Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl−]i) and hydrogen ([H+] i) the construct, called ClopHensor, which consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H+]i and [Cl−]i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H+]i and [Cl−]i under different experimental conditions including changing of external concentrations of ions (Ca2+, Cl−, K+, Na+) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl− and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl− and pH.


Introduction
Neuronal activity is accompanied by dynamical changes in the intra-and extracellular concentration of ions. A number of studies demonstrated that activation or inhibition of neurons can cause a rapid shift of hydrogen (H + ) and chloride (Cl − ) [1][2][3]. The physiological intracellular pH range in different eukaryotic cells is 6.5-8.0 [4], which corresponds to a very low free H + concentration, from 10 nM to 300 nM. In the mammalian brain the intracellular pH is 7.0-7.4 [5,6] reflecting the importance of maintaining the intracellular H + concentration ([H + ] i ) in a narrow range.
Intracellular Cl − concentration ([Cl − ] i ) in different cell types varies from 3 mM to 60 mM, being around 5-10 mM in the majority of mammalian neurons [7]. Deviations from this physiological range can alter the excitability of cells, modulate the function of a variety of proteins including ion channels [8][9][10]. Abnormal changes in concentrations of these ions are associated with the development of pathological processes and some disorders including neurodegeneration, epilepsy and brain ischemia [11][12][13].
To maintain H + and Cl − in physiological ranges, various transporters, cotransporters, and other ion regulating proteins are expressed in cells of biological organisms. The (1) Examples of fluorescence at different excitation wavelengths are shown in Figure 1C. Details of calibration are presented in Section 4.  In the present study, we recorded fluorescence at slightly different excitation wavelengths (455 nm, 505 nm and 590 nm) and estimated changes in concentrations of ions using the following ratios: R Cl = ∆F 590nm /∆F 455nm (2) Examples of fluorescence at different excitation wavelengths are shown in Figure 1C. Details of calibration are presented in Section 4.

Effect of Bicuculline on Synaptically Induced Cl − and pH-Transients in Hippocampal Neurons
To examine the properties of intracellular Cl − and H + transients during synaptic activation, we performed experiments on hippocampal slices of transgenic mice expressing ClopHensor. The first task was to establish how the changes in intracellular concentrations of Cl − and H + , evoked by electrical stimulation, are amenable to pharmacological modulation of synaptic transmission. Fluorescent emission signals were recorded from the hippocampal CA1 area upon sustained tetanic stimulation of Schaffer's collaterals (100 Hz for 20 s). Stimulus intensities ranged from 20 µA to 320 µA. The experiments were carried out on adult transgenic mice aged 2-8 months.
High-frequency stimulation of Schaffer's collaterals caused strong changes of fluorescence signals excited at 455 nm and 505 nm without effect on excitation at 590 nm (not shown). Ratiometric analysis revealed that these changes correspond to an elevation of both [Cl − ] i and [H + ] i . In this set of experiments, in hippocampal CA1 neurons, the base level of [Cl − ] i was 8.0 ± 1.1 mM (n = 5) and the mean intracellular pH was 7.30 ± 0.02 (n = 11). High frequency synaptic stimulation increased the [Cl − ] i to 0.8 ± 0.3 mM (n = 5) and acidification of neurons to 0.024 ± 0.006 units pH (n = 11).
In order to test the assumption that during synaptic stimulation, the accumulation of Cl − in the hippocampal neurons is, at least partially, due to Cl − influx via activated GABAA receptors, we applied bicuculline, an antagonist of these receptors [34]. The traces shown in Figure 2A illustrate that upon the synaptic stimulation, bicuculline strongly decreased the amplitude of synaptically induced changes of [Cl − ] i . On average, the mean amplitude of Cl − influx decreased to 41.2 ± 12.2% ( Figure 2B, n = 7, p < 0.01). In contrast, upon application of bicuculline the amplitude of synaptically induced pH transients increased by 90.7 ± 17.4% ( Figure 2C, n = 8, p < 0.01). Picrotoxin, a blocker of Cl − -selective GABA receptor channels, caused similar changes in the amplitudes of Cl − and pH transients (data not shown).
The augmentation of pH transients can be, at least partially, explained by the fact that bicuculline blocks inhibitory transmission, which leads to an increase in the general excitability [35]. To check the involvement of this mechanism in the potentiation of synaptically induced H + responses upon inhibition of GABA receptors, we analyzed the effect of bicuculline on the amplitude of evoked local field potentials (eLFP). After stimulation of Schaffer's collaterals, a population of CA1 neurons are activated simultaneously and fire an action potential in synchrony, giving rise to a single eLFP. As illustrated in Figure 2D, the addition of bicuculline (40 µM) led to an elevation of eLFPs. The mean potentiation was 24 ± 3.8% ( Figure 2E). Consequently, in the presence of bicuculline, the effectiveness of the stimulation increased, causing stronger depolarization and augmentation of pH transients. A competitive AMPA/kainate receptor antagonist (CNQX, 40 µm) completely abolished stimulation-induced changes of ratiometric emission signals, confirming that they have emerged from synaptic activation (not shown).
Altogether, these data demonstrate that: (i) bicuculline modulates in opposite ways the synaptically evoked Cl − -and pH-transients in neurons expressing ClopHensor; (ii) bicuculline only partially inhibits synaptically induced elevation of [Cl − ] i ; (iii) inhibition of GABA receptor by bicuculline causes potentiation of eLFPs.

