Activity-dependent changes in transporter and ...

Brain Research Bulletin 136 (2018) 37–43

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Original Research Article

Activity-dependent changes in transporter and potassium currents in hippocampal astrocytes Albina Lebedevaa,1, Alex Plataa,1, Olga Nosovaa, Olga Tyurikovaa,b, Alexey Semyanova,c, a b c

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UNN Institute of Neuroscience, University of Nizhny Novgorod, Nizhny Novgorod, Russia UCL Institute of Neurology, London, UK Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

A R T I C L E I N F O

A B S T R A C T

Keywords: Astrocytes Potassium Glutamate uptake

Astrocytes are involved in maintenance of synaptic microenvironment by glutamate uptake and K+ clearance. These processes are associated with net charge transfer across the membrane and therefore can be recorded as glutamate transporter (IGluT) and K+ (IK) currents. It has been previously shown that the blockade of IK with BaCl2 enhances the IGluT. Here we show that activity-dependent facilitation (5 stimuli at 50 Hz) of IGluT was not significantly different in BaCl2 compared to facilitation of IGluT isolated by post-hoc subtraction of IK. Nevertheless, BaCl2 abolished the activity-dependent prolongation of τdecay, which was observed for IGluT isolated by post-hoc subtraction of IK. This finding suggests that activity-dependent accumulation of extracellular K+ ([K+]o) causes astrocytic depolarization, which is responsible for the increase in τdecay of IGluT. The blockade of inward rectifying K+ channels (Kir) with BaCl2 makes astrocytic membrane potential insensitive to [K+]o elevation and thus abolishes this increase. Blockade of IGluT with glutamate transporter blocker, DL-threo-βbenzyloxyaspartic acid (TBOA) did not significantly affect the amplitude of IK but decreased its τdecay. However, activity dependent facilitations of both amplitude and τdecay of IK were larger in TBOA, than in the control conditions. We suggest that activity-dependent accumulation of extracellular glutamate can enhance release of K+. Thus activity-dependent changes in [K+]o can affect glutamate dwell-time in the synaptic cleft, and vice versa, extracellular glutamate accumulation can affect [K+]o time-course. Our finding is important for understanding of the astrocytic mechanisms in glutamate excitotoxicity and in diseases related to disruption of K+ homeostasis (e.g. stroke, migraine, and epilepsy).

1. Introduction Astrocytes are actively involved in various aspects of synaptic transmission and have been recognized as a key element of the ‘synaptic triate’, the term suggested by Kettenmann et al. in (1996), which later became known as ‘tripartite synapse’ (Araque et al., 1999; Nedergaard and Verkhratsky, 2012). This idea has however recently evolved into a concept of ‘astroglial cradle’ (Verkhratsky and Nedergaard, 2014). Lamellae – like perisynaptic astrocytic processes (PAPs) form this cradle by filling the space between synapse. The PAPs isolate synapses from each other, and interact with them by releasing gliotransmitters and by controlling synaptic microenvironment (Semyanov and Verkhratsky, 2016; Zorec et al., 2012). For example, release of D-serine by astrocytes promotes synaptic long-term potentiation, while release of glycine promotes long-term depression (Henneberger et al., 2012; Henneberger et al., 2010). PAPs uptake synaptically released neurotransmitters such

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as glutamate and GABA (Danbolt 2001; Kersante et al., 2013; Song et al., 2013) and clear [K+]o that accumulates during synaptic transmission (Poolos et al., 1987; Bergles and Jahr, 1997; Kofuji and Newman, 2004; Meeks and Mennerick, 2007). These processes can be regulated both by densities of transporters, channels and pumps in PAPs, and by morphological plasticity of PAPs. Neuronal and astrocytic neurotransmitter transporters belong to different types. In neurons glutamate uptake is mediated predominantly by excitatory amino acid transporters (EAATs) types 3,4 and 5; while in astrocytes by types 1 and 2 (Danbolt, 2001). The density of glutamate transporters is also significantly higher in PAPs, than in axons or dendritic spines. Therefore, most of glutamate uptake is carried by astrocytes. For example, astrocytic EAAT2 is responsible for 95% of glutamate uptake in the hippocampus (Haugeto et al., 1996; Holmseth et al., 2012). Similarly, GABA transporters (GAT) are expressed in cell type specific manner (Scimemi, 2014). GAT3 is confined to astrocytes, while

Corresponding author at: UNN Institute of Neuroscience, University of Nizhny Novgorod, Nizhny Novgorod, Russia. E-mail address: [email protected] (A. Semyanov). Equally contributed authors.

http://dx.doi.org/10.1016/j.brainresbull.2017.08.015 Received 10 May 2017; Received in revised form 29 August 2017; Accepted 30 August 2017 Available online 07 September 2017 0361-9230/ © 2017 Elsevier Inc. All rights reserved.


