Antonio Novellino

A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals.

The spike represents the fundamental bit of information transmitted by the neurons within a network in order to communicate. Then, given the importance of the spike rate as well as the spike time for coding the activity generated at the level of a cell assembly, a relevant issue in extracellular electrophysiology is the correct identification of the spike in multisite recordings from brain areas or neuronal networks. In this paper, we present a novel spike detection algorithm, named Precise Timing Spike Detection (PTSD), aimed at (i) reducing the number of false positives and false negatives, in order to optimize the rate code, and (ii) improving the time precision of the identified spike, in order to optimize the spike timing. The PTSD algorithm considers consecutive portions of the signal and looks for the Relative Maximum/Minimum whose peak-to-peak amplitude is above a defined differential threshold and responds to specific requirements. To validate the algorithm, the presented spike detection has been compared with other methods either commercially available or proposed in the literature by using two benchmarking procedures: (i) visual inspection by a group of experts of a portion of signal recorded from a rat cortical culture and (ii) detection of the spikes generated by a realistic neuronal network model. In both cases our algorithm produced the best performances in terms of efficiency and precision. The ROC curve analysis further proved that the best results are reached by the application of the PTSD.

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 (IC50s) 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.

Burst detection algorithms for the analysis of spatio-temporal patterns in cortical networks of neurons

Cortical neurons extracted from the developing rat central nervous system and put in culture, show, after a few days, spontaneous activity with a typical electrophysiological pattern ranging from stochastic spiking to synchronized bursting. Using microelectrode arrays (MEA), on which dissociated cultures can be grown for long-term measurements, we recorded the electrophysiological activity of cortical networks during development, in order to monitor their responses at different stages of the maturation process. Employing algorithms for detection and analysis of bursts in single-channel spike trains and of synchronized network bursts in multi-channel spike trains, significant changes have been revealed in the firing dynamics at different stages of the developmental process.

Application of micro-electrode arrays (MEAs) as an emerging technology for developmental neurotoxicity: Evaluation of domoic acid-induced effects in primary cultures of rat cortical neurons

Due to lack of knowledge only a few industrial chemicals have been identified as developmental neurotoxicants. Current developmental neurotoxicity (DNT) guidelines (OECD and EPA) are based entirely on in vivo studies that are both time consuming and costly. Consequently, there is a high demand to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. One of the most promising tools for neurotoxicity assessment is the measurement of neuronal electrical activity using micro-electrode arrays (MEAs) that provides a functional and neuronal specific endpoint that until now has been used mainly to detect acute neurotoxicity. Here, electrical activity measurements were evaluated to be a suitable endpoint for the detection of potential developmental neurotoxicants. Initially, primary cortical neurons grown on MEA chips were characterized for different cell markers over time, using immunocytochemistry. Our results show that primary cortical neurons could be a promising in vitro model for DNT testing since some of the most critical neurodevelopment processes such as progenitor cell commitment, proliferation and differentiation of astrocytes and maturation of neurons are present. To evaluate if electrical activity could be a suitable endpoint to detect chemicals with DNT effects, our model was exposed to domoic acid (DomA), a potential developmental neurotoxicant for up to 4 weeks. Long-term exposure to a low concentration (50nM) of DomA increased the basal spontaneous electrical activity as measured by spike and burst rates. Moreover, the effect induced by the GABA(A) receptor antagonist bicuculline was significantly lower in the DomA treated cultures than in the untreated ones. The MEA measurements indicate that chronic exposure to DomA changed the spontaneous electrical activity leading to the possible neuronal mal functioning. The obtained results suggest that the MEAs could be a useful tool to identify compounds with DNT potential.

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.

The effects of rate and sequence complexity on repetitive finger movements

We studied the effects of movement rate and sequence complexity on the execution of externally paced finger movements. Simple thumb-index opposition movements (SEQ1), oppositions of thumb to index, medium, ring and little fingers (SEQ2), and oppositions of thumb to index, ring, medium and little fingers (SEQ3) were paced by a metronome at rates that ranged from 0.5 to 5 Hz. At rates higher than 2.5 Hz touch duration, as well as spatial and timing accuracy changed, although with a different pattern, for the sequences. Delayed movements were mostly present at lowest rates in SEQ1; at 0.5 Hz and at frequencies higher than 3.5 Hz in SEQ2; at rates higher than 3.5 Hz in SEQ3. Syncopation occurred at rates higher than 3 Hz but only for SEQ2 and SEQ3 when movements are delayed. Power spectrum analysis of timing error series indicated that SEQ1 performance is influenced by memory-related processes at all movement rates. On the other hand, for the other two sequences, at rates higher than 4 Hz the timing error series exhibit a slight but significant reduction of its long-range correlation characteristics. These findings suggest that different strategies are used for sensorimotor processing when the movement rate and sequence complexity are increased.

