Michela Chiappalone

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

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

SELF-ORGANIZATION AND NEURONAL AVALANCHES IN NETWORKS OF DISSOCIATED CORTICAL NEURONS

Dissociated cortical neurons from rat embryos cultured onto micro-electrode arrays exhibit characteristic patterns of electrophysiological activity, ranging from isolated spikes in the first days of development to highly synchronized bursts after 3-4 weeks in vitro. In this work we analyzed these features by considering the approach proposed by the self-organized criticality theory: we found that networks of dissociated cortical neurons also generate spontaneous events of spreading activity, previously observed in cortical slices, in the form of neuronal avalanches. Choosing an appropriate time scale of observation to detect such neuronal avalanches, we studied the dynamics by considering the spontaneous activity during acute recordings in mature cultures and following the development of the network. We observed different behaviors, i.e. sub-critical, critical or super-critical distributions of avalanche sizes and durations, depending on both the age and the development of cultures. In order to clarify this variability, neuronal avalanches were correlated with other statistical parameters describing the global activity of the network. Criticality was found in correspondence to medium synchronization among bursts and high ratio between bursting and spiking activity. Then, the action of specific drugs affecting global bursting dynamics (i.e. acetylcholine and bicuculline) was investigated to confirm the correlation between criticality and regulated balance between synchronization and variability in the bursting activity. Finally, a computational model of neuronal network was developed in order to interpret the experimental results and understand which parameters (e.g. connectivity, excitability) influence the distribution of avalanches. In summary, cortical neurons preserve their capability to self-organize in an effective network even when dissociated and cultured in vitro. The distribution of avalanche features seems to be critical in those cultures displaying medium synchronization among bursts and poor random spiking activity, as confirmed by chemical manipulation experiments and modeling studies.

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.

Importance of Shank3 Protein in Regulating Metabotropic Glutamate Receptor 5 (mGluR5) Expression and Signaling at Synapses

Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

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.

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 transparent organic transistor structure for bidirectional stimulation and recording of primary neurons

Real-time stimulation and recording of neural cell bioelectrical activity could provide an unprecedented insight in understanding the functions of the nervous system, and it is crucial for developing advanced in vitro drug screening approaches. Among organic materials, suitable candidates for cell interfacing can be found that combine long-term biocompatibility and mechanical flexibility. Here, we report on transparent organic cell stimulating and sensing transistors (O-CSTs), which provide bidirectional stimulation and recording of primary neurons. We demonstrate that the device enables depolarization and hyperpolarization of the primary neuron membrane potential. The transparency of the device also allows the optical imaging of the modulation of the neuron bioelectrical activity. The maximal amplitude-to-noise ratio of the extracellular recording achieved by the O-CST device exceeds that of a microelectrode array system on the same neuronal preparation by a factor of 16. Our organic cell stimulating and sensing device paves the way to a new generation of devices for stimulation, manipulation and recording of cell bioelectrical activity in vitro and in vivo.

Network plasticity in cortical assemblies.

To investigate distributed synaptic plasticity at the cell assembly level, we used dissociated cortical networks from embryonic rats grown on grids of 60 extracellular substrate‐embedded electrodes (micro‐electrode arrays). We developed a set of experimental plasticity protocols based on the pairing of tetanic bursts (20 Hz) with low‐frequency stimuli (≤ 1 Hz), delivered through two separate channels of the array (i.e. associative tetanic stimulation). We tested our protocols on a large data set of 26 stable cultures, selected on the basis of both their initial level of spontaneous firing and the capability of low‐frequency test stimuli to evoke spikes. Our main results are summarized as follows: (i) low‐frequency stimuli produce neither short‐ nor long‐term changes in the evoked response of the network; (ii) associative tetanic stimulation is able to induce plasticity in terms of a significant increase or decrease of the evoked activity in the whole network; (iii) the amount of change (i.e. increase or decrease of the evoked firing) strongly depends on the specific features of the applied protocols; and (iv) the potentiation induced by a specific associative protocol can last several hours. The results obtained demonstrate that large in vitro cortical assemblies display long‐term network potentiation, a mechanism considered to be involved in the memory formation at cellular level. This pilot study could represent a relevant step towards understanding plastic properties at the neuronal population level.

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.