Microfluidic Bi-Layer Platform to Study Functional Interaction between Co-Cultured Neural Networks with Unidirectional Synaptic Connectivity.

The complex synaptic connectivity architecture of neuronal networks underlies cognition and brain function. However, studying the spiking activity propagation and processing in heterogeneous networks in vivo poses significant challenges. In this study, we present a novel two-layer PDMS chip that facilitates the culturing and examination of the functional interaction of two interconnected neural networks. We utilized cultures of hippocampal neurons grown in a two-chamber microfluidic chip combined with a microelectrode array. The asymmetric configuration of the microchannels between the chambers ensured the growth of axons predominantly in one direction from the Source chamber to the Target chamber, forming two neuronal networks with unidirectional synaptic connectivity. We showed that the local application of tetrodotoxin (TTX) to the Source network did not alter the spiking rate in the Target network. The results indicate that stable network activity in the Target network was maintained for at least 1-3 h after TTX application, demonstrating the feasibility of local chemical activity modulation and the influence of electrical activity from one network on the other. Additionally, suppression of synaptic activity in the Source network by the application of CPP and CNQX reorganized spatio-temporal characteristics of spontaneous and stimulus-evoked spiking activity in the Target network. The proposed methodology and results provide a more in-depth examination of the network-level functional interaction between neural circuits with heterogeneous synaptic connectivity.

Experimental Platform to Study Spiking Pattern Propagation in Modular Networks In Vitro.

The structured organization of connectivity in neural networks is associated with highly efficient information propagation and processing in the brain, in contrast with disordered homogeneous network architectures. Using microfluidic methods, we engineered modular networks of cultures using dissociated cells with unidirectional synaptic connections formed by asymmetric microchannels. The complexity of the microchannel geometry defined the strength of the synaptic connectivity and the properties of spiking activity propagation. In this study, we developed an experimental platform to study the effects of synaptic plasticity on a network level with predefined locations of unidirectionally connected cellular assemblies using multisite extracellular electrophysiology.

Functional Scaffolding for Brain Implants: Engineered Neuronal Network by Microfabrication and iPSC Technology.

Neuroengineering methods can be effectively used in the design of new approaches to treat central nervous system and brain injury caused by neurotrauma, ischemia, or neurodegenerative disorders. During the last decade, significant results were achieved in the field of implant (scaffold) development using various biocompatible and biodegradable materials carrying neuronal cells for implantation into the injury site of the brain to repair its function. Neurons derived from animal or human induced pluripotent stem (iPS) cells are expected to be an ideal cell source, and induction methods for specific cell types have been actively studied to improve efficacy and specificity. A critical goal of neuro-regeneration is structural and functional restoration of the injury site. The target treatment area has heterogeneous and complex network topology with various types of cells that need to be restored with similar neuronal network structure to recover correct functionality. However, current scaffold-based technology for brain implants operates with homogeneous neuronal cell distribution, which limits recovery in the damaged area of the brain and prevents a return to fully functional biological tissue. In this study, we present a neuroengineering concept for designing a neural circuit with a pre-defined unidirectional network architecture that provides a balance of excitation/inhibition in the scaffold to form tissue similar to that in the injured area using various types of iPS cells. Such tissue will mimic the surrounding niche in the injured site and will morphologically and topologically integrate into the brain, recovering lost function.

Closed-Loop Systems and In Vitro Neuronal Cultures: Overview and Applications.

One of the main limitations preventing the realization of a successful dialogue between the brain and a putative enabling device is the intricacy of brain signals. In this perspective, closed-loop in vitro systems can be used to investigate the interactions between a network of neurons and an external system, such as an interacting environment or an artificial device. In this chapter, we provide an overview of closed-loop in vitro systems, which have been developed for investigating potential neuroprosthetic applications. In particular, we first explore how to modify or set a target dynamical behavior in a network of neurons. We then analyze the behavior of in vitro systems connected to artificial devices, such as robots. Finally, we provide an overview of biological neuronal networks interacting with artificial neuronal networks, a configuration currently offering a promising solution for clinical applications.

Theta rhythm-like bidirectional cycling dynamics of living neuronal networks in vitro.

