Development and Application of a Synthetic Near Infrared Fluorescent Probe for Imaging Modulatory Neurotransmitters

Dopamine neurotransmission plays critical roles in brain function in both health and/ndisease and aberrations in dopamine neurotransmission are implicated in several/npsychiatric and neurological disorders, including schizophrenia, depression, anxiety, and/nParkinson’s disease. Until recently, measuring the dynamics of dopamine and other/nneurotransmitters of this class could not be achieved at spatiotemporal resolutions/nnecessary to study how dopamine regulates the plasticity and function of neurons and neural/ncircuits, and how dysfunctions in this regulation lead to disease. Probes that satisfy critical/nattributes in spatiotemporal resolution and chemical selectivity are needed to facilitate/ninvestigations of dopamine neurochemistry./nTo address this need, this dissertation describes the synthesis and implementation of/nan ultrasensitive near-infrared “turn-on” nanosensor (nIRCat) for the catecholamine/nneuromodulators dopamine and norepinephrine. To guide probe development, we present/nresults from a computational model that offers insight into the spatiotemporal dynamics of/ndopamine in the striatum, a subcortical structure that is enriched in dopamine. With this/nmodel, we elucidated the kinetic requirements for a prototypical optical indicator as well as/noptimal imaging frame rates needed for measuring dopamine neurochemical dynamics./nStochastic modeling of dopamine dynamics, driven by kinetic phenomena of vesicular/nrelease, diffusion and clearance, provide a platform to evaluate dopaminergic volume/ntransmission arising from a single terminal or ensemble terminal activity. With this work,/nwe illustrate that only probes with kinetic parameters in a particular range are feasible for/ndopamine imaging at spatiotemporal scales likely to be encountered in brain tissue./nIn two subsequent chapters, we describe the development and in vitro/ncharacterization of nIRCats, synthesized from functionalized single wall carbon nanotubes/n(SWCNT) that fluoresce in the near infrared range of the spectrum. We show that nIRCats/nexhibit maximal relative change in fluorescence intensity (ΔF/F0) of up to 35-fold in/nresponse to catecholamines and have optimal dynamic range that span physiological/nconcentrations of their target brain analytes. Through a combination of experimental and/nmolecular dynamics approaches, we elucidate the photophysical principles and intermolecular/ninteractions that govern the molecular recognition and fluorescence modulation of nIRCats by dopamine./nFinally, we demonstrate that nIRCat can be used to measure electrically and/noptogenetically evoked release of dopamine in striatal brain slices, revealing hotspots of/nactivity with a median size of 2 μm, and exhibiting a log-normal size distribution that extends/nup to 10 μm. Moreover, nIRCats are shown to be compatible with dopamine pharmacology/nand permit studies of how receptor-targeting drugs modulate evoked dopamine release. Our/nresults suggest nIRCats may uniquely support similar explorations of processes that regulate/ndopamine neuromodulation at the level of individual synapses, and exploration of the effects/nof receptor agonists and antagonists that are commonly used as psychiatric drugs and/npsychoactive molecules that modulate the release and clearance profiles of dopamine. We/nconclude that nIRCats and other nanosensors of this class can serve as versatile synthetic/noptical tools to monitor interneuronal chemical signaling in the brain extracellular space at/nspatial and temporal scales pertinent to the encoded information.

Advisor: Landry, Markita