Functional Inhibitory Connections Modulate the Electrophysiological Activity Patterns of Cortical-Hippocampal Ensembles.

The brain is a complex organ composed of billions of neurons connected through excitatory and inhibitory synapses. Its structure reveals a modular topological organization, where neurons are arranged in interconnected assemblies. The generated patterns of electrophysiological activity are shaped by two main factors: network heterogeneity and the topological properties of the underlying connectivity that strongly push the dynamics toward different brain-states. In this work, we exploited an innovative polymeric structure coupled to Micro-Electrode Arrays (MEAs) to recreate in vitro heterogeneous interconnected (modular) neuronal networks made up of cortical and hippocampal neurons. We investigated the propagation of spike sequences between the two interconnected subpopulations during the networks' development, correlating functional and structural connectivity to dynamics. The simultaneous presence of two neuronal types shaped the features of the functional connections (excitation vs. inhibition), orchestrating the emerging patterns of electrophysiological activity. In particular, we found that hippocampal neurons mostly project inhibitory connections toward the cortical counterpart modulating the temporal scale of the population events (network bursts). In contrast, cortical neurons establish a larger amount of intrapopulation connections. Moreover, we proved topological properties such as small-worldness, degree distribution, and modularity of neuronal assemblies were favored by the physical environment where networks developed and matured.

Electrophysiological features of cortical 3D networks are deeply modulated by scaffold properties.

Three-dimensionality (3D) was proven essential for developing reliable models for different anatomical compartments and many diseases. However, the neuronal compartment still poses a great challenge as we still do not understand precisely how the brain computes information and how the complex chain of neuronal events can generate conscious behavior. Therefore, a comprehensive model of neuronal tissue has not yet been found. The present work was conceived in this framework: we aimed to contribute to what must be a collective effort by filling in some information on possible 3D strategies to pursue. We compared directly different kinds of scaffolds (i.e., PDMS sponges, thermally crosslinked hydrogels, and glass microbeads) in their effect on neuronal network activity recorded using micro-electrode arrays. While the overall rate of spiking activity remained consistent, the type of scaffold had a notable impact on bursting dynamics. The frequency, density of bursts, and occurrence of random spikes were all affected. The examination of inter-burst intervals revealed distinct burst generation patterns unique to different scaffold types. Network burst propagation unveiled divergent trends among configurations. Notably, it showed the most differences, underlying that functional variations may arise from a different 3D spatial organization. This evidence suggests that not all 3D neuronal constructs can sustain the same level of richness of activity. Furthermore, we commented on the reproducibility, efficacy, and scalability of the methods, where the beads still offer superior performances. By comparing different 3D scaffolds, our results move toward understanding the best strategies to develop functional 3D neuronal units for reliable pre-clinical studies.

Cortical, striatal, and thalamic populations self-organize into a functionally connected circuit with long-term memory properties.

The human brain is a complex organ with an intricate neuronal connectivity and diverse functional regions. Neurological disorders often disrupt the delicate balance among these anatomical compartments, resulting in severe impairments. The available therapeutic options constitute an incomplete solution as many patients respond partially, highlighting the need for continued research into causes and treatments. Bottom-up approaches, like in vitro models, offer insights into brain functions as they recreate the in vivo microenvironment that allows studying how specific features affect physiological and pathological conditions. In this work, we engineered the cortical-striatal-thalamic (CST) circuit, involved in many brain functions such as action initiation and selection, using a three-compartment polymeric device. We characterized the emerging spontaneous electrophysiological activity by using Micro-Electrode Arrays (MEAs). Cortical neurons exhibited complex bursting activity, which influenced the entire circuit. Striatal and thalamic neurons displayed predominantly tonic firing when isolated, while interconnections with the cortex synchronized and organized their neuronal activity, highlighting the cortical pivotal role in bursting activity and information processing. The CST circuit demonstrated self-organization abilities and displayed high entropy values, indicative of dynamic richness and information encoding potential. Furthermore, we proved the CST's involvement in learning and memory. Our CST model provides a platform for further exploration into brain circuitry and potential therapeutic interventions, underscoring the necessity of realistic in vitro models to fully understand neurological diseases' pathophysiology.

Investigating the interplay between segregation and integration in developing cortical assemblies.

