Adenosine 5’ triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF₁-ATP synthase ε subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.

Body weight is regulated by interoceptive neural circuits that track energy need, but how the activity of these circuits is altered in obesity remains poorly understood. Here we describe the in vivo dynamics of hunger-promoting AgRP neurons during the development of diet-induced obesity in mice. We show that high-fat diet attenuates the response of AgRP neurons to an array of nutritionally-relevant stimuli including food cues, intragastric nutrients, cholecystokinin and ghrelin. These alterations are specific to dietary fat but not carbohydrate or protein. Subsequent weight loss restores the responsiveness of AgRP neurons to exterosensory cues but fails to rescue their sensitivity to gastrointestinal hormones or nutrients. These findings reveal that obesity triggers broad dysregulation of hypothalamic hunger neurons that is incompletely reversed by weight loss and may contribute to the difficulty of maintaining a reduced weight.