in vitro. The phase-modulated laser beam can illuminate the sample concurrently or independently from above or below assuring compatibility with different types of microelectrode array and patch-clamp electrophysiology. By combining electrophysiological and optical tools, neural activity in response to localized stimuli or injury can be studied and quantified at sub-cellular, cellular, and network level.

Microelectrode electrophysiology has become a widespread technique for the extracellular recording of bioelectrical signals. To date, electrodes are made of metals or inorganic semiconductors, or hybrids thereof. We demonstrate that these traditional conductors can be completely substituted by highly flexible electroconductive polymers. Pursuing a two-level replica-forming strategy, conductive areas for electrodes, leads and contact pads are defined as microchannels in poly(dimethylsiloxane) (PDMS) as a plastic carrier and track insulation material. These channels are coated by films of organic conductors such as polystyrenesulfonate-doped poly(3,4-ethylenedioxy-thiophene) (PEDOT:PSS) or filled with a graphite-PDMS (gPDMS) composite, either alone or in combination. The bendable, somewhat stretchable, non-cytotoxic and biostable all-polymer microelectrode arrays (polyMEAs) with a thickness below 500 μm and up to 60 electrodes reliably capture action potentials (APs) and local field potentials (LFPs) from acute preparations of heart muscle cells and retinal whole mounts, in vivo epicortical and epidural recordings as well as during long-term in vitro recordings from cortico-hippocampal co-cultures.

An imbalance in medium osmolarity is a determinant that affects cell culture longevity. Even in humidified incubators, evaporation of water leads to a gradual increase in osmolarity over time. We present a simple replica-moulding strategy for producing self-sealing lids adaptable to standard, small-size cell-culture vessels. They are made of polydimethylsiloxane (PDMS), a flexible, transparent and biocompatible material, which is gas-permeable but largely impermeable to water. Keeping cell cultures in a humidified 5% CO2 incubator at 37 degrees C, medium osmolarity increased by +6.86 mosmol/kg/day in standard 35 mm Petri dishes, while PDMS lids attenuated its rise by a factor of four to changes of +1.72 mosmol/kg/ day. Depending on the lid membrane thickness,pH drifts at ambient CO2 levels were attenuated by a factor of 4 to 9. Comparative evaporation studies at temperatures below 60 degrees C yielded a 10-fold reduced water vapour flux of 1.75 g/day/ dm 2 through PDMS lids as compared with 18.69 g/day/dm 2 with conventional Petri dishes. Using such PDMS lids,about 2/3 of the cell cultures grew longer than 30 days in vitro. Among these,the average survival time was 69 days with the longest survival being 284 days under otherwise conventional cell culture conditions.

We report on the system integration of a CMOS chip that is capable of bidirectionally communicating (stimulation and recording) with electrogenic cells such as neurons or cardiomyocytes and that is targeted at investigating electrical signal propagation within cellular networks in vitro. The overall system consists of three major subunits: first, the core component is a 6.5 mm × 6.5 mm CMOS chip, on top of which the cells are cultured. It features 128 bidirectional electrodes, each equipped with dedicated analog filters and amplification stages and a stimulation buffer. The electrodes are sampled at 20 kHz with 8-bit resolution. The measured input-referred circuitry noise is 5.9V root mean square (10 Hz to 100 kHz), which allows to reliably detect the cell signals ranging from 1 mVpp down to 40Vpp. Additionally, temperature sensors, a digital-to-analog converter for stimulation, and a digital interface for data transmission are integrated. Second, there is a reconfigurable logic device, which provides chip control, event detection, data buffering and an USB interface, capable of processing the 2.56 million samples per second. The third element includes software that is running on a standard PC performing data capturing, processing, and visualization. Experiments involving the stimulation of neurons with two different spatio-temporal patterns and the recording of the triggered spiking activity have been carried out. The response patterns have been successfully classified (83% correct) with respect to the different stimulation patterns. The advantages over current microelectrode arrays, as has been demonstrated in the experiments, include the capability to stimulate (voltage stimulation, 8 bit, 60 kHz) spatio-temporal patterns on arbitrary sets of electrodes and the fast stimulation reset mechanism that allows to record neuronal signals on a stimulating electrode 5 ms after stimulation (instantaneously on all other electrodes). Other advantages of the overall system include the small number of needed electrical connections due to the digital interface and the short latency time that allows to initiate a stimulation less than 2 ms after the detection of an action potential in closed-loop configurations. © 2007 Elsevier B.V. All rights reserved.

