Low glycemic index diet restrains epileptogenesis in a gender-specific fashion.

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2-3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

Identification of excitatory-inhibitory links and network topology in large-scale neuronal assemblies from multi-electrode recordings.

Functional-effective connectivity and network topology are nowadays key issues for studying brain physiological functions and pathologies. Inferring neuronal connectivity from electrophysiological recordings presents open challenges and unsolved problems. In this work, we present a cross-correlation based method for reliably estimating not only excitatory but also inhibitory links, by analyzing multi-unit spike activity from large-scale neuronal networks. The method is validated by means of realistic simulations of large-scale neuronal populations. New results related to functional connectivity estimation and network topology identification obtained by experimental electrophysiological recordings from high-density and large-scale (i.e., 4096 electrodes) microtransducer arrays coupled to in vitro neural populations are presented. Specifically, we show that: (i) functional inhibitory connections are accurately identified in in vitro cortical networks, providing that a reasonable firing rate and recording length are achieved; (ii) small-world topology, with scale-free and rich-club features are reliably obtained, on condition that a minimum number of active recording sites are available. The method and procedure can be directly extended and applied to in vivo multi-units brain activity recordings.

A topological study of repetitive co-activation networks in in vitro cortical assemblies.

To address the issue of extracting useful information from large data-set of large scale networks of neurons, we propose an algorithm that involves both algebraic-statistical and topological tools. We investigate the electrical behavior of in vitro cortical assemblies both during spontaneous and stimulus-evoked activity coupled to Micro-Electrode Arrays (MEAs). Our goal is to identify core sub-networks of repetitive and synchronous patterns of activity and to characterize them. The analysis is performed at different resolution levels using a clustering algorithm that reduces the network dimensionality. To better visualize the results, we provide a graphical representation of the detected sub-networks and characterize them with a topological invariant, i.e. the sequence of Betti numbers computed on the associated simplicial complexes. The results show that the extracted sub-populations of neurons have a more heterogeneous firing rate with respect to the entire network. Furthermore, the comparison of spontaneous and stimulus-evoked behavior reveals similarities in the identified clusters of neurons, indicating that in both conditions similar activation patterns drive the global network activity.

Electrical and chemical modulation of homogeneous and heterogeneous human-iPSCs-derived neuronal networks on high density arrays.

The delicate "Excitatory/Inhibitory balance" between neurons holds significance in neurodegenerative and neurodevelopmental diseases. With the ultimate goal of creating a faithful in vitro model of the human brain, in this study, we investigated the critical factor of heterogeneity, focusing on the interplay between excitatory glutamatergic (E) and inhibitory GABAergic (I) neurons in neural networks. We used high-density Micro-Electrode Arrays (MEA) with 2304 recording electrodes to investigate two neuronal culture configurations: 100% glutamatergic (100E) and 75% glutamatergic / 25% GABAergic (75E25I) neurons. This allowed us to comprehensively characterize the spontaneous electrophysiological activity exhibited by mature cultures at 56 Days in vitro, a time point in which the GABA shift has already occurred. We explored the impact of heterogeneity also through electrical stimulation, revealing that the 100E configuration responded reliably, while the 75E25I required more parameter tuning for improved responses. Chemical stimulation with BIC showed an increase in terms of firing and bursting activity only in the 75E25I condition, while APV and CNQX induced significant alterations on both dynamics and functional connectivity. Our findings advance understanding of diverse neuron interactions and their role in network activity, offering insights for potential therapeutic interventions in neurological conditions. Overall, this work contributes to the development of a valuable human-based in vitro system for studying physiological and pathological conditions, emphasizing the pivotal role of neuron diversity in neural network dynamics.

Electrophysiological and morphological modulation of neuronal-glial network by breast cancer and nontumorigenic mammary cell conditioned medium.

