Publications

Purpose: To interrogate animal physiology in vivo, there is a lack of non-genetic methods to control the activity of endogenous proteins with pharmacological and spatiotemporal precision. To address this need, we recently developed targeted covalent photoswitchable (TCP) compounds that enable the remote control of endogenous glutamate receptors (GluRs) using light. Methods: We combine the photopharmacological effector TCP9 with neuronal activity sensors to demonstrate all-optical reversible control of endogenous GluRs across multiple spatiotemporal scales in rat brain tissue ex vivo and in Xenopus tadpole brains in vivo. Findings: TCP9 allows photoactivation of neuronal ensembles, individual neurons, and single synapses in ex vivo tissue and in intact brain in vivo, which is challenging using optogenetics and neurotransmitter uncaging. TCP9 covalently targets AMPA and kainate receptors, maintaining their functionality and photoswitchability for extended periods (>8 h) after a single compound application. This allows tracking endogenous receptor physiology during synaptic plasticity events such as the reduction of functional AMPA receptors during long-term depression in hippocampal neurons. Conclusion: TCP9 is a unique non-invasive tool for durable labeling, reversible photoswitching, and functional tracking of native receptors in brain tissue without genetic manipulation.

JTD Keywords: 2-photon, Ampa receptors, Ampar, Azobenzene, Caged glutamate, Calcium imaging, Covalent drug, Dendritic spines, Hippocampus, Kainate, Long-term depression, Optical control, Optopharmacology, Photopharmacology, Photoswitch, Plasticity, Proteins, Pulse-chase, Rat, Subunit, Surface expression, Synaptic ampa, Xenopus

Xenopus tadpoles are an emerging model for developmental, genetic and behavioral studies. A small size, optical accessibility of most of their organs, together with a close genetic and structural relationship to humans make them a convenient experimental model. However, there is only a limited toolset available to measure behavior and organ function of these animals at medium or high-throughput. Herein, we describe an imaging-based platform to quantify body and autonomic movements of Xenopus tropicalis tadpoles of advanced developmental stages. Animals alternate periods of quiescence and locomotor movements and display buccal pumping for oxygen uptake from water and rhythmic cardiac movements. We imaged up to 24 animals in parallel and automatically tracked and quantified their movements by using image analysis software. Animal trajectories, moved distances, activity time, buccal pumping rates and heart beat rates were calculated and used to characterize the effects of test compounds. We evaluated the effects of propranolol and atropine, observing a dose-dependent bradycardia and tachycardia, respectively. This imaging and analysis platform is a simple, cost-effective high-throughput in vivo assay system for genetic, toxicological or pharmacological characterizations.

JTD Keywords: Xenopus tropicalis, Animal behavior, Cardiac imaging, Motion analysis, Animal tracking, Hhigh-throughput in vivo assay