Spiking Signatures of Spontaneous Activity Bursts in Hippocampal Cultures

Dense dissociated hippocampal cultures are known to generate spontaneous bursting electrical activity which can be recorded by multielectrode arrays. We have analyzed spatio-temporal profiles of the distribution of spikes in the bursts recorded after 2 weeks in vitro. We have found a statistically significant similarity between the spiking patterns in sequential bursting events, we refer to these spiking patterns as spiking signatures. Such spiking signatures may appear in different parts of the bursts, including the activation patterns – the first spike times in the bursts, and deactivation patterns – the last spike times in the bursts. Moreover, these patterns may display apparent time scaling, e.g., they may be replayed in the subsequent bursts at different speeds, while preserving the spiking order. We discuss how such properties of the bursts may be associated with the formation of repeatable signaling pathways in cultured networks in vitro

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

Functional characterization of human pluripotent stem cell-derived cortical networks differentiated on laminin-521 substrate : comparison to rat cortical cultures

Human pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.Peer reviewe

Burst analysis tool for developing neuronal networks exhibiting highly varying action potential dynamics

In this paper we propose a firing statistics based neuronal network burst detection algorithm for neuronal networks exhibiting highly variable action potential dynamics. Electrical activity of neuronal networks is generally analyzed by the occurrences of spikes and bursts both in time and space. Commonly accepted analysis tools employ burst detection algorithms based on predefined criteria. However, maturing neuronal networks, such as those originating from human embryonic stem cells (hESCs), exhibit highly variable network structure and time-varying dynamics. To explore the developing burst/spike activities of such networks, we propose a burst detection algorithm which utilizes the firing statistics based on interspike interval (ISI) histograms. Moreover, the algorithm calculates ISI thresholds for burst spikes as well as for pre-burst spikes and burst tails by evaluating the cumulative moving average (CMA) and skewness of the ISI histogram. Because of the adaptive nature of the proposed algorithm, its analysis power is not limited by the type of neuronal cell network at hand. We demonstrate the functionality of our algorithm with two different types of microelectrode array (MEA) data recorded from spontaneously active hESC-derived neuronal cell networks. The same data was also analyzed by two commonly employed burst detection algorithms and the differences in burst detection results are illustrated. The results demonstrate that our method is both adaptive to the firing statistics of the network and yields successful burst detection from the data. In conclusion, the proposed method is a potential tool for analyzing of hESC-derived neuronal cell networks and thus can be utilized in studies aiming to understand the development and functioning of human neuronal networks and as an analysis tool for in vitro drug screening and neurotoxicity assays

Effects of short-term plasticity in UP-DOWN cortical dynamics

Neuronal dynamics are strongly influenced by short-term plasticity (STP), that is, changes in synaptic efficacy that occur on a short (from milliseconds to seconds) time scale. Depending on the brain areas considered, STP can be dominated by short-term depression (STD), short-term facilitation (STF), or both mechanisms can coexist simultaneously. These two plasticity mechanisms modulate particular patterns of electrophysiological activity characterized by alternating UP and DOWN states. In this work, we develop a network model made up of excitatory and inhibitory multi-compartment neurons endowed with both mechanisms (STD and STF), spatially arranged to emulate the connectivity circuitry observed experimentally in the visual cortex. Our results reveal that both depression and facilitation can be involved in the switching process between different activity patterns, from an alternation of UP and DOWN states (for relatively low levels of depression and high levels of facilitation) to an asynchronous firing regime (for relatively high levels of depression and low levels of facilitation). For STD and STF, we identify the critical levels of depression and facilitation that push the network into the different regimes. Furthermore, we also find that these critical levels separate different growth rates of the mean synaptic conductances of the whole network with respect to the depression levels. This latter data is paramount to understanding how excitation and inhibition are organized to generate different brain activity regimes. Finally, after observing the changes in the trajectories of excitatory and inhibitory instantaneous firing rates near these critical boundaries, we identify dynamic patterns that shed light on the type of bifurcations that should arise in a rate model for this complex network"AG has been funded by Catalan Research Agency (AGAUR) grant 2017-SGR-1049, by the Spanish Ministerio de Ciencia e Innovación grant PID2021-122954-I00 and by the Spanish State Research Agency through the Severo Ochoa and María de Maeztu Program for Centers and Units of Excellence in R&D (CEX2020-001084-M)."Peer ReviewedPostprint (published version