The phenomena of synchronization, rhythmogenesis and coherence observed in brain networks are believed to be a dynamic substrate for cognitive functions such as learning and memory. However, researchers are still debating whether the rhythmic activity emerges from the network morphology that developed during neurogenesis or as a result of neuronal dynamics achieved under certain conditions. In the present study, we observed self-organized spiking activity that converged to long, complex and rhythmically repeated superbursts in neural networks formed by mature hippocampal cultures with a high cellular density. The superburst lasted for tens of seconds and consisted of hundreds of short (50-100 ms) small bursts with a high spiking rate of 139.0 ± 78.6 Hz that is associated with high-frequency oscillations in the hippocampus. In turn, the bursting frequency represents a theta rhythm (11.2 ± 1.5 Hz). The distribution of spikes within the bursts was non-random, representing a set of well-defined spatio-temporal base patterns or motifs. The long superburst was classified into two types. Each type was associated with a unique direction of spike propagation and, hence, was encoded by a binary sequence with random switching between the two "functional" states. The precisely structured bidirectional rhythmic activity that developed in self-organizing cultured networks was quite similar to the activity observed in the in vivo experiments.

Design of Cultured Neuron Networks in vitro with Predefined Connectivity Using Asymmetric Microfluidic Channels.

The architecture of neuron connectivity in brain networks is one of the basic mechanisms by which to organize and sustain a particular function of the brain circuitry. There are areas of the brain composed of well-organized layers of neurons connected by unidirectional synaptic connections (e.g., cortex, hippocampus). Re-engineering of the neural circuits with such a heterogeneous network structure in culture may uncover basic mechanisms of emergent information functions of these circuits. In this study, we present such a model designed with two subpopulations of primary hippocampal neurons (E18) with directed connectivity grown in a microfluidic device with asymmetric channels. We analysed and compared neurite growth in the microchannels with various shapes that promoted growth dominantly in one direction. We found an optimal geometric shape features of the microchannels in which the axons coupled two chambers with the neurons. The axons grew in the promoted direction and formed predefined connections during the first 6 days in vitro (DIV). The microfluidic devices were coupled with microelectrode arrays (MEAs) to confirm unidirectional spiking pattern propagation through the microchannels between two compartments. We found that, during culture development, the defined morphological and functional connectivity formed and was maintained for up to 25 DIV.

Selectivity of stimulus induced responses in cultured hippocampal networks on microelectrode arrays.

Sensory information can be encoded using the average firing rate and spike occurrence times in neuronal network responses to external stimuli. Decoding or retrieving stimulus characteristics from the response pattern generally implies that the corresponding neural network has a selective response to various input signals. The role of various spiking activity characteristics (e.g., spike rate and precise spike timing) for basic information processing was widely investigated on the level of neural populations but gave inconsistent evidence for particular mechanisms. Multisite electrophysiology of cultured neural networks grown on microelectrode arrays is a recently developed tool and currently an active research area. In this study, we analyzed the stimulus responses represented by network-wide bursts evoked from various spatial locations (electrodes). We found that the response characteristics, such as the burst initiation time and the spike rate, can be used to retrieve information about the stimulus location. The best selectivity in the response spiking pattern could be found for a small subpopulation of neurones (electrodes) at relatively short post-stimulus intervals. Such intervals were unique for each culture due to the non-uniform organization of the functional connectivity in the network during spontaneous development.

Adaptive enhancement of learning protocol in hippocampal cultured networks grown on multielectrode arrays.

Learning in neuronal networks can be investigated using dissociated cultures on multielectrode arrays supplied with appropriate closed-loop stimulation. It was shown in previous studies that weakly respondent neurons on the electrodes can be trained to increase their evoked spiking rate within a predefined time window after the stimulus. Such neurons can be associated with weak synaptic connections in nearby culture network. The stimulation leads to the increase in the connectivity and in the response. However, it was not possible to perform the learning protocol for the neurons on electrodes with relatively strong synaptic inputs and responding at higher rates. We proposed an adaptive closed-loop stimulation protocol capable to achieve learning even for the highly respondent electrodes. It means that the culture network can reorganize appropriately its synaptic connectivity to generate a desired response. We introduced an adaptive reinforcement condition accounting for the response variability in control stimulation. It significantly enhanced the learning protocol to a large number of responding electrodes independently on its base response level. We also found that learning effect preserved after 4-6 h after training.