A high degree of connectivity and the coordinated electrical activity of neural cells or networks are believed to be the reason that the brain is capable of highly sophisticated information processing. Likewise, the effectiveness of an animal heart largely depends on such coordinated cell activity. To advance our understanding of these complex biological systems, high spatiotemporal-resolution techniques to monitor the cell electrical activity and an ideally seamless interaction between cells and recording devices are desired. Here we present a monolithic microsystem in complementary metal oxide semiconductor (CMOS) technology that provides bidirectional communication (stimulation and recording) between standard electronics technology and cultured electrogenic cells. The microchip can be directly used as a substrate for cell culturing, it features circuitry units per electrode for stimulation and immediate cell signal treatment, and it provides on-chip signal transformation as well as a digital interface so that a very fast, almost real-time interaction (2 ms loop time from event recognition to, e.g., a defined stimulation) is possible at remarkable signal quality. The corresponding spontaneous and stimulated electrical activity recordings with neuronal and cardiac cell cultures will be presented. The system can be used to, e.g., study the development of neural networks, reveal the effects of neuronal plasticity and study cellular or network activity in response to pharmacological treatments.

In the context of interfacing biological entities to technical substrates, proof of principle results on patterning cell culturing substrates by inkjet deposition of the cell adhesion mediator polyethyleneimine are presented. Pattern fidelity, cell adhesion, and cell differentiation of chick neurons and glia cells on such interfaces were evaluated microscopically. The exemplary results demonstrate that piezo inkjet printing of biologically active compounds has the potential of becoming an easy to handle and versatile strategy for applying a wide variety of substances in any combination and concentration with cellular to subcellular resolution onto a wide selection of substrates. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Our current understanding of brain function is still too limited to take advantage of the computational power of even the simplest biological nervous systems. To fill this gap, the Si elegans project (www.si-elegans.eu) aims at developing a computational framework that will replicate the nervous system and rich behavior of the nematode Caenorhabditis elegans , a tiny worm with just 302 neurons. One key element of this emulation testbed is an electro-optical, micromirrorbased connectome. Unlike any other current ICT communication protocol, we expect it to accurately mimic the parallel information transfer between neurons. This strategy promises to give new insights into the nature of two hypothesized key mechanisms - the parallel and precisely timed information flow - that make brains excel von-Neumann-type machines. In this contribution, we briefly introduce the overall Si elegans concept to then describe the requirements for designing a light-based connectome within the given boundary conditions imposed by the hardware infrastructure it will be integrated into.

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO 2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO 2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

Replica-casting finds wide application in soft lithography and microfluidics. Most commonly, structures are molded with micro- to nano-patterned photoresists as master casts into polydimethylsiloxane (PDMS). PDMS features many favorable properties. It reproduces geometric details with nanometer fidelity, has low cytotoxicity and is transparent in the visible spectrum. It is furthermore biostable both in vitro and in vivo, can be plasma-bonded to itself, has low water permeability and is easy to handle and process. After curing, the PDMS can be peeled from the master and latter usually be reused if patterns are not undercut. Here, we describe the straightforward replica-molding process for devices that can be exploited either as perforated microchannel scaffolds for the in vitro use in axonal guidance and regeneration studies on microelectrode arrays (MEAs) or for the production of tissue-conformal in vivo MEAs for neuroprosthetic applications.

A CMOS metal-electrode-based micro system for bidirectional communication (stimulation and recording) with neuronal cells in vitro is presented. The chip overcomes the interconnect challenge that limits today's bidirectional microelectrode arrays. The microsystem has been fabricated in an industrial CMOS technology with several post-CMOS processing steps to realize 128 biocompatible electrodes and to ensure chip stability in physiological saline. The system comprises all necessary control circuitry and on-chip A/D and D/A conversion. A modular design has been implemented, where individual stimulation- and signal-conditioning circuitry units are associated with each electrode. Stimulation signals with a resolution of 8 bits can be sent to any subset of electrodes at a rate of 60 kHz, while all electrodes of the chip are continuously sampled at a rate of 20 kHz. The circuitry at each electrode can be individually reset to its operating point in order to suppress artifacts evoked by the stimulation pulses. Biological measurements from cultured neuronal networks originating from dissociated cortical tissue of fertilized chicken eggs with amplitudes of up to 500 muVpp are presented

