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.

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.

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