Effect of Bicuculline on Intracellular pH Changes after Tetanic Stimulation, Assessed Using BCECF-AM
To ensure the specificity of the pH changes when using ClopHsensor, we performed a series of experiments with the pH-sensitive dye BCECF-AM [36]. This compound is well tolerated by cells and offers ratiometric estimation of pH values.
We determined the pH changes by calculating the ratio of two emission signals obtained after illumination by light at 455 nm and 505 nm. The intracellular pH changes after tetanic stimulation and its modulation by bicuculline were analyzed. Experiments were conducted on wild type juvenile mice (P10-P12). Registration conditions were similar to those performed on slices from transgenic ClopHensor mice. Examples of CA1 hippocampal neurons loaded with BCECF and excited at different wavelengths are presented in Figure 3A. Evoked pH-specific ratiometric emission signals were obtained by giving the same pattern of electrical stimulation (100 Hz for 20s); the stimulus intensities ranged from 200 µA to 250 µA. Similar to the observations with ClopHensor, on neurons loaded with BCECF-AM, the addition of bicuculline resulted in an increase of the pH transients evoked by tetanic stimulation ( Figure 3B). Summary data from 5 slices showed that upon addition of bicuculline, the mean amplitude of the pH signals increased to 208.6 ± 48.8% ( Figure 3C, n = 5, p < 0.05).
These results demonstrate that: (i) sustained high-frequency stimulation induces acidification of cells and bicuculline potentiates the amplitude of pH transients; (ii) pH-specific emission signals are similar at recording either with BCECF-AM or using ClopHensor.

Analysis of the Decay Kinetics of Cl − and pH Transients
We then performed a comparative analysis of the kinetics of synaptically induced Cl − and pH transients. It was evaluated by determining the decay time constants, i.e., the time during which the peak amplitude of Cl − and H + components decreases by e-times (τ decay ).
The τ decay of pH-fluorescent signals recorded from hippocampal slices of mice expressing ClopHensor was 157 ± 20.3 s, n = 16 ( Figure 4A,B). This was close to values obtained on slices from wild type mice loaded with BCECF (τ decay =188.6 ± 16.5 s, n = 14; Figure  4A,B). The concentration of intracellular Cl − ions was restored considerably faster than the pH after high-frequency stimulation of synaptic inputs ( Figure 4A, red trace). The mean τ decay of Cl − transients was 35 ± 4.2 s (n = 9, Figure 4B), i.e., about 4.5 times shorter than that of pH signals. These results suggest that the mechanisms involved in the withdrawing of ions from neurons operate much faster for Cl − than for H + and different mechanisms are involved in the maintenance of normal physiological transmembrane concentrations of these ions.
Next, we analyzed how changes of physiologically important ions, involved in regulation of Cl − and H + transporters, modulate the amplitude of Cl − and pH transients induced by depolarization.

Changes of [H + ] i -and [Cl − ] i -Induced by High [K + ] o -Depolarization
Several studies used [K + ] o -induced depolarization as a tool for stimulation of pH changes in different cell types [37][38][39][40]. We used this approach to analyze the properties of pH i and [Cl − ] i transients in neurons of hippocampal slices under different experimental conditions.
In all experiments, the high [K + ] o solution contained 20 mM, i.e., 17.5 mM of potassium gluconate was added to normal ASCF containing 2.5 mM K + . Depolarization induced by application of high [K + ] o ASCF caused a remarkable elevation of [Cl − ] i in all slices, with an average of 11.4 ± 1.1 mM (n = 11). Concerning pH, two types of responses were observed. In the majority of slices, [K + ] o -induced depolarization caused acidification of neurons by, on average, 0.15 ± 0.02 pH units (n = 23). In 8 cases, biphasic pH i -shifts composed of an early short-lasting alkalinisation that turned into a longer-lasting acidification were observed. We have not analyzed yet the reasons for this difference in the pH responses.

[K + ] o -Induced Changes of pH and Cl − in a Ca 2+ -Free ACSF
Observations of cells in culture conditions demonstrated that elevation of external K + causes strong depolarization of neurons, accompanied, in addition to acidification, by a rise in intracellular Ca 2+ [38,39]. In these studies, acidic [K + ] o -induced pH responses were either completely prevented by the use of a Ca 2+ -free medium [38], or even inversed and became alkalizing [39], suggesting a key role of Ca 2+ ions in depolarization-induced pH changes.
We performed an analysis of the removal of external Ca 2+ on depolarization-induced transients of [Cl − ] i and pH i in brain slices of ClopHensor mice. Under control conditions, when high K + was applied to slices for 3-6 min, an elevation in [Cl − ] i and decrease in pH were observed ( Figure 5A). In this set of experiments, [K + ] o -induced intracellular acidification occurred, on average by 0.12 ± 0.11 pH (n = 4, Figure 5C), and an increase in [Cl − ] i occurred by 10.12 ± 1.21 mM (n = 4, Figure 5B). After washing, [Cl − ] i recovered toward the baseline, while the base level of pH i sometimes followed to more alkaline values ( Figure 5A).
Applying a Ca 2+ -free ACSF containing 1 mM EGTA did not change the base level of [Cl − ] i , while a decrease in pH, i.e., acidification of the cell cytoplasm was observed ( Figure  5A). On average, the base level of pH i decreased by 0.09 ± 0.02 рН( Figure 5D Figure 5B). Surprisingly, unlike reports on preparations in culture [38,39], in our conditions, the pH responses to high K + were acidifying, only with reduced amplitudes compared to the control. On average, the [K + ] o -induced pH decrease was by 0.10 ± 0.08 pH (n = 4, Figure 5C).  Figure  6A). On average, [Cl − ] i diminished by 7.1 ± 1.1 mM (p < 0.05, n = 5) and рН i increased by 0.19 ± 0.01 рНunits (p < 0.05, n = 5; Figure 6D,E).
During the first minutes after establishing new steady-state levels in "low-Cl − " external solution, high-frequency stimulation of Schaffer's collaterals caused the acidification directed pH responses, in which the amplitude was much higher than in control ACSF ( Figure 6A). On average, ∆pH i increased by 5 times in comparison with that recorded in the normal ACSF and became 0.063 ± 0.013 units pH (p < 0.05, n = 5, Figure 6C). The Cl − transients on stimulation were nearly completely suppressed ( Figure 6A,B). With an increase in the duration of incubation of slices in the "low-Cl − " ACSF, the amplitude of the evoked pH responses decreased until they completely disappeared (data not shown). Our preliminary observations suggest that the reason for this elimination of pH responses is a continuous strong decrease in the amplitude of evoked local field potentials upon transition to "low-Cl − " ACSF conditions. when high K + was applied to slices for 3-6 min, an elevation in [Cl − ]i and decrease in pH were observed ( Figure 5A). In this set of experiments, [K + ]o-induced intracellular acidification occurred, on average by 0.12 ± 0.11 pH (n = 4, Figure 5C), and an increase in [Cl − ]i occurred by 10.12 ± 1.21 mM (n = 4, Figure 5B). After washing, [Cl − ]i recovered toward the baseline, while the base level of pHi sometimes followed to more alkaline values ( Figure  5A). Applying a Ca 2+ -free ACSF containing 1 mM EGTA did not change the base level of [Cl − ]i, while a decrease in pH, i.e., acidification of the cell cytoplasm was observed ( Figure  5A). On average, the base level of pHi decreased by 0.09 ± 0.02 рН ( Figure 5D, n = 8, p < 0.01). [K + ]o-induced depolarization caused an elevation of [Cl − ]i by 12.75 ± 0.83 mM (n = 4, Figure 5B). Surprisingly, unlike reports on preparations in culture [38,39], in our condi-