A novel Three-Dimensional Micro-Electrode Array for in-vitro electrophysiological applications

Microelectrode arrays (MEAs) represent a powerful and popular tool to study in vitro neuronal networks and acute brain slices. The research standard for MEAs is planar or 2D-MEAs, which have been in existence for over 30 years and used for extracellular recording and stimulation from cultured neuronal cells and tissue slices. However, planar MEAs suffer from rapid data attenuation in the z-direction when stimulating/recording from 3D in-vitro neuronal cultures or brain slices. The existing proposed 3D in-vitro neuronal models allow to record the electrophysiological activity of the 3D network only from the bottom layer (i.e. the one directly coupled to the planar MEAs). Thus, to further develop and optimize such 3D neuronal network systems and to study and understand how the 3D neuronal network dynamics changes in different layers of the 3D structure, new three-dimensional microelectrodes arrays (3D-MEAs) are required. Early attempts in this field resulted in interesting integrated approaches toward protruding or spiked 3D-MEAs. Although these first prototypes could be successfully employed with brain slices, the limited heights of the electrodes (up to max 70 \u3bcm) and the peculiar shape of the recording areas made them not an ideal solution for 3D neuronal cultures. Moreover, a convenient and versatile method for the fabrication of multilevel 3D microelectrode arrays has yet to be obtained, due to the usually complicated and expensive designs and a lack of a full compatibility with standard MEAs both in terms of materials and recording area dimensions. To overcome the afore-mentioned challenges, in this work, I present the design, microfabrication, and characterization of a new 3D-MEA composed of pillar-shaped gold 3D structures with heights of more than 100 \u3bcm that can be used, in principle, on every kind of MEA, both custom-made and commercial. I successfully demonstrate the capability and ability of such 3D-MEA to record electrophysiological spontaneous activity from 3D engineered in-vitro neuronal networks and both 4-AP-induced epileptiform-like and electrically-evoked activity from mouse acute brain slices. I also demonstrate how the developed 3D-MEA allows better recording and stimulating conditions while interfacing with acute brain slices as compared to planar electrode arrays and previously reported 3D MEA technologies

Multifunctional nanostructures for intracellular delivery and sensing in electrogenic cells

