Dissecting inter-region communication in human organoid models with dual-color optogenetic probes - PROJECT SUMMARY/ABSTRACT To make full impact of organoid technology, it is key to build organoid disease models with regionalized and interconnected compartments. Such regional specificity and the associated inter-region communication are essential calibers for evaluating functional maturation and structural integrity of the organoid models. Besides the considerations of culturing yield and structural complexity, it is technically non-trivial to probe the functional connectivity and pathways among sub-regions of 3D-structured organoids with high spatiotemporal resolutions. While 3D electrophysiology platforms are suited for recording intact organoid activities, they are yet to combine with high-resolution, cell-type-specific circuit manipulation to fully examine interregional circuits under definitive stimulus patterns and substantiate the analysis of functional connectivity. To this end, optogenetic control over cell activity has been noted for its combined advantages of temporal precision, cell-type specificity, and bi- directionality over other ways of cell modulation. Optoelectronic probes assembled with light sources together with an MEA have emerged as a powerful tool to optogenetically modulate the cell activity in a variety of settings. In this project, we will tailor design one high-precision optogenetic probing system, based on monolithic integration of close-packed dual-color LEDs and MEA, to dissect inter-region communication within regionalized organoid disease models. Leveraging our efforts on optoelectronic probes and organoid patterning methods, we will investigate if our probes with scalable pixel counts/pitches could advance organoid disease modeling via cellular-precision, tissue-level optogenetic electrophysiology. The proposed work will establish a new set of organoid probing systems that enable cellular resolution bi- directional optogenetic control of inter-region circuits and electrophysiology across multiple regions and depths of the organoids, which cannot be achieved by existing RNA sequencing, 3D organoid electro-physiology, or cell imaging methods. Such capabilities will deepen our understandings on regional circuits, functional connectivity, and organoid disease models. Given their scalable form, these probes could also be applied to transplanted organoids, cortical spheroids, and assembloids. Moving forward, our technology may provide insight into functional maturation, developmental stages of diseases, and even tissue engineering via optogenetic control of gene expression in select regions of the organoids. In particular, this project is comprised of two research aims: Aim 1. Bi-directional optogenetic probing of region-specific, depth-dependent organoid dynamics. We will develop the probe structures with the LED/MEA pitches/counts tailor designed to optogenetically access depth-dependent activity in targeted regions of SCZ organoid disease models and its control group. Aim 2. Bi-directional optogenetic probing of inter-region communication within living organoids. We will develop multi-shank structured probes to access cell activities across multiple organoid regions, with the focus on identifying inter-region connectivity and pathways via bi-directional optogenetics.
