Combining Microfluidics, Optogenetics and Calcium Imaging to Study Neuronal Communication In Vitro

Abstract

In this paper we report the combination of microfluidics, optogenetics and calcium imaging as a cheap and convenient platform to study synaptic communication between neuronal populations in vitro. We first show that Calcium Orange indicator is compatible in vitro with a commonly used Channelrhodopsine-2 (ChR2) variant, as standard calcium imaging conditions did not alter significantly the activity of transduced cultures of rodent primary neurons. A fast, robust and scalable process for micro-chip fabrication was developed in parallel to build micro-compartmented cultures. Coupling optical fibers to each micro-compartment allowed for the independent control of ChR2 activation in the different populations without crosstalk. By analyzing the post-stimuli activity across the different populations, we finally show how this platform can be used to evaluate quantitatively the effective connectivity between connected neuronal populations.

μNeurocircuitry: Establishing in vitro models of neurocircuits with human neurons

“…1,5a) or suitable to be used for functional analysis using an inverted microscope. Previous studies have shown the ability of compartmentalized systems to record inter-chamber communication using calcium imaging 12,21 . While our system has the capacity for calcium imaging, we designed the system to have an large open well to allow for electrophysiology, which measures high temporal resolution (in millisecond scale) neuronal firing or synaptic transmission.…”

“…These microfluidic devices have enabled studies of axonal injury 15–17 , axon pathfinding 18 and cellular migration 19 . They have also been utilized to investigate synaptogenesis 20 and circuit connectivity 21–24 . A compartmentalized model of synaptic competition has been devised allowing the study of differences in individual neurons by chemically treating one chamber and performing synapse counting in another 22 .…”

“…Renault et al . have demonstrated the use of optogenetics and calcium imaging as tools to evaluate neuronal communication in a compartmentalized device 21 . These approaches have provided a proof of concept for microfluidics as a strategy to compartmentalize culture in brain circuit studies, but thus far they have only used primary neuronal cultures or are limited to human induced neurons of a single subtype 24 .…”

Bridging the gap: microfluidic devices for short and long distance cell–cell communication

“…The unique ability to study single cell signaling or small populations in microfluidics with be important in future understanding of cell heterogeneity, and it will have wide implications in many topics including cancer stem cells, metastasis, immunology, neuroscience, and more. Immunology has been one of the biggest directions for cell-cell communication and single cell analysis devices 34 , while neuroscience has been widely popular for long-distance communication 140–142 . Despite this, there are still many unexplored biological areas where research could benefit from microfluidic cell-cell signaling devices.…”

Microfluidic Models of Neuromuscular Junctions

“…The device geometry helps in preventing the cross talk between neuronal populations. 14 Li et al 15 have exploited the use of microfluidic device for amperometric measurements of neurotransmitter release. The microscale environment of the device helps in the formation of in vitro neural networks between superior cervical ganglion and effector smooth muscle cells.…”