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50 mM sucrose; 87 mM NaCl; 2.5 mM KCl; 8.48 mM MgSO4; 1.24 mM NaH2PO4; 26.2 mM NaHCO3; 0.5 mM CaCl2; 22 mM D-Glucose. Hippocampi from both hemispheres were isolated, then transverse slices (350 μm) were cut with a vibrating microtome (Microm HM 650 V; Thermo Fisher Scientific) and left to recover at 34° C for 1 h in an interfaced chamber with ‘storage’ solution containing: 119 mM NaCl; 2.5 mM KCl; 1.3 mM MgSO4; 1 mM NaH2PO4; 26.2 mM NaHCO3; 1 mM CaCl2; 1.6 mM MgCl2; 22 mM D-Glucose. Then the slices were transferred to the recording chamber and were continuously perfused with a solution containing: 119 mM NaCl; 2.5 mM KCl; 1.3 mM MgSO4; 1 mM NaH2PO4; 26.2 mM NaHCO3; 2 mM CaCl2; 11 D-Glucose. All solutions were saturated with Gas mixture containing 95% O2 and 5% CO2. Osmolarity was 292 ± 5 mOsm, pH 7.4.

GAT1 is expressed in both astrocytes and neurons (Kersante et al., 2013; Song et al., 2013). Astrocytes possess several mechanisms for K+ clearance (Walz, 2000). When [K+]o accumulates, it shifts reversal potential for this ion. Then K+ enters astrocytes trough K+ channels (predominantly, Kir) according to the driving force (Kofuji and Newman, 2004; Cheung et al., 2015). [K+]o can be also removed by Na+/K+/2Cl− cotransporter of the subtype 1 (NKCC1) and by Na+/K+ ATPase (Cheung et al., 2015; Walz, 2000). Morphological properties of PAPs are also very important for effective glutamate uptake and K+ clearance. In contrast to main astrocytic processes, PAPs have lamellae-like structure, which significantly increases their surface-to-volume ratio (SVR) (Patrushev et al., 2013). PAPs fill the space between synapses achieving large surface of their membrane exposed to glutamate and K+ within small volume of the tissue (Lehre and Rusakov, 2002). High SVR is achieved by a reduction of cytoplasmic volume, and therefore PAPs are devoid of organelles such as endoplasmic reticulum or mitochondria commonly thought to be essential for neuron triggered Ca2+ responses in astrocytes (Patrushev et al., 2013; Bernardinelli et al., 2014a,b; Reichenbach et al., 2010). On the other hand, such morphological organization of PAPs may be beneficial for their morphological plasticity that also regulates glutamate uptake and K+ clearance (Tanaka et al., 2013; Bernardinelli et al., 2014; Heller and Rusakov, 2015). Stoichiometry of glutamate transporters has been well documented: together with 1 glutamate−, 3 Na+ and 1 H+ taken into the cell while 1 K+ transported outside (Kanai et al., 1995; Billups and Attwell 1996; Kanner and Bendahan, 1982). In sum, two net positive charges are moved into the cell each cycle. Thus, both glutamate transporter current (IGluT) and K+ current (IK) can be detected in patch-clamped astrocytes as two components of astrocytic current in response to synaptic stimulation (Isyn). IGluT appears as fast initial transient of Isyn. The amplitude and time-course of IGluT depend on the number of synapses recruited by synaptic stimulation, glutamate release probability, density and kinetics of transporters, distance between PAP and the synapse etc (Thomas et al., 2011; Scimemi and Diamond, 2013). Therefore, such processes as presynaptic facilitation and glutamate spillover can be estimated from the IGluT analysis. IK appears as a slow component of Isyn (Cheung et al., 2015; Dallerac et al. 2013; Shih et al., 2013). The amplitude of IK largely depends on K+ efflux through postsynaptic AMPA and NMDA receptors (Ge and Duan, 2007; Shih et al., 2013; De Saint Jan and Westbrook, 2005; Pannasch et al., 2011), therefore it can be used to estimate the postsynaptic response. This opens attractive possibility to monitor processes in pre- and postsynaptic parts of the synapse from the astrocytic recordings. However, the major obstacle in such analysis is separation of IGluT and IK. Although two currents have very different time-courses, they partially overlap. Moreover, the Isyn is sometimes contaminated by reflection of field potentials (Pannasch et al., 2012). Indeed, because of very low input resistance astrocyte can ‘operate’ as an extracellular electrode. Recording of field potentials has been suggested as an additional method to monitor synaptic signaling during astrocyte recording (Henneberger et al., 2010). Here we used two different approaches to isolate IGluT and IK, either by blocking Kir with 200 μM BaCl2 or by blocking glutamate transporters with 50 μM TBOA. These methods revealed how activity-dependent changes in IGluT and IK are influenced by ambient glutamate or [K+]o acomulation.

