Tight Coupling of Astrocyte pH Dynamics to Epileptiform Activity Revealed by Genetically Encoded pH Sensors

Abstract

Astrocytes can both sense and shape the evolution of neuronal network activity and are known to possess unique ion regulatory mechanisms. Here we explore the relationship between astrocytic intracellular pH dynamics and the synchronous network activity that occurs during seizure-like activity. By combining confocal and two-photon imaging of genetically encoded pH reporters with simultaneous electrophysiological recordings, we perform pH measurements in defined cell populations and relate these to ongoing network activity. This approach reveals marked differences in the intracellular pH dynamics between hippocampal astrocytes and neighboring pyramidal neurons in rodent in vitro models of epilepsy. With three different genetically encoded pH reporters, astrocytes are observed to alkalinize during epileptiform activity, whereas neurons are observed to acidify. In addition to the direction of pH change, the kinetics of epileptiform-associated intracellular pH transients are found to differ between the two cell types, with astrocytes displaying significantly more rapid changes in pH. The astrocytic alkalinization is shown to be highly correlated with astrocytic membrane potential changes during seizure-like events and mediated by an electrogenic Na(+)/HCO3 (-) cotransporter. Finally, comparisons across different cell-pair combinations reveal that astrocytic pH dynamics are more closely related to network activity than are neuronal pH dynamics. This work demonstrates that astrocytes exhibit distinct pH dynamics during periods of epileptiform activity, which has relevance to multiple processes including neurometabolic coupling and the control of network excitability.

Significance statement: Dynamic changes in intracellular ion concentrations are central to the initiation and progression of epileptic seizures. However, it is not known how changes in intracellular H(+) concentration (ie, pH) differ between different cell types during seizures. Using recently developed pH-sensitive proteins, we demonstrate that astrocytes undergo rapid alkalinization during periods of seizure-like activity, which is in stark contrast to the acidification that occurs in neighboring neurons. Rapid astrocytic pH changes are highly temporally correlated with seizure activity, are mediated by an electrogenic Na(+)/HCO3- cotransporter, and are more tightly coupled to network activity than are neuronal pH changes. As pH has profound effects on signaling in the nervous system, this work has implications for our understanding of seizure dynamics.

Keywords: astrocytes; epileptiform activity; intracellular pH; seizures.

Figure 1.

Astrocytes experience rapid intracellular alkalinization during epileptiform activity. A, Schematic (left) showing the experimental setup in which a hippocampal pyramidal neuron expressing a genetically encoded pH reporter was imaged, while a simultaneous patch-clamp recording was performed from a neighboring neuron. Dynamic intracellular pH measurements from a representative CA3 pyramidal neuron expressing E²GFP (right, blue trace) and current-clamp recording from a neighboring pyramidal neuron (black trace; somata <200 μm apart), which provided a readout of the epileptiform activity in Mg²⁺-free ACSF. Acidic intracellular pH shifts were closely associated with SLEs (onset indicated by vertical dashed lines). B, Schematic (left) showing a similar setup as in A for the measurement of pH dynamics in hippocampal astrocytes during SLEs. Intracellular pH measurements made from a representative astrocyte expressing E²GFP (right, red trace, same astrocyte as in C) and current-clamp recording from a neighboring pyramidal neuron (black trace), which provided a readout of the epileptiform activity in Mg²⁺-free ACSF. Individual SLEs of different durations are closely associated with rapid intracellular alkaline transients in the astrocyte. C, Confocal image (top) of an astrocyte expressing E²GFP. After performing pH imaging (shown in B), this astrocyte was targeted for whole-cell patch-clamp recording and confirmed to exhibit a low membrane resistance (Rm = 20.5 MΩ) and lack voltage-gated conductances in response to current injection (inset). Confocal image (bottom) of a neuron expressing E²GFP, which exhibited action potentials in response to current injection (inset). D, Population data demonstrating a significant difference in pre-SLE intracellular pH between hippocampal neurons and astrocytes. ***p < 0.0001, t test. E, Population data showing the peak shift in intracellular pH as a function of the duration of the epileptiform activity (data from 23 neurons, blue, and 24 astrocytes, red). For neurons, the peak change in pH was negatively correlated with the duration of the SLE (r = −0.6719, p < 0.0001, Pearson correlation). For astrocytes, the peak pH shift was positively correlated with the length of the SLE (r = 0.5766, p < 0.0001, Pearson correlation).