Changes of [Cl ]i and pHi during [K + ] -Induced Depolarization in the Low-Cl ACSF
We then tested how a decrease in extracellular Cl affects the amplitude of [Cl ]i and pHi changes caused by the [K + ] -induced depolarization. Similarly to previously described observations (Section 2.5), the addition of 17.5 mM K + in normal ACSF induced reversible transients of acidification and synchronous elevation of [Cl ]i in neurons ( Figure 7A). In this set of experiments, the mean K + -induced decrease in pHi was 0.15 ± 0.02 pH and the elevation of [Cl ]i was 4.36 ± 0.77 mM (n = 5).   Figure 7A). In this set of experiments, the mean K + -induced decrease in pH i was 0.15 ± 0.02 pH and the elevation of [Cl − ] i was 4.36 ± 0.77 mM (n = 5).
As previously described, upon perfusion of slices with "low Cl − " ACSF, the base level of pH increased and [Cl − ] i synchronously decreased ( Figure 7A). In "low Cl − " ACSF conditions, depolarization caused by the addition of 17.5 mM K + , resulted in a significant increase in the amplitude of pH acidification responses, while [Cl − ] i responses were nearly completely suppressed or even oppositely directed, i.e., showed a small efflux of intracellular Cl − ( Figure 7A). On average, the mean amplitude of "high K + "-induced pH i responses was 0.33 ± 0.06 (p < 0.05, n = 5), i.e., about 2 times higher than in the control ACSF, while the mean change of [Cl − ] i was −0.14 ± 0.03 (p < 0.05, n = 5), i.e., about 30 times smaller in comparison with control conditions. After washing with normal ACSF, the base levels of pH i and [Cl − ] i returned to initial values; in addition, the amplitudes of K + -induced transients were fully recovered ( Figure 7B,C).
These results are in accord with the above-described effect of lowering external Cl − on synaptically induced changes of pH i and [Cl − ] i . As previously described, upon perfusion of slices with "low Cl − " ACSF, the base level of pH increased and [Cl − ]i synchronously decreased ( Figure 7A). In "low Cl − " ACSF conditions, depolarization caused by the addition of 17.5 mM K + , resulted in a significant increase in the amplitude of pH acidification responses, while [Cl − ]i responses were nearly completely suppressed or even oppositely directed, i.e., showed a small efflux of intracellular Cl − ( Figure 7A). On average, the mean amplitude of "high K + "-induced pHi responses was 0.33 ± 0.06 (p < 0.05, n = 5), i.e., about 2 times higher than in the control ACSF, while the mean change of [Cl − ]i was −0.14 ± 0.03 (p < 0.05, n = 5), i.e., about 30 times smaller in comparison with control conditions. After washing with normal ACSF, the base levels of pHi and [Cl − ]i returned to initial values; in addition, the amplitudes of K + -induced transients were fully recovered ( Figure 7B,C).
These results are in accord with the above-described effect of lowering external Cl − on synaptically induced changes of pHi and [Cl − ]i.

Changes in [Cl − ]i and [H + ]i during [K + ]о-Induced Depolarization in the Low-Na + ACSF
Extracellular Na + is one of the important participants and regulators of transporters of intracellular proton concentration [3]. For instance, the electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) contributes to intracellular as well as extracellular acid/base homeostasis in the brain and its dysfunctions are associated with pathophysiological states [18,41,42]. In addition, the Na + -dependent 2HCO3 − /Cl − -exchanger (NDCBE) represents an important pH-Cl − -coupled physiological controller of ions [17].
Lastly, we analyzed how a decrease in external Na + affects changes in pHi and [Cl − ]i caused by high K + depolarization. Similar to the above-presented results, in ASCF containing normal Na + concentration, K + -induced depolarization caused intracellular elevation of both H + and Cl − ions ( Figure 8A). On average, the increase in [Cl − ]i was 12.06 ± 1.19 mM ( Figure 8B) and acidification was 0.18 ± 0.04 pH ( Figure 8C).
Changing the solution to a low-Na + ASCF (replacement of NaCl with choline Cl) led to a decrease in the base level of intracellular Cl − and H + ( Figure 8A). The mean changes were for [Cl − ]i by 1.98 ± 0.15 mM (n = 5, p < 0.05, 6 months) and for H+ by 0.08 ± 0.01 pH units (n = 5, p < 0.05, 6 months). In low-Na + ASCF, responses to K + -induced depolarization