2.2. Electrophysiology Cells were visually identified under infrared DIC using the Olympus BX51WI microscope. Whole-cell voltage clamp recordings in CA1 str.radiatum astrocytes were obtained using patch electrodes filled with a solution containing: 130 mM KCH3SO3, 10 mM HEPES, 10 mM Na2phosphocreatine, 8 mM NaCl, 3 mM L-ascorbic acid, 2 mM Mg-GTP (pH was adjusted to 7.2 with KOH; osmolarity to 292 mOsm) and with a resistance of 3–5 MΩ. Astrocytes were identified by small soma size (about 10 μm in diameter), resting membrane potential about −80 mV (mean RMP: −80 ± 2 mV, n = 6, recorded in current clamp). Passive cell properties were confirmed by linear I–V characteristics. The recordings were done with the patch-clamp amplifier Multiclamp 700 B (Molecular Devices), filtered at 2.8 kHz, and digitized at 5 kHz with the NI PCI-6221 card (National Instruments). The data were visualized and stored with the software WinWCP (supplied free of charge to academic users by Dr. John Dempster, University of Strathclyde, UK). Bipolar extracellular tungsten electrodes (FHC, Bowdoinham, USA) were placed in CA1 str.radiatum about 100 μm from the recorded astrocyte. 1, 4 and 5 stimuli at 50 Hz were used to induce Isyn in astrocytes. For current recordings the cells were held in voltage clamp mode at −80 mV. Holding current (Ihold = −16 ± 52 pA, n = 6) was measured directly, while input resistance (Ri = 25 ± 7 MΩ, n = 6) and membrane capacitance (Cm = 28 ± 9, pF, n = 6) were calculated from the currents recorded in response to hyperpolarizing voltage steps (−5 mV) applied to the astrocyte. Ihold and series resistance (Rs) were monitored throughout the experiment. If they changed by more than 20% regardless of drug application the recordings were discarded. 2.3. Analysis of currents

2. Materials and methods

The recordings were analyzed with custom-made code written in Matlab. First, Ihold was subtracted from the recordings. Then the traces were averaged in the baseline conditions and during drug application. IK was recorded in the presence of 50 μM DL-TBOA. Then it was tail fitted to Isyn. IGluT was obtained by subtraction of IK from Isyn. IGluT and IK to the fifth stimulus were calculated by subtracting the current to 4 stimuli from the current to 5 stimuli. Because 50 μM DL-TBOA may not block completely EAAT1 we have performed additional recording with 30 μM TBF-TBOA and found similar result (data not shown).

2.1. Hippocampal slice preparation

2.4. Statistical analysis

All the experiments were performed in 30- to 45-day-old SpragueDawley rats. All procedures were done in accordance with University of Nizhny Novgorod regulations. Animals were anesthetized with 1-Chlor2,2,2-trifluorethyl-difluormethylether and then decapitated. The brains were exposed, and then chilled with ice-cold solution containing:

All data are presented as mean ± standard error of mean (SEM). Statistical significance was assessed using nonparametric MannWhitney test for unpaired samples, one-sample Wilcoxon signed rank test and paired-sample Wilcoxon signed rank test for paired samples. A P < 0.05 was considered statistically significant. 38


