An improved platform for cultured neuronal network electrophysiology: multichannel optogenetics integrated with MEAs.

Cultured neuronal networks (CNNs) are powerful tools for studying how neuronal representation and adaptation emerge in networks of controlled populations of neurons. To ensure the interaction of a CNN and an artificial setting, reliable operation in both open and closed loops should be provided. In this study, we integrated optogenetic stimulation with microelectrode array (MEA) recordings using a digital micromirror device and developed an improved research tool with a 64-channel interface for neuronal network control and data acquisition. We determined the ideal stimulation parameters including light intensity, frequency, and duty cycle for our configuration. This resulted in robust and reproducible neuronal responses. We also demonstrated both open and closed loop configurations in the new platform involving multiple bidirectional channels. Unlike previous approaches that combined optogenetic stimulation and MEA recordings, we did not use binary grid patterns, but assigned an adjustable-size, non-binary optical spot to each electrode. This approach allowed simultaneous use of multiple input-output channels and facilitated adaptation of the stimulation parameters. Hence, we advanced a 64-channel interface in that each channel can be controlled individually in both directions simultaneously without any interference or interrupts. The presented setup meets the requirements of research in neuronal plasticity, network encoding and representation, closed-loop control of firing rate and synchronization. Researchers who develop closed-loop control techniques and adaptive stimulation strategies for network activity will benefit much from this novel setup.

Adult mouse dorsal root ganglia neurons form aberrant glutamatergic connections in dissociated cultures.

Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.