Mariateresa Tedesco

Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development.

In vitro cultured neuronal networks coupled to microelectrode arrays (MEAs) constitute a valuable experimental model for studying changes in the neuronal dynamics at different stages of development. After a few days in culture, neurons start to connect each other with functionally active synapses, forming a random network and displaying spontaneous electrophysiological activity. The patterns of collective rhythmic activity change in time spontaneously during in vitro development. Such activity-dependent modifications play a key role in the maturation of the network and reflect changes in the synaptic efficacy, fact widely recognized as a cellular basis of learning, memory and developmental plasticity. Getting advantage from the possibilities offered by the MEAs, the aim of our study is to analyze and characterize the natural changes in dynamics of the electrophysiological activity at different ages of the culture, identifying peculiar steps of the spontaneous evolution of the network. The main finding is that between the second and the third week of culture, the network completely changes its electrophysiological patterns, both in terms of spiking and bursting activity and in terms of cross-correlation between pairs of active channels. Then the maturation process can be characterized by two main phases: modulation and shaping in the synaptic functional connectivity of the network (within the first and second week) and general moderate correlated activity, spread over the entire network, with connections properly formed and stabilized (within the fourth and fifth week).

Active pixel sensor array for high spatio-temporal resolution electrophysiological recordings from single cell to large scale neuronal networks

This paper presents a chip-based electrophysiological platform enabling the study of micro- and macro-circuitry in in-vitro neuronal preparations. The approach is based on a 64x64 microelectrode array device providing extracellular electrophysiological activity recordings with high spatial (21 microm of electrode separation) and temporal resolution (from 0.13 ms for 4096 microelectrodes down to 8 micros for 64 microelectrodes). Applied to in-vitro neuronal preparations, we show how this approach enables neuronal signals to be acquired for investigating neuronal activity from single cells and microcircuits to large scale neuronal networks. The main elements of the platform are the metallic microelectrode array (MEA) implemented in Complementary Metal Oxide Semiconductor (CMOS) technology similar to a light imager, the in-pixel integrated low-noise amplifiers (11 microVrms) and the high-speed random addressing logic. The chip is combined with a real-time acquisition system providing the capability to record at 7.8 kHz/electrode the whole array and to process the acquired signals.

Opposite Changes in Glutamatergic and GABAergic Transmission Underlie the Diffuse Hyperexcitability of Synapsin I–Deficient Cortical Networks

Synapsins (Syns) are synaptic vesicle (SV) phosphoproteins that play a role in synaptic transmission and plasticity. Mutation of the SYN1 gene results in an epileptic phenotype in mouse and man, implicating SynI in the control of network excitability. We used microelectrode array and patch-clamp recordings to study network activity in primary cortical neurons from wild-type (WT) or SynI knockout (KO) mice. SYN1 deletion was associated with increased spontaneous and evoked activities, with more frequent and sustained bursts of action potentials and a high degree of synchronization. Blockade of GABA(A) (gamma-aminobutyric acid(A)) receptors with bicuculline attenuated, but did not completely abolish, the differences between WT and SynI KO networks in both spontaneous and evoked activities. Patch-clamp recordings on cortical autaptic neurons revealed a reduced amplitude of evoked inhibitory postsynaptic currents (PSCs) and a concomitantly increased amplitude of evoked excitatory PSCs in SynI KO neurons, in the absence of changes in miniature PSCs. Cumulative amplitude analysis revealed that these effects were attributable to opposite changes in the size of the readily releasable pool of SVs. The results indicate distinct roles of SynI in GABAergic and glutamatergic neurons and provide an explanation for the high susceptibility of SynI KO mice to epileptic seizures.

Cortical cultures coupled to micro-electrode arrays: a novel approach to perform in vitro excitotoxicity testing.

In vitro neuronal cultures exhibit spontaneous electrophysiological activity that can be modulated by chemical stimulation and can be monitored over time by using Micro-Electrode Arrays (MEAs), devices composed by a glass substrate and metal electrodes. Dissociated networks respond to transmitters, their blockers and many other pharmacological substances, including neurotoxic compounds. In this paper we present results related to the effects, both acute (i.e. 1 hour after the treatment) and chronic (3 days after the treatment), of increasing glutamatergic transmission induced by the application of rising concentrations of glutamate and its agonists (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid - AMPA, N-methyl-D-aspartate - NMDA and AMPA together with cyclothiazide - CTZ). Increase of available glutamate was obtained in two ways: 1) by direct application of exogenous glutamate and 2) by inhibiting the clearance of the endogenously released glutamate through DL-threo-β-benzyloxyaspartate (TBOA). Our findings show that fine modulations (i.e. low concentrations of drug) of the excitatory synaptic transmission are reflected in the electrophysiological activation of the network, while intervention leading to excessive direct stimulation of glutamatergic pathways (i.e. medium and high concentrations of drug) results in the abolishment of the electrophysiological activity and eventually cell death. The results obtained by means of the MEA recordings have been compared to the analysis of cell viability to confirm the excitotoxic effect of the applied drug. In conclusion, our study demonstrates that MEA-coupled cortical networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic transmission and might provide sensitive endpoints to detect acute and chronic neurotoxic effects of chemicals and drugs for predictive toxicity testing.

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.

Mechanical and morphological properties of living 3T6 cells probed via scanning force microscopy

Scanning Force Microscopy (SFM) is utilized to study living confluent 3T6 cells. Images based on mechanical contrast are obtained and related morphological details, mostly regarding the cell cytoskeleton, are analyzed. Moreover, numerical estimates of the local mechanical properties of the living cells are given, by extensive use of the “force‐vs.‐distance” operation mode. On the basis of the results obtained, the potentialities of SFM as an optimal new technique available for probing the cell cytoskeleton of unstained living cells, and assessing related models, are shortly discussed. Microsc. Res. Tech. 36:165–171, 1997.

Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology

Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

Brain-machine interfaces (BMI) were born to control “actions from thoughts” in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI—a neuromorphic chip for brain repair—to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary “bottom-up” approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of “finite size networks” which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

Multiscale functional connectivity estimation on low-density neuronal cultures recorded by high-density CMOS Micro Electrode Arrays

We used electrophysiological signals recorded by CMOS Micro Electrode Arrays (MEAs) at high spatial resolution to estimate the functional-effective connectivity of sparse hippocampal neuronal networks in vitro by applying a cross-correlation (CC) based method and ad hoc developed spatio-temporal filtering. Low-density cultures were recorded by a recently introduced CMOS-MEA device providing simultaneous multi-site acquisition at high-spatial (21 μm inter-electrode separation) as well as high-temporal resolution (8 kHz per channel). The method is applied to estimate functional connections in different cultures and it is refined by applying spatio-temporal filters that allow pruning of those functional connections not compatible with signal propagation. This approach permits to discriminate between possible causal influence and spurious co-activation, and to obtain detailed maps down to cellular resolution. Further, a thorough analysis of the links strength and time delays (i.e., amplitude and peak position of the CC function) allows characterizing the inferred interconnected networks and supports a possible discrimination of fast mono-synaptic propagations, and slow poly-synaptic pathways. By focusing on specific regions of interest we could observe and analyze microcircuits involving connections among a few cells. Finally, the use of the high-density MEA with low density cultures analyzed with the proposed approach enables to compare the inferred effective links with the network structure obtained by staining procedures.