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Fig. 1. Isolation of IGluT by IK subtraction (TBOA experiment) and by IK blockade (BaCl2 experiment). A. Sample recording of Isyn(1) (black trace) in response to single stimulus with superimposed IK(1) (red trace), obtained in the presence of TBOA (A1) and IGluT(1) (black trace) obtained by subtraction of IK(1) from Isyn(1) (A2). B. Same as in A. Sample recordings of Isyn(1–4) and IK(1–4) in response to 4 stimuli (B1) and IGluT(1–4) obtained by subtraction of IK(1–4) from Isyn(1–4) (B2). C. Same as in A. Sample recordings of Isyn(1–5) and IK(1–5) in response to 5 stimuli (C1) and IGluT(1–5) obtained by subtraction of IK(1–5) from Isyn(1–5) (C2). Red trace in C2 is IGluT(5) obtained by subtraction of IGluT(1–4) from IGluT(1–5). D. Sample recording of Isyn(1) (black trace) in response to single stimulus with superimposed IGluT(1) (red trace), obtained in the presence of BaCl2. E. and F. same as D. but for 4 and 5 stimuli, respectively.

3. Results

proportionally increases membrane time constant. However, 20% increase in membrane time constant cannot be fully responsible for observed changes in τdecay of IGluT in BaCl2. In fact, the increased amplitude and the slowdown of IGluT may be mediated by accumulation of [K+]o upon blockade of astrocytic K+ clearance by BaCl2. Elevated [K+]o depolarizes presynaptic terminals increasing the release probability (Hori and Takahashi, 2009; Shih et al., 2013). Consequently, stimulation of Schaffer collaterals triggers more release events at their synapses located within the same astrocytic domain. Thus, the amplitude of IGluT recorded in this astrocyte increases. Moreover, depolarization of the astrocytes by BaCl2 and buildup of [K+]o can directly affect glutamate uptake which is both electrogenic and K+-dependent (Grewer and Rauen, 2005). [K+]o can accumulate in the synaptic cleft in activity-dependent manner because of large K+ efflux through postsynaptic ionotropic glutamate receptors (Ge and Duan, 2007; De Saint Jan and Westbrook, 2005; Pannasch et al., 2011). Increased [K+]o depolarizes presynaptic terminal triggering frequency dependent facilitation of glutamate release (Shih et al., 2013; Contini et al., 2017). BaCl2 blocks K+ clearance, and therefore can enhance activity-dependent facilitation of IGluT. Surprisingly, there we did not observe a significant difference in activity-dependent IGluT facilitation in BaCl2 (IGluT(5)/IGluT(1): 1.7 ± 0.2, n = 6 in TBOA experiment; and 1.4 ± 0.2, n = 6 in BaCl2; P = 0.37, Mann-Whitney test; Fig. 3A–B). Possibly elevated baseline [K+]o in the presence of BaCl2 had already increased the baseline synaptic release probability limiting its further facilitation. Alternatively, the summation of elevated baseline [K+]o with the activity dependent-increase in [K+]o can reach a level that produces inactivation of presynaptic Ca2+ channels (Hori and Takahashi, 2009). We also observed activity-dependent increase in the τdecay of IGluT in TBOA experiment (τdecay of IGluT(5)/τdecay of IGluT (1): 1.5 ± 0.1, n = 6, P = 0.003, one-sample Wilcoxon signed rank test; Fig. 3C). This funding is consistent with decreased efficiency of glutamate uptake because of astrocyte depolarization caused by activity-dependent [K+]o accumulation. Indeed, in the presence of BaCl2 astrocyte membrane potential becomes insensitive to changes in [K+]o, and therefore no