Caenorhabditis elegans features one of the simplest nervous systems in nature, yet its biological information processing still evades our complete understanding. The position of its 302 neurons and almost its entire connectome has been mapped. However, there is only sparse knowledge on how its nervous system codes for its rich behavioral repertoire. The EU-funded Si elegans project aims at reverse-engineering C. elegans‘ nervous system function by its emulation. 302 in parallel interconnected field-programmable gate array (FPGA) neurons will interact through their sensory and motor neurons with a biophysically accurate soft-body representation of the nematode in a virtual behavioral arena. Each FPGA will feature its own reprogrammable neural response model that researchers world-wide will be able to modify to test their neuroscientific hypotheses. In a closed-feedback loop, any sensory experience of the virtual nematode in its virtual environment will be processed by sensory and subsequently interconnected neurons to result in motor commands at neuromuscular junctions at the hardware-software interface to actuate virtual muscles of the virtual nematode. Postural changes in the virtual world will lead to a new sensory experience and thus close the loop. In this contribution we present the overall concepts with special focus on the virtual embodiment of the nematode. For further information and recent news please visit http://www.si-elegans.eu.

Biological neural systems are powerful, robust and highly adaptive computational entities that outperformconventional computers in almost all aspects of sensory-motor integration. Despite dramatic progress ininformation technology, there is a big performance discrepancy between artificial computational systemsand brains in seemingly simple orientation and navigation tasks. In fact, no system exists that can faithfullyreproduce the rich behavioural repertoire of the tiny worm Caenorhabditis elegans which features one of thesimplest nervous systems in nature made of 302 neurons and about 8000 connections. The Si elegans projectaims at providing this missing link. This article is sketching out the main platform components.

In the context of a neurobionic approach to chemical analysis and sensorics, this article depicts the development of a miniaturized autonomous perfusion chamber setup for the growth and the electrical as well as optical investigation of (neural) cell cultures in vitro. We suggest an autonomous, modular, temperature-controlled, transparent, and sealed perfusion cell culture housing adaptable to various mounts, sizes and different needs. The design includes the electronics of a temperature and medium supply control unit. The setup combines the possibility of uninterrupted cell culturing with simultaneous microscopic and analytical investigation of variable amounts of cells or organs of human, animal, or plant origin under sterile conditions on different substrates without the need of an external incubator or a sterile working environment. Its use is demonstrated exemplarily with neuronal cultures from embryonic chicken that were cultured in a prototype system for 3 weeks. It turned out that cell survival in such a chamber was prolonged with timed medium flow rather than continuous perfusion.

The brain is perhaps the most advanced and robust computation system known. We are creating a method to study how information is processed and encoded in living cultured neuronal networks by interfacing them to a computer-generated animal, the Neurally-Controlled Animat, within a virtual world. Cortical neurons from rats are dissociated and cultured on a surface containing a grid of electrodes (multi-electrode arrays, or MEAs) capable of both recording and stimulating neural activity. Distributed patterns of neural activity are used to control the behavior of the Animat in a simulated environment. The computer acts as its sensory system providing electrical feedback to the network about the Animat's movement within its environment. Changes in the Animat's behavior due to interaction with its surroundings are studied in concert with the biological processes (e.g., neural plasticity) that produced those changes, to understand how information is processed and encoded within a living neural network. Thus, we have created a hybrid real-time processing engine and control system that consists of living, electronic, and simulated components. Eventually this approach may be applied to controlling robotic devices, or lead to better real-time silicon-based information processing and control algorithms that are fault tolerant and can repair themselves.

In classic monolayer cell culture, the world is flat. In contrast, tissue-embedded cells experience a three-dimensional context to interact with. We assessed a selection of natural gelling agents of non-animal origin (ι- and κ-carrageenan, gellan gum, guar gum, locust bean gum, sodium alginate, tragacanth and xanthan gum) in serum-free medium at 1–4% (w/v) concentration for their suitability as a more natural 3D culture environment for brain-derived cells. Their biophysical properties (viscosity, texture, transparency, gelling propensity) resemble those of the extracellular matrix (ECM). Gels provide the neurons with a 3D scaffold to interact with and allow for an increase of the overall cell density compared to classical monolayer 2D culture. They not only protect neurons in cell culture from shear forces and medium evaporation, but stabilize the microenvironment around them for efficient glial proliferation, tissue-analog neural differentiation and neural communication. We report on their properties (viscosity, transparency), their ease of handling in a cell culture context and their possible use modalities (cell embedment, as a cell cover or as a cell culture substrate). Among the selected gels, guar gum and locust bean gum with intercalated laminin allowed for cortical cell embedment. Neurons plated on and migrating into gellan gum survived and differentiated even without the addition of laminin. Sodium alginate with laminin was a suitable cell cover. Finally, we exemplarily demonstrate how guar gum supported the functional survival of a cortical culture over a period of 79 days in a proof-of-concept long-term microelectrode array (MEA) electrophysiology study.

