Lighting Up Neurons - All Optical Interrogation of Designed Neuronal Networks

Abstract

Introduction. The brain’s ability to process information relies on its tightly interconnected and modularlyorganized networks of neurons. Neuronal activity is coordinated in a way that leads to periods of synchronicityand asynchronicity. Most in vitro experiments on MEAs [1] or in microfluidic systems [2] usuallywork with large, random neuronal networks and heavily undersample the complete population of neurons.Therefore, we combine optogenetic techniques with our well-established patterned neuronal cell cultures torecord and manipulate the large majority of neurons within a well-defined small-scale network. Specifically,we analyzed the impact of one population with unidirectional output onto downstream single cells (CT2WT)and larger unstructured networks (fComIn). With this idea, we try to test the theory that synchronousnetwork activity originates from regions of high connectivity [1].Materials And Methods. We based the designs for our patterns on previously published triangular structureswith unidirectional output [3]. To grow patterned, primary rat neuronal cultures cell adhesive aminoacid chains were covalently bound onto a cell repellent surface via an improved version of microcontactprinting. During three weeks of cell culture, the neurons were transduced (at day 9-10) with two adenoassociatedviruses containing the light-activated ion channel channelrhodopsin and the red calcium indicatorRCamP1b. This combination of optogenetic tools enables us to record and stimulate neurons at highspatial precision using a fluorescence microscope equipped with a high-speed camera and a fluorescencerecovery after photobleaching (FRAP) laser.Results And Outlook. In both patterns, we used two modalities of optogenetic stimulation: a) an individualstimulation of neurons within different subpopulations or b) a near-simultaneous stimulation of the sameneurons.In CT2WT structures, we previously found, using patch-clamp recordings, that near-simultaneous stimulationof the larger input triangle can increase the spontaneous firing of the output neuron. We nowinvestigated the same structures with the all-optical system. We can now use the resulting data for furtheranalysis of the mechanisms of network signal integration in neurons.In fComIn structures, we show that the preferred directionality of the triangular structures is kept to a certainextent even after three weeks of cell culture. Moreover, the different modalities and locations of stimulationslead to different neuronal responses within the patterned population. We further compared the firing patternsfor spatio-temporal activity patterns within spontaneous and stimulated neuronal firing. Additionally,we analyzed the networks for special cell types like hub- and leader cells.In the future, this well observable and highly controllable network system can be compared with equallydetailed computational models