Breast cancer is a significant global health concern, with the overexpression of human epidermal growth factor receptor 2 (HER2/ERBB2) being a driver oncogene in 20%-30% of cases. Indeed, HER2/ERBB2 plays a crucial role in regulating cell growth, differentiation, and survival via a complex signaling network. Overexpression of HER2/ERBB2 is associated with more aggressive behavior and increased risk of brain metastases, which remains a significant clinical challenge for treatment. Recent research has highlighted the role of breast cancer secretomes in promoting tumor progression, including excessive proliferation, immune invasion, and resistance to anti-cancer therapy, and their potential as cancer biomarkers. In this study, we investigated the impact of ERBB2+ breast cancer SKBR-3 cell line compared with MCF10-A mammary non-tumorigenic cell conditioned medium on the electrophysiological activity and morphology of neural networks derived from neurons differentiated from human induced pluripotent stem cells. Our findings provide evidence of active modulation of neuronal-glial networks by SKBR-3 and MCF10-A conditioned medium. These results provide insights into the complex interactions between breast cancer cells and the surrounding microenvironment. Further research is necessary to identify the specific factors within breast cancer conditioned medium that mediate these effects and to develop targeted therapies that disrupt this interaction.

Selective modulation of chemical and electrical synapses of Helix neuronal networks during in vitro development.

BACKGROUND: A large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed. RESULTS: In this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs. CONCLUSIONS: Networks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.

Investigating the reliability of the evoked response in human iPSCs-derived neuronal networks coupled to micro-electrode arrays.

In vitro models of neuronal networks have emerged as a potent instrument for gaining deeper insights into the intricate mechanisms governing the human brain. Notably, the integration of human-induced pluripotent stem cells (hiPSCs) with micro-electrode arrays offers a means to replicate and dissect both the structural and functional elements of the human brain within a controlled in vitro environment. Given that neuronal communication relies on the emission of electrical (and chemical) stimuli, the employment of electrical stimulation stands as a mean to comprehensively interrogate neuronal assemblies, to better understand their inherent electrophysiological dynamics. However, the establishment of standardized stimulation protocols for cultures derived from hiPSCs is still lacking, thereby hindering the precise delineation of efficacious parameters to elicit responses. To fill this gap, the primary objective of this study resides in delineating effective parameters for the electrical stimulation of hiPSCs-derived neuronal networks, encompassing the determination of voltage amplitude and stimulation frequency able to evoke reliable and stable responses. This study represents a stepping-stone in the exploration of efficacious stimulation parameters, thus broadening the electrophysiological activity profiling of neural networks sourced from human-induced pluripotent stem cells.

Deepening the role of excitation/inhibition balance in human iPSCs-derived neuronal networks coupled to MEAs during long-term development.

Objective.The purpose of this study is to investigate whether and how the balance between excitation and inhibition ('E/I balance') influences the spontaneous development of human-derived neuronal networksin vitro. To achieve that goal, we performed a long-term (98 d) characterization of both homogeneous (only excitatory or inhibitory neurons) and heterogeneous (mixed neuronal types) cultures with controlled E/I ratios (i.e. E:I 0:100, 25:75, 50:50, 75:25, 100:0) by recording their electrophysiological activity using micro-electrode arrays.Approach.Excitatory and inhibitory neurons were derived from human induced pluripotent stem cells (hiPSCs). We realized five different configurations by systematically varying the glutamatergic and GABAergic percentages.Main results.We successfully built both homogeneous and heterogeneous neuronal cultures from hiPSCs finely controlling the E/I ratios; we were able to maintain them for up to 3 months. Homogeneity differentially impacted purely inhibitory (no bursts) and purely excitatory (few bursts) networks, deviating from the typical traits of heterogeneous cultures (burst dominated). Increased inhibition in heterogeneous cultures strongly affected the duration and organization of bursting and network bursting activity. Spike-based functional connectivity and image-based deep learning analysis further confirmed all the above.Significance.Healthy neuronal activity is controlled by a well-defined E/I balance whose alteration could lead to the onset of neurodevelopmental disorders like schizophrenia or epilepsy. Most of the commonly usedin vitromodels are animal-derived or too simplified and thus far from thein vivohuman condition. In this work, by performing a long-term study of hiPSCs-derived neuronal networks obtained from healthy human subjects, we demonstrated the feasibility of a robustin vitromodel which can be further exploited for investigating pathological conditions where the E/I balance is impaired.

Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.

Methods for studying brain function and disease heavily rely onin vivoanimal models,ex-vivotissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects ofin vivohuman brain are needed as additional preclinical tools. In this context, 3D hydrogel-basedin vitromodels of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-ß-glycerophosphate (CHITO-ß-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-ß-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-ß-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

An efficient deep learning approach to identify dynamics in in vitro neural networks.