During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we developed a system integrating an optical tweezers and a laser dissector to manipulate the sample. A sub-nanosecond pulsed UVA laser was used to inflict a partial damage to the axon of mouse hippocampal neurons at early days in vitro. Partial axonal transections were performed in a highly controlled and reproducible way without affecting the regeneration process. Force spectroscopy measurements, during and after the ablation of the axon, were performed by optical tweezers with a bead attached to the neuronal membrane. Thus, the release of tension in the neurite could be analyzed in order to quantify the inflicted damage. After dissection, we monitored the viscoelastic properties of the axonal membrane, the cytoskeleton reorganization, and the dynamics of the newly formed growth cones during regeneration. In order to follow cytoskeleton dynamics in a long time window by tracking a bead attached to the neuron, we developed a real-time control of the microscope stage position with sub-millisecond and nanometer resolution. Axonal regeneration was documented by long-term (24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion.

Neural prostheses are devices that interface with the central or peripheral nervous system. They target at the capture, modulation or elicitation of neural activity, in most cases to record the information flow within a neural pathway for its online or later decoding, or to mimic or replace neural functionality that has been compromised or lost. While in theory any information carrying modality of the neuron could be tapped into (e.g., concentrations of neurotransmitters in the synaptic cleft, of energy carriers such as ATP or glucose, or of ions or oxygen; optical properties; ionic currents; membrane potential), most devices sample or alter the membrane potential (or a proportional quantity1 thereto). Since the discovery and description of ‘body electricity’ by Luigi Galvani and Alessandro Volta in their studies on voltage-induced muscle contraction (Volta, 1793), metal electrodes have been used for establishing a bidirectional communication link between electrically excitable cells2 and stimulation or recording apparatuses. From an engineering point of view, this has always been the least challenging and technologically least demanding approach. It just requires bringing a locally deinsulated conductor into close vicinity of a neuron to capture or induce fluctuations in its surrounding electrical field. Given the multitude of conductive materials to choose from and the ever growing number of technological possibilities for their structuring and processing into any desired shape and arrangement, historically, the interface of choice for neuroprosthetics has become the electrode array despite some of its fundamental conceptional shortcomings3. It has evolved into other clinically relevant

Holographic microscopy is increasingly recognized as a promising tool for the study of the central nervous system. Here we present a "holographic module", a simple optical path that can be combined with commercial scanheads for simultaneous imaging and uncaging with structured two-photon light. The present microscope is coupled to two independently tunable lasers and has two principal configurations: holographic imaging combined with galvo-steered uncaging and holographic uncaging combined with conventional scanning imaging. We applied this flexible system for simultaneous two-photon imaging and photostimulation of neuronal cells with complex light patterns, opening new perspectives for the study of brain function in situ and in vivo.

During differentiation, cell processes initiate exploratory motion to create connections with other cells thereby creating a tissue architecture that is capable of performing complex tasks. The interplay between mechanical and chemical stimuli seems necessary for triggering the proper biochemical reactions that eventually lead to the functional organization of cells and tissue[1].There are different approaches for studying the focused mechanical-chemical transduction, either at single cell[2] level or at tissue[3] level. To better understand tissue development ( cell differentiation, cells contact formation, tissue organization), we would like to bridge the gap between experiments on single cells and complex tissues. Therefore we are developing a system for combining optical techniques such as optical tweezers[4] and a laser dissector with electrophysiological tools. Optical tweezers permit to apply localized mechano-chemical stimuli onto cells[5] while a laser dissector can alter individual neuronal connections[6]. By adopting neuronal networks as a biological model, neural signal transmission affected by such external stimuli can be recorded non-invasively by microelectrode arrays.Ongoing work is targeted at correlating the temporary or lasting changes in neural networks to the type and site of the stimulus.1. Pampaloni, F. et al. Nat. Rev. Mol. Cell Biol. 8, (2007).2. Peyton, S.R. et al. Cell Biochemistry and Biophysics 47, (2007).3. Judex, S. et al. J. Biomech. 30, (1997).4. Neuman, K.C. & Block,S.M. Review of Scientific Instruments 75, (2004).5. Cojoc, D. et al. PLoS. ONE. 2, (2007).6. Colombelli J. et al. Methods Cell Biol. 82, 267(2007).