Changes in [Cl − ] i and [H + ] i during [K + ] o -Induced Depolarization in the Low-Na + ACSF
Extracellular Na + is one of the important participants and regulators of transporters of intracellular proton concentration [1]. For instance, the electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) contributes to intracellular as well as extracellular acid/base homeostasis in the brain and its dysfunctions are associated with pathophysiological states [16,41,42]. In addition, the Na + -dependent 2HCO 3 − /Cl − -exchanger (NDCBE) represents an important pH-Cl − -coupled physiological controller of ions [15].
Lastly, we analyzed how a decrease in external Na + affects changes in pH i and [Cl − ] i caused by high K + depolarization. Similar to the above-presented results, in ASCF containing normal Na + concentration, K + -induced depolarization caused intracellular elevation of both H + and Cl − ions ( Figure 8A). On average, the increase in [Cl − ] i was 12.06 ± 1.19 mM ( Figure 8B) and acidification was 0.18 ± 0.04 pH ( Figure 8C).
Changing the solution to a low-Na + ASCF (replacement of NaCl with choline Cl) led to a decrease in the base level of intracellular Cl − and H + ( Figure 8A). The mean changes were for [Cl − ] i by 1.98 ± 0.15 mM (n = 5, p < 0.05, 6 months) and for H+ by 0.08 ± 0.01 pH units (n = 5, p < 0.05, 6 months). In low-Na + ASCF, responses to K + -induced depolarization were potentiated by more than 2 times for both ions, resulting in the mean changes of 25.77 ± 2.74 mM and 0.40 ± 0.05 pH units (n = 5, Figure 8B,C). were potentiated by more than 2 times for both ions, resulting in the mean changes of 25.77 ± 2.74 mM and 0.40 ± 0.05 pH units (n = 5, Figure 8B,C).

Discussion
Our study provides evidence that the optosensoric reporter, ClopHensor, expressed in transgenic mice, represents an efficient tool for non-invasive monitoring of intracellular Cl − and H + ions. Applying different conditions for modulation of neuronal activity in brain slices from transgenic mice, we demonstrated the ability of ClopHensor to simultaneously monitor and analyze changes in [Cl − ]i and [H + ]i during artificial changes of its equilibrium.
Maintaining physiologically relevant concentrations of H + and Cl − has a pivotal role in controlling neuronal excitability in the adult brain and during development, and is likely to be crucial in pathophysiological conditions. Both ions play an important role in many cellular processes, including neurotransmission, regulation of membrane potential, cell volume and water-salt balance, modulation of the functions of different proteins, including voltage-gated and receptor-operated channels [3,14,[43][44][45][46]. Because of high metabolic activity, accompanied by depolarization, neurons may be susceptible to acidification-induced injury, as can happen in excessive synaptic activation or during conditions of anoxia or ischemia.
The steady-state physiologically relevant dynamic range of pHi and [Cl − ]i is determined by the balance of both passive transport, through ion channels and by active mechanisms via exchangers or cotransporters [3,46,47].