Feasibility Assessment of Micro-Electrode Chip Assay as a Method of Detecting Neurotoxicity in vitro

Detection and characterization of chemically induced toxic effects in the nervous system represent a challenge for the hazard assessment of chemicals. In vivo, neurotoxicological assessments exploit the fact that the activity of neurons in the central and peripheral nervous system has functional consequences. And so far, no in vitro method for evaluating the neurotoxic hazard has yet been validated and accepted for regulatory purpose. The micro-electrode array (MEA) assay consists of a culture chamber into which an integrated array of micro-electrodes is capable of measuring extracellular electrophysiology (spikes and bursts) from electro-active tissues. A wide variety of electrically excitable biological tissues may be placed onto the chips including primary cultures of nervous system tissue. Recordings from this type of in vitro cultured system are non-invasive, give label free evaluations and provide a higher throughput than conventional electrophysiological techniques. In this paper, 20 substances were tested in a blinded study for their toxicity and dose–response curves were obtained from fetal rat cortical neuronal networks coupled to MEAs. The experimental procedure consisted of evaluating the firing activity (spiking rate) and modification/reduction in response to chemical administration. Native/reference activity, 30 min of activity recording per dilution, plus the recovery points (after 24 h) were recorded. The preliminary data, using a set of chemicals with different mode-of-actions (13 known to be neurotoxic, 2 non-neuroactive and not toxic, and 5 non-neuroactive but toxic) show good predictivity (sensitivity: 0.77; specificity: 0.86; accuracy: 0.85). Thus, the MEA with a neuronal network has the potency to become an effective tool to evaluate the neurotoxicity of substances in vitro.

A multi-laboratory evaluation of microelectrode array-based measurements of neural network activity for acute neurotoxicity testing ☆

HIGHLIGHTSFour laboratories tested 6 compounds for neuroactivity using microelectrode arrays.All four laboratories were able to correctly identify the three neurotoxic compounds.Three non‐neuroactive compounds were correctly identified 10/12 times.Despite methodological differences, results were consistent across laboratories.These results support use of microelectrode arrays for neurotoxicity screening. ABSTRACT There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non‐neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non‐neurotoxic (“negative controls”), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration‐dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration‐response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function.

Behaviors from an electrically stimulated spinal cord neuronal network cultured on microelectrode arrays

Abstract The spontaneous electrophysiological activity of neural networks seems to play an important role in the Central Nervous System (CNS) developing, subsequent maturation and learning. Learning a new behavior is an exploration process that involves the modulation and the formation of association set between stimuli and responses. Here, we analyze how the electrophysiological activity of cultured spinal cord neurons (14 DIV) from the chick embryo is affected by electrical stimulation. Active neurons show a typical high frequency activity pattern called burst. Induced changes in the patterns of electrophysiological activity are described.

An engineered glove for investigating the neural correlates of finger movements using functional magnetic resonance imaging

Objective measurement of concomitant finger motor performance is recommended for functional magnetic resonance imaging (fMRI) studies investigating brain activity during finger tapping tasks, because performance modality and ability can influence the selection of different neural networks. In this study, we present a novel glove system for quantitative evaluation of finger opposition movements during fMRI (called Glove Analyzer for fMRI, GAF). Several tests for magnetic resonance (MR) compatibility were performed concerning magnet forces, image artifacts and right functioning of the system. Then, pilot fMRI of finger opposition tasks were conducted at 1.5T and 3T to investigate the neural correlates of sequences of finger opposition movements with the right hand, with simultaneous behavioral recording by means of GAF. All the MR compatibility tests succeeded, and the fMRI analysis revealed mainly the activation of the left sensorimotor areas and right cerebellum, regions that are known to be involved in finger movements. No artifactual clusters were detected in the activation maps. At the same time, through the parameters calculated by GAF it was possible to describe the sensorimotor strategy adopted by the subjects during the required task. Thus, the proposed device resulted to be MR compatible and can be useful for future fMRI studies investigating the neural correlates of finger opposition movements, allowing follow-up studies and comparisons among different groups of patients.