Assessing functional connectivity across 3D tissue engineered axonal tracts using calcium fluorescence imaging

Micro-tissue engineered neural networks (micro-TENNs) are anatomically-inspired constructs designed to structurally and functionally emulate white matter pathways in the brain. These 3D neural networks feature long axonal tracts spanning discrete neuronal populations contained within a tubular hydrogel, and are being developed to reconstruct damaged axonal pathways in the brain as well as to serve as physiologically-relevant in vitro experimental platforms. The goal of the current study was to characterize the functional properties of these neuronal and axonal networks.

Bidirectional micro-TENNs were transduced to express genetically-encoded calcium indicators, and spontaneous fluorescence activity was recorded using real-time microscopy at 20 Hz from specific regions-of-interest in the neuronal populations. Network activity patterns and functional connectivity across the axonal tracts were then assessed using various techniques from statistics and information theory including Pearson cross-correlation, phase synchronization matrices, power spectral analysis, directed transfer function, and transfer entropy.

Pearson cross-correlation, phase synchronization matrices, and power spectral analysis revealed high values of correlation and synchronicity between the spatially segregated neuronal clusters connected by axonal tracts. Specifically, phase synchronization revealed high synchronicity of  >0.8 between micro-TENN regions of interest. Normalized directed transfer function and transfer entropy matrices suggested robust information flow between the neuronal populations. Time varying power spectrum analysis revealed the strength of information propagation at various frequencies. Signal power strength was visible at elevated peak levels for dominant delta (1-4 Hz) and theta (4-8 Hz) frequency bands and progressively weakened at higher frequencies. These signal power strength results closely matched normalized directed transfer function analysis where near synchronous information flow was detected between frequencies of 2-5 Hz.

To our knowledge, this is the first report using directed transfer function and transfer entropy methods based on fluorescent calcium activity to estimate functional connectivity of distinct neuronal populations via long-projecting, 3D axonal tracts in vitro. These functional data will further improve the design and optimization of implantable neural networks that could ultimately be deployed to reconstruct the nervous system to treat neurological disease and injury.