Discussion
Our study provides evidence that the optosensoric reporter, ClopHensor, expressed in transgenic mice, represents an efficient tool for non-invasive monitoring of intracellular Cl − and H + ions. Applying different conditions for modulation of neuronal activity in brain slices from transgenic mice, we demonstrated the ability of ClopHensor to simultaneously monitor and analyze changes in [Cl − ] i and [H + ] i during artificial changes of its equilibrium.
Maintaining physiologically relevant concentrations of H + and Cl − has a pivotal role in controlling neuronal excitability in the adult brain and during development, and is likely to be crucial in pathophysiological conditions. Both ions play an important role in many cellular processes, including neurotransmission, regulation of membrane potential, cell volume and water-salt balance, modulation of the functions of different proteins, including voltage-gated and receptor-operated channels [1,12,[43][44][45][46]. Because of high metabolic activity, accompanied by depolarization, neurons may be susceptible to acidification-induced injury, as can happen in excessive synaptic activation or during conditions of anoxia or ischemia.
The steady-state physiologically relevant dynamic range of pH i and [Cl − ] i is determined by the balance of both passive transport, through ion channels and by active mechanisms via exchangers or cotransporters [1,46,47].
The intracellular Cl − in neurons of the mammalian brain is primary regulated by two cation-Cl − cotransporters, the Na + /K + /2Cl − cotransporter 1 (NKCC-1) and the K + /Cl − co-transporter 2 (KCC-2) [46][47][48][49]. NKCC1 pumps Cl − into neurons, while KCC2 uses the energy of the K + gradient to extrude Cl − from neurons and maintain relatively low [Cl − ] i ( Figure 9). Dysfunction or changes in the expression of these cotransporters is critically linked to the etiology of several neurologic disorders including epilepsy, acute trauma, ischemia, autoimmune disorders, and neuropathic pain [48,50,51]. Similarly, a modest shift of intra-and extracellular H + ion concentration can significant effects on brain functions, such as neuronal excitability, synaptic transm and metabolism [3,52]. This is the result of the sensitivity of a large number of proc to protons, such as ion channel gating and conductance, synaptic transmission, cell-t communication via gap junctions, and enzymatic activity in brain energy metabolism 55]. The major pH regulating transporters identified in the mammalian brain so far prise the Na + /H + exchanger (NHE1), electrogenic Na + /HCO3 − cotransporter 1 (NB Na + -dependent Cl -/HCO3 − exchange (NDCBE), and Na + -independent anion exch (AE3) (Figure 9; [3,17,56]. NBce-1, NDCBE and AE3 transporters perform translocat HCO3 − , thus controlling the physiological range of pHi. The electrogenic Na + /HCO transporter NBCe1 is one of the major regulators of [H + ]i and is expressed in most cell types, with the most prominent expression being astrocytes [3]. It has been show NBCe1 is not only an acid extruder/base loader, as suggested in early studies [57,58 also an acid loader/base extruder [18]. Anion Cl − /HCO3 − exchange (AE3) is known important acid-loading performer in brain cells [18,59]. The finding that hippoca neurons of knockdown AE3 mice (Ae3 -/-) lack detectable Cl − /HCO3 − exchange activit suggests that AE3 plays a critical role in the maintenance of the Cl − equilibrium pot and/or pHi in neurons.
The role of monocarboxylate transporters (MCTs) in the regulation of the func integrity of synaptic transmission has been demonstrated. In excitatory synapses, constitutively supports synaptic transmission, even under conditions when there is ficient amount of glucose and intracellular ATP [61]. Monocarboxylates cause a dec in the pH of neurons, which is associated with changes in bioelectric activity [62]. M involvement in Cl modulation requires future analysis. Similarly, a modest shift of intra-and extracellular H + ion concentration can have significant effects on brain functions, such as neuronal excitability, synaptic transmission and metabolism [1,52]. This is the result of the sensitivity of a large number of processes to protons, such as ion channel gating and conductance, synaptic transmission, cell-to-cell communication via gap junctions, and enzymatic activity in brain energy metabolism [52][53][54][55]. The major pH regulating transporters identified in the mammalian brain so far comprise the Na + /H + exchanger (NHE1), electrogenic Na + /HCO 3 − cotransporter 1 (NBCe1), Na + -dependent Cl − /HCO 3 − exchange (NDCBE), and Na + -independent anion exchanger (AE3) (Figure 9; [1,15,56]. NBce-1, NDCBE and AE3 transporters perform translocation of HCO 3 − , thus controlling the physiological range of pH i . The electrogenic Na + /HCO 3 − cotransporter NBCe1 is one of the major regulators of [H + ] i and is expressed in most brain cell types, with the most prominent expression being astrocytes [1]. It has been shown that NBCe1 is not only an acid extruder/base loader, as suggested in early studies [57,58], but also an acid loader/base extruder [16]. Anion Cl − /HCO 3 − exchange (AE3) is known as an important acid-loading performer in brain cells [16,59]. The finding that hippocampal neurons of knockdown AE3 mice (Ae3 -/-) lack detectable Cl − /HCO 3 − exchange activity [60] suggests that AE3 plays a critical role in the maintenance of the Cl − equilibrium potential and/or pH i in neurons.
The role of monocarboxylate transporters (MCTs) in the regulation of the functional integrity of synaptic transmission has been demonstrated. In excitatory synapses, MCT constitutively supports synaptic transmission, even under conditions when there is a sufficient amount of glucose and intracellular ATP [61]. Monocarboxylates cause a decrease in the pH of neurons, which is associated with changes in bioelectric activity [62]. MCT's involvement in Cl modulation requires future analysis.
These features of transporters emphasize the need for simultaneous monitoring of [Cl − ] i and [H + ] i when analyzing the mechanisms of ion homeostasis and neuronal activ-ity, particularly using neuropathological models. A genetically encoded sensor, named ClopHensor, was proposed for simultaneous measurement of Cl − and H + ion concentrations [27]. This construct has shown its effectiveness at the heterologous expression in cell lines, demonstrating good sensitivity and high stability to bleaching during long fluorescence measurements [28]. We recently presented a line of transgenic mice expressing ClopHensor in neurons and obtained a detailed map of its distribution in the mouse brain [32]. Expression of this probe in transgenic mice was found to be highly specific and reproducible in different animals, suggesting that this experimental model represents a promising tool for analysis of dynamic changes in [Cl − ] i and pH i .
In the present study, we analyzed the effectiveness of ClopHensor expressed in transgenic mice for monitoring [Cl − ] i and [H + ] i in neurons of hippocampal slices upon changing the ionic equilibrium by pharmacological modulation of neuronal activity, changes in extracellular concentrations of Ca 2+ , Cl − or Na + , and by depolarization caused by high-frequency synaptic stimulation or the use of high K + .

Kinetics of Synaptically Induced Transients of [Cl − ] i and [H + ] i
High frequency induced stimulation of Schaffer's collaterals resulted in the elevation of [Cl − ] i and [H + ] i and slow recovery. Importantly, the decay kinetics of Cl − transients was nearly 5-fold faster than pH. The experiments on wild type mice using the chemical pH sensor, BCECF, confirmed remarkably slow kinetics of pH i recovery. These differences in the kinetics of recovery of Cl − and H + responses may reflect the involvement of different cellular participants in control of these ions' homeostasis.
In the study using the ClopHensorN indicator heterologously expressed in cultured hippocampal slices, about 1.5 times faster decay of intracellular Cl − concentrations were indicated [29]. A profound analysis is necessary to reveal relationships and efficacy of different transporters and other mechanisms involved in the recovery of [Cl − ] i and [H + ] i from synaptically induced disequilibrium.

