Rapid generation of functional engineered 3D human neuronal assemblies: network dynamics evaluated by micro-electrodes arrays.

Objective.In this work we adapted a protocol for the fast generation of human neurons to build 3D neuronal networks with controlled structure and cell composition suitable for systematic electrophysiological investigations.Approach.We used biocompatible chitosan microbeads as scaffold to build 3D networks and to ensure nutrients-medium exchange from the core of the structure to the external environment. We used excitatory neurons derived from human-induced pluripotent stem cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2 differentiation protocol, we obtained 3D engineered networks with good control over cell density, volume and cell composition. We coupled the 3D neuronal networks to 60-channel micro electrode arrays (MEAs) to monitor and characterize their electrophysiological development. In parallel, we generated two-dimensional neuronal networks cultured on chitosan to compare the results of the two models.Main results.We sustained samples until 60 din vitro(DIV) and 3D cultures were healthy and functional. From the structural point of view, the hiPSC derived neurons were able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to the connections among cells. From a functional point of view, neuronal networks showed spontaneous activity after a couple of weeks.Significance.We presented a particular method to generate 3D engineered cultures for the first time with human-derived neurons coupled to MEAs, overcoming some of the limitations related to 2D and 3D neuronal networks and thus increasing the therapeutic target potential of these models for biomedical applications.

An organic neurophysiological tool for neuronal metabolic activity monitoring.

Monitoring cell metabolism in vitro is considered a relevant methodology in several scientific fields ranging from fundamental biology research to neuro-toxicology. In the last 20 years, several in vitro neuro-pharmacological and neuro-toxicological approaches have been developed, with the intent of addressing the increasing demand for real-time, non-invasive in vitro systems capable of continuously and reliably monitoring cellular activity. In this paper, an Organic Charge Modulated Field Effect Transistor-based device is proposed as a promising tool for neuro-pharmacological applications, thanks to its ultra-high pH sensitivity and a simple fabrication technology. The preliminary characterization of this versatile organic device with primary neuronal cultures shows how these remarkable properties can be exploited for the realization of ultra-sensitive metabolic probes, which are both reference-less and low cost. These features, together with the already assessed capability of this sensor to also monitor the electrical activity of electrogenic cells, could provide important advances in the fabrication of multi-sensing lab-on-chip devices, thus opening up interesting perspectives in the neuro-pharmacological field.

An organic transistor-based system for reference-less electrophysiological monitoring of excitable cells.

In the last four decades, substantial advances have been done in the understanding of the electrical behavior of excitable cells. From the introduction in the early 70's of the Ion Sensitive Field Effect Transistor (ISFET), a lot of effort has been put in the development of more and more performing transistor-based devices to reliably interface electrogenic cells such as, for example, cardiac myocytes and neurons. However, depending on the type of application, the electronic devices used to this aim face several problems like the intrinsic rigidity of the materials (associated with foreign body rejection reactions), lack of transparency and the presence of a reference electrode. Here, an innovative system based on a novel kind of organic thin film transistor (OTFT), called organic charge modulated FET (OCMFET), is proposed as a flexible, transparent, reference-less transducer of the electrical activity of electrogenic cells. The exploitation of organic electronics in interfacing the living matters will open up new perspectives in the electrophysiological field allowing us to head toward a modern era of flexible, reference-less, and low cost probes with high-spatial and high-temporal resolution for a new generation of in-vitro and in-vivo monitoring platforms.

Development of micro-electrode array based tests for neurotoxicity: assessment of interlaboratory reproducibility with neuroactive chemicals.

