Abstract
Studying intracellular dynamics in neurons is crucial to better understand how brain circuits communicate and adapt to environmental changes. In neurons, axonal secretory vesicles underlie various functions from growth during development to plasticity in the mature brain. Similarly, transport of mitochondria, the power plant of the cell, regulates both axonal development and synaptic homeostasis. However, because of their submicrometric size and rapid velocities, studying the kinetics of these organelles in projecting axons in vivo is technically challenging. In parallel, primary neuronal cultures are adapted to study axonal transport but they lack the physiological organization of neuronal networks, which in turn may bias observations. We previously developed a microfluidic platform to reconstruct a physiologically-relevant and functional corticostriatal network in vitro that is compatible with high-resolution videorecording of axonal trafficking. Here, using this system we report progressive changes in axonal transport kinetics of both dense core vesicles and mitochondria that correlate with network development and maturation. Interestingly, axonal flow of both types of organelles change in opposite directions, with rates increasing for vesicles and decreasing for mitochondria. Overall, our observations highlight the need for a better spatiotemporal control for the study of intracellular dynamics in order to avoid misinterpretations and improve reproducibility.
Highlights
Understanding mechanisms that drive the establishment, maturation, function and dysfunction of neuronal networks on a subcellular level requires microscopic approaches that are often technically challenging in the in vivo context
Because of the spatiotemporal feature of the platform, each cellular compartment of the neuron can be systematically identified at each stage of the culture, which allows to establish a temporal map of intracellular dynamics
Because the standardized architecture of the platform allows space and time compartmentalization of neuronal networks, we identified the different stages of network maturation using systematic analyses with selective markers and live reporters (SYP/PSD95, iGluSnFR, GCaMP6f)
Summary
Understanding mechanisms that drive the establishment, maturation, function and dysfunction of neuronal networks on a subcellular level requires microscopic approaches that are often technically challenging in the in vivo context. The motile nature and submicrometric size of cellular organelles make their study extremely difficult in vivo because it requires technology with high spatial and temporal resolution that have yet to be developed This is true for axonal trafficking of dense core vesicles (DCV) that transports key elements for neuronal growth and transmission. Because of the standardized architecture and specific physical and chemical constraints of the microfluidic platform, neuronal networks develop with specific kinetics that are similar through different devices In this configuration, network development can be synchronized between different conditions, facilitating systematic analyses and reproducibility[5,6,7]. Network development can be synchronized between different conditions, facilitating systematic analyses and reproducibility[5,6,7] Using these spatiotemporal features, we cross-compared axonal trafficking of two motile organelles, dense core vesicles and mitochondria, throughout network maturation. Trafficking kinetics of vesicles and mitochondria evolved in opposite directions, as demonstrated by the progressive acceleration and densification of anterograde vesicles compared to the dramatic reduction in motile mitochondria in mature axons
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