Adenosine 5′ triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Learning and memory formation involve long-term potentiation (LTP) of synaptic strength. A fundamental feature of LTP induction in the brain is the need for coincident pre- and postsynaptic activity. This restricts LTP expression to activated synapses only (homosynaptic LTP) and leads to its input specificity. In the spinal cord, we discovered a fundamentally different form of LTP that is induced by glial cell activation and mediated by diffusible, extracellular messengers, including d-serine and tumor necrosis factor (TNF), and that travel long distances via the cerebrospinal fluid, thereby affecting susceptible synapses at remote sites. The properties of this gliogenic LTP resolve unexplained findings of memory traces in nociceptive pathways and may underlie forms of widespread pain hypersensitivity.

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source 1 and an evolutionarily ancient 2 extracellular signal 3-5 . Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR 1.0 responded to relevant ATP concentrations (30 µM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. iATPSnFRs represent promising new reagents for imaging ATP dynamics.Given widespread roles in energy homeostasis and cell signaling 3-5 , several methods have been used to detect ATP using small-molecule chemical approaches 6 , firefly luciferase 7,8 , ion channel-expressing "sniffer" cells 9-11 , voltammetry 12 , and different types of microelectrodes 13,14 . However, these methods lack spatial resolution, cannot be easily used in tissue slices or in vivo, respond to off-target ligands, need unwieldy photon-counting cameras, are imprecise and/or unavoidably damage tissue during probe placement. Importantly, none of these methods can be genetically targeted to specific cells, sub-cellular compartments or whole organisms, and they all lack cellular-scale spatial resolution. Although luciferase is genetically encoded 15 , it requires the exogenous substrate luciferin, addition of which complicates use in tissue slices and in vivo. It also saturates at nanomolar ATP, far lower than concentrations expected during ATP signaling (~1 µM to ~1 mM). Additionally, luciferase's bioluminescent output yields low photon fluxes and rules out cellular-resolution imaging. To address these issues, genetically-encoded fluorescent ATP sensors have been developed, including Perceval and PercevalHR 16,17 , ATeam 18 , and QUEEN 19 . These sensors are valuable, but they have limitations. They either respond to ADP/ATP ratio instead of ATP concentration or are susceptible to optical overlap with cellular sources of auto-fluorescence, and all are incompatible with single-wavelength fluorescence imaging, the workhorse of functional fluorescence microscopy 20 .Our goals were to: 1) develop an ATP sensor that could be used in routine fluorescence imaging, e.g. at GFP's 488 nm excitation and ~525 nm emission and 2) deploy such a sensor on the cell surface and within cells to image ATP. QUEEN is an excitation ratio sensor, ATeam is a fluorescence resonance energy transfer (FRET) sensor, and the Perceval sensors are fluorescence lifetime indicators -all of these methods require customized equipment, substantially slow down imaging rates, complicate use with other reagents, and typically have lower signal-to-noise ratio (SNR)...

Astrocytes are indispensable for proper neuronal functioning. Given the diverse needs of neuronal circuits and the variety of tasks astrocytes perform, the perceived homogeneous nature of astrocytes has been questioned. In the spinal dorsal horn, complex neuronal circuitries regulate the integration of sensory information of different modalities. The dorsal horn is organized in a distinct laminar manner based on termination patterns of high‐ and low‐threshold afferent fibers and neuronal properties. Neurons in laminae I (L1) and II (L2) integrate potentially painful, nociceptive information, whereas neurons in lamina III (L3) and deeper laminae integrate innocuous, tactile information from the periphery. Sensory information is also integrated by an uncharacterized network of astrocytes. How these lamina‐specific characteristics of neuronal circuits of the dorsal horn are of functional importance for properties of astrocytes is currently unknown. We addressed if astrocytes in L1, L2, and L3 of the upper dorsal horn of mice are differentially equipped for the needs of neuronal circuits that process sensory information of different modalities. We found that astrocytes in L1 and L2 were characterized by a higher density, higher expression of GFAP, Cx43, and GLAST and a faster coupling speed than astrocytes located in L3. L1 astrocytes were more responsive to Kir4.1 blockade and had higher levels of AQP4 compared to L3 astrocytes. In contrast, basic membrane properties, network formation, and somatic intracellular calcium signaling were similar in L1–L3 astrocytes. Our data indicate that the properties of spinal astrocytes are fine‐tuned for the integration of nociceptive versus tactile information.