Neuronal assemblies within the nervous system produce electrical activity that can be recorded in terms of action potential patterns. Such patterns provide a sensitive endpoint to detect effects of a variety of chemical and physical perturbations. They are a function of synaptic changes and do not necessarily involve structural alterations. In vitro neuronal networks (NNs) grown on micro-electrode arrays (MEAs) respond to neuroactive substances as well as the in vivo brain. As such, they constitute a valuable tool for investigating changes in the electrophysiological activity of the neurons in response to chemical exposures. However, the reproducibility of NN responses to chemical exposure has not been systematically documented. To this purpose six independent laboratories (in Europe and in USA) evaluated the response to the same pharmacological compounds (Fluoxetine, Muscimol, and Verapamil) in primary neuronal cultures. Common standardization principles and acceptance criteria for the quality of the cultures have been established to compare the obtained results. These studies involved more than 100 experiments before the final conclusions have been drawn that MEA technology has a potential for standard in vitro neurotoxicity/neuropharmacology evaluation. The obtained results show good intra- and inter-laboratory reproducibility of the responses. The consistent inhibitory effects of the compounds were observed in all the laboratories with the 50% Inhibiting Concentrations (IC(50)s) ranging from: (mean ± SEM, in µM) 1.53 ± 0.17 to 5.4 ± 0.7 (n = 35) for Fluoxetine, 0.16 ± 0.03 to 0.38 ± 0.16 µM (n = 35) for Muscimol, and 2.68 ± 0.32 to 5.23 ± 1.7 (n = 32) for Verapamil. The outcome of this study indicates that the MEA approach is a robust tool leading to reproducible results. The future direction will be to extend the set of testing compounds and to propose the MEA approach as a standard screen for identification and prioritization of chemicals with neurotoxicity potential.

Tracking burst patterns in hippocampal cultures with high-density CMOS-MEAs.

In this work, we investigate the spontaneous bursting behaviour expressed by in vitro hippocampal networks by using a high-resolution CMOS-based microelectrode array (MEA), featuring 4096 electrodes, inter-electrode spacing of 21 µm and temporal resolution of 130 µs. In particular, we report an original development of an adapted analysis method enabling us to investigate spatial and temporal patterns of activity and the interplay between successive network bursts (NBs). We first defined and detected NBs, and then, we analysed the spatial and temporal behaviour of these events with an algorithm based on the centre of activity trajectory. We further refined the analysis by using a technique derived from statistical mechanics, capable of distinguishing the two main phases of NBs, i.e. (i) a propagating and (ii) a reverberating phase, and by classifying the trajectory patterns. Finally, this methodology was applied to signal representations based on spike detection, i.e. the instantaneous firing rate, and directly based on voltage-coded raw data, i.e. activity movies. Results highlight the potentialities of this approach to investigate fundamental issues on spontaneous neuronal dynamics and suggest the hypothesis that neurons operate in a sort of 'team' to the perpetuation of the transmission of the same information.

Low-frequency stimulation enhances burst activity in cortical cultures during development.

The intact brain is continuously targeted by a wealth of stimuli with distinct spatio-temporal patterns which modify, since the very beginning of development, the activity and the connectivity of neuronal networks. In this paper, we used dissociated neuronal cultures coupled to microelectrode arrays (MEAs) to study the response of cortical neuron assemblies to low-frequency stimuli constantly delivered over weeks in vitro. We monitored the spontaneous activity of the cultures before and after the stimulation sessions, as well as their evoked response to the stimulus. During in vitro development, the vast majority of the cultures responded to the stimulation by significantly increasing the bursting activity and a widespread stabilization of electrical activity was observed after the third week of age. A similar trend was present between the spontaneous activity of the networks observed over 30 min after the stimulus and the responses evoked by the stimulus itself, although no significant differences in spontaneous activity were detected between stimulated and non-stimulated cultures belonging to the same preparations. The data indicate that the stimulation had a delayed effect modulating responsiveness capability of the network without directly affecting its intrinsic in vitro development.

Extracellular recordings from locally dense microelectrode arrays coupled to dissociated cortical cultures.

