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Guidance of neuronal extensions is a complex process essential for linking neurons into complex functional networks underlying the workings of the neural system. Decades of research have suggested the ability of neuronal growth cones to integrate multiple types of cues during the extension process, but also have raised numerous still unanswered questions about synergy or antagonism between the superimposed chemical and mechanical signaling inputs. In this study, using a novel microfabricated analysis platform, we investigate the response of primary mouse embryonic hippocampal neurons to superimposed topographic and soluble chemical cues. We find that an optimal spatial frequency of topographic cues exists, maximizing the precision of the neurite extension. This optimal frequency can help the extending neurites navigate a topographically complex environment, providing pronounced directional selectivity. We also demonstrate that this cue can synergistically enhance attractive and suppress repulsive guidance by the bi-functional soluble cue Netrin-1, and eliminate the repulsive guidance by a chemorepellent Semaphorin3A (Sema3A). These results suggest that topographic cues can provide optimal periodic input into the guidance signaling processes involved in growth cone chemoattraction and can synergistically interact with chemical gradients of soluble guidance cues, shedding light on complex events accompanying the development of the functional nervous system.

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.

Simultaneous electrical stimulation and recording are used to gain insights into the function of neuronal circuitry. However, artifacts produced by the electrical stimulation pulses prevent the recording of neural responses during, and a short period after, the stimulation duration. In this work, we describe a mixed-signal recording topology with template subtraction for removing the artifact during the stimulation pulse. Emulated artifacts generated from a lumped electrical circuit model and experimental artifacts in cardiac cell cultures are used to evaluate the topology. The simulations show that delays between the emulated artifact and its estimated compensation template represent the largest error source of the analog template subtraction. The quantization error appears like random noise and determines the threshold level for the action potential detection. Simulations show that removal of the artifacts is possible, allowing the detection of action potentials during the stimulation pulsing period, even for high-amplitude saturating artifacts. Measurement results with artifacts elicited in cardiac cell cultures show feasible applications of this topology. The proposed topology therefore promisingly opens up a previously unavailable detection window for improving the analysis of the neuronal activity.

Embryonic cells are very robust in surviving dissection and culturing protocols and easily adapt to their in vitro environment. Despite these advantages, research in the olfactory field on cultured embryonic olfactory neurons is sparse. In this study, two primary rat olfactory explant cultures of different embryonic d (E17 and E20) were established, comprising epithelium and bulb. The functionality of these neurons was tested by measuring intracellular calcium responses to cAMP-inducing agents forskolin (FSK) and 3-isobutyl-1-methylxanthine (IBMX) with fluorescence microscopy. For E17, the responsive cell fraction increased over time, from an initial 3% at the 1 d in vitro (DIV) to a maximum of 19% at 11 DIV. The response of E20 neurons fluctuated over time around a more or less stable 13%. A logistic regression analysis indicated a significant difference between both embryonic d in the response to FSK + IBMX. In addition, of these functional neurons, 23.3% of E17 and 54.3% of E20 cultures were responsive to the odorant isoamyl acetate.

The reliable integration of carbon nanotube (CNT) electrodes in future neural probes requires a proper embedding of the CNTs to prevent damage and toxic contamination during fabrication and also to preserve their mechanical integrity during implantation. Here we describe a novel bottom-up embedding approach where the CNT microelectrodes are encased in SiO(2) and Parylene C with lithographically defined electrode openings. Vertically aligned CNTs are grown on microelectrode arrays using low-temperature plasma-enhanced chemical vapor deposition compatible with wafer-scale CMOS processing. Electrodes with 5, 10, and 25 μm diameter are realized. The CNT electrodes are characterized by electrochemical impedance spectroscopy and cyclic voltammetry and compared against cofabricated Pt and TiN electrodes. The superior performance of the CNTs in terms of impedance (≤4.8 ± 0.3 kΩ at 1 kHz) and charge-storage capacity (≥513.9 ± 61.6 mC/cm(2)) is attributed to an increased wettability caused by the removal of the SiO(2) embedding in buffered hydrofluoric acid. Infrared spectroscopy reveals an unaltered chemical fingerprint of the CNTs after fabrication. Impedance monitoring during biphasic current pulsing with increasing amplitudes provides clear evidence of the onset of gas evolution at CNT electrodes. Stimulation is accordingly considered safe for charge densities ≤40.7 mC/cm(2). In addition, prolonged stimulation with 5000 biphasic current pulses at 8.1, 40.7, and 81.5 mC/cm(2) increases the CNT electrode impedance at 1 kHz only by 5.5, 1.2, and 12.1%, respectively. Finally, insertion of CNT electrodes with and without embedding into rat brains demonstrates that embedded CNTs are mechanically more stable than non-embedded CNTs.