Isyn was recorder in CA1 stratum (str.) radiatum astrocytes of rat hippocampal slices in response to extracellular Shaffer collateral stimulation (1, 4 and 5 stimuli at 50 Hz; Fig. 1). TBOA abolished IGluT (initial fast transient in Isyn) and left pharmacologically isolated IK. Then the IK was tail fit and subtracted from Isyn to isolate pure IGluT. To estimate activity-dependent changes in glutamate uptake, IGluT to 4 stimuli (IGluT(1–4)) was subtracted from IGluT to 5 stimuli (IGluT(1–5)). Resulting current, IGluT(5) was then compared to IGluT(1) induced by single stimulus. This approach allows obtaining IGluT without possible adverse effects of K+ channel blockers, including buildup of [K+]o and changes in electrical properties of the membrane (e.g. resistance, time constant) (Afzalov et al., 2013; Dvorzhak et al., 2016). Nevertheless, pharmacological isolation of IGluT with Kir blocker, BaCl2, has been used in some previous reports (D’Ambrosio et al., 2002; De Saint Jan and Westbrook, 2005). Although, BaCl2 abolished slow IK (Fig. 1D–F), it also significantly potentiated the amplitude of IGluT (IGluT(1)/Isyn(1): 0.63 ± 0.06, n = 6 in TBOA experiment; and 1.63 ± 0.37, n = 6 in BaCl2; P = 0.013, Mann-Whitney test; Fig. 2A–B). This finding is in agreement with previous demonstrations that BaCl2 can potentiate the transporter currents (Afzalov et al., 2013; Dvorzhak et al., 2016). In addition, BaCl2 increased Ri by about 20 % of baseline (ΔRi = 5 ± 3 M Ω, n = 6, P = 0.03, one-sample Wilcoxon signed rank test), and shifted Ihold towards more negative values (ΔIhold = −144 ± 37 pA, n = 6, P = 0.02, one-sample Wilcoxon signed rank test), consistent with depolarization of the cell upon blockade of K+ conductance. We also observed significantly different time-courses of IGluT isolated by subtraction of IK and isolated pharmacologically with BaCl2. Both time-to-peak and exponential decay time constant (τdecay) were several fold larger in BaCl2 (time-to-peak of IGluT(1): 5.2 ± 0.4 ms, n = 6 in TBOA experiment; and 16.2 ± 0.6 ms, n = 6 in BaCl2; P = 0.005, Mann-Whitney test; Fig. 2C; and τdecay of IGluT(1): 7.1 ± 0.9 ms, n = 6 in TBOA experiment; and 28 ± 2 ms, n = 6 in BaCl2; P = 0.005, Mann-Whitney test; Fig. 2D). This result can be partially explained by the increase in Ri produced by BaCl2, which 39


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Fig. 2. Blockade of Kir with BaCl2 affects the amplitude and time-course of IGluT. A. Sample recordings of Isyn(1) (black traces) with superimposed IGluT(1) (red traces) in response to single stimulus. Top − IGluT(1) was obtained by subtraction of IK (TBOA experiment). Bottom − IGluT(1) was obtained by blocking IK (BaCl2 experiment). B. Summary graph of IGluT(1)/Isyn(1) showing an increase in IGluT(1) in BaCl2. C. and D. Summary graphs showing difference in time-to-peak and τdecay of IGluT(1) obtained in TBOA experiment and in BaCl2 experiment. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, Mann-Whitney test.

activation of these receptors. To test this hypothesis we measured IK in the control (from Isyn) and in the presence of TBOA (from isolated IK) at 200 ms after the stimulus (Fig. 4A). At this time point in the control IGluT is ended and the current is mediated purely by K+ (Shih et al., 2013). We did not observed a significant change in the amplitude of IK in response single stimulus in the presence of TBOA (IK(1) in TBOA normalized to control Isyn: 0.88 ± 0.12, n = 6, P = 0.2, one-sample Wilcoxon signed rank test; Fig. 4B). Notably, the τdecay of IK(1) became significantly smaller in TBOA (τdecay of IK normalized to control Isyn: 0.82 ± 0.08, n = 6 P = 0.02, one-sample Wilcoxon signed rank test;

activity-dependent increase but even some decrease (although nonsignificant) in τdecay of IGluT was detected (τdecay of IGluT(5)/τdecay of IGluT(1): 0.80 ± 0.07, n = 6, P = 0.07, one-sample Wilcoxon signed rank test, and P = 0.005 for difference with TBOA experiment, MannWhitney test; Fig. 3C). The above experiments suggest that activity-dependent accumulation of [K+]o can affect both presynaptic glutamate release by depolarizing presynaptic terminal and glutamate uptake depolarizing PAPs. Because postsynaptic receptors are the major source of K+, TBOA can also affect [K+]o by prolonging glutamate dwell-time and thus

Fig. 3. Activity dependent changes in IGluT. A. Sample recordings of IGluT(1) in response to single stimulus (black traces) with superimposed IGluT(5) response to fifth stimulus in the train of 5 stim. x 50 Hz (red traces). Top − Currents obtained by IK subtraction (TBOA experiment). Bottom − Currents obtained by IK blockade (BaCl2 experiment). B. Summary graph of IGluT(5)/IGluT(1) showing no significant difference in activity-dependent facilitation of IGluT between TBOA and BaCl2 experiments. C. Summary graph of τdecay of IGluT(5)/τdecay of IGluT(1) showing that BaCl2 abolishes activity-dependent prolongation of IGluT. Data presented as mean ± SEM. N.S. (non-significant) P > 0.05, **P < 0.01, Mann-Whitney test.