Effect of GABA Receptor Inhibition on Synaptically Induced Transients of [Cl − ] i and [H + ] i
Results of our study demonstrate that in hippocampal slices of transgenic mice, high frequency stimulation of Shaffer's collaterals causes an intracellular increase of both ions, Cl − and H + . Synaptic stimulation causes the release of neurotransmitters, primary glutamate and GABA, which activates corresponding receptors, leading to the opening of cation and anion-selective ion channels and resulting depolarization of neurons.
As predicted, inhibition of GABA-mediated chloride conductance by bicuculline resulted in a decrease of the synaptically induced influx of Cl − . On the other side, this decrease was accompanied by potentiation of acidific [H + ] i transients. This could be a consequent of two main things: (i) increased synaptic stimulation due to blockade of inhibitory GABA receptors; (ii) changes in the activity of transporters.
In support of the first possibility, the electrophysiological analysis showed that bicuculline causes an elevation in the amplitude of evoked local field potentials. On the other side, experiments that lowered the extracellular Cl showed that in spite of the strong reduction of evoked local field potentials, the amplitude of synaptically induced pH responses was potentiated nearly 10 times in comparison with control.
We suggest that this may be a consequence of the weakening of the acidifying activity of the Cl − HCO 3 − exchange. This assumption was tested in experiments with high K +induced depolarization and changes of external concentrations of ion participating in the functioning of transporters.

Analysis of High K + -Induced Depolarization on Changes of [Cl − ] i and [H + ] i in External Ca 2+ -Free Conditions
Depolarization induced by application of high K + or by other means has been used in a number of studies for analysis of pH i changes in cultured and freshly dissociated neurons, and brain slices [37][38][39][40]63]. In most studies, depolarization induced by application of high external K + was accompanied by elevation of intracellular Ca 2+ and decrease of pH, i.e., acidification [37,39]. Early observations performed on cultured cells showed that under external Ca 2+ -free conditions, the acidification responses were either completely prevented [38] or even inversed, i.e., exhibited an increase in pH i [39]. Moreover, a blocker of voltage-gated Ca 2+ channels inhibited K + -induced responses, suggesting that an increase in [Ca 2+ ] i is a key factor for acidosis to occur [38].
We tested this suggestion in experiments on brain slices of ClopHensor-expressing mice and obtained distinct results. In our experiments, transition to external Ca 2+ -free conditions alone caused a decrease in base pH, i.e., acidification. Surprisingly, K + -induced depolarization produced additional acidification. The responses were only partially attenuated in comparison with control, suggesting an only partial contribution of Ca 2+ -dependent processes in pH i responses induced by membrane depolarization.
The reasons for these contrast observations have to be clarified. Among them may be the use of HEPES solution in cell culture experiments and different energy states of neurons.

Decreasing Extracellular Na +
Since the NHE, NBCe1 and NDCBE transporters operate on a Na + gradient, a decrease in the extracellular Na + concentration should lead to a decline in the operation of these pumps and, as a result, to a weakening of protons extruding from the cytoplasm, and a decrease in intracellular HCO 3 − We assume that for these reasons, in our experiments, there was a decrease in the pH i of neurons during the transition to low Na + and a potentiation of acidic responses to depolarization caused by increased K + . In the future, these processes will be analyzed in detail.
In conclusion, the baseline level and fluctuations of intracellular Cl − and pH play a crucial role for intercellular and intracellular signaling, as well as for cellular and synaptic plasticity. Our study demonstrates that transgenic mice expressing ClopHensor provide ample opportunities for studying the homeostasis of chloride and hydrogen not only under normal conditions, but also in pathology.
In particular, Cl − gradients are disrupted in epilepsy, especially in early childhood, which significantly complicates treatment. Hydrogen gradients in the central nervous system are disrupted by neuroinflammatory processes of various origins and ischemia. Research in these areas is relevant today, and ClopHensor mice can be a reliable tool for in-depth analysis of the mechanisms controlling the physiological ranges of concentrations of these ions.

Animals
Experiments were conducted on laboratory ICR CD-1 outbreed mice of both sexes at postnatal days 10-12 and adult transgenic mice, aged 2-6 months, strain C57BL/6N expressing ClopHensor (Diuba et al., 2020). Use of animals was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No. 123; 1985). All animal protocols and experimental procedures were approved by the Local Ethics Committee of Kazan State Medical University. Mice had free access to food and water and were kept under natural day length fluctuations. Animals were not involved in any previous procedures.

Preparation of Brain Slices
Sagittal 350 µm thick sections of the cerebral hemispheres containing the hippocampus were obtained with the use of a vibratome of Model NVSLM1, World Precision Instruments. Mice were euthanized by decapitation. The brain was quickly removed and placed in a Petri dish filled with ice-cold high-K + solution. The cerebellum and olfactory bulbs were cut off using a scalpel; the cerebral hemispheres were separated by cutting along the longitudinal fissure and mounted onto the vibratome specimen disc using superglue, orienting them downward with the sagittal cut surface and the cortex facing the razor blade. Sagittal 350 µm thick sections were prepared in ice-cold high K + solution. After being cut, slices were incubated for 15 min at room temperature in a resting high magnesium oxygenated ACSF. Then, slices were placed in a chamber filled with oxygenated ACSF. Before use, slices were allowed to recover for at least 1 h at room temperature.
Experiments were conducted during the period of 1-6 h after slicing. For the recordings, the brain slices were placed in a chamber perfused with an oxygenated ACSF. Recordings were carried out at 30-31 • C with the speed of perfusion of 25 mL/min.