For historical reasons, the signal transduction interface of bioelectronic devices is commonly based on metals or inorganic (semi-)conductors. This also applies to application areas where artificial components such as biomedical screening devices, in vitro microelectrode arrays and in vivo neuroprosthetics come into direct contact with biological tissue. In a proof-of-concept microelectrode array design study, we present an alternative all-polymer approach for the low-cost fabrication of bioelectrical signal transduction devices with adjustable flexibility, electrical impedance and transparency. The fabrication process entailed three steps. Firstly, by means of a replica-moulding strategy, different types of transparent polymers were microstructured by two-level SU-8 masters to create vias for contact pads and electrodes, and indentations for interconnecting microchannels. Secondly, recesses in the insulating polymer sheets were filled with conductive polymer composites based on quasi-transparent polystyrenesulfonate doped poly(3,4-ethylenedioxy-thiophene) (PEDOT:PSS). In a last step, the passive microelectrode arrays were backside-insulated by a second layer of a transparent polymer. The electrical properties of the resulting polymer microelectrode arrays were characterized by impedance spectroscopy, baseline noise measurements and recordings of bioelectrical signals from acute preparations of chicken cardiomyocytes. Biocompatibility was tested with in vitro cultures of cortical neurons derived from embryonic chicken.

Peptide design for unassisted passive water/lipid translocation remains a challenge, notwithstanding its importance for drug delivery. We introduce a design paradigm based on conformational switches operating as passive translocation vehicles. The interfacial behavior of the molecular prototype, probed in single-molecule AFM experiments, reveals a near-barrierless translocation. The associated free-energy agrees with mesoscopic measurements, and the in vitro behavior is quantitatively reproduced in cellular assays. The prototypes herald the advent of novel nano-biomaterials for passive translocation.

A monolithic microsystem in CMOS (complementary metal oxide semiconductor) technology is presented that provides bidirectional communication (stimulation and recording) between standard microelectronics and cultured electrogenic cells. The 128-electrode chip can be directly used as a substrate for cell culturing. It features circuitry units for stimulation and immediate cell signal treatment near each electrode. In addition, it provides on-chip A/D conversion as well as a digital interface so that a fast interaction is possible at good signal quality. Spontaneous and stimulated electrical activity recordings with neuronal and cardiac cell cultures will be presented. The system can be used to, e.g., study the behavior and development of neural networks in vitro, to reveal the effects of neuronal plasticity and to study network activity in response to pharmacological treatments.

We report on the system integration of a CMOS chip that is capable of bidirectionally communicating (stimulation and recording) with electrogenic cells such as neurons or cardiomyocytes, and that is targeted at investigating electrical signal propagation within cellular networks in vitro. Experiments including the stimulation of neurons with two different spatio-temporal patterns and the recording of the triggered spiking activity have been carried out. The neuronal response patterns have been successfully classified (83% correct classifications) with respect to the different stimulation patterns.

We report on a microelectrode array for bidirectional communication (stimulation and recording) with electrogenic cells, for e.g., observing neuronal information processing in vitro. The chip overcomes the interconnect challenge that limits today's bidirectional microelectrode arrays. The array has been fabricated in industrial CMOS-technology with post-CMOS processing and comprises all necessary control circuitry and on-chip A/D-conversion. Measurements from neural cell cultures with amplitudes of 500 /spl mu/V are presented.

Signal degradation and an array size dictated by the number of available interconnects are the two main limitations inherent to standalone microelectrode arrays (MEAs). A new biochip consisting of an array of microelectrodes with fully-integrated analog and digital circuitry realized in an industrial CMOS process addresses these issues. The device is capable of on-chip signal filtering for improved signal-to-noise ratio (SNR), on-chip analog and digital conversion, and multiplexing, thereby facilitating simultaneous stimulation and recording of electrogenic cell activity. The designed electrode pitch of 250 m significantly limits the space available for circuitry: a repeated unit of circuitry associated with each electrode comprises a stimulation buffer and a bandpass filter for readout. The bandpass filter has corner frequencies of 100 Hz and 50 kHz, and a gain of 1000. Stimulation voltages are generated from an 8-bit digital signal and converted to an analog signal at a frequency of 120 kHz. Functionality of the read-out circuitry is demonstrated by the measurement of cardiomyocyte activity. The microelectrode is realized in a shifted design for flexibility and biocompatibility. Several microelectrode materials (platinum, platinum black and titanium nitride) have been electrically characterized. An equivalent circuit model, where each parameter represents a macroscopic physical quantity contributing to the interface impedance, has been successfully fitted to experimental results. © 2004 Elsevier B.V. All rights reserved.