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Fig. 4. Blockade of glutamate transporters with TBOA affects the time-course of IK. A. Sample recordings of Isyn(1) in response to single stimulus (black traces) with superimposed IK(1) recorded in TBOA (red traces, TBOA). Both used for analysis of K+ current in control and upon blockade of glutamate transporters. Inset − magnified initial part of the currents. Grey bar indicates the time point (200 ms from the stimulus) where amplitudes and decays of the currents were measured in Ctrl and TBOA. B. Summary graph of IK normalized to Isyn shows no significant difference between current amplitudes in Ctrl and in TBOA. B. Summary graph of τdecay of IK normalized to τdecay of Isyn shows significant decrease of IK decay in TBOA. Data presented as mean ± SEM. N.S. (non-significant) P > 0.05,*P < 0.05, Mann-Whitney test.

Fig. 4C). This finding was rather unexpected and is consistent with reduced K+ release during single pulse stimulation upon blockade of glutamate transporters. Next, we investigated if TBOA can affect activity-dependent changes in [K+]o. To estimate activity-dependent changes in [K+]o, IK to 4 stimuli (IK(1–4)) was subtracted from IK to 5 stimuli (IK(1–5)). Resulting currents, IK(5) was then compared to IK(1). The same protocol was applied to IK in TBOA and to Isyn to estimate K+ current without TBOA. In the control conditions, we observed activity-dependent depression of K+ current in Isyn (Isyn(5)/Isyn(1): 0.63 ± 0.04, n = 6; P = 0.01, onesample Wilcoxon signed rank test; Fig. 5A–B). In agreement with our hypothesis, IK(5) in the presence of TBOA was enhanced relatively to Isyn(5) (IK(5)/IK(1): 1.1 ± 0.2, n = 6, P = 0.4, one-sample Wilcoxon signed rank test; P = 0.03 for difference with Isyn(5)/Isyn(1), pairedsample Wilcoxon signed rank test; Fig. 5A–B). There was no significant activity-dependent change in τdecay of Isyn in control conditions measured from 200 ms point (τdecay of Isyn(5)/ τdecay of Isyn(1): 1.0 ± 0.1, n = 6, P = 0.4, one-sample Wilcoxon signed rank test; Fig. 5C). Similarly to the IK amplitude, we observed significant activity-dependent increase in τdecay of IK in TBOA (τdecay of IK(5)/τdecay of IK(1): 1.5 ± 0.2, n = 6, P = 0.03, one-sample Wilcoxon signed rank test; P = 0.02 for difference with τdecay of Isyn(5)/τdecay of Isyn(1), paired-sample Wilcoxon signed rank test; Fig. 5C). These results suggest that blockade of glutamate uptake leads to enhancement of activity-dependent K+ efflux. This can be because of longer glutamate dwell-time in the synaptic cleft and consequently longer activation of postsynaptic receptors.

