Gap Junction Coupling Confers Isopotentiality on ...

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Oct 7, 2015 - Baofeng Ma,1 Richard Buckalew,2 Yixing Du,1 Conrad M. Kiyoshi,1 .... The C57/BL6 mice of both genders older than P21 (Charles River),.
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Gap junction coupling confers isopotentiality on astrocyte syncytium ARTICLE in GLIA · OCTOBER 2015 Impact Factor: 6.03 · DOI: 10.1002/glia.22924

10 AUTHORS, INCLUDING: Baofeng Ma

Min Zhou

The Ohio State University

The Ohio State University

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Available from: Min Zhou Retrieved on: 07 October 2015

RESEARCH ARTICLE

Gap Junction Coupling Confers Isopotentiality on Astrocyte Syncytium Baofeng Ma,1 Richard Buckalew,2 Yixing Du,1 Conrad M. Kiyoshi,1 Catherine C. Alford,1 Wei Wang,1 Dana M. McTigue,1 John J. Enyeart,1 David Terman,3 and Min Zhou1 Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K1 free pipette solutions that alter the K1 equilibrium potential to non-physiological voltages. We show that an astrocyte’s associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte’s VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K1 and thereby maintains a sustained driving force for highly efficient K1 uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits. Key words: electrical coupling, coupling coefficient, membrane potential, K1 clearance

Introduction

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stablishment of a syncytium through gap junction coupling is a prominent feature of astrocytes in the central nervous system (Brightman and Reese, 1969; Dermietzel and Spray, 1993; Giaume et al., 2010; Ransom, 1996). Gap junction coupling is known to mediate the exchange of small molecules (< 1.2 kDa). This facilitates important homeostatic and signaling functions of astrocytes, such as spatial buffering of K1 and Na1 ions and the long-range redistribution of nutrients, metabolites and signaling molecules for the coordination of neuronal activity and brain energy metabolism (Kuga et al., 2011; Langer et al., 2012; Lin et al., 1998; Newman, 2001; Orkand et al., 1966; Rose and Ransom, 1997; Rouach et al., 2008; Simard et al., 2003; Wang et al., 2012). Gap junctions also enable electrical coupling to minimize membrane potential (VM) differences among interconnected

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cells in a mutual and rapid manner. For pairs of coupled cells, the strength of electrical coupling, termed the coupling coefficient (CC), is commonly evaluated by the ratio of voltage in coupled to that of injected cell (transjunctional voltage to input voltage) using the dual patch recording method. However, the CC between any two nearest neighboring astrocytes in the intact brain has been a long-standing but unresolved question. Although the transjunctional voltage analysis has been used to explore this question between astroyctes (Ceelen et al., 2001; Kettenmann and Ransom, 1988; Meme et al., 2009; Muller et al., 1996; Ransom and Kettenmann, 1990; Xu et al., 2010), the CC cannot be accurately measured due to an extremely low membrane resistance that shunts the experimentally injected currents through astrocyte’s low resistance membrane (6 MX) (Ma et al., 2014). At the syncytial level, each hippocampal astrocyte is directly coupled with 11 nearest neighbors, and several

View this article online at wileyonlinelibrary.com. DOI: 10.1002/glia.22924 Published online Month 00, 2015 in Wiley Online Library (wileyonlinelibrary.com). Received July 1, 2015, Accepted for publication Sep 11, 2015. Address correspondence to Min Zhou, 4066C Graves Hall, 333 West 10th Avenue, Columbus, OH 43210. E-mail: [email protected] From the 1Department of Neuroscience, the Ohio State University Wexner Medical Center, Columbus, Ohio 43210; 2Mathematical Biosciences Institute, the Ohio State University, Columbus, Ohio 43210; 3Department of Mathematics, the Ohio State University, Columbus, Ohio 43210 B.M. and M.Z. designed experiments; B.M. and Y.D. performed experiments; D.M.M. contributed the transgenic mice; B.M., R.B., D.T., and J.J.E. performed theoretical analysis; B.M., J.J.E., R.B., D.T., and M.Z. wrote the manuscript. All authors discussed the results and commented on the manuscript. Wei Wang is currently at Department of Physiology, Huazhong University of Science and Technology, Tongji Medical College, Wuhan, Hubei 430030, China Additional Supporting Information may be found in the online version of this article.