New strategies for spatially controllable cell adhesion have been developed for brain cells from embryonic chicken. They are based on electrochemically active phenol and pyrrole derivatives, and can be used for the selective coverage of electroconductive substrates. Besides mimicking standard laminin-related adhesion promoting mechanisms by means of an electroactive monomer-linked 18-peptide segment from laminin (SRARKQAASIKVAVSADR), electrochemically generated thin (6-30 nm) polymer films of 3-hydroxybenzyl-hydrazine (3HBH) and 2-(3-hydroxyphenyl)-ethanol (2(3HP)E) with and without mechanically entrapped or covalently linked D-lysine have proved to promote cell adhesion in serum-free medium on indium-doped tin oxide (ITO) substrates during the first 6 culturing days in vitro. The effectiveness of the peptide was strongly density-dependent. Unexpectedly, laminin itself or a combination of laminin and poly-D-lysine (PDL) did not promote cell adhesion and neuron differentiation in serum-free cultures on ITO. However, they worked perfectly well on regular polystyrene substrates in serum-free medium or on ITO when medium with serum was used. This finding might suggest that the adhesion efficiency of laminin does not depend only on the kind of medium supplement but also on the type of substrate. In contrast, the adhesion-promoting properties of "artificial" polymeric films seemed to be based on a more direct cell-film interaction, with the film masking the substrate properties.

It has long been assumed that the learning process changes the shape and connectivity of the cells in our brains. But no one has been able to observe this process while it is happening. By combining advanced microscopic imaging techniques with multi-electrode array recording and stimulation, we can determine the relationships between the morphology of brain cells and their electrical activity. We developed a system for studying learning and memory in cultured neuronal networks (Fig.1). By using in vitro approaches, one has access to every cell in the neural circuit, something that is not possible in living animals.

Regeneration of functional connectivity within a neural network after different degrees of lesion is of utmost clinical importance. To test pharmacological approaches aimed at recovering from a total or partial damage of neuronal connections within a circuit, it is necessary to develop a precise method for controlled ablation of neuronal processes. We combined a UV laser microdissector to ablate neural processes in vitro at single neuron and neural network level with infrared holographic optical tweezers to carry out force spectroscopy measurements. Simultaneous force spectroscopy, down to the sub-pico-Newton range, was performed during laser dissection to quantify the tension release in a partially ablated neurite. Therefore, we could control and measure the damage inflicted to an individual neuronal process. To characterize the effect of the inflicted injury on network level, changes in activity of neural subpopulations were monitored with subcellular resolution and overall network activity with high temporal resolution by concurrent calcium imaging and microelectrode array recording. Neuronal connections have been sequentially ablated and the correlated changes in network activity traced and mapped. With this unique combination of electrophysiological and optical tools, neural activity can be studied and quantified in response to controlled injury at the subcellular, cellular, and network level.

We report on a CMOS-based microelectrode-array chip (6.5 by 6.5 mm2) for bidirectional communication (stimulation and recording) with electrogenic cells such as cardiomyocytes or neurons targeted at investigating electrical signal propagation within cellular networks in vitro. The integration of on-chip circuitry, which includes analog signal amplification and filtering stages, analog-to-digital converters, a digital-to-analog converter, stimulation buffers, temperature sensors, and a digital interface for data transmission notably improves the overall system performance. Additionally, the interconnect challenge that limits the size of currently used microelectrode arrays is overcome. Measurements with cardiomyocytes and neuronal cells were successfully carried out, and the circuitry characterization evidenced a total equivalent input noise of 11.7 µ V RMS (0.1 Hz -100 kHz) at a gain of 1,000.

Recordings have been performed with a CMOS-based microelectrode array (MEA) featuring 11'016 metal electrodes and 126 channels, each of which comprises recording and stimulation electronics for extracellular, bidirectional communication with electrogenic cells. The important features of the device include (i) high spatial resolution at (sub) cellular level with 3'200 electrodes per mm(2) (diameter 7 microm, pitch 18 microm), (ii) a reconfigurable routing of the electrodes to the 126 channels, and (iii) low noise levels. Recordings from neonatal rat cardiomyocytes forming confluent layers and microtissues are shown. Moreover, signals from dissociated rat hippocampal neurons and from neurons in an acute cerebellar slice preparation are presented.