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Summary Three-dimensional (3D) in vitro cultures recapitulate key features of the brain including morphology, cell-cell and cell-extracellular matrix interactions, gradients of factors, and mechanical properties. However, there remains a need for experimental and computational tools to investigate network functions in these 3D models. To address this need, we present an experimental system based on 3D scaffold-based cortical neuron cultures in which we expressed the genetically encoded calcium indicator GCaMP6f to record neuronal activity at the millimeter-scale. Functional neural network descriptors were computed with graph-theory-based network analysis methods, showing the formation of functional networks at 3 weeks of culture. Changes to the functional network properties upon perturbations to glutamatergic neurotransmission or GABAergic neurotransmission were quantitatively characterized. The results illustrate the applicability of our 3D experimental system for the study of brain network development, function, and disruption in a biomimetic microenvironment.

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long‐term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk‐based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic‐like indicators of increased pathological β‐amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long‐term stability of the platform is suited to study chronic brain disease including neurodegeneration.

As neurodegenerative conditions are increasingly linked to mitochondrial dysfunction, methods for studying brain cell metabolism at high spatial resolution are needed to elucidate neurodegeneration mechanisms. Two-photon excited fluorescence (TPEF) imaging is a non-destructive, high-resolution technique for studying cell metabolism via endogenous fluorescence of reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). We employed TPEF to study the metabolism of primary rat astrocyte and neuronal cultures under normal growth conditions and in response to manganese (Mn) treatment. Histograms of pixel-wise optical redox ratio, defined as FAD/(FAD + NAD(P)H), revealed three distinct redox distributions and significant differences in their relative weights between astrocytes and neurons. When treated with Mn, both cell types exhibited redox ratio shifts consistent with increased oxidative stress. However, the manner in which the redox distributions was affected was distinct for the two cell types. Furthermore, NAD(P)H fluorescence lifetime imaging revealed an increase in bound NAD(P)H fraction upon Mn treatment for neurons, consistent with enhanced apoptosis. Astrocytes showed a decrease in bound fraction, possibly due to a shift towards glycolytic metabolism in response to impaired respiration. These results exhibit TPEF’s utility for characterizing detailed metabolic changes of different brain cell types in response to neurotoxins.