High-density microelectrode arrays (MEAs) enabled by recent developments of microelectronic circuits (CMOS-MEA) and providing spatial resolutions down to the cellular level open the perspective to access simultaneously local and overall neuronal network activities expressed by in vitro preparations. The short inter-electrode separation results in a gain of information on the micro-circuit neuronal dynamics and signal propagation, but requires the careful evaluation of the time resolution as well as the assessment of possible cross-talk artifacts. In this respect, we have realized and tested Pt high-density (HD)-MEAs featuring four local areas with 10microm inter-electrode spacing and providing a suitable noise level for the assessment of the high-density approach. First, simulated results show how possible artifacts (duplicated spikes) can be theoretically observed on nearby microelectrodes only for very high-shunt resistance values (e.g. R(sh)=50 kOmega generates up to 60% of false positives). This limiting condition is not compatible with typical experimental conditions (i.e. dense but not confluent cultures). Experiments performed on spontaneously active cortical neuronal networks show that spike synchronicity decreases by increasing the time resolution and analysis results show that the detected synchronous spikes on nearby electrodes are likely to be unresolved (in time) fast local propagations. Finally, functional connectivity analysis results show stronger local connections than long connections spread homogeneously over the whole network demonstrating the expected gain in detail provided by the spatial resolution.

Self-organization and neuronal avalanches in networks of dissociated cortical neurons.

Dissociated cortical neurons from rat embryos cultured onto micro-electrode arrays exhibit characteristic patterns of electrophysiological activity, ranging from isolated spikes in the first days of development to highly synchronized bursts after 3-4 weeks in vitro. In this work we analyzed these features by considering the approach proposed by the self-organized criticality theory: we found that networks of dissociated cortical neurons also generate spontaneous events of spreading activity, previously observed in cortical slices, in the form of neuronal avalanches. Choosing an appropriate time scale of observation to detect such neuronal avalanches, we studied the dynamics by considering the spontaneous activity during acute recordings in mature cultures and following the development of the network. We observed different behaviors, i.e. sub-critical, critical or super-critical distributions of avalanche sizes and durations, depending on both the age and the development of cultures. In order to clarify this variability, neuronal avalanches were correlated with other statistical parameters describing the global activity of the network. Criticality was found in correspondence to medium synchronization among bursts and high ratio between bursting and spiking activity. Then, the action of specific drugs affecting global bursting dynamics (i.e. acetylcholine and bicuculline) was investigated to confirm the correlation between criticality and regulated balance between synchronization and variability in the bursting activity. Finally, a computational model of neuronal network was developed in order to interpret the experimental results and understand which parameters (e.g. connectivity, excitability) influence the distribution of avalanches. In summary, cortical neurons preserve their capability to self-organize in an effective network even when dissociated and cultured in vitro. The distribution of avalanche features seems to be critical in those cultures displaying medium synchronization among bursts and poor random spiking activity, as confirmed by chemical manipulation experiments and modeling studies.

High-resolution MEA platform for in-vitro electrogenic cell networks imaging.

A platform based on an active-pixel-sensor electrode array (APS-MEA) for high-resolution imaging of in-vitro electrogenic cell cultures is presented, characterized and validated under culture conditions. The system enables full frame acquisition at 8 kHz from 4096 microelectrodes integrated with separations of 21 microm and zoomed area acquisition with temporal resolutions down to 8 micros. This bi-modal acquisition feature opens new perspectives in particular for neuronal activity analysis and for the correlation of micro-scale and macro-scale behaviors. The low-noise performances of the integrated amplifier (11 microVRMS) combined with a hardware implementation reflecting image-/video-concepts enable high-resolution acquisitions with real-time processing capabilities adapted to the handling of the large amount of acquired data.

Connecting neurons to a mobile robot: an in vitro bidirectional neural interface.

One of the key properties of intelligent behaviors is the capability to learn and adapt to changing environmental conditions. These features are the result of the continuous and intense interaction of the brain with the external world, mediated by the body. For this reason "embodiment" represents an innovative and very suitable experimental paradigm when studying the neural processes underlying learning new behaviors and adapting to unpredicted situations. To this purpose, we developed a novel bidirectional neural interface. We interconnected in vitro neurons, extracted from rat embryos and plated on a microelectrode array (MEA), to external devices, thus allowing real-time closed-loop interaction. The novelty of this experimental approach entails the necessity to explore different computational schemes and experimental hypotheses. In this paper, we present an open, scalable architecture, which allows fast prototyping of different modules and where coding and decoding schemes and different experimental configurations can be tested. This hybrid system can be used for studying the computational properties and information coding in biological neuronal networks with far-reaching implications for the future development of advanced neuroprostheses.

