Key points: Employing quantitative Na⁺‐imaging and Förster resonance energy transfer‐based imaging with ATeam1.03^YEMK(ATeam), we studied the relation between activity‐induced Na⁺influx and intracellular ATP in CA1 pyramidal neurons of the mouse hippocampus. Calibration of ATeamin situenabled 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 mmin 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]. 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⁺]_isignalling and ATP in mouse hippocampal CA1 pyramidal neurons by Na⁺imaging with sodium‐binding benzofurane isophthalate (SBFI) and employing the genetically encoded nanosensor ATeam1.03^YEMK(ATeam).In situcalibrations demonstrated a sigmoidal dependence of the ATeam Förster resonance energy transfer ratio on the intracellular ATP concentration ([ATP]_i) with an apparentK_Dof 2.6 mm, indicating its suitability for [ATP]_imeasurement. Induction of recurrent network activity resulted in global [Na⁺]_ioscillations with amplitudes of ∼10 mm, encompassing somata and dendrites. These were accompanied by a steady decline in [ATP]_iby 0.3–0.4 mmin both compartments. Global [Na⁺]_itransients, induced by afferent fibre stimulation or bath application of glutamate, caused delayed, transient decreases in [ATP]_ias well. Brief focal glutamate application that evoked transientlocalNa⁺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⁺]_itransients 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⁺clearance relies on the K⁺uptake by astrocytes and the subsequent diffusion of K⁺through the astrocyte network. When astrocytes become uncoupled, K⁺clearance becomes hindered. Accumulation of extracellular K⁺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⁺transients in response to glutamate became prolonged. Na⁺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-E²GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na⁺/HCO₃^–cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification bothin vitroandin vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENTWe aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K⁺from the extracellular space was impaired. Epileptiform activity activated inward Na⁺/HCO₃^–cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K⁺clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.

Fluorescence lifetime imaging microscopy (FLIM) with fluorescent ion sensors enables the measurement of ion concentrations based on the detection of photon emission events after brief excitation with a pulsed laser source. In contrast to intensity-based imaging, it is independent of dye concentration, photobleaching, or focus drift and has thus been successfully employed for quantitative analysis of, e.g., calcium levels in different cell types and cellular microdomains. Here, we tested the suitability of CoroNaGreen for FLIM-based determination of sodium concentration ([Na⁺]) inside cells. In vitro measurements confirmed that fluorescence lifetimes of CoroNaGreen (CoroNaFL) increased with increasing [Na⁺]. Moreover, CoroNaFL was largely independent of changes in potassium concentration or viscosity. Changes in pH slightly affected FL in the acidic range (pH ≤ 5.5). For intracellular determination of [Na⁺], HEK293T cells were loaded with the membrane-permeable form of CoroNaGreen. Fluorescence decay curves of CoroNaGreen, derived from time-correlated single-photon counting, were approximated by a bi-exponential decay. In situ calibrations revealed a sigmoidal dependence of CoroNaFL on [Na⁺] between 0 and 150 mM, exhibiting an apparent K_d of ∼80 mM. Based on these calibrations, a [Na⁺] of 17.6 mM was determined in the cytosol. Cellular nuclei showed a significantly lower [Na⁺] of 13.0 mM, whereas [Na⁺] in perinuclear regions was significantly higher (26.5 mM). Metabolic inhibition or blocking the Na⁺/K⁺-ATPase by removal of extracellular K⁺ caused significant [Na⁺] increases in all cellular subcompartments. Using an alternative approach for data analysis (“Ratio FLIM”) increased the temporal resolution and revealed a sequential response to K⁺ removal, with cytosolic [Na⁺] increasing first, followed by the nucleus and finally the perinuclear regions. Taken together, our results show that CoroNaGreen is suitable for dynamic, FLIM-based determination of intracellular [Na⁺]. This approach thus represents a valuable tool for quantitative determination of [Na⁺] and changes thereof in different subcellular compartments.

