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Brain function is substantially linked to the highly organized structure of neuronal networks. Emerging three-dimensional (3D) neuronal cell culture technologies attempt to mimic the complexity of brain circuits asin vitromicrophysiological systems. Nevertheless, structures ofin vitroassembled neuronal circuits often varies between samples and changes over time that makes it challenging to reliably record network functional output and link it to the network structure. Hence, engineering neuronal structures with pre- defined geometry and reproducible functional features are essential to modelin vivoneuronal circuits in a robust way. Here, we engineered thin microchannel devices to assemble 2D and 3D modular networks. Microchannel devices were coupled with multi-electrode array (MEA) electrophysiology system to enable long-term electrophysiology recordings from microengineered circuits. Each network was composed of 64 micromodules which were connected through micron size channels to their adjacent modules. Microstructures physically confined neurons to the recording electrodes that considerably enhanced the electrophysiology readout efficiency. In addition, microstructures preserved modular network structure over weeks. Modular circuits within microfluidic devices showed consistent spatial patterns of activity over weeks, which was missing in the randomly formed circuits. Number of physical connections per module was shown to be influencing the measured activity and functional connectivity parameters, that represents the impact of network structure on its functional output. We show that microengineered 3D modular networks with a profound activity and higher number of functional connections recapitulate key functional features of developing cortex. Structurally and functionally stable 2D and 3D network mimic the modular architecture of brain circuits and offers a robust and reproduciblein vitromicrophysiolopgical system to serve basic and translational neuroscience research.

Phosphodiesterase (PDE) inhibitors have been safely and effectively used in the clinic and increase the concentration of intracellular cyclic nucleotides (cAMP/cGMP). These molecules activate downstream mediators, including the cAMP response element-binding protein (CREB), which controls neuronal excitability and growth responses. CREB gain of function enhances learning and allocates neurons into memory engrams. CREB also controls recovery after stroke. PDE inhibitors are linked to recovery from neural damage and to stroke recovery in specific sites within the brain. PDE2A is enriched in cortex. In the present study, we use a mouse cortical stroke model in young adult and aged male mice to test the effect of PDE2A inhibition on functional recovery, and on downstream mechanisms of axonal sprouting, tissue repair, and the functional connectivity of neurons in recovering cortex. Stroke causes deficits in use of the contralateral forelimb, loss of axonal projections in cortex adjacent to the infarct, and functional disconnection of neuronal networks. PDE2A inhibition enhances functional recovery, increases axonal projections in peri-infarct cortex, and, through two-photonin vivoimaging, enhances the functional connectivity of motor system excitatory neurons. PDE2A inhibition after stroke does not have an effect on other aspects of tissue repair, such as angiogenesis, gliogenesis, neurogenesis, and inflammatory responses. These data suggest that PDE2A inhibition is an effective therapeutic approach for stroke recovery in the rodent and that it simultaneously enhances connectivity in peri-infarct neuronal populations.SIGNIFICANCE STATEMENTInhibition of PDE2A enhances motor recovery, axonal projections, and functional connectivity of neurons in peri-infarct tissue. This represents an avenue for a pharmacological therapy for stroke recovery.