Kenneth S. Hettie

Three-Input Logic Gates with Potential Applications for Neuronal Imaging

Convenient methods for the direct visualization of neurotransmitter trafficking would bolster investigations into the development of neurodegenerative diseases. Here, tunable fluorescent molecular logic gates with applications to neuronal imaging have been developed. The three-input AND molecular logic gates are based on the coumarin-3-aldehyde scaffold and designed to give a turn-on fluorescence response upon the corelease of glutamate and zinc from secretory vesicles via exocytosis. Spectroscopic studies reveal an 11-fold fluorescence enhancement under conditions mimicking exocytosis. Methylation of the scaffold was used to optimize the spectral profile of the sensors toward desired excitation wavelengths. A binding study that elucidates the sensor-analyte interactions is presented. These sensors serve as a proof-of-concept toward the direct imaging of neurotransmitters released upon exocytosis using fluorescent molecular logic gates.

Selective Catecholamine Recognition with NeuroSensor 521: A Fluorescent Sensor for the Visualization of Norepinephrine in Fixed and Live Cells

A method for the selective labeling and imaging of catecholamines in live and fixed secretory cells is reported. The method integrates a tailored approach using a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that can exploit the high concentration of neurotransmitters and acidic environment within secretory vesicles for the selective recognition of norepinephrine and dopamine. The utility of the method was demonstrated by selectively labeling and imaging norepinephrine in secretory vesicles such that discrimination between norepinephrine- and epinephrine-enriched populations of chromaffin cells was observed. This method was validated in fixed cells by co-staining with an anti-PNMT antibody.

ExoSensor 517: a dual-analyte fluorescent chemosensor for visualizing neurotransmitter exocytosis.

A dual-analyte fluorescent chemosensor (ExoSensor 517) for the direct visualization of neurotransmitters released upon exocytosis is presented. The sensor exploits the high concentration of neurotransmitters (e.g., glutamate, norepinephrine, and dopamine) and the pH gradient between the vesicle and synaptic cleft. The cooperative recognition elements require both binding and a change in environmental pH to afford a fluorescence response which makes ExoSensor 517 one of the first integrated molecular logic gates to be used for biological applications.

Coumarin‐3‐Aldehyde as a Scaffold for the Design of Tunable PET‐Modulated Fluorescent Sensors for Neurotransmitters

NeuroSensor 521 (NS521) is a fluorescent sensor for primary-amine neurotransmitters based on a platform that consists of an aryl moiety appended to position C4 of the coumarin-3-aldehyde scaffold. We demonstrate that sensors based on this platform behave as a directly linked donor-acceptor system that operates through an intramolecular acceptor-excited photoinduced electron transfer (a-PET) mechanism. To evaluate the PET process, a series of benzene- and thiophene-substituted derivatives were prepared and the photophysical properties, binding affinities, and fluorescence responses toward glutamate, norepinephrine, and dopamine were determined. The calculated energy of the highest occupied molecular orbital (EHOMO ) of the pendant aryl substituents, along with oxidation and reduction potential values derived from the calculated molecular orbital energy values of the platform components, allowed for calculation of the fluorescence properties of the benzene sensor series. Interestingly, the thiophene derivatives did not fit the typical PET model, highlighting the limitations of the method. A new sensor, NeuroSensor 539, displayed enhanced photophysical properties aptly suited for biological imaging. NeuroSensor 539 was validated by selectively labeling and imaging norepinephrine in secretory vesicles of live chromaffin cells.

Tunable Molecular Logic Gates Designed for Imaging Released Neurotransmitters

Tunable dual-analyte fluorescent molecular logic gates (ExoSensors) were designed for the purpose of imaging select vesicular primary-amine neurotransmitters that are released from secretory vesicles upon exocytosis. ExoSensors are based on the coumarin-3-aldehyde scaffold and rely on both neurotransmitter binding and the change in environmental pH associated with exocytosis to afford a unique turn-on fluorescence output. A pH-functionality was directly integrated into the fluorophore π-system of the scaffold, thereby allowing for an enhanced fluorescence output upon the release of labeled neurotransmitters. By altering the pH-sensitive unit with various electron-donating and -withdrawing sulfonamide substituents, we identified a correlation between the pKa of the pH-sensitive group and the fluorescence output from the activated fluorophore. In doing so, we achieved a twelvefold fluorescence enhancement upon evaluating the ExoSensors under conditions that mimic exocytosis. ExoSensors are aptly suited to serve as molecular imaging tools that allow for the direct visualization of only the neurotransmitters that are released from secretory vesicles upon exocytosis.

Turn-On Near-Infrared Fluorescent Sensor for Selectively Imaging Serotonin

A molecular imaging tool that provides for the direct visualization of serotonin would significantly aid in the investigation of neuropsychiatric disorders that are attributed to its neuronal dysregulation. Here, the design, synthesis, and evaluation of NeuroSensor 715 (NS715) is presented. NS715 is the first molecular sensor that exhibits a turn-on near-infrared fluorescence response toward serotonin. Density functional theory calculations facilitated the design of a fluorophore based on a coumarin-3-aldehyde scaffold that derives from an electron-rich 1,2,3,4-tetrahydroquinoxaline framework, which provides appropriate energetics to prevent the hydroxyindole moiety of serotonin from quenching its fluorescence emission. Spectroscopic studies revealed that NS715 produces an 8-fold fluorescence enhancement toward serotonin with an emission maximum at 715 nm. Accompanying binding studies indicated NS715 displays a 19-fold selective affinity for serotonin and a modest affinity for catecholamines over other primary-amine neurotransmitters. The utility of NS715 toward neuroimaging applications was validated by selectively labeling and directly imaging norepinephrine within secretory vesicles using live chromaffin cells, which serve as a model system for specialized neurons that synthesize, package, and release only a single, unique type of neurotransmitter. In addition, NS715 effectively differentiated between cell populations that express distinct neurotransmitter phenotypes.

Neurosensor 521: A Fluorescent Sensor for Selective Labeling of Norepinephrine-Containing Vesicles

We have developed a novel fluorescence-based turn-on molecular sensor (NeuroSensor 521) that selectively binds to primary amines via iminium ion formation. The fluorescence increases > 5-fold upon binding norepinephrine and is accompanied by a red shift in the absorption maximum from 448 to 488 nm. Norepinephrine and dopamine bind with a ∼10-fold higher affinity (∼10 mM) than other alkyl amines such as glycine, but secondary amines such as epinephrine do not have a measurable binding affinity. We used confocal microscopy to image chromaffin cells labeled with NS521. Chromaffin cells were separated into norepinephrine-enriched and epinephrine-enriched fractions by centrifugation on a Percoll gradient. The norepinephrine-enriched cell population shows strong, punctate fluorescence compared to the epinephrine-enriched cell population, which exhibits only marginal fluorescence. To further validate the selective labeling of norepinephrine-containing vesicles, we labeled fixed cells using an antibody against phenylethanolamine N-methyltransferase (PNMT). The NE-enriched cell population stains brightly with NS521 and weakly with the fluorescent PNMT secondary antibody while the opposite is true for the EP-enriched cell population. Next, we used Total Internal Reflection Fluorescence Microscopy (TIRFM) to image the bottom surface of chromaffin cells loaded with NS521. Clear punctate fluorescence consistent with labeling of individual norepinephrine-containing granules was observed. We conclude that NeuroSensor 521 represents a convenient method to selectively stain norepinephrine and dopamine in neurosecretory vesicles. Supported by the NSF (CHE-1112194) and the NIH (R43 MH096650).