In vitro cortical neuronal networks as a new high-sensitive system for biosensing applications.

By taking advantages of the main features of the microelectrode array (MEA) technology (i.e. multisite recordings, stable and long-term coupling with the biological preparation), we analyzed the changes in activity patterns induced by applying specific substances to dissociated cortical neurons from rat-embryos (E18). Data were recorded simultaneously from 60 electrodes, and the electrophysiological behavior was investigated during the third week in vitro, both at the spike and burst level. The analysis of the electrophysiological activity modulation, by applying agonists of the ionotropic glutamate receptors at low (i.e. 0.2-1-5 microM) and high (i.e. 50-100 microM) concentrations, is presented. Preliminary results show that the dynamics of the in vitro cortical neurons is very sensitive to pharmacological manipulation of the glutamatergic transmission and the effects on the network behavior are strictly dependent from the drug concentration. In particular, the addition of a high-dose of agonist determined a global and irreversible depression of the network activity, while, in the low-concentration case, the electrophysiological behavior showed different results, depending on the type of receptor involved. From these observations, we are encouraged to think of a more engineered system, based on in vitro cortical neurons, as a novel sensitive system for drug (pre)-screening and neuropharmacological evaluations.

Networks of neurons coupled to microelectrode arrays: a neuronal sensory system for pharmacological applications.

Two main features make microelectrode arrays (MEAs) a valuable tool for electrophysiological measurements under the perspective of pharmacological applications, namely: (i) they are non-invasive and permit, under appropriate conditions, to monitor the electrophysiological activity of neurons for a long period of time (i.e. from several hours up to months); (ii) they allow a multi-site recording (up to tens of channels). Thus, they should allow a high-throughput screening while reducing the need for animal experiments. In this paper, by taking advantages of these features, we analyze the changes in activity pattern induced by the treatment with specific substances, applied on dissociated neurons coming from the chick-embryo spinal cord. Following pioneering works by Gross and co-workers (see e.g. Gross and Kowalski, 1991. Neural Networks, Concepts, Application and Implementation, vol. 4. Prentice Hall, NJ, pp. 47-110; Gross et al., 1992. Sensors Actuators, 6, 1-8.), in this paper analysis of the drugs' effects (e.g. NBQX, CTZ, MK801) to the collective electrophysiological behavior of the neuronal network in terms of burst activity, will be presented. Data are simultaneously recorded from eight electrodes and besides variations induced by the drugs also the correlation between different channels (i.e. different area in the neural network) with respect to the chemical stimuli will be introduced (Bove et al., 1997. IEEE Trans. Biomed. Eng., 44, 964-977.). Cultured spinal neurons from the chick embryo were chosen as a neurobiological system for their relative simplicity and for their reproducible spontaneous electrophysiological behavior. It is well known that neuronal networks in the developing spinal cord are spontaneously active and that the presence of a significant and reproducible bursting activity is essential for the proper formation of muscles and joints (Chub and O'Donovan, 1998. J. Neurosci., 1, 294-306.). This fact, beside a natural variability among different biological preparations, allows a comparison also among different experimental session giving reliable results and envisaging a definition of a bioelectronic 'neuronal sensory system'.

Development of ISFET array-based microsystems for bioelectrochemical measurements of cell populations.

Monitoring the bioelectrochemical activity of living cells with sensor array-based microsystems represents an emerging technique in a large area of biomedical applications, ranging from basic research to various fields of pharmacological analyses. The main appeal is the ability of these miniaturised microsystems to perform, in real time, non-invasive in-vitro investigations of the physiological state of a cell population. In this paper, we present two different microsystems designed for multisite monitoring of the physiological state of a cell population. The first microsystem, intended for cellular metabolism monitoring, consists of an array of 12 spatially distributed ISFETs to detect small pH variations induced by the cell population. The second microsystem consists of an array of 40 ISFETs and 20 gold microelectrodes and it has been designed to monitor the electrical activity of neurons. This is achieved by direct coupling of the neuronal culture with the ISFET sensitive layer and by utilising gold microelectrodes for neuronal electrical stimulation.

