Complex network topologies represent the necessary substrate to support complex brain functions. In this work, we reviewed in vitro neuronal networks coupled to Micro-Electrode Arrays (MEAs) as biological substrate. Networks of dissociated neurons developing in vitro and coupled to MEAs, represent a valid experimental model for studying the mechanisms governing the formation, organization and conservation of neuronal cell assemblies. In this review, we present some examples of the use of statistical Cluster Coefficients and Small World indices to infer topological rules underlying the dynamics exhibited by homogeneous and engineered neuronal networks.

Cerebral (or brain) organoids derived from human cells have enormous potential as physiologically relevant downscaled in vitro models of the human brain. In fact, these stem cell-derived neural aggregates resemble the three-dimensional (3D) cytoarchitectural arrangement of the brain overcoming not only the unrealistic somatic flatness but also the planar neuritic outgrowth of the two-dimensional (2D) in vitro cultures. Despite the growing use of cerebral organoids in scientific research, a more critical evaluation of their reliability and reproducibility in terms of cellular diversity, mature traits, and neuronal dynamics is still required. Specifically, a quantitative framework for generating and investigating these in vitro models of the human brain is lacking. To this end, the aim of this review is to inspire new computational and technology driven ideas for methodological improvements and novel applications of brain organoids. After an overview of the organoid generation protocols described in the literature, we review the computational models employed to assess their formation, organization and resource uptake. The experimental approaches currently provided to structurally and functionally characterize brain organoid networks for studying single neuron morphology and their connections at cellular and sub-cellular resolution are also discussed. Well-established techniques based on current/voltage clamp, optogenetics, calcium imaging, and Micro-Electrode Arrays (MEAs) are proposed for monitoring intra- and extra-cellular responses underlying neuronal dynamics and functional connections. Finally, we consider critical aspects of the established procedures and the physiological limitations of these models, suggesting how a complement of engineering tools could improve the current approaches and their applications.

Objective. Our goal is to re-introduce an optimized version of the partial correlation to infer structural connections from functional-effective ones in dissociated neuronal cultures coupled to microelectrode arrays. Approach. We first validate our partialization procedure on in silico networks, mimicking different experimental conditions (i.e., different connectivity degrees and number of nodes) and comparing the partial correlation’s performance with two gold-standard methods: cross-correlation and transfer entropy. Afterwards, to infer the structural connections in in vitro neuronal networks where the ground truth is unknown, we propose a thresholding heuristic approach. Then, to validate whether the partialization process correctly reconstructs macroscopic features of the network structure, we extract a modularity index from segregated in silico and in vitro models. Finally, as a case study, we apply our partialization procedure to analyze connectivity and topology on spontaneous developing and electrically stimulated in vitro cultures. Main results. In simulated networks, partial correlation outperforms cross-correlation and transfer entropy at low and medium connectivity degrees, not only in relatively small (60 nodes) but also in larger (120–240 nodes) assemblies. Furthermore, partial correlation correctly identifies interconnected neuronal sub-populations and allows one to derive network topology in in vitro cortical networks. Significance. Our results support the idea that partial correlation is a good method for connectivity studies and can be applied to derive topological and structural features of neuronal assemblies.

Objective . Functions ascribed to the hippocampal sub-regions for encoding episodic memories include the separation of activity patterns propagated from the entorhinal cortex (EC) into the dentate gyrus (DG) and pattern completion in CA3 region. Since a direct assessment of these functions is lacking at the level of specific axonal inputs, our goal is to directly measure the separation and completion of distinct axonal inputs in engineered pairs of hippocampal sub-regional circuits. Approach . We co-cultured EC-DG, DG-CA3, CA3-CA1 or CA1-EC neurons in a two-chamber PDMS device over a micro-electrode array (MEA60), inter-connected via distinct axons that grow through the micro-tunnels between the compartments. Taking advantage of the axonal accessibility, we quantified pattern separation and completion of the evoked activity transmitted through the tunnels from source into target well. Since pattern separation can be inferred when inputs are more correlated than outputs, we first compared the correlations among axonal inputs with those of target somata outputs. We then compared, in an analog approach, the distributions of correlation distances between rate patterns of the axonal inputs inside the tunnels with those of the somata outputs evoked in the target well. Finally, in a digital approach, we measured the spatial population distances between binary patterns of the same axonal inputs and somata outputs. Main results . We found the strongest separation of the propagated axonal inputs when EC was axonally connected to DG, with a decline in separation to CA3 and to CA1 for both rate and digital approaches. Furthermore, the digital approach showed stronger pattern completion in CA3, then CA1 and EC. Significance . To the best of our knowledge, these are the first direct measures of pattern separation and completion for axonal transmission to the somata target outputs at the rate and digital population levels in each of four stages of the EC-DG-CA3-CA1 circuit.

Nowadays, the use of in vitro reduced models of neuronal networks to investigate the interplay between structural-functional connectivity and the emerging collective dynamics is a widely accepted approach. In this respect, a relevant advance for this kind of studies has been given by the recent introduction of high-density large-scale Micro-Electrode Arrays (MEAs) which have favored the mapping of functional connections and the recordings of the neuronal electrical activity. Although several toolboxes have been implemented to characterize network dynamics and derive functional links, no specifically dedicated software for the management of huge amount of data and direct estimation of functional connectivity maps has been developed. TOOLCONNECT offers the implementation of up to date algorithms and a user-friendly Graphical User Interface (GUI) to analyze recorded data from large scale networks. It has been specifically conceived as a computationally efficient open-source software tailored to infer functional connectivity by analyzing the spike trains acquired from in vitro networks coupled to MEAs. In the current version, TOOLCONNECT implements correlation- (cross-correlation, partial-correlation) and information theory (joint entropy, transfer entropy) based core algorithms, as well as useful and practical add-ons to visualize functional connectivity graphs and extract some topological features. In this work, we present the software, its main features and capabilities together with some demonstrative applications on hippocampal recordings.

High-frequency electrical stimulation (tetanus) promotes global synaptic potentiation in dissociated cortical networks coupled to multi-electrode arrays (MEAs). Since little is known about the functional changes induced by this protocol, this work aims to investigate the statistical dependences between the time series (i.e. functional links) of the network nodes involved pre- and post-tetanus. Specifically, we first show a strong reshaping of the functional connections induced by the stimulation and possibly associated with the global plasticity. Then, we find that about 30% of the nodes linked before and after electrical perturbation show high-connectivity degree (⩾9 links), occupying a central role in the neuronal communication. Finally, we observe that these functional units drive the global network plasticity showing more synaptic potentiation than the other nodes involved in the connectivity reshaping.