Introduction: The human brain is an intricate structure composed of interconnected modular networks, whose organization is known to balance the principles of segregation and integration, enabling rapid information exchange and the generation of coherent brain states. Segregation involves the specialization of brain regions for specific tasks, while integration facilitates communication among these regions, allowing for efficient information flow. Several factors influence this balance, including maturation, aging, and the insurgence of neurological disorders like epilepsy, stroke, or cancer. To gain insights into information processing and connectivity recovery, we devised a controllable in vitro model to mimic and investigate the effects of different segregation and integration ratios over time. Methods: We designed a cross-shaped polymeric mask to initially establish four independent sub-populations of cortical neurons and analyzed how the timing of its removal affected network development. We evaluated the morphological and functional features of the networks from 11 to 18 days in vitro (DIVs) with immunofluorescence techniques and micro-electrode arrays (MEAs). Results: The removal of the mask at different developmental stages of the network lead to strong variations in the degree of intercommunication among the four assemblies (altering the segregation/integration balance), impacting firing and bursting parameters. Early removal (after 5 DIVs) resulted in networks with a level of integration similar to homogeneous controls (without physical constraints). In contrast, late removal (after 15 DIVs) hindered the formation of strong inter-compartment connectivity, leading to more clustered and segregated assemblies. Discussion: A critical balance between segregation and integration was observed when the mask was removed at DIV 10, allowing for the formation of a strong connectivity among the still-separated compartments, thus demonstrating the existence of a time window in network development in which it is possible to achieve a balance between segregation and integration.

How 3D scaffolds with different mechanical properties affect the activity of neuronal networks in in vitro models.

Three-dimensionality has been proven extensively to be critical in the development of a reliable model for different anatomical compartments and for many diseases. Currently, we can produce implantable structures that help in the regeneration of different tissues such as bone and heart. Different is the situation when we consider the neuronal compartment. As it is still difficult to understand exactly how the brain computes, to conceive how the complex chain of neuronal events can generate conscious behavior, a comprehensive and workable model of neuronal tissue still has to be found. In this perspective, in the present work, we developed and compared different 3D scaffolds to understand the effects produced by the mechanical and material properties of four different scaffolds on a 3D neuronal network. To help in preclinical testing procedure, the scalability and ease-of-use of the different approaches were also taken into consideration.Clinical Relevance- By comparing different 3D scaffolds for the creation of neuronal constructs, the results in this paper move towards understanding the best strategy to develop functional 3D neuronal units for reliable pre-clinical studies.

Modularity and neuronal heterogeneity: Two properties that influence in vitro neuropharmacological experiments.

Introduction: The goal of this work is to prove the relevance of the experimental model (in vitro neuronal networks in this study) when drug-delivery testing is performed. Methods: We used dissociated cortical and hippocampal neurons coupled to Micro-Electrode Arrays (MEAs) arranged in different configurations characterized by modularity (i.e., the presence of interconnected sub-networks) and heterogeneity (i.e., the co-existence of neurons coming from brain districts). We delivered increasing concentrations of bicuculline (BIC), a neuromodulator acting on the GABAergic system, and we extracted the IC50 values (i.e., the effective concentration yielding a reduction in the response by 50%) of the mean firing rate for each configuration. Results: We found significant lower values of the IC50 computed for modular cortical-hippocampal ensembles than isolated cortical or hippocampal ones. Discussion: Although tested with a specific neuromodulator, this work aims at proving the relevance of ad hoc experimental models to perform neuropharmacological experiments to avoid errors of overestimation/underestimation leading to biased information in the characterization of the effects of a drug on neuronal networks.

Developmental conditions and culture medium influence the neuromodulated response of in vitro cortical networks.

Goal of this work is to show how the developmental conditions of in vitro neuronal networks influence the effect of drug delivery. The proposed experimental neuronal model consists of dissociated cortical neurons plated to Micro-Electrode Arrays (MEAs) and grown according to different conditions (i.e., by varying both the adopted culture medium and the number of days needed to let the network grow before performing the chemical modulation). We delivered rising amount of bicuculline (BIC), a competitive antagonist of GABAA receptors, and we computed the firing rate dose-response curve for each culture. We found that networks matured in BrainPhys for 18 days in vitro exhibited a decreasing firing trend as a function of the BIC concentration, quantified by an average IC50 (i.e., half maximal inhibitory concentration) of 4.64 ± 4.02 µM. On the other hand, both cultures grown in the same medium for 11 days, and ones matured in Neurobasal for 18 days displayed an increasing firing rate when rising amounts of BIC were delivered, characterized by average EC50 values (i.e., half maximal excitatory concentration) of 0.24 ± 0.05 µM and 0.59 ± 0.46 µM, respectively.Clinical Relevance- This research proves the relevance of the experimental factors that can influence the network development as key variables when developing a neuronal model to conduct drug delivery in vitro, simulating the in vivo environment. Our findings suggest that not considering the consequences of the chosen growing conditions when performing in vitro pharmacological studies could lead to incomplete predictions of the chemically induced alterations.