Figure 2.

Multiple genetically encoded pH sensors reveal astrocytic alkalinization during epileptiform activity. A, Calibration curve relating the fluorescence ratio of DeGFP4-expressing cells to their intracellular pH. This revealed the reporter to have a pKa of 7.4. Similarly, calibration of E²GFP (B) and Cl-sensor (C) revealed a pKa of 7.5 and 7.7 for these genetically encoded pH sensors, respectively. D, An astrocyte expressing the pH sensor DeGFP4 was imaged for pH (red trace), while 0 Mg²⁺ induced epileptiform activity was monitored via a simultaneous current-clamp recording from a nearby pyramidal neuron (black trace). Epileptiform activity was associated with a pronounced increase in the astrocyte's pH. E, pH imaging from an astrocyte expressing the pH sensor E²GFP revealed intracellular alkalinization (red trace) during periods of epileptiform activity induced with 0 Cl⁻ ACSF (black trace). F, pH imaging from an astrocyte expressing the pH-sensitive Cl-sensor also reveals intracellular alkalinization (red trace) during periods of epileptiform activity induced with 0 Cl⁻ ACSF (black trace).

Figure 3.

Cell-type-specific changes in pH during epileptiform activity. A, Astrocytes (red) and neurons (blue) imaged with the pH sensor E²GFP, showed opposing pH transients during 0 Mg²⁺ induced SLEs (20–40 s duration). Neurons exhibited transient acidic shifts, whereas astrocytes exhibited transient alkaline shifts during periods of epileptiform activity. Similar differences were observed when intracellular pH was imaged with E²GFP during 0 Cl⁻-induced SLEs (B), or DeGFP4 during 0 Mg²⁺-induced SLEs (C), or Cl-sensor during 0 Cl⁻-induced SLEs (D). ***p < 0.0001, t test.

Figure 4.

Alkaline transients in astrocytes during epileptiform activity are mediated by depolarization and the sodium-bicarbonate cotransporter NBCe1. A, Dual whole-cell current-clamp recording from a pair of CA3 pyramidal neurons (somata <200 μm apart) during 0 Mg²⁺-induced epileptiform activity. Dashed line indicates onset of a SLE. The membrane potential of the two neurons was highly synchronous, as indicated by the tight correlation in the cells' membrane potentials (r = 0.92, Pearson correlation). Action potential generation in response to somatic current injection (insets) confirmed both cells were neuronal. B, Dual whole-cell current-clamp recording from a pyramidal neuron (black trace) and a nearby astrocyte (red trace; somata <200 μm apart). Note that the astrocytic membrane potential depolarization during the SLE is tightly correlated with neuronal membrane potential (r = 0.82, Pearson correlation). Response to somatic current injection confirmed the identity of the cells (insets). C, Population data revealed a high correlation in the membrane potential between pyramidal neurons (r = 0.91 ± 0.01, Pearson correlation), and between pyramidal neurons and astrocytes (r = 0.80 ± 0.02, Pearson correlation) during SLEs. D, Under control conditions intracellular pH imaging with E²GFP (red trace) revealed transient alkalinizations that were associated with 0 Mg²⁺-induced epileptiform activity, as monitored from a nearby pyramidal neuron (black trace). E, Bath application of the selective Na⁺/HCO₃⁻ cotransporter blocker S0859 (50 μm) to the same cells as in D, resulted in a marked attenuation of the astrocytic alkalinizations. F, Population data demonstrate that S0859 blocks the astrocytic alkalinizations and eliminates the correlation between SLE duration and the size of the alkaline shift (r = 0.4289, p = 0.08 vs r = 0.69, p < 0.0001, Pearson correlation). G, For SLEs of comparable duration (<35 s duration), S0859 significantly reduced the peak change in astrocytic pH_i associated with 0 Mg²⁺-induced SLEs (left) and 0 Cl⁻-induced SLEs (center). Low HCO₃⁻ ACSF also reduced SLE-associated astrocytic alkaline shifts in 0 Cl⁻ ACSF (right). *p < 0.05, **p < 0.01, ***p < 0.0001, t test.