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hundreds of astrocytes are coupled in a syncytium (D’Ambrosio et al., 1998; Xu et al., 2010). An intriguing question is how the aggregate coupling strength affects the VM and other physiological behavior of any individual astrocyte in a syncytium. No methodology is, as yet, available to tackle these fundamentally important questions. In this study, we combine electrophysiological and computational modeling methods to investigate the mechanisms by which nearest neighboring astrocytes coordinate the VM’s in a syncytium. The study is conceived based on a basic feature of astrocytes: they behave as perfect K1 electrodes as a result of their predominant expression of leak type K1 conductances (Kuffler et al., 1966; Ransom and Goldring, 1973). Therefore, one can experimentally lower the intracellular K1 concentration ([K1]i) using reduced or K1-free pipette solutions to establish a depolarized K1 equilibrium potential (EK) in a recorded astrocyte to levels that can be predicted by the membrane equilibrium potential (EM) from the GoldmanHodgkin-Katz (GHK) equation. The nearest neighbors should then act to hyperpolarize the recorded astrocyte by minimizing the differences in K1 concentrations (ionic coupling) and membrane potential (electrical coupling). Consequently, the deviation of measured VM from the experimentally established EM in the recorded astrocyte can serve as a reliable indicator of the coupling strength among astrocytes in a syncytium (See mathematic modeling in Suppl. info.). We hypothesized that a strong gap junction coupling among hippocampal CA1 astrocytes suppresses the VM depolarization in recordings made with reduced or free K1 pipette solutions.

Materials and Methods Animals The C57/BL6 mice of both genders older than P21 (Charles River), and PDGFRA-driven eGFP transgenic mice (Hesp et al., 2015) were used in this study according to the guidelines of the Institutional Animal Care and Use Committee, The Ohio State University.

Hippocampal Slice Preparation For slice recording, hippocampal slices were prepared as described previously (Ma et al., 2014). In brief, after anesthesia with 8% chloral hydrate in 0.9% NaCl, the mouse brain was rapidly removed from the skull and submerged into ice-cold oxygenated (95% O2/ 5% CO2) cutting solution containing (in mM): 125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 0.1 CaCl2, 3 MgCl2, and 10 glucose. Coronal hippocampal slices (250 lm) were cut at 48C with a Vibratome (Pelco 1500) and transferred to the normal aCSF (in mM): Abbreviations

CC GHK IR-DIC MFA

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Coupling coefficient Goldman-Hodgkin-Katz Infrared differential interference Meclofenamic acid

125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, and 10 glucose (osmolality, 295 6 5 mOsm; pH 7.3–7.4) at room temperature (20–228C). Slices were kept in aCSF with continuous oxygenation for at least 1 hour before recording.

Sulforhodamine 101 Staining For sulforhodamine 101 (SR101, Invitrogen, New York, NY) staining (Nimmerjahn et al., 2004), the slices were transferred to a sliceholding basket containing 0.6 lM SR101 in aCSF at 348C for 30 min. Then, the basket was transferred back to the normal aCSF at room temperature before the experiment.

Fresh Dissociation of Hippocampal Tissues At the animal age older than P21, the spatially distinct domains, electrical coupling, and K1 channel expression have all reached maturity (Bushong et al., 2004; Xu et al., 2010; Zhou et al., 2006). To dissociate single and multiple astrocytes with intact domain and functional gap junctional coupling, a new hippocampal tissue dissociation method was used (Du et al., 2015). Briefly, after incubation with SR-101 for 30 min, the CA1 regions were dissected out from slices, cut into small pieces (1 mm2) and then placed in oxygenated aCSF containing 24U/ml papain and 0.8 mg/ml L-cysteine for 7 min. After papain digestion, a fire polished glass pipette, diameter 150 lm, was used to triturate the loosened tissues five to seven times into a tissue suspension and then transferred into the recording chamber. The dissociated cells were allowed 3-5 min to touch down to the bottom of the chamber before switching on a constant aCSF perfusion at flow rate of 2 ml/min. The viable astrocytes, with their elaborate processes and well-preserved domain shape, are similar to their counterparts in situ (Du et al., 2015).

