Development and Application of a Synthetic Near Infrared Fluorescent Probe for Imaging Modulatory Neurotransmitters
- Author(s): Beyene, Abraham G
- Advisor(s): Landry, Markita
- et al.
Dopamine neurotransmission plays critical roles in brain function in both health and
disease and aberrations in dopamine neurotransmission are implicated in several
psychiatric and neurological disorders, including schizophrenia, depression, anxiety, and
Parkinson’s disease. Until recently, measuring the dynamics of dopamine and other
neurotransmitters of this class could not be achieved at spatiotemporal resolutions
necessary to study how dopamine regulates the plasticity and function of neurons and neural
circuits, and how dysfunctions in this regulation lead to disease. Probes that satisfy critical
attributes in spatiotemporal resolution and chemical selectivity are needed to facilitate
investigations of dopamine neurochemistry.
To address this need, this dissertation describes the synthesis and implementation of
an ultrasensitive near-infrared “turn-on” nanosensor (nIRCat) for the catecholamine
neuromodulators dopamine and norepinephrine. To guide probe development, we present
results from a computational model that offers insight into the spatiotemporal dynamics of
dopamine in the striatum, a subcortical structure that is enriched in dopamine. With this
model, we elucidated the kinetic requirements for a prototypical optical indicator as well as
optimal imaging frame rates needed for measuring dopamine neurochemical dynamics.
Stochastic modeling of dopamine dynamics, driven by kinetic phenomena of vesicular
release, diffusion and clearance, provide a platform to evaluate dopaminergic volume
transmission arising from a single terminal or ensemble terminal activity. With this work,
we illustrate that only probes with kinetic parameters in a particular range are feasible for
dopamine imaging at spatiotemporal scales likely to be encountered in brain tissue.
In two subsequent chapters, we describe the development and in vitro
characterization of nIRCats, synthesized from functionalized single wall carbon nanotubes
(SWCNT) that fluoresce in the near infrared range of the spectrum. We show that nIRCats
exhibit maximal relative change in fluorescence intensity (ΔF/F0) of up to 35-fold in
response to catecholamines and have optimal dynamic range that span physiological
concentrations of their target brain analytes. Through a combination of experimental and
molecular dynamics approaches, we elucidate the photophysical principles and intermolecular
interactions that govern the molecular recognition and fluorescence modulation of nIRCats by dopamine.
Finally, we demonstrate that nIRCat can be used to measure electrically and
optogenetically evoked release of dopamine in striatal brain slices, revealing hotspots of
activity with a median size of 2 μm, and exhibiting a log-normal size distribution that extends
up to 10 μm. Moreover, nIRCats are shown to be compatible with dopamine pharmacology
and permit studies of how receptor-targeting drugs modulate evoked dopamine release. Our
results suggest nIRCats may uniquely support similar explorations of processes that regulate
dopamine neuromodulation at the level of individual synapses, and exploration of the effects
of receptor agonists and antagonists that are commonly used as psychiatric drugs and
psychoactive molecules that modulate the release and clearance profiles of dopamine. We
conclude that nIRCats and other nanosensors of this class can serve as versatile synthetic
optical tools to monitor interneuronal chemical signaling in the brain extracellular space at
spatial and temporal scales pertinent to the encoded information.