Abstract
Sporadic spontaneous network activity emerges during early central nervous system (CNS) development and, as the number of neuronal connections rises, the maturing network displays diverse and complex activity, including various types of synchronized patterns. These activity patterns have major implications on both basic research and the study of neurological disorders, and their interplay with network morphology tightly correlates with developmental events such as neuronal differentiation, migration and establishment of neurotransmitter phenotypes. Although 2D neural cultures models have provided important insights into network activity patterns, these cultures fail to mimic the complex 3D architecture of natural CNS neural networks and its consequences on connectivity and activity. A 3D in-vitro model mimicking early network development while enabling cellular-resolution observations, could thus significantly advance our understanding of the activity characteristics in the developing CNS. Here, we longitudinally studied the spontaneous activity patterns of developing 3D in-vitro neural network “optonets,” an optically-accessible bioengineered CNS model with multiple cortex-like characteristics. Optonet activity was observed using the genetically encodable calcium indicator GCaMP6m and a 3D imaging solution based on a standard epi-fluorescence microscope equipped with a piezo-electric z-stage and image processing-based deconvolution. Our results show that activity patterns become more complex as the network matures, gradually exhibiting longer-duration events. This report characterizes the patterns over time, and discusses how environmental changes affect the activity patterns. The relatively high degree of similarity between the network's spontaneously generated activity patterns and the reported characteristics of in-vivo activity, suggests that this is a compelling model system for brain-in-a chip research.
Highlights
During the early stages of central nervous system development, neuronal connections are formed
To characterize optonet neuronal activity, volumetric activity movies (3 planes, 80 μm stack), from which ∆F/F signal traces were calculated for each cell (Figure 1), were captured from E18 cortical neuron-embedded Matrigel scaffolds over a 24-day period
The optonet cultures were transfected with a GCaMP6m associated virus (AAV) virus driven by a synapsin promoter to express the probe only in neurons and 5 days after transfection they expressed sufficient amounts of the indicator to allow imaging
Summary
During the early stages of central nervous system development, neuronal connections are formed. High-density 2D cultures of dissociated cortical cells tend to exhibit spontaneous activity patterns that develop in a stereotyped manner. They begin as uncorrelated sporadic action potentials toward the end of the first week in culture, followed by single-cell activity that combines sporadic and clustered action potentials (Kamioka et al, 1996) and which later develops into synchronized bursting of the network. By 22–33 days, more complicated and diverse patterns appear and the activity becomes synchronized, non-periodic, and clustered (Habets et al, 1987; Maeda et al, 1995) This activity pattern persists for more than 2 months, and is considered to be the mature state of the neural network (Marom and Shahaf, 2002)
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