Understanding and discriminating the spatiotemporal patterns of activity generated by in vitro and in vivo neuronal networks is a fundamental task in neuroscience and neuroengineering. The state-of-the-art algorithms to describe the neuronal activity mostly rely on global and local well-established spike and burst-related parameters. However, they are not able to capture slight differences in the activity patterns. In this work, we introduce a deep-learning-based algorithm to automatically infer the dynamics exhibited by different neuronal populations. Specifically, we demonstrate that our algorithm is able to discriminate with high accuracy the dynamics of five different populations of in vitro human-derived neural networks with an increasing inhibitory to excitatory neurons ratio.

A micropatterned thermoplasmonic substrate for neuromodulation of in vitro neuronal networks.

Understanding how the spatial organization of a neural network affects its activity represents a leading issue in neuroscience. Thanks to their accessibility and easy handling, in vitro studies remain an essential tool to investigate the relationship between the structure and function of a neuronal network. Among all the patterning techniques, ink-jet printing acquired great interest thanks to its direct-write approach, which allows the patterned substrate realization without mold, leading to a considerable saving of both cost and time. However, the inks commonly used give the possibility to control only the structure of a neuronal network, leaving aside the functional aspect. In this work, we synthesize a photosensitive ink combining the rheological and bioadhesive properties of chitosan with the plasmonic properties of gold nanorods, obtaining an ink able to control both the spatial organization of a two-dimensional neuronal network and its activity through photothermal effect. After the ink characterization, we demonstrate that it is possible to print, with high precision, different geometries on a microelectrode array. In this way, it is possible obtaining a patterned device to control the structure of a neuronal network, to record its activity and to modulate it via photothermal effect. Finally, to our knowledge, we report the first evidence of photothermal inhibition of human neurons activity. STATEMENT OF SIGNIFICANCE: Patterned cell cultures remain the most efficient and simple tool for linking structural and functional studies, especially in the neuronal field. Ink-jet printing is the technique with which it is possible to realize patterned structures in the fastest, simple, versatile and low-cost way. However, the inks currently used permit the control only of the neuronal network structure but do not allow the control-modulation of the network activity. In this study, we realize and characterize a photosensitive bioink with which it is possible to drive both the structure and the activity of a neuronal network. Moreover, we report the first evidence of activity inhibition by the photothermal effect on human neurons as far as we know.

Human-Derived Cortical Neurospheroids Coupled to Passive, High-Density and 3D MEAs: A Valid Platform for Functional Tests.

With the advent of human-induced pluripotent stem cells (hiPSCs) and differentiation protocols, methods to create in-vitro human-derived neuronal networks have been proposed. Although monolayer cultures represent a valid model, adding three-dimensionality (3D) would make them more representative of an in-vivo environment. Thus, human-derived 3D structures are becoming increasingly used for in-vitro disease modeling. Achieving control over the final cell composition and investigating the exhibited electrophysiological activity is still a challenge. Thence, methodologies to create 3D structures with controlled cellular density and composition and platforms capable of measuring and characterizing the functional aspects of these samples are needed. Here, we propose a method to rapidly generate neurospheroids of human origin with control over cell composition that can be used for functional investigations. We show a characterization of the electrophysiological activity exhibited by the neurospheroids by using micro-electrode arrays (MEAs) with different types (i.e., passive, C-MOS, and 3D) and number of electrodes. Neurospheroids grown in free culture and transferred on MEAs exhibited functional activity that can be chemically and electrically modulated. Our results indicate that this model holds great potential for an in-depth study of signal transmission to drug screening and disease modeling and offers a platform for in-vitro functional testing.

Simultaneous recording of electrical and metabolic activity of cardiac cells in vitro using an organic charge modulated field effect transistor array.

In vitro electrogenic cells monitoring is an important objective in several scientific and technological fields, such as electrophysiology, pharmacology and brain machine interfaces, and can represent an interesting opportunity in other translational medicine applications. One of the key aspects of cellular cultures is the complexity of their behavior, due to the different kinds of bio-related signals, both chemical and electrical, that characterize these systems. In order to fully understand and exploit this extraordinary complexity, specific devices and tools are needed. However, at the moment this important scientific field is characterized by the lack of easy-to-use, low-cost devices for the sensing of multiple cellular parameters. To the aim of providing a simple and integrated approach for the study of in vitro electrogenic cultures, we present here a new solution for the monitoring of both the electrical and the metabolic cellular activity. In particular, we show here how a particular device called Micro Organic Charge Modulated Array (MOA) can be conveniently engineered and then used to simultaneously record the complete cell activity using the same device architecture. The system has been tested using primary cardiac rat myocytes and allowed to detect the metabolic and electrical variations thar occur upon the administration of different drugs. This first example could lay the basis for the development of a new generation of multi-sensing tools that can help to efficiently probe the multifaceted in vitro environment.

