The same molecule can play different roles inside and outside a cell. Adenosine connects energy metabolism with intercellular communication, yet research has more readily tracked its extracellular changes. A study published on May 7, 2025 in Nature Communications develops the green fluorescent sensor HypnoS to look inside cells. It reveals adenosine waves following calcium waves in a seizure model, different signal-decay rates in neurons and astrocytes, and sleep-related intracellular dynamics more varied than a simple steady accumulation.

01 / Looking inside

An extracellular message can begin inside

Adenosine sits at the intersection of metabolism and communication. Extracellular adenosine can arise from extracellular ATP breakdown or from intracellular production followed by membrane transport. Measuring homogenized tissue mixes cell identity and molecular location, obscuring who produces or receives the signal and where changes begin.

HypnoS links an adenosine-sensing protein to a green fluorescent protein, changing fluorescence when the molecule binds. Targeting the sensor to a particular cell type lets researchers observe intracellular adenosine in that population. Processes previously blended into tissue averages become distinct cellular stories.

Original figure 1Turning adenosine binding into fluorescence

02 / Making changes visible

Fast molecular sensing, distinct cellular timing

In purified-protein tests, HypnoS shows a fluorescence-response increase exceeding ninefold, with subsecond binding and dissociation responses. Tested molecules including ATP, ADP, AMP and inosine do not evoke the same response. The sensor also lacks the adenosine-deaminase activity of its original sensing protein, so it does not directly catalyze adenosine breakdown as that enzyme does.

The sensor reacts quickly to molecules, while actual intracellular changes can last tens of seconds or longer. A fluorescent control that does not respond to adenosine helps identify molecule-associated changes. In cultured cells, intracellular adenosine can also be compared with changes in energy state.

Original figure 2Energy state and intracellular adenosine

03 / Different cellular rhythms

A calcium rise need not mean an adenosine rise

When cultured neuronal metabolism is perturbed or activity increases, intracellular adenosine rises as the ATP/ADP ratio falls. Astrocytic changes are smaller under corresponding conditions; some stimuli evoke clear calcium responses without a corresponding adenosine increase. The two molecules track different processes, and the two cell types respond differently metabolically.

The link between activity and adenosine extends to mouse brain slices and living flies. Increasing activity in mushroom-body Kenyon cells raises intracellular adenosine signals, with stronger activation producing larger responses. Across cultures, tissue and intact animals, the sensor follows activity-associated changes.

Original figure 4Following activity-associated changes in living flies

04 / Waves in sequence

Adenosine waves follow calcium waves

In a chemically induced mouse seizure model, cortical calcium and intracellular adenosine are recorded together. The waves begin at approximately the same location and propagate in similar directions, but the adenosine wave follows calcium by about twenty seconds and decays more slowly, making their temporal relationship visible.

Hippocampal recordings in freely moving mice also capture seizure-associated adenosine increases, appearing near seizure termination in EEG recordings. During successive seizures, the second response is smaller. Adenosine dynamics vary with events in their onset, delay and duration, rather than simply rising at a single fixed moment.

Original figure 5Timing of cortical calcium and adenosine waves

05 / One event, different cells

Astrocytic signals recede faster

At single-cell resolution, both neurons and astrocytes show seizure-associated adenosine increases, but astrocytic signals decay faster. The same brain state does not produce identical dynamics in every cell: cell identity influences how long the intracellular signal persists.

Faster decay is consistent with differences in adenosine metabolism between the two cell types. It also illustrates why a tissue total cannot describe the full process: at one moment, signals have receded in some cells while remaining elevated in others, a timing difference that an average can conceal.

Original figure 6Different decay rates in two cell types

06 / Fluctuations during sleep

Intracellular signals are higher in wakefulness and REM

Adenosine is often linked to sleep pressure after wakefulness, but intracellular signals in the basal forebrain are more complex than high while awake and low while asleep. HypnoS recordings show higher signals during wakefulness and REM than during NREM, with changes unfolding over tens of seconds around state transitions.

Although REM is sleep, its molecular state differs from NREM. Intracellular adenosine dynamically tracks brain state, emphasizing the value of distinguishing internal metabolic signals from extracellular communication when asking how each participates in sleep.

Original figure 8Sleep states and intracellular adenosine

07 / Across the membrane

Reduced transport, opposite intracellular changes

ENT1/2 are important adenosine membrane transporters. In mice with impaired transporter function involving the basal forebrain, neuronal intracellular signals increase during wakefulness and REM, with no clear NREM difference. Astrocytic signals decrease in all three states. The shared transporter perturbation produces different net effects in the two cell types.

Cultured neuronal experiments further show that reducing transport weakens responses to external adenosine while increasing intracellular accumulation during activity or metabolic perturbation. Membrane traffic and cellular metabolism jointly shape the molecule’s distribution. Fluorescence also depends on measurement conditions; HypnoS provides a spatially resolved window that turns an adenosine increase into a question of which cells and when.

Original figure 10Cell-type differences after transporter perturbation