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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.
Abstract

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.

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