On the way back from 3D to 2D: Chitosan promotes adhesion and development of neuronal networks onto culture supports.

Most in vitro functional and morphological studies for developing nervous system have been performed using traditional monolayer cultures onto supports modified by extracellular matrix components or synthetic biopolymers. These biomolecules act as adhesion factors essential for neuronal growth and differentiation. In this study, the use of chitosan as adhesion factor was investigated. Primary rat neurons and neurons differentiated from human induced pluripotent stem cells were cultured onto chitosan and standard adhesion factors modified supports. The initiation, elongation and branching of neuritic processes, synaptogenesis and electrophysiological behavior were studied. The biopolymers affected neurites outgrowth in a time dependent manner; in particular, chitosan promoted neuronal polarity in both cell cultures. These results indicate chitosan as a valid adhesion factor alternative to the standard ones, with the advantage that it can be used both in 2D and 3D cultures, acting as a bridge between these in vitro models.

A new integrated system combining atomic force microscopy and micro-electrode array for measuring the mechanical properties of living cardiac myocytes.

In this paper we present a new experimental set-up which combines the surface characterization capabilities of atomic force microscopy at the sub-micrometer scale with non-invasive electrophysiological measurements obtained by using planar micro-electrode arrays. In order to show the potential of the combined measurements we studied the changes in cell topography and elastic properties of cardiac muscle cells as during the contraction-relaxation cycle. The onset of each beating cycle was precisely identified by the use of the extracellular potential signal, allowing us to combine nanomechanical measurements from multiple cardiomyocyte contractions in order to analyze the time-dependent variation of cell morphology and elasticity. Moreover, by estimating the elastic modulus at different indentation depths in a single location on the cell membrane, we observed a dynamic mechanical behavior that could be related to the underlying myofibrillar structure dynamics.

A self-adapting approach for the detection of bursts and network bursts in neuronal cultures.

Dissociated networks of neurons typically exhibit bursting behavior, whose features are strongly influenced by the age of the culture, by chemical/electrical stimulation or by environmental conditions. To help the experimenter in identifying the changes possibly induced by specific protocols, we developed a self-adapting method for detecting both bursts and network bursts from electrophysiological activity recorded by means of micro-electrode arrays. The algorithm is based on the computation of the logarithmic inter-spike interval histogram and automatically detects the best threshold to distinguish between inter- and intra-burst inter-spike intervals for each recording channel of the array. An analogous procedure is followed for the detection of network bursts, looking for sequences of closely spaced single-channel bursts. We tested our algorithm on recordings of spontaneous as well as chemically stimulated activity, comparing its performance to other methods available in the literature.

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.

A three-dimensional micro-electrode array for in-vitro neuronal interfacing.

OBJECTIVE: In this paper, we report on the development of an easy-to-fabricate three-dimensional Micro-Electrode Array (3D-MEA) specifically designed for brain-on-a-dish applications. APPROACH: The proposed device consists of pillar-shaped gold microelectrodes realized by electroplating directly on top of a standard MEA, making this approach highly versatile and convenient for batch fabrication. Moreover, with this simple technique, it is possible to obtain electrodes with a height of more than 100 µm onto different kind of substrates, ranging from glass to flexible plastic ones. MAIN RESULTS: This novel 3D-MEA structure has been validated with acute brain slices, successfully recording both epileptiform-like discharges (upon the administration of 4-AP), and electrically-evoked neuronal activity. The preliminary validation showed a substantial improvement in the signals amplitude with respect to both commercial and custom planar electrodes thanks to a better coupling offered by the peculiar shape of the three-dimensional electrodes. SIGNIFICANCE: Beside the versatility of the fabrication approach, which allows to obtain 3D MEA devices onto both rigid and flexible substrates, the reported validation showed how the pillar approach can outperform standard planar MEA recordings in terms of signal amplitude. Moreover, thanks to the possibility of obtaining multi-level 3D structures within the same device, the proposed fabrication technique offers an interesting and flexible approach for the development of a new family of electrophysiological tools for 3D in vitro electrophysiology, in particular for acute brain slices and 3D neuronal cultures for brain-on-a-dish applications.

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.

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.