PROJECT SUMMARY Objectives. The primary objective of this proposal is to develop chemically specific electrochemical sensors to provide rapid and direct measurements of physiologically relevant chemical messenger release (technology development). The secondary objective is to apply these sensors to determine signaling heterogeneity in astrocytes from different brain regions (biological hypothesis). Significance and Knowledge Gap. Astrocytes act as integrators across many circuits and environments in the brain and how they interact with other cell types can vary in time and space. Rigorous prior research demonstrates that astrocytes are heterogenous, varying by brain region and circuit. Heterogeneity can be affected by transcriptional control related to synapse function, plasticity, molecular transmission and protein machinery and organelles that underly gliotransmission. Astrocyte heterogeneity also affects how this class of cells respond to insults and age and can potentially be predictive of disease vulnerability. Astrocytes have typically been studied as a homogenous population, thus there is a need to study functional heterogeneity of astrocytes (e.g., signaling) to determine functional roles of astrocytes in development, response to injuries, and neurodegenerative disease and how this response influences local circuit function and homeostasis. Solution and Specific Aims. A major barrier to studying gliotransmission is the lack of measurement tools that possess the combined spatiotemporal and chemical specificity to study dynamic molecular signaling from astrocytes. We aim to develop a sensor platform that overcomes this barrier by providing direct and rapid measurement of gliotransmission over broad spatial and temporal ranges. Through a collaborative proposal we propose to leverage the universal and specific chemical detection abilities of electrochemical, aptamer-based (E-AB) sensors with innovative measurement science for rapid determination of gliotransmitter dynamics and heterogeneity. With this new measurement technology, we aim to test the hypothesis that gliotransmitter signaling varies in terms of the frequency, amount, and identity of transmitters released with circuit-, and inter- and intraregional specificity. We will 1) Develop electrochemical, aptamer-based (E-AB) sensors to monitor real-time release of gliotransmitters from a cell population in 3D culture. 2) Develop recessed, microscale E-AB sensors to monitor the release of gliotransmitters from single astrocytes. 3) Develop recessed, nanoscale sensors to monitor the release of gliotransmitters from sub-cellular regions. 4) Select and characterize highly specific structure-switching nucleic acid aptamers as binding partners for glutamate, GABA, and D-serine with nM affinity for use in aims 1-3.

PROJECT NARRATIVE The goal of this proposal is to develop sensors that will provide mechanistic insight into how non-neuronal cells, astrocytes, communicate in the brain in a region- and circuit-specific manner. This layer of information about functional astrocyte heterogeneity will contribute to the field by providing quantitative metrics to assess diversity. The new knowledge of how astrocytes communicate in the brain generated by our sensors can potentially lead to paradigm-shifting therapeutic approaches to neurological and developmental disorders.