Three-dimensional highly porous hydrogel scaffold for neural circuit dissection and modulation

Biomimetic brain structures and artificial neural networks have provided a simplified strategy for quantitatively investigating the complex structural and functional characteristics of highly interconnected neural networks. To achieve this, three-dimensional (3D) cell culture approaches have attracted much attention, which can mimic cell-cell interactions at the organism level and help better understand the function of specific neurons and neuronal networks than traditional two-dimensional cell culture methods. However, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. To resolve this, 3D hydrogel scaffolds can be fabricated via an innovative thermal treatment followed by an esterification process. A highly porous microstructure was formed within the bulk hydrogel scaffold, which showed a high porosity of 91% and a low Young's modulus of 6.11 kPa. Due to the merits of the fabricated hydrogel scaffolds, we constructed 3D neural networks and detected spontaneous action potentials in vitro. We successfully induced seizure-like waveforms in 3D cultured neurons and suppressed hyperactivated discharges by selectively activating γ-aminobutyric acid-ergic (GABAergic) interneurons. These results prove the advantages of our hydrogel scaffolds and demonstrate their application potential in the accurate dissection of neural circuits, which may help develop effective treatments for various neurological disorders. STATEMENT OF SIGNIFICANCE: While 3D cell culture approaches have attracted much attention and offer more advantages than two-dimensional cell culture methods, 3D scaffolds similar to the natural extracellular matrix to support the culturing, recording, and manipulation of neurons have long been an unresolved challenge. Herein, we developed a simplified and low-cost strategy for fabricating highly porous and cytocompatible hydrogel scaffolds for the construction of three-dimensional (3D) neural networks in vitro. The cultured 3D neural networks can mimic the in vivo connection among different neuron subgroups and help accurately dissect and manipulate the structure and function of specific neural circuits.