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks., (This journal is © The Royal Society of Chemistry 2012)

Since a few decades, micro-fabricated neural probes are being used, together with microelectronic interfaces, to get more insight in the activity of neuronal networks. The need for higher temporal and spatial recording resolutions imposes new challenges on the design of integrated neural interfaces with respect to power consumption, data handling and versatility. In this paper, we present an integrated acquisition system for in vitro and in vivo recording of neural activity. The ASIC consists of 16 low-noise, fully-differential input channels with independent programmability of its amplification (from 100 to 6000 V/V) and filtering (1-6000 Hz range) capabilities. Each channel is AC-coupled and implements a fourth-order band-pass filter in order to steeply attenuate out-of-band noise and DC input offsets. The system achieves an input-referred noise density of 37 nV/√Hz, a NEF of 5.1, a CMRR > 60 dB, a THD < 1% and a sampling rate of 30 kS/s per channel, while consuming a maximum of 70 μA per channel from a single 3.3 V. The ASIC was implemented in a 0.35 μm CMOS technology and has a total area of 5.6 × 4.5 mm². The recording system was successfully validated in in vitro and in vivo experiments, achieving simultaneous multichannel recordings of cell activity with satisfactory signal-to-noise ratios.

The signal-to-noise ratio of in vivo extracellular neural recordings with microelectrodes is influenced by many factors including the impedance of the electrode-tissue interface, the noise of the recording equipment and biological background noise from distant neurons. In this work we study the different noise sources affecting the quality of neural signals. We propose a simplified noise model as an analytical tool to predict the noise of an electrode given its geometrical dimensions and impedance characteristics. With this tool we are able to quantify different noise sources, which is important to determine realistic noise specifications for the design of electronic neural recording interfaces.

