UC San Diego

From moment to moment, dynamic patterns of neurotransmitter release and surface receptor activity determine the function of the central nervous system. Fundamental to neuroscience is the ability to measure these patterns. However, the tools to make these measurements at a physiologically relevant temporal and spatial resolution are largely unavailable. Recent advances in genetically encoded optical biosensors have begun to allow direct visualization of molecular events previously only indirectly inferable. The aim of the work presented in this dissertation is to develop and characterize a novel in vivo cellular biosensor that takes advantage of current optical biosensor technology as well as standard cell-based assays used in high-throughput screening by the pharmaceutical industry. The biosensors are named Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs). In this initial realization, we express the M1 muscarinic acetylcholine receptor and the fluorescent Ca²⁺ indicator TN-XXL in cultured HEK293 cells. Activation of the M1 receptor by acetylcholine leads to an increase in HEK293 cytosolic Ca²⁺ then reported by TN-XXL. In vivo, M1-CNiFERs are implanted with control-CNiFERs: HEK293 cells expressing TN -XXL and the non-functional fluorescent protein mCherry, but not M1. M1- but not control-CNiFERs respond robustly, up to 40% signal change, to endogenous acetylcholine release evoked via electrical stimulation of Nucleus Basalis Magnocellularis (NBM). The response is a single peak initiated within 2 s with a half-maximal rise time as short as 1̃ s and a full width at half maximal amplitude of < 10 s. As expected for NBM stimulation, we observe an inverse correlation between M1-CNiFER response and electrocorticogram delta band power. The cholinergic nature of this response is further verified by enhancement with the acetylcholinesterase inhibitor physostigmine and suppression with the muscarinic antagonist atropine. The pharmaceutical industry has made such extensive use of the cell-based assay in part because of its modularity. By simply expressing a different receptor, CNiFERs can be built to detect the release of a large number of endogenous neurotransmitters and signaling molecules implicated in a wide variety of central nervous system functions