Recent evidence indicates that the concentration of ATP remains stable during neuronal activity due to activity-dependent ATP production. However, the mechanisms of activity-dependent ATP production remain controversial. To stabilize the ATP concentration, feedforward mechanisms, which may rely on calcium or the sodium-potassium pump, do not require changes in the ATP and ADP concentrations. On the other hand, feedback mechanisms could be triggered by changes in the concentration of the adenine nucleotides. To test the possibility of feedback mechanisms, we quantified the ATP concentration in presynaptic terminals during synaptic activity in acute brain slices from mice stably expressing a genetically encoded ATP sensor. We first focused on the cerebellar mossy fiber bouton (cMFB) as a large presynaptic terminal that is specialized for high-frequency synaptic transmission. At physiological temperature and metabolite concentrations, the resting ATP concentration was in the range of approximately 2.5–2.7 mM. During strong, presumably non-physiological activity, the ATP concentration decreased within a few seconds. Experiments with blockade of ATP production indicated that ATP production increased ~10-fold during neuronal activity. Weaker stimulation resembling physiological activity at this synapse caused a decrease in ATP concentration by ~150 μM. We found similar results with in vivo-recorded spike sequences at the calyx of Held, another central glutamatergic synapse tuned for high-frequency synaptic activity. At conventional small synapses of cultured hippocampal neurons, weak stimulations also caused a decrease in ATP concentrations. Finally, quantitative modeling indicated that a pure ADP-based feedback mechanism can explain the activity-dependent ATP production when assuming a three-times higher maximal rate of ATP production compared to our measured rate of ATP production during high-frequency transmission. Our data reveal ATP reduction in presynaptic terminals during physiological-like activity, provide quantitative constraints on feedback mechanisms, and suggest that the ATP concentration can decrease during signaling, at least in some neuronal compartments of our brain