APP.V717I and Tau.P301L transgenic mice develop Alzheimer's disease pathology comprising important aspects of human disease including increased levels of amyloid peptides, cognitive and motor impairment, amyloid plaques and neurofibrillary tangles. The combined model, APP.V717I×Tau.P301L bigenic mice (biAT mice) exhibit aggravated amyloid and tau pathology with severe cognitive and behavioral defects. In the present study, we investigated early changes in synaptic function in the CA1 and CA3 regions of acute hippocampal slices of young APP.V717I, Tau.P301L and biAT transgenic animals. We have used planar multi-electrode arrays (MEA) and improved methods for simultaneous multi-site recordings from two hippocampal sub-regions. In the CA1 region, long-term potentiation (LTP) was severely impaired in all transgenic animals when compared with age-matched wild-type controls, while basal synaptic transmission and paired-pulse facilitation were minimally affected. In the CA3 region, LTP was normal in Tau.P301L and APP.V717I but clearly impaired in biAT mice. Surprisingly, frequency facilitation in CA3 was significantly enhanced in Tau.P301L mice, while not affected in APP.V717I mice and depressed in biAT mice. The findings demonstrate important synaptic changes that differ considerably in the hippocampal sub-regions already at young age, well before the typical amyloid or tau pathology is evident., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The fields of bioscience and nanomedicine demand precise thermometry for nanoparticle heat characterization down to the nanoscale regime. Since current methods often use indirect and less accurate techniques to determine the nanoparticle temperature, there is a pressing need for a direct and reliable element-specific method. In-situ extended X-ray absorption fine structure (EXAFS) spectroscopy is used to determine the thermo-optical properties of plasmonic branched gold nanoparticles upon resonant laser illumination. With EXAFS, the direct determination of the nanoparticle temperature increase upon laser illumination is possible via the thermal influence on the gold lattice parameters. More specifically, using the change of the Debye-Waller term representing the lattice disorder, the temperature increase is selectively measured within the plasmonic branched nanoparticles upon resonant laser illumination. In addition, the signal intensity shows that the nanoparticle concentration in the beam more than doubles during laser illumination, thereby demonstrating that photothermal heating is a dynamic process. A comparable temperature increase is measured in the nanoparticle suspension using a thermocouple. This good correspondence between the temperature at the level of the nanoparticle and at the level of the suspension points to an efficient heat transfer between the nanoparticle and the surrounding medium, thus confirming the potential of branched gold nanoparticles for hyperthermia applications. This work demonstrates that X-ray absorption spectroscopy-based nanothermometry could be a valuable tool in the fast-growing number of applications of plasmonic nanoparticles, particularly in life sciences and medicine., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The electrochemistry of 50 μm diameter Pt electrodes used for neural stimulation was studied in vitro by reciprocal derivative chronopotentiometry. This differential method provides well-defined electrochemical signatures of the various polarization phenomena that occur at Pt microelectrodes and are generally obscured in voltage transients. In combination with a novel in situ coulometric approach, irreversible H(2) and O(2) evolution, Pt dissolution and reduction of dissolved O(2) were detected. Measurements were performed with biphasic, charge-balanced, cathodic-first and anodic-first current pulses at charge densities ranging from 0.07 to 1.41 mC/cm(2) (real surface area) in phosphate buffered saline (PBS) with and without bovine serum albumin (BSA). The extent to which O(2) reduction occurs under the different stimulation conditions was compared in O(2)-saturated and deoxygenated PBS. Adsorption of BSA inhibited Pt dissolution as well as Pt oxidation and oxide reduction by blocking reactive sites on the electrode surface. This inhibitory effect promoted the onset of irreversible H(2) and O(2) evolution, which occurred at lower charge densities than those in PBS. Reduction of dissolved O(2) on Pt electrodes accounted for 19-34% of the total injected charge in O(2)-saturated PBS, while a contribution of 0.4-12% was estimated for in vivo stimulation. These result may prove important for the interpretation of histological damage induced by neural stimulation and therefore help define safer operational limits.

Very-large scale integration and micro-machining have enabled the development of novel platforms for advanced and automated examination of cells and tissues in vitro. In this paper, we present a CMOS chip designed in a commercial 0.18 μm technology with integrated micro-syringes combined with micro-nail shaped electrodes and readout electronics. The micro-syringes could be individually addressed by a through-wafer micro-fluidic channel with an inner diameter of 1 μm. We demonstrated the functionality of the micro-fluidic access by diffusion of fluorescent species through the channels. Further, hippocampal neurons were cultured on top of an array of micro-syringes, and focused ion beam-scanning electron microscopy cross-sections revealed protrusion of the cells inside the channels, creating a strong interface between the membrane and the chip surface. This principle demonstrates a first step towards a novel type of automated in vitro platforms, allowing local delivery of substances to cells or advanced planar patch clamping.

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 μm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The amyloid peptides Aβ(40) and Aβ(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ(42) seems more pathogenic than Aβ(40), the reason for this is not well understood. We show here that small alterations in the Aβ(42):Aβ(40) ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ(42):Aβ(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

In neurophysiological and pharmaceutical research, parallel and individual access to a dense population of in-vitro cultured neurons is a key feature for analyzing networks of neurons. This paper presents a 0.18µm CMOS chip containing a dense array of micro-nail electrodes, a 128×128 sensor/actuator matrix with in-situ differential amplification circuits, pico-Ampere current stimulation, and impedance measurement circuits. Measurements on packaged chips show successful impedance measurements matching the simulation model and electrical recordings of in-vitro cultured cardiomyocytes, correlated with recorded changes in intra-cellular calcium concentrations. This system is a first step towards a high-throughput neuron/chip interface.

