The ATP:ADP ratio is a critical parameter of cellular energy status that regulates many metabolic activities. Here we report an optimized genetically encoded fluorescent biosensor, PercevalHR, that senses the ATP:ADP ratio. PercevalHR is tuned to the range of intracellular ATP:ADP expected in mammalian cells, and it can be used with one-or two-photon microscopy in live samples. We use PercevalHR to visualize activity-dependent changes in ATP:ADP when neurons are exposed to multiple stimuli, demonstrating that it is a sensitive reporter of physiological changes in energy consumption and production. We also use PercevalHR to visualize intracellular ATP:ADP while simultaneously recording currents from ATP-sensitive potassium (K ATP ) channels in single cells, showing that PercevalHR enables the study of coordinated variation in ATP:ADP and K ATP channel open probability in intact cells. With its ability to monitor changes in cellular energetics within seconds, PercevalHR should be a versatile tool for metabolic research.

Summary Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phospho-regulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive KATP channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the KATP channel, implicating the BAD-KATP axis in metabolic control of neuronal excitation and seizure responses.

ATP-sensitive potassium channels (KATP channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in non-glucosensing central neurons is less well understood. To examine a possible role for KATP channels in modulating excitability in hippocampal circuits, we recorded the activity of single KATP channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (popen) of single KATP channels over repeated trials of stimulated spike activity showed a transient increase in popen in response to action potential firing. Channel currents were identified as KATP channels through blockade with glibenclamide, and by comparison with recordings from Kir6.2 knockout mice. The transient elevation in KATP popen may arise from submembrane ATP depletion by the Na+-K+ ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated popen of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect KATP activity. We also found using perforated-patch recording that KATP channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of KATP channels may help granule cells act as a “seizure gate” in the hippocampus, and that ketone body mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

When neurons engage in intense periods of activity, the consequent increase in energy demand can be met by the coordinated activation of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. However, the trigger for glycolytic activation is unknown and the role for Ca2+ in the mitochondrial responses has been debated. Using genetically encoded fluorescent biosensors and NAD(P)H autofluorescence imaging in acute hippocampal slices, here we find that Ca2+ uptake into the mitochondria is responsible for the buildup of mitochondrial NADH, probably through Ca2+ activation of dehydrogenases in the TCA cycle. In the cytosol, we do not observe a role for the Ca2+/calmodulin signaling pathway, or AMPK, in mediating the rise in glycolytic NADH in response to acute stimulation. Aerobic glycolysis in neurons is triggered mainly by the energy demand resulting from either Na+ or Ca2+ extrusion, and in mouse dentate granule cells, Ca2+ creates the majority of this demand.