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Key points Employing quantitative Na+‐imaging and Förster resonance energy transfer‐based imaging with ATeam1.03YEMK (ATeam), we studied the relation between activity‐induced Na+ influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeam in situ enabled a quantitative estimate of changes in intracellular ATP concentrations. Different paradigms of stimulation that induced global Na+ influx into the entire neuron resulted in decreases in [ATP] in the range of 0.1–0.6 mm in somata and dendrites, while Na+ influx that was locally restricted to parts of dendrites did not evoke a detectable change in dendritic [ATP]. Our data suggest that global Na+ transients require global cellular activation of the Na+/K+‐ATPase resulting in a consumption of ATP that transiently overrides its production. For recovery from locally restricted Na+ influx, ATP production as well as fast intracellular diffusion of ATP and Na+ might prevent a local drop in [ATP]. Abstract Excitatory neuronal activity results in the influx of Na+ through voltage‐ and ligand‐gated channels. Recovery from accompanying increases in intracellular Na+ concentrations ([Na+]i) is mainly mediated by the Na+/K+‐ATPase (NKA) and is one of the major energy‐consuming processes in the brain. Here, we analysed the relation between different patterns of activity‐induced [Na+]i signalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na+ imaging with sodium‐binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03YEMK (ATeam). In situ calibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]i) with an apparent KD of 2.6 mm, indicating its suitability for [ATP]i measurement. Induction of recurrent network activity resulted in global [Na+]i oscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]i by 0.3–0.4 mm in both compartments. Global [Na+]i transients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]i as well. Brief focal glutamate application that evoked transient local Na+ influx into a dendrite, however, did not result in a measurable reduction in [ATP]i. Our results suggest that ATP consumption by the NKA following global [Na+]i transients temporarily overrides its availability, causing a decrease in [ATP]i. Locally restricted Na+ transients, however, do not result in detectable changes in local [ATP]i, suggesting that ATP production, together with rapid intracellular diffusion of both ATP and Na+ from and to unstimulated neighbouring regions, counteracts a local energy shortage under these conditions.

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K 1 clearance relies on the K 1 uptake by astrocytes and the subsequent diffusion of K 1 through the astrocyte network. When astrocytes become uncoupled, K 1 clearance becomes hindered. Accumulation of extracellular K 1 leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K 1 transients in response to glutamate became prolonged. Na 1 imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E 2 GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na 1 /HCO 3 cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.

Fluorescence imaging is an indispensable method for analysis of diverse cellular and molecular processes, enabling, for example, detection of ions, second messengers, or metabolites. Intensity-based approaches, however, are prone to artifacts introduced by changes in fluorophore concentrations. This drawback can be overcome by fluorescence lifetime imaging (FLIM) based on time-correlated single-photon counting. FLIM often necessitates long photon collection times, resulting in strong temporal binning of dynamic processes. Recently, rapidFLIM was introduced, exploiting ultra-low dead-time photodetectors together with rapid electronics. Here, we demonstrate the applicability of rapidFLIM, combined with new and improved correction schemes, for spatiotemporal fluorescence lifetime imaging of low-emission fluorophores in a biological system. Using tissue slices of hippocampi of mice of either sex, loaded with the Na 1 indicator ING2, we show that improved rapidFLIM enables quantitative, dynamic imaging of neuronal Na 1 signals at a full-frame temporal resolution of 0.5 Hz. Induction of transient chemical ischemia resulted in unexpectedly large Na 1 influx, accompanied by considerable cell swelling. Both Na 1 loading and cell swelling were dampened on inhibition of TRPV4 channels. Together, rapidFLIM enabled the spatiotemporal visualization and quantification of neuronal Na 1 transients at unprecedented speed and independent from changes in cell volume. Moreover, our experiments identified TRPV4 channels as hitherto unappreciated contributors to neuronal Na 1 loading on metabolic failure, suggesting this pathway as a possible target to ameliorate excitotoxic damage. Finally, rapidFLIM will allow faster and more sensitive detection of a wide range of dynamic signals with other FLIM probes, most notably those with intrinsic low-photon emission.

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