Figure 5.

The kinetics of pH transients differ between astrocytes and neurons during epileptiform activity. A, A confocal image of a CA3 pyramidal neuron and neighboring astrocyte both expressing E²GFP. The expanded region shows a magnified view of the astrocyte and adjacent apical dendrite of the pyramidal neuron. Dashed boxes indicate regions of interest used for pH measurements. B, Current-clamp recording of a nearby neuron (somata <200 μm apart) provides a readout of 0 Mg²⁺-induced epileptiform activity (black trace). Simultaneous pH imaging from the regions of interest indicated by the dashed lines in A. The astrocyte (red) exhibited a transient increase in pH that was closely aligned with the SLE (gray bar), while the neuron (blue) exhibited a more prolonged decrease in pH. The arrowheads indicate the point of maximal pH shift (filled arrowheads) and the point at which pH recovered to baseline (empty arrowheads). C, Representative changes in intracellular pH during 0 Mg²⁺-induced epileptiform activity in an astrocyte (red traces) and a CA3 pyramidal neuron (blue traces) within the same hippocampal slice. The astrocyte exhibited a more rapid alkalinization, which peaked and then recovered more quickly than the more sustained acidification of the neuron. D, Similar differences in the kinetics of cell-specific pH shifts were observed when epileptiform activity was induced with 0 Cl⁻ ACSF. E, Population data show that astrocytes reached their peak pH shift significantly faster than neurons in both 0 Mg²⁺ and 0 Cl⁻-induced epileptiform activity. F, The time for pH to recover to baseline was also significantly faster in astrocytes than neurons in both 0 Mg²⁺ and 0 Cl⁻-induced epileptiform activity. ***p < 0.0001, t test.

Figure 6.

Simultaneous recordings from different cell-pair combinations reveal that astrocytic pH dynamics closely reflect both astrocytic and neuronal membrane potential dynamics. A, Paired pH imaging (green trace) and whole-cell patch-clamp recording (black trace) from a pair of astrocytes in stratum radiatum of CA3 revealed a close correlation between changes in astrocytic pH and changes in astrocytic membrane potential during epileptiform activity (r = 0.72, Pearson correlation). B, A similar experiment on an astrocyte–neuron pair also revealed a high degree of correlation between changes in astrocytic pH and neuronal membrane potential (r = 0.49, Pearson correlation). C, In contrast, a similar experiment on a neuron–neuron pair revealed a much lower correlation between the change in neuronal pH and the change in neuronal membrane potential (r = 0.17, Pearson correlation).

Figure 7.

Astrocytic pH dynamics are more strongly coupled to network activity than neuronal pH dynamics. A, The absolute correlation between astrocytic pH and astrocytic membrane potential was highest (r = 0.71 ± 0.02), followed by the correlation between astrocytic pH and neuronal membrane potential (r = 0.43 ± 0.04), and the weakest correlation was observed between neuronal −pH and neuronal membrane potential (r = 0.15 ± 0.05). B, Cross-correlation analysis demonstrated that the maximum correlation between traces was observed when pH measurements were shifted with a negative lag, consistent with pH changes occurring in response to network activity. Shaded regions depict SEM. C, The highest cross-correlation values (calculated after shifting pH relative to membrane potential, as in B) were observed between astrocytic pH and astrocytic membrane potential (r = 0.77 ± 0.02). The next highest cross-correlation values were observed between astrocytic pH and neuronal membrane potential (r = 0.68 ± 0.02). These correlation values were both significantly greater than the cross-correlation values observed between neuronal −pH and neuronal membrane potential (r = 0.45 ± 0.04). *p < 0.05, ***p < 0.0001, t test.