Monitoring of [Cl − ] i and pH on Brain Slices ClopHensor Mice
Fluorescence images were obtained using an upright microscope Olympus BX51WI equipped with the iXon Life 897 EMCCD camera (Andor, Oxford Instruments, Abingdon, UK), a 4-Wavelength LED Source (LED4D001, Thorlabs, Newton, NJ, USA) accompanied with a Four-Channel LED Driver (DC4100, Thorlabs), a quad-band filter set (Cat# 9403, Chroma, Foothill Ranch, CA, USA), and a water-immersion objective (60× magnification, 1 numerical aperture; LumPlanFL N, Olympus, Tokyo, Japan) ( Figure 10). with a Four-Channel LED Driver (DC4100, Thorlabs), a quad-band filter set (Cat# 9403, Chroma, Foothill Ranch, CA, USA), and a water-immersion objective (60× magnification, 1 numerical aperture; LumPlanFL N, Olympus, Tokyo, Japan) ( Figure 10). The 4-Wavelength LED Source was supplied by light-emitting diodes (LEDs) at 365 nm, 455 nm, 505 nm and 590 nm, the last three of which were used in our study. The capacity of lighting and the LED switching order were adjusted via the DC4100 driver. Cyan channel fluorescence (455 nm excitation) was detected from 460 nm to 485 nm, green channel (505 nm excitation) from 527 nm to 551 nm, and red channel (590 nm excitation) from 600 nm to 680 nm.
All peripheral hardware control, image acquisition and the average fluorescence intensity measurement were achieved using DriverLed software (KSMU, Kazan). Regions of interest (ROI) were set around pyramidal cell bodies of the CA1 hippocampal region. The average fluorescence intensity of the ROI was tracked online at the time of the realtime fluorescence imaging. Numerical data were output by the DriverLed software on the computer in the form of text documents, which were subsequently parsed using Excel 2016 (Microsoft). The 4-Wavelength LED Source was supplied by light-emitting diodes (LEDs) at 365 nm, 455 nm, 505 nm and 590 nm, the last three of which were used in our study. The capacity of lighting and the LED switching order were adjusted via the DC4100 driver. Cyan channel fluorescence (455 nm excitation) was detected from 460 nm to 485 nm, green channel (505 nm excitation) from 527 nm to 551 nm, and red channel (590 nm excitation) from 600 nm to 680 nm.
All peripheral hardware control, image acquisition and the average fluorescence intensity measurement were achieved using DriverLed software (KSMU, Kazan). Regions of interest (ROI) were set around pyramidal cell bodies of the CA1 hippocampal region. The average fluorescence intensity of the ROI was tracked online at the time of the real-time fluorescence imaging. Numerical data were output by the DriverLed software on the computer in the form of text documents, which were subsequently parsed using Excel 2016 (Microsoft).
Excitation of E2GFP was provided by light-emitting diodes (LEDs) at 455 nm and 505 nm, and a LED at 590 nm provided excitation of DsRed. The duration of excitation at each wavelength was usually 70-110 ms. Examples of the images taken are shown in Figure 1C. Fluorescent emission was recorded continuously from the hippocampal CA1 pyramidal cells with a sampling interval of 10 s. Evoked emission signals were obtained by stimulation of Schaffer's collaterals by glass bipolar electrode, filled with ACSF, placed in the stratum radiatum of the CA2 hippocampal area (100 Hz for 20 s, 20-320 µA, single pulse width 200 µs).

Monitoring of pH Using BCECF
As an additional control of proper pH monitoring of intracellular pH in hippocampal neurons, we used the pH-sensitive dye, 2 ,7 -Bis (2-carboxyethyl)-5 (6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) ( Figure 11A). The fluorescent dye, BCECF, was introduced for measuring cytoplasmic pH by Roger Tsien and co-workers [36]. Presently used BCECF-AM is a mixture of three types of cell-permeable non-fluorescent molecules, which are converted to fluorescent non-membrane-penetrating form by intracellular esterases [21]. This dye provides dual-excitation ratiometric monitoring of intracellular pH.
Excitation of E2GFP was provided by light-emitting diodes (LEDs) at 455 nm and 505 nm, and a LED at 590 nm provided excitation of DsRed. The duration of excitation at each wavelength was usually 70-110 ms. Examples of the images taken are shown in Figure  1C. Fluorescent emission was recorded continuously from the hippocampal CA1 pyramidal cells with a sampling interval of 10 s. Evoked emission signals were obtained by stimulation of Schaffer's collaterals by glass bipolar electrode, filled with ACSF, placed in the stratum radiatum of the CA2 hippocampal area (100 Hz for 20 s, 20-320 μA, single pulse width 200 μs).