We used reciprocal derivative chronopotentiometry (RDC) with platinum electrodes of 50 microm diameter in 0.15 M phosphate buffered saline solution to identify the various electrochemical processes occurring at the electrode during biphasic current pulsing. RDC allowed to determine the limits of water hydrolysis based on the specific (dt/dE)-E data representation employed in this technique resulting in curves similar to the voltammetric i-E response. Current stimulation was performed by either varying the pulse amplitude or pulse width. We found that the limits for H(2) and O(2) evolution for constant-amplitude pulses lied at 0.51 mC/cm(2) and 0.67 mC/cm(2), respectively, while for constant-width pulses they occurred at slightly lower values of 0.49 mC/cm(2) and 0.61 mC/cm(2), respectively. We could also extract values for the anodic and cathodic overvoltages associated with gas evolution. The cathodic overvoltage for H(2) evolution was 1.43 V for both constant-amplitude and constant-width pulses, while the anodic overpotentials for O(2) evolution were 2.45 V in the first and 2.24 V in the latter case. These values are clearly larger than the gas evolution limits generally found with steady-state voltammetry.

In drug screening and pharmaceutical research, high-throughput systems that are able to perform single-cell measurements are highly desired. Micro-electrode arrays try to answer this need but still suffer from significant drawbacks such as a small amount of electrodes and the inability to address single cells. Here, we present a novel multi-transistor array chip with 16,384 subcellular-sized electrodes based on 0.18 µm CMOS technology. We show that single-cell stimulation is possible by applying voltage pulses on the electrode to stimulate the cells lying on top. Electroporation of the cell membrane is observed using the whole-cell patch clamp technique and fluorescent dye-based live imaging. This technology could be used for high-throughput, single-cell manipulations for the purpose of large-scale drug screening and the investigation of fundamental cell processes.

Closed loop systems, in which stimulation parameters are adjusted according to recorded signals would reduce the occurrence of side effects of stimulation and broaden current therapeutic options. As a step towards a closed-loop clinical system, we developed a single electrode stimulation / recording system for an in vitro microelectrode array. The system was used in vitro to simultaneously stimulate and record cardiac cells. Results indicated that stimulation artifacts depend on the distance between recording electrode and stimulating electrode and on the voltage amplitude. No artifact reduction algorithm was required for detecting cardiac action potentials 2ms after stimulation if the stimulation pulses were less than or equal to ± 1.5 V, and the distance from stimulation site was more than 200 µm. Cardiac signal propagation was also investigated with this system.

Interfacing neurons with micro- and nano-electronic devices has been a subject of intense study over the last decade. One of the major problems in assembling efficient neuro-electronic hybrid systems is the weak electrical coupling between the components. This is mainly attributed to the fundamental property of living cells to form and maintain an extracellular cleft between the plasma membrane and any substrate to which they adhere. This cleft shunts the current generated by propagating action potentials and thus reduces the signal-to-noise ratio. Reducing the cleft thickness, and thereby increasing the seal resistance formed between the neurons and the sensing surface, is thus a challenge and could improve the electrical coupling coefficient. Using electron microscopic analysis and field potential recordings, we examined here the use of gold micro-structures that mimic dendritic spines in their shape and dimensions to improve the adhesion and electrical coupling between neurons and micro-electronic devices. We found that neurons cultured on a gold-spine matrix, functionalized by a cysteine-terminated peptide with a number of RGD repeats, readily engulf the spines, forming tight apposition. The recorded field potentials of cultured Aplysia neurons are significantly larger using gold-spine electrodes in comparison with flat electrodes.

This work investigates the feasibility of transducing molecular-recognition events into a measurable potentiometric signal. It is shown for the first time that biorecognition of acetylcholine (ACh) can be translated to conformational changes in the enzyme, acetylcholine-esterase (AChE), which in turn induces a measurable change in surface potential. Our results show that a highly sensitive detector for ACh can be obtained by the dilute assembly of AChE on a floating gate derived field effect transistor (FG-FET). A wide concentration range response is observed for ACh (10(-2)-10(-9)M) and for the inhibitor carbamylcholine CCh (10(-6)-10(-11)M). These enhanced sensitivities are modeled theoretically and explained by the combined response of the device to local pH changes and molecular dipole variations due to the enzyme-substrate recognition event.