4. Discussion Astrocytes remove glutamate and clear K+ which are released during synaptic transmission. Typically, these processes are studied by measuring astrocytic IGluT and IK, respectively. The amplitudes and time-courses of these currents can be influenced by a number of factors. Here we show that activity-depended accumulation of K+ affects IGluT, while activity dependent‐accumulation of glutamate affects IK. To decrease the efficiency of [K+]o clearance we blocked Kir with BaCl2. Under these conditions, multiple stimulation (5 stim × 50 Hz) should produce more [K+]o accumulation in the synaptic cleft than in control recordings. Consequently, this should depolarize the presynaptic terminal and increase presynaptic release probability and IGluT to a greater degree (Contini et al., 2017; Shih et al., 2013). Surprisingly, the IGluT(5)/IGluT(1) ratio was not increased, but decreased in BaCl2. This finding is consistent with previous report suggesting that moderate increase in [K+]o increases presynaptic release probability, while large increase depresses presynaptic release (Hori and Takahashi, 2009). In addition, BaCl2 can directly affect IGluT (Patrushev et al., 2013) [K+]o accumulation may not only depolarize presynaptic terminals, it may affect astrocytic glutamate uptake too. Firstly, K+ is one of the ions transported by EAATs, and change in K+ gradient can potentially affect glutamate transport (Grewer et al., 2008; Grewer and Rauen, 2005). Secondly, [K+]o accumulation depolarizes PAPs, which can affect voltage dependent stages of glutamate transport (Grewer et al., 2008; Grewer and Rauen, 2005). There is evidence that glutamate dwell-time in the synaptic cleft is largely determined by voltage-dependent substrate translocation stage (Mennerick et al., 1999) Indeed, we observed significant increase τdecay of IGluT(5) compare to τdecay of IGluT(1) in TBOA experiments. Notably, τdecay of IGluT(5) was not Fig. 5. Activity dependent changes in IK. A. Sample currents in response to single stimulus (black traces) with superimposed current to fifth stimulus in the train of 5 stim. x 50 Hz (red traces). Top − Control experiment (Ctrl), Isyn(1) and Isyn(5) without any drugs. Bottom − IK(1) and IK(5) isolated by TBOA application (TBOA). Grey bars indicate the time point (200 ms from the stimulus) where amplitudes and decays of the currents were measured in Ctrl and TBOA. B. Summary graph of Isyn(5)/Isyn(1) for Ctrl and IK(5)/IK(1) for TBOA showing activity dependent enhancement of IK in TBOA relatively to Ctrl. C. Summary graph of τdecay of Isyn(5)/τdecay of Isyn(1) for Ctrl and τdecay of IK(5)/τdecay of IK(1) for TBOA showing activity dependent enhancement of τdecay of IK in TBOA relatively to Ctrl. Data presented as mean ± SEM. *P < 0.05, MannWhitney test.

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increased in BaCl2 when astrocytes could not be depolarized by [K+]o elevation. This suggests that cell depolarization rather than K+ gradient may suppress astrocytic glutamate uptake. This result is of particular importance because several pathological processes (e.g. epilepsy) can be associated both with enhanced K+ efflux and with reduced K+ clearance (Bedner et al., 2015; Frohlich et al., 2008). Our findings predict reduced glutamate uptake and longer glutamate dwell-time in these conditions. Consequently, longer glutamate presence in the synaptic cleft can increase K+ efflux though glutamatergic AMPA and NMDA receptors serving as the major sources of K+ release during synaptic transmission (Ge and Duan, 2007; Shih et al., 2013; De Saint Jan and Westbrook, 2005; Pannasch et al., 2011). Consistent with this hypothesis blocking glutamate uptake with TBOA triggered significant facilitation of IK(5) amplitude and τdecay compare to control recordings. In summary, we suggest that glutamate release probability, efficiency of glutamate uptake, postsynaptic K+ efflux and astrocytic K+ clearance during synaptic transmission interact to each other in activity-dependent manner. Because K+ release and clearance may be significantly altered in several neurological conditions such as stroke, migraine, epilepsy (Bedner et al., 2015; Frohlich et al., 2008; Dreier and Reiffurth, 2015), our findings suggest a causal link between altered K+ dynamics and changes in glutamate release and uptake in these diseases. The future work should consider the role of other astrocytic signaling routes shaping extracellular glutamate and [K+]o dynamics. Synaptic activity can trigger Ca2+ elevations in astrocytes or change properties (duration and size) of spontaneous Ca2+ events in astrocytes (Wu et al., 2014; Zorec et al., 2012; Rusakov et al., 2011). Astrocytic Ca2+ promotes K+ clearance by activation of Ca2+ dependent K+ channels (Wang et al., 2012). Astrocytic Ca2+ regulates morphological plasticity in astrocytes (Tanaka et al., 2013). When astrocytic Ca2+ dynamics is suppressed synaptic coverage by PAPs is also reduced. This leads to activity-dependent prolongation of IGluT and enhanced glutamate spillover. Such morphological remodeling of astrocytes is characteristic to some physiological conditions such as lactation (Oliet et al. 2001) and pathological conditions such as epilepsy, Alzheimer’s disease and Huntington disease (Pekny et al., 2016; Verkhratsky et al., 2014).

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