Monitoring of pH Using BCECF
As an additional control of proper pH monitoring of intracellular pH in hippocampal neurons, we used the pH-sensitive dye, 2′,7′-Bis (2-carboxyethyl)-5 (6)-carboxyfluorescein-acetoxymethyl ester (BCECF-AM) ( Figure 11A). The fluorescent dye, BCECF, was introduced for measuring cytoplasmic pH by Roger Tsien and co-workers [36]. Presently used BCECF-AM is a mixture of three types of cell-permeable non-fluorescent molecules, which are converted to fluorescent non-membrane-penetrating form by intracellular esterases [23]. This dye provides dual-excitation ratiometric monitoring of intracellular pH. We determined the pH changes by calculating the ratio of two emission signals obtained after illumination by light at 455 nm and 505 nm ( Figure 11B). The intracellular pH changes after tetanic stimulation and its modulation by bicuculline were analyzed. Experiments were conducted on wild type juvenile mice (P10-P12). Registration conditions were similar to those performed on slices from transgenic ClopHensor mice. Evoked pH-specific ratiometric emission signals were obtained by giving the same pattern of electrical stimulation (100 Hz for 20s); the stimulus intensities ranged from 200 μA to 250 μA.
Hippocampal slices were stained with 10 µM BCECF-AM for 40 min in the oxygenated ACSF. Then the dye was washed out and slices were superfused with normal ACSF for at least 40 min to allow esterases to cleave AM and stabilise pHi. For fluorescent monitoring, slices were transferred to the optical recording chamber, which was mounted on the stage of an upright microscope (Olympus BX51WI) ( Figure 10).
The setup consisted of a microscope (Olympus BX51WI equipped with the iXon Life 897 EMCCD camera (Andor camera)), a 4-Wavelength Thorlabs LED Source (Light We determined the pH changes by calculating the ratio of two emission signals obtained after illumination by light at 455 nm and 505 nm ( Figure 11B). The intracellular pH changes after tetanic stimulation and its modulation by bicuculline were analyzed. Experiments were conducted on wild type juvenile mice (P10-P12). Registration conditions were similar to those performed on slices from transgenic ClopHensor mice. Evoked pHspecific ratiometric emission signals were obtained by giving the same pattern of electrical stimulation (100 Hz for 20s); the stimulus intensities ranged from 200 µA to 250 µA.
Hippocampal slices were stained with 10 µM BCECF-AM for 40 min in the oxygenated ACSF. Then the dye was washed out and slices were superfused with normal ACSF for at least 40 min to allow esterases to cleave AM and stabilise pHi. For fluorescent monitoring, slices were transferred to the optical recording chamber, which was mounted on the stage of an upright microscope (Olympus BX51WI) ( Figure 10).
The setup consisted of a microscope (Olympus BX51WI equipped with the iXon Life 897 EMCCD camera (Andor camera)), a 4-Wavelength Thorlabs LED Source (Light source), and a quad-band filter set (excitation filter). Excitation light with wavelengths of 505 nm, 455 nm and 590 nm, each for 70-100 ms, was sequentially applied to slices and the emission signals from CA1 pyramidal cells were registered by an EMCCD camera. The stimulation electrode was placed in the stratum radiatum of the CA2 hippocampal area. The evoked local field potentials were recorded using a glass micropipette electrode, placed in the stratum radiatum of the CA1 hippocampal area and an HEKA EPC 10 Patch Clamp Amplifier (not shown).
Slices were alternately illuminated at 455 nm and 505 nm with duration of 100 ms. Light from both wavelengths was passed through appropriate excitation and emission filters. Fluorescence image pairs were captured every 10 s by an intensified camera. Evoked emission signals were obtained in the same way as for ClopHensor: by sustained highfrequency electrical stimulation of Schaffer's collaterals (100 Hz for 20 s, 20-320 µA, single pulse width 200 µs).

Electrophysiological Recording
Evoked local field potentials (eLFP) were recorded in the stratum radiatum of the CA1 hippocampal region using glass micropipette electrodes filled with ACSF (resistance 1-2 MΩ) and an HEKA EPC 10 Patch Clamp Amplifier (HEKA Elektronik, Lambrecht, Germany). The DS3 Constant Current Isolated Stimulator (Digitimer, Welwyn Garden, UK) and a bipolar stimulating electrode placed on the Schaffer's collaterals at the hippocampal area CA2 were used for the induction of eLFPs. Current pulses 20-300 µA in amplitude and 200 µs in duration were applied to obtain reliable eLFPs. Single eLFPs were recorded continuously every 20 s. PatchMaster software (HEKA Electronik, Lambrecht, Germany) was used to record eLFPs, control the HEKA EPC 10 Patch Clamp Amplifier and the DS3 Constant Current Isolated Stimulator.

Intracellular pH and Cl − Calibration of ClopHensor on Hippocampal Slices
To perform pH and Cl − calibration, we used a double ionophore technique [64]. The brain slices were exposed to the antibiotic nigericin, which acts as H + /K + antiporter and the Cl − /OH − antiporter tributyltin, which forms pores in the cell membrane and allows external Cl − to equilibrate with intracellular Cl − [65]. For the action of the compounds, before fluorescent monitoring, the hippocampal slices were kept for 1-28 h at +4 • C in the following solution: 150 mM K-Gluconate, 20 mM HEPES, and 10 mM D-glucose) with addition of nigericin (20 mkM, pH = 7.22-7.25) and tributyltin (20 mkM).
For pH calibration, the solutions with different pH values (from 6.23 pH to 8.04 pH) were prepared by adding KOH for alkalization. For calibration of Cl − , the solutions were prepared at pH = 7.25 and contained a different concentration of Cl − (0, 3, 10, 30, 100 mM). To keep the osmolality of the solutions, KCl was correspondently substituted by the Kgluconate (150, 147, 140, 120, 50 mM). All of these solutions contained 20 mM HEPES, and 10 mM D-glucose.
Fluorescent signals were recorded from the CA1 zone of the hippocampus with the following lighting parameters in the Drive LED program: Exposition 70 ms, LED on 100 ms, binning 2, Voltage (590 nm) 70 mV, Voltage (505 nm) 10 mV, Voltage (455 nm) 15 mV. The slices were incubated in each solution until the fluorescence stabilized, usually for 20-30 min.
All coefficients were obtained by fitting data using the Igor Pro 6.02 software.

Data Analysis and Statistics
Amplitudes of evoked local field potentials were measured using the Online Analysis function of PatchMaster software (HEKA Electronik, Lambrecht, Germany). Superimposed average traces of evoked local field potentials were generated in PatchMaster software and processed for further presentation in Igor Pro 6.02 software (WaveMetrics, Tigard, OR, USA).
The average emission intensity of the region of interest (ROI) was measured online at the time of the real-time fluorescence imaging or, if necessary, recalculated using DriverLed software. Excel 2016 (Microsoft) software was used to compute and plot Cl − and pHdependent ratios of emission intensities and to measure the amplitudes evoked by tetanic stimulation pH-and Cl − -specific ratiometric fluorescence emission signals.
Origin 15 software was used to perform a statistical analysis of the data, to plot the graphs and compute the decay times of the evoked pH-and Cl − -specific fluorescence emission signals.
Data are represented as means ± SEM. Statistical significance was determined using Paired Sample Wilcoxon Signed Rank Test and Mann Whitney U test. Differences were considered significant at p < 0.05.