Significance The neurotransmitter dopamine controls normal behavior and dopaminergic dysfunction is prevalent in multiple brain diseases. To reach a detailed understanding of how dopamine release and signaling are regulated at the subcellular level, we developed a near infrared fluorescent dopamine nanosensor 'paint' (AndromeDA) to directly image dopamine release and its spatiotemporal characteristics. With AndromeDA, we can ascribe discrete DA release events to defined axonal varicosities, directly assess the heterogeneity of DA release events across such release sites, and determine the molecular components of the DA release machinery. AndromeDA thus provides a new method for gaining fundamental insights into the core mechanisms of dopamine release, which with greatly benefit our knowledge of dopamine biology and pathobiology.

Cells use biomolecules to convey information. For instance, neurons communicate by releasing chemicals called neurotransmitters, including several monoamines. The information transmitted by neurons is, in part, coded in the type and amount of neurotransmitter released, the spatial distribution of release sites, the frequency of release events, and the diffusion range of the neurotransmitter. Therefore, quantitative information about neurotransmitters at the (sub)cellular level with high spatiotemporal resolution is needed to understand how complex cellular networks function. So far, various analytical methods have been developed and used to detect neurotransmitter secretion from cells. However, each method has limitations with respect to chemical, temporal and spatial resolution. In this review, we focus on emerging methods for optical detection of neurotransmitter release and discuss fluorescent sensors/probes for monoamine neurotransmitters such as dopamine and serotonin. We focus on the latest advances in near infrared fluorescent carbon nanotube‐based sensors and engineered fluorescent proteins for monoamine imaging, which provide high spatial and temporal resolution suitable for examining the release of monoamines from cells in cellular networks.

The neurotransmitter dopamine is released from discrete axonal structures called varicosities. Its release is essential in behaviour and is critically implicated in prevalent neuropsychiatric diseases. Existing dopamine detection methods are not able to detect and distinguish discrete dopamine release events from multiple varicosities. This prevents an understanding of how dopamine release is regulated across populations of discrete varicosities. Using a near infrared fluorescent (980 nm) dopamine nanosensor 'paint' (AndromeDA), we show that action potential-evoked dopamine release is highly heterogeneous across release sites and also requires molecular priming. Using AndromeDA, we visualize dopamine release at up to 100 dopaminergic varicosities simultaneously within a single imaging field with high temporal resolution (15 images/s). We find that 'hotspots' of dopamine release are highly heterogeneous and are detected at only ~17% of all varicosities. In neurons lacking Munc13 proteins, which prime synaptic vesicles, dopamine release is abolished during electrical stimulation, demonstrating that dopamine release requires vesicle priming. In summary, AndromeDA reveals the spatiotemporal organization of dopamine release.