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

Abstract

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. KEYWORDS: Fluorescent sensor, catecholamine, norepinephrine, cell imaging, chromaffin T he catecholamines dopamine, norepinephrine, and epinephrine are the principal neurotransmitters in the sympathetic nervous system. 1 In particular, norepinephrine regulates many critical functions that include attention, memory, learning, emotion, and autonomic and cardiovascular function. In the periphery, norepinephrine increases heart rate, cardiac contractility, vascular tone, renin-angiotensin system activity, and renal sodium reabsorption. 2 Norepinephrine is secreted by chromaffin cells, which package catecholamines at high concentrations (0.5−1.0 M) and at low pH (5.0−5.5) in neurosecretory vesicles. 3 Chromaffin cells possess approximately 30 000 large dense-core vesicles (LDCV) with norepinephrine and epinephrine. 4 Chromaffin cells that store and release mainly epinephrine can be separated from those that utilize mainly norepinephrine through density-gradient centrifugation, though a third subpopulation which secrete both epinephrine and norepinephrine has been identified via cyclic voltametry. 5 Over the years, chromaffin cells have become a standard platform for the study of processes related to exocytosis. Thus, chromaffin cells appeared to be an ideal platform for the study of novel sensors for neurotransmitters.Currently, catecholamines can be studied via electrochemical and chromatographic techniques that provide characterization and quantification, although these techniques can only provide crude spatial information. 6 Recently, fluorescent false neurotransmitters (FFNs) have been developed which are selectively loaded into vesicles that express neuronal vesicular monoamine transporter (VMAT) and represent an optical approach for labeling vesicles containing catecholamines and imaging catecholamine release at the single-vesicle level. 7 However, FFNs are loaded into all secretory vesicles expressing the VMAT protein without discrimination to cell type and thus, the approach cannot distinguish distinct cell populations that secrete a particular neurotransmitter.Fluorescent sensors remain a compelling technology for approaching the general problem of selective neurotransmitter detection. In recent years, a number of catecholamine sensors have been reported including RNA aptamers, fluorescen...

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

“…More recently, we developed NeuroSensor 521 (NS521) as a turn‐on fluorescent sensor for the catecholamines norepinephrine and dopamine (Figure 2). 6…”

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