Biological studies on in vitro cell cultures are of fundamental importance for investigating cell response to external stimuli, such drugs for specific treatments, or for studying communication between cells. In the electrophysiology field, multielectrode array devices (MEA) are the gold standard for the study of large ensambles of electrogenic cells. Thus, their improvement is a central topic nowadays in neuroscience and cardiology [1]. In the last decades, thanks to the adoption of nanotechnologies, the study of physiological and pathological conditions of electro-active cells in culture have becomes increasingly accurate[2], allowing for monitoring action potentials from many cells simultaneously. In fact, nanoscale biomaterials were able to overcome the limitations of previous technologies, paving the way to the development of platforms for interfacing the electrogenic cells at unprecedented spatiotemporal scales. These devices, together with microfluidics, are starting to be used for drug screening and pharmaceutical drug development since they represent a powerful tool for monitoring cell response when cultures are stimulated by target compounds. Many pharmaceutical agents, however, including various large molecules (enzymes, proteins, antibodies) and even drug-loaded pharmaceutical nanocarriers, need to be delivered intracellularly to exercise their therapeutic action inside the cytoplasm[3]. Nanoscale electrodes offer individual cell access and non-destructive poration of the cellular membrane enabling high capability in the delivery of biomolecules. Among all the techniques, electroporation have proven encouraging potential as alternative to the carrier mediated methods for molecular delivery into cultured cells[4]. In this regard, different groups [5][6][7] exploited the integration of nanostructures with delivering capabilities with single-cell specificity and high throughput in biosensing platforms. These efforts provided powerful tools for advancing applications in therapeutics, diagnostics, and drug discovery, in order to reach an efficient and localized delivery on a chip. Despite these new tactics, there is still a critical need for the development of a functional approach that combines recording capabilities of nanostructured biosensors with intracellular delivery. The device should provide for tight contact between cells and electrode so as to enable highly localized delivery and optimal recording of action potentials in order to attain a high degree of prediction for the disease modeling and drug discovery. This \u201con-chip\u201d approach will help to gain deeper insight in several bio-related studies and analyses, providing a comprehensive knowledge of the entire cellular dynamics when selectively stimulated by the desired bio-molecules. In the first part of this dissertation, a solution will be proposed in order to fill this gap and respond to this need in the biology field. In the first chapter, I will describe briefly the principles of action potentials and how neurons and cardiomyocyte are composed, together with the development of electrophysiology and the advent of multielectrode arrays. In the second chapter, more details about fabrication and cell-electrode system modelling will be explained. In the same chapter, I will explore the development of multielectrode arrays up to the present days, along with the advent of nanotechnologies and the related techniques for improving the previous platforms. The different cell poration techniques will be described in order to reach the best recording capabilities without damaging cells. Electroporation, optoporation and spontaneous poration will be presented and the chosen technique for our application (electroporation) will be reviewed more in detail. In the third chapter, different methodologies for intracellular delivery will be explained, focusing also on the electroporation technique. A small paragraph about the integration of these techniques on chip will be inserted to illustrate the state of the art of these devices. The fourth chapter will explicate in details the Microfluidic multielectrode array idea, the approach used in order to fabricate this novel platform from scratch, the experiments carried out to verify its capabilities and the associated results. In the last paragraph, I will discuss how the proposed platform could became suitable for the day to day uses in research activity by employing nanoporous materials. In fact, big efforts are carried out in order to find appropriate metamaterials as substitutes of the 3D counterparts so as to decrease the cost of device manufacturing that makes them unfitting with research activity. As a novel electrode material, nanoporous metals possess unique properties, such as a low fabrication cost, high plasmonic enhancement and large surface-volume ratio[8]. Nanoporous gold behaves like a metamaterial whose effective dielectric response can be tuned accordingly to the wanted use. These properties make the material suitable for multiple biosensing application, from a high-performance and reliable SERS (surface enhanced raman scattering) substrate [9] to an electrode in CMOS MEAs capable of intracellular recordings[10]. All these properties were explored in the last years, but it could be interesting to further study if the characteristics of this material could make it a good photoelectrical modulating material for eliciting electrogenic cells firing activity. In this way, this technology could be in principle easily implemented on commercial CMOS devices, consenting stimulation and recording at single cell level with high-resolution sensors, opening the way to new methodologies for studying electrogenic cells and tissues. Electrical stimulation of excitable cells is the basis for many implantable devices in cardiac treatment and in neurological studies for treating debilitating neurological syndromes. In order to make the technique less invasive, optical stimulation was widely investigated [11]. The non-genetic photostimulation is starting to make its way in the field since it allows to avoid changing the biological framework by using transient thermal or electrochemical outputs from synthetic materials attached to the target cells[12]. If stimulated with impinging light these materials could inject free charges into the solution resulting in an ionic current at the interface able to eliciting of neurons[13] or cardiomyocyte action potentials. Plasmonic porous materials have all the suitable properties to be considered as an appealing tools for charge injection and consequently for stimulation of electrically active cells [14]. Thus, the second part of this dissertation will exploit the capabilities of these plasmonic metamaterials, placing particular emphasis on the possibility of photoelectrochemical modulation. In particular, in the fifth and last chapter I will describe all the properties and application of the porous material and the mechanism of photoemission. In the experimental paragraphs, the free charge photoemission properties of porous gold will be explored together with plasmonic non-genetic photostimulation of the cardiac cells on commercial CMOS MEAs