Coupling of organotypic brain slice cultures to silicon-based arrays of electrodes.

Fetal or early postnatal brain tissue can be cultured in viable and healthy condition for several weeks with development and preservation of the basic cellular and connective organization as so-called organotypic brain slice cultures. Here we demonstrate and describe how it is possible to establish such hippocampal rat brain slice cultures on biocompatible silicon-based chips with arrays of electrodes with a histological organization comparable to that of conventional brain slice cultures grown by the roller drum technique and on semiporous membranes. Intracellular and extracellular recordings from neurons in the slice cultures show that the electroresponsive properties of the neurons and synaptic circuitry are in accordance with those described for cells in acutely prepared slices of the adult rat hippocampus. Based on the recordings and the possibilities of stimulating the cultured cells through the electrode arrays it is anticipated that the setup eventually will allow long-term studies of defined neuronal networks and provide valuable information on both normal and neurotoxicological and neuropathological conditions.

A simple microfluidic system for patterning populations of neurons on silicon micromachined substrates.

The purpose of this paper is to describe a low-cost simple technique based on the hydraulically driven deposition of adhesion molecules for patterning populations of neurons on silicon micromachined substrates. First, the design and fabrication process of the silicon micromachined substrates and the design of a flow-through chamber for the localised deposition of adhesive proteins are described. The experimental protocol for the deposition of the adhesive proteins is then presented. Finally, the results of experiments of 'entrapment' of chick embryo spinal cord neurons in microstructures of the silicon substrates and of formation of patterned biological neural networks are shown.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Analysis of the signals generated by networks of neurons coupled to planar arrays of microtransducers in simulated experiments.

Planar microelectrode arrays can be used to characterize the dynamics of networks of neurons reconstituted in vitro. In this paper simulations related to experiments of the electrical activity recording by means of planar arrays of microtransducers coupled to networks of neurons are described. First a detailed model of single and synaptically connected neurons is given, appropriate to computer simulate the action potentials of neuronal populations. Then 'realistic' signals are generated. These signals are intended to reproduce, both in shape and intensity, those recorded by a microelectrode array. Typical experimental conditions are considered, and a detailed analysis given, of the bioelectronic coupling and of its influence on the shape of the recorded signals. Finally, simulated experiments dealing with dorsal root ganglia neurons are described and analysed in comparison with experimental results reported in the literature and obtained in our own laboratory. The effectiveness of the planar microelectrode technique is briefly discussed.

Silicon neuron simulation with SPICE: tool for neurobiology and neural networks.

The paper deals with computer simulations of 'silicon neurons', which are assemblies of CMOS circuits that generate the equivalents of the ionic currents and of the action potentials of real (biological) neurons. The circuit simulation program SPICE is used to simulate the generation of action potentials by a silicon neuron. Moreover, the equivalent circuits of silicon synapses are described and the behaviours of simple two- and three-neuron networks are analysed. Implications for the areas of neurobiology and formal neural networks are briefly considered.

Realistic simulations of neurons by means of an ad hoc modified version of SPICE.

This paper describes an ad hoc modified version of the electrical circuit analysis program SPICE, which has been optimized for detailed simulations of the behaviour of neurons. An equivalent-circuit description of the simulation building blocks is provided, and the SPICE modifications are specified. These modifications, in contrast to previous uses of SPICE, allows one to simulate the behaviour of neurons of Hodgkin-Huxley type (excitable membrane) and of postsynaptic membranes without any approximations. Simulation results are reported and compared, both with data previously analysed in the literature by other authors and with experimental data recently obtained by coupling neurons to planar extracellular microelectrodes. Details of the circuit elements used in the simulations are reported. The improvements of our proposed model are discussed in comparison with a previous SPICE-based model described in the literature.

A general-purpose system for long-term recording from a microelectrode array coupled to excitable cells.

A PC-based system for acquisition and processing of data from excitable cells on a microelectrode array is described. Simple and low-cost amplification and filtering custom stages are used. A software package for processing acquired data is proposed.