In this paper, we describe the localized and selective electrical stimulation of single cells using a three-dimensional electrode array. The chip consisted of 84 nail-like electrodes with a stimulation surface of 0.8 microm(2) and interelectrode distances as small as 3 microm. N2A cells were used to compare bipolar stimulation between one electrode in- and one outside the cell on the one hand, and two electrodes in the same cell on the other hand. Selective and localized stimulation of primary embryonic cardiomyocytes showed the possibility to use this chip with excitable cells. The response of the cells to applied electrical fields was monitored using calcium imaging whereas assessment of electroporation was determined following influx of propidium iodide. Arrays of these three-dimensional electrodes could eventually be used as a tool to selectively electroporate the membrane of single cells for genetic manipulation or to obtain electrical access to the inner compartment of the cell.

The electrochemical behavior of neural implants with 50 microm-diameter platinum electrodes was tested during acute implantations in the motor cortex of anesthetized rats. Custom Ag|AgCl reference electrodes were prepared that could be co-implanted with the probes. The results obtained in vivo are compared with in vitro measurements performed in buffered saline solution (PBS) with and without the addition of bovine serum albumin (BSA). The presence of BSA clearly altered the performance of the electrodes which was studied by means of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), voltage transient measurements (VT) and monitoring of the open circuit potential (OCP). We found that hydrogen gas evolved at 1.22 A/cm(2) in BSA-free PBS whereas in BSA-containing PBS it occurred already at 0.51 A/cm(2).

In this paper, we demonstrate the feasibility of selective extracellular electrical stimulation at the (sub)cellular level in dissociated cultured cells. Using a CMOS-compatible process, we have fabricated an electrode array with sub-micrometer nail probes. Due to their particular configuration, the nails are strongly engulfed by the cellular membrane. By measuring the calcium signals, we found that electrical stimulation via the micronails activates the cell locally, in a dose-dependent manner, with very low applied currents. The results suggest the applicability of the device in pharmacological or signal propagation studies.

This paper describes the fabrication and in vitro testing of a device capable of chemically stimulating individual neurons. Electrophoretic actuation is used to locally induce the release of the neurotransmitter L-glutamate in a network of hippocampal neurons cultured on top of the device. Cell activation by the neurotransmitter is visualized using calcium imaging. Single-cell stimulation is demonstrated close to the release site after application of a voltage of 500 mV. Such a device could provide a useful tool in both basic and clinical neuroscience. In a broader context, this device can be used to locally release small amounts of chemical compounds in Lab-on-Chip and drug delivery applications.

Extracellular, high signal-to-noise ratio recordings from electrogenic cells require a tight coupling between the cellular membrane and the recording electrode. Self assembled monolayers (SAMs) of alkanethiols functionalized with peptides were used in combination with micro- and nano-structured features on the sensor surface. This combination of surface chemistry and topography triggers a phagocytosis-like engulfment and ensures tight coupling. In this paper we report the results concerning usage of different SAMs and the influence of the peptide concentration towards cell adhesion and outgrowth. Later on, the optimized peptide functionalized SAMs were applied on micro- and nano-structured sensor surfaces. As a result, phagocytosis-like events could be shown using focused ion beam SEM and confocal fluorescence imaging.

The use of organic thin-film transistors (OTFTs) in sensorics is relatively new. Although electronic noses, electronic textiles and disposable biochemical sensors appear to be viable applications for this type of devices, the benefits of the technology still have to be proven. This paper aims to provide a review of the recent advances in the area of chemically sensitive field-effect devices based on organic thin-film transistors (OTFTs), with emphasis on bioanalytical applications. Detection principle, device configuration, materials and fabrication processes as well as sensor performances will be discussed, with emphasis on the potential for implementation in real applications and the important challenges ahead.