With the recent surge in the development of highly selective probes, fluorescence microscopy has become one of the most widely used approaches to studying cellular properties and signaling in living cells and tissues. Traditionally, microscopy image analysis heavily relies on manufacturer-supplied software, which often demands extensive training and lacks automation capabilities for handling diverse datasets. A critical challenge arises if the fluorophores employed exhibit low brightness and a low signal-to-noise ratio (SNR). Consequently, manual intervention may become a necessity, introducing variability in the analysis outcomes even for identical samples when analyzed by different users. This leads to the incorporation of blinded analysis, which ensures that the outcome is free from user bias to a certain extent but is extremely time-consuming. To overcome these issues, we developed a tool called DL-SCAN that automatically segments and analyzes fluorophore-stained regions of interest such as cell bodies in fluorescence microscopy images using deep learning. We demonstrate the program’s ability to automate cell identification and study cellular ion dynamics using synthetic image stacks with varying SNR. This is followed by its application to experimental Na⁺ and Ca²⁺ imaging data from neurons and astrocytes in mouse brain tissue slices exposed to transient chemical ischemia. The results from DL-SCAN are consistent, reproducible, and free from user bias, allowing efficient and rapid analysis of experimental data in an objective manner. The open-source nature of the tool also provides room for modification and extension to analyze other forms of microscopy images specific to the dynamics of different ions in other cell types.

Astrocytes maintain extracellular ion and transmitter homeostasis, with the Na⁺ inward gradient playing a crucial role. Earlier studies suggested a rather low, uniform Na⁺ distribution in astrocytes, consistent with the view that these basic homeostatic properties are well-protected. Here, we employed multi-photon fluorescence lifetime imaging to quantitatively determine astrocytic [Na ⁺ ] in mouse brain tissue slices and in vivo. Our data reveals a significant subcellular and cellular heterogeneity in astrocytic [Na ⁺ ], accompanied by differences in the capacity for Na ⁺ /K ⁺ -ATPase (NKA)-mediated uptake of extracellular K ⁺ . RNAscope and immunohistochemistry indicate differential spatial expression patterns of NKA ß1 and ß2 subunits in astrocytes. Biophysical modeling of differential NKA expression together with varying strength of Na ⁺ influx replicate the experimentally observed heterogeneity in astrocytic [Na ⁺ ]. Altogether, our results suggest the existence of functionally distinct astrocytes and astrocyte subdomains in which Na ⁺ homeostasis is locally adapted to the specific requirements of surrounding neural networks.

Sodium ions (Na ⁺ ) are major charge carriers mediating neuronal excitation and play a fundamental role in brain physiology. Glutamatergic synaptic activity is accompanied by large transient Na ⁺ increases, but the spatiotemporal dynamics of Na ⁺ signals and properties of Na ⁺ diffusion within dendrites are largely unknown. To address these questions, we employed multiphoton Na ⁺ imaging combined with whole-cell patch clamp in dendrites of CA1 pyramidal neurons in tissue slices from mice of both sexes. Fluorescence lifetime microscopy revealed a dendritic baseline Na ⁺ concentration of ∼10 mM. Using intensity-based line scan imaging, we found that local, glutamate-evoked Na ⁺ signals spread rapidly within dendrites, with peak amplitudes decreasing and latencies increasing with increasing distance from the site of stimulation. Spread of Na ⁺ along dendrites was independent of dendrite diameter, order, or overall spine density in the ranges measured. Our experiments also show that dendritic Na ⁺ readily invades spines and suggest that spine necks may represent a partial diffusion barrier. Experimental data were well reproduced by mathematical simulations assuming normal diffusion with a diffusion coefficient of D _Na+ = 600 µm ² /s. Modeling moreover revealed that lateral diffusion is key for the clearance of local Na ⁺ increases at early time points, whereas when diffusional gradients are diminished, Na ⁺ /K ⁺ -ATPase becomes more relevant. Taken together, our study thus demonstrates that Na ⁺ influx causes rapid lateral diffusion of Na ⁺ within spiny dendrites. This results in an efficient redistribution and fast recovery from local Na ⁺ transients which is mainly governed by concentration differences.