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.

Lack of Epileptogenic Effects of the Creatine Precursor Guanidinoacetic Acid on Neuronal Cultures In Vitro.

The creatine precursor Guanidinoacetic Acid (GAA) accumulates in the genetic deficiency of the GuanidinoAcetate Methyl Transferase (GAMT) enzyme and it is believed to cause the seizures that often occur in this condition. However, evidence that it is indeed epileptogenic is scarce and we previously found that it does not cause neuronal hyperexcitation in in vitro brain slices. Here, we used Micro-Electrode Arrays (MEAs) to further investigate the electrophysiological effects of its acute and chronic administration in the networks of cultured neurons, either neocortical or hippocampal. We found that: (1) GAA at the 1 µM concentration, comparable to its concentration in normal cerebrospinal fluid, does not modify any of the parameters we investigated in either neuronal type; (2) at the 10 µM concentration, very similar to that found in the GAMT deficiency, it did not affect any of the parameters we tested except the bursting rate of neocortical networks and the burst duration of hippocampal networks, both of which were decreased, a change pointing in a direction opposite to epileptogenesis; (3) at the very high and unphysiological 100 µM concentration, it caused a decrease in all parameters, a change that again goes in the direction opposite to epileptogenesis. Our results confirm that GAA is not epileptogenic.

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.

AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

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.

Three-dimensionality shapes the dynamics of cortical interconnected to hippocampal networks.

OBJECTIVE: The goal of this work is to develop and characterize an innovative experimental framework to design interconnected (i.e. modular) heterogeneous (cortical-hippocampal) neuronal cultures with a three-dimensional (3D) connectivity and to record their electrophysiological activity using micro-electrode arrays (MEAs). APPROACH: A two-compartment polymeric mask for the segregation of different neuronal populations (cortex and hippocampus) was coupled to the MEA surface. Glass microbeads were used as a scaffold to mimic the 3D brain micro-architecture. MAIN RESULTS: We built a fully functional heterogeneous 3D neuronal network. From an electrophysiological point of view, we found that the heterogeneity induces a global increase of the activity rate, while the 3D connectivity modulates the duration and the organization of the bursting activity. SIGNIFICANCE: In vivo, studies of network dynamics and interactions between neuronal populations are often time-consuming, low-throughput, complex, and suffer from reproducibility. On the other hand, most of the commonly used in vitro brain models are too simplified and thus far from the in vivo situation. The achieved results demonstrate the feasibility to build a more realistic and controllable experimental in vitro model of interconnected brain regions on-a-chip whose applications may have impacts on the study of neurological disorders that impair the connectivity between brain areas (e.g. Parkinson disease).

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.

Simultaneous AFM Investigation of the Single Cardiomyocyte Electro-Chemo-Mechanics During Excitation-Contraction Coupling.

The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.

From MEAs to MOAs: The Next Generation of Bioelectronic Interfaces for Neuronal Cultures.

Since their introduction in the early 1970s, microelectrode arrays (MEAs) have been dominating the electrophysiology market thanks to their reliability, extreme robustness, and usability. Over the past 40 years, silicon technology has also played a role in the advancement of the field, and CMOS-based in vitro and in vivo systems are now able to achieve unprecedented spatial resolutions, giving the possibility to unveil hidden behavior of cellular aggregates down to the subcellular level. However, both the MEAs and silicon-based electronic devices present unavoidable problems such as their expensiveness, the usual rigidity of the employed materials, and the need of an (usually bulky) external reference electrode. Possible interesting alternatives to these incredibly useful devices unexpectedly lie in the field of organic electronics, thanks to the fast-growing pace of improvement that this discipline has undergone in the last 10-15 years. In this chapter, a particular organic transistor called organic charge-modulated field-effect transistor (OCMFET) will be presented as a promising bio-electronic interface, and a complete description of its employment as a detector of cellular electrical activity and as an ultrasensitive pH sensor will be provided, together with the discussion about the possibility of using such a device as an innovative multisensing tool for both electrophysiology and (neuro)pharmacology.

Exosomes From Astrocyte Processes: Signaling to Neurons.

It is widely recognized that extracellular vesicles subserve non-classical signal transmission in the central nervous system. Here we assess if the astrocyte processes, that are recognized to play crucial roles in intercellular communication at the synapses and in neuron-astrocyte networks, could convey messages through extracellular vesicles. Our findings indicate, for the first time that freshly isolated astrocyte processes prepared from adult rat cerebral cortex, can indeed participate to signal transmission in central nervous system by releasing exosomes that by volume transmission might target near or long-distance sites. It is noteworthy that the exosomes released from the astrocyte processes proved ability to selectively target neurons. The astrocyte-derived exosomes were proven positive for neuroglobin, a protein functioning as neuroprotectant against cell insult; the possibility that exosomes might transfer neuroglobin to neurons would add a mechanism to the potential astrocytic neuroprotectant activity. Notably, the exosomes released from the processes of astrocytes maintained markers, which prove their parental astrocytic origin. This potentially allows the assessment of the cellular origin of exosomes that might be recovered from body fluids.

A Neuromorphic Prosthesis to Restore Communication in Neuronal Networks.

Recent advances in bioelectronics and neural engineering allowed the development of brain machine interfaces and neuroprostheses, capable of facilitating or recovering functionality in people with neurological disability. To realize energy-efficient and real-time capable devices, neuromorphic computing systems are envisaged as the core of next-generation systems for brain repair. We demonstrate here a real-time hardware neuromorphic prosthesis to restore bidirectional interactions between two neuronal populations, even when one is damaged or missing. We used in vitro modular cell cultures to mimic the mutual interaction between neuronal assemblies and created a focal lesion to functionally disconnect the two populations. Then, we employed our neuromorphic prosthesis for bidirectional bridging to artificially reconnect two disconnected neuronal modules and for hybrid bidirectional bridging to replace the activity of one module with a real-time hardware neuromorphic Spiking Neural Network. Our neuroprosthetic system opens avenues for the exploitation of neuromorphic-based devices in bioelectrical therapeutics for health care.

Acoustic stimulation can induce a selective neural network response mediated by piezoelectric nanoparticles.

OBJECTIVE: We aim to develop a novel non-invasive or minimally invasive method for neural stimulation to be applied in the study and treatment of brain (dys)functions and neurological disorders. APPROACH: We investigate the electrophysiological response of in vitro neuronal networks when subjected to low-intensity pulsed acoustic stimulation, mediated by piezoelectric nanoparticles adsorbed on the neuronal membrane. MAIN RESULTS: We show that the presence of piezoelectric barium titanate nanoparticles induces, in a reproducible way, an increase in network activity when excited by stationary ultrasound waves in the MHz regime. Such a response can be fully recovered when switching the ultrasound pulse off, depending on the generated pressure field amplitude, whilst it is insensitive to the duration of the ultrasound pulse in the range 0.5 s-1.5 s. We demonstrate that the presence of piezoelectric nanoparticles is necessary, and when applying the same acoustic stimulation to neuronal cultures without nanoparticles or with non-piezoelectric nanoparticles with the same size distribution, no network response is observed. SIGNIFICANCE: We believe that our results open up an extremely interesting approach when coupled with suitable functionalization strategies of the nanoparticles in order to address specific neurons and/or brain areas and applied in vivo, thus enabling remote, non-invasive, and highly selective modulation of the activity of neuronal subpopulations of the central nervous system of mammalians.

An automated microdrop delivery system for neuronal network patterning on microelectrode arrays.

The aim of this work is to present a new technique for defining interconnected sub-populations of cultured neurons on microelectrode arrays (MEAs). An automated microdrop delivery technique allows to design and realize spatially distributed neuronal ensembles by depositing sub-nanoliter volumes of adhesion molecules in which neurons grow and develop. Electrophysiological tests demonstrate that functionally interconnected clusters are obtained and experimental results (both spontaneous and stimulus evoked activity recordings) attesting the feasibility of the proposed approach are presented. By means of the automated system, different and specific architectures can be easily designed and functionally studied. In the presented system the speed of drop deposition is about 30 drops/min; the mean diameter is 147 microm; typical cell survival time is 4-5 weeks. By changing drop size and spacing, investigations about how the network dynamics is related to the network structure can be systematically carried out.