Electrophysiology To record astrocytes in CA1 stratum radium, individual slices were transferred to a recording chamber (RC-22, Warner Instruments, Holliston, MA), mounted on a Olympus BX51WI microscope equipped with infrared differential interference (IR-DIC), and were perfused with oxygenated aCSF (2.5 mM/min) at room temperature. Astrocytes in situ were identified based on SR101 staining; they typically exhibited an irregular soma shape, diameter around 10 lm, with several visible primary processes stemming from the soma. The NG2 glial cells were identified by GFP from PDGFRA-driven eGFP transgenic mice (Hesp et al., 2015). The interneurons were identified based on their large soma size and shapes. After establishing whole-cell configuration, the mature astrocytes were unequivocally identified by their expression of passive membrane K1 conductance (Zhou et al., 2006). Interneurons expressed large inward Na1 currents in voltage clamp recording and fired action potentials spontaneously when switched to current clamp recording (Fig. 1F). Recording pipettes were fabricated from borosilicate capillaries (1.5/0.86 mm outer/inner diameter, Warner Instruments, Holliston, MA) using a Flaming/Brown Micropipette Puller (Model P-87, Sutter Instrument). When filled with pipette solution noted below, the pipettes had the open tip resistance of 2.5-3.5 MX. The standard pipette solution contained (in mM): 140 KCl or Kgluconate, 0.5

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FIGURE 1: Physiological VM can be well-maintained in astrocyte recordings with reduced or K1 free pipette solutions in situ. (A) SR101 staining of astrocytes in CA1 region. (B and C) Astrocyte VM recordings, first in cell-attached, then in whole-cell mode after membrane patch breakthrough (arrows). The resting VM’s were comparable between [K1]p and [Na1]p solutions. (D and E) An NG2 glial cell identified from PDGF-driven GFP transgenic mouse CA1 region in situ and its VM recording with [Na1]p. The VM showed an initial hyperpolarization and a following depolarization. (F) The VM recording from an interneuron with [Na1]p, showing an initially hyperpolarization (240 mV), followed by a depolarization (~0 mV). A burst of spikes appeared shortly after patch breakthrough (upper inset). (G) Summary of the VM values for the cell types and conditions indicated (one-way ANOVA with post-hoc F test). (H) Summary of the VM values from astrocytes recorded with various pipette NMDG1 concentrations in situ (one-way ANOVA with post-hoc F test). (I) Gap junction inhibition by 100 lM MFA depolarized VM and the subsequent Kir4.1 inhibition (100 mM Ba21) hyperpolarized VM as predicted by the mathematical model (Supp. Info. Fig. 4). (J) Summary of the VM values in MFA, MFA1Ba21 and a predicted value for the latter from the model (paired sample t test). *P < 0.05. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

CaCl2, 1 MgCl2, 5 EDTA, 10 HEPES, 3 Mg21-ATP and 0.3 Na1 2 GTP that was titrated with KOH to pH 7.25-7.27. The final osmolality was 280 mOsm. The 140 mM KCl was substituted by NaCl, or NMDGCl either partially or fully in the referred experiments. Whole-cell membrane current or membrane potential was amplified by a MultiClamp 700A or MultiClamp 700B amplifiers,

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and the data acquisition was controlled by PClamp 9.2 (Molecular Devices, Sunnyvale, CA) installed on Dell personal computers. DIGIDATA 1322A interface was used to convert digital-analog signals between amplifier and computer. A minimum of 2 GX seal resistance was required before rupturing the membrane for whole-cell configuration. The total membrane resistance, or input resistance

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(Rin), was measured by using the “Membrane test” protocol built into the pClampex. The access resistance (Ra) was not compensated for in all the voltage clamp recordings for low RM astrocytes (Ma et al., 2014). The membrane potential (VM) was read either in “I 5 0” mode or measured directly in current clamp mode with no holding currents. Astrocytes with a resting membrane potential more positive than 270 mV in both brain slices and freshly isolated single astrocytes were discarded. All the experiments were conducted at room temperature (20 6 28C). The liquid junction potential was compensated for before establishment of cell-attached mode in all the recordings and confirmed to be  0 mV after experiments by withdrawal of recording pipette. SR101 positive staining was used to confirm the astrocytic identity and determine the number of astrocytes in a freshly dissociated tissue block. The same patch clamp set-up and procedure, for slice astrocyte recording, was used for freshly dissociated astrocyte recording. In the experiment for manipulating coupling strength, brain slices were pretreated with aCSF containing 100 mM meclofenamic acid (MFA) for one hour before recording and perfused with the same solution during recording. When VM was stable, 100 mM MFA plus 100 mM Ba21 was bath applied. In the experiment for determining effect of 100 lM MFA 1 100 lM Ba21 on the RM, dual patch recording was used as we previous reported (Ma et al., 2014).

Local High K1 Application We used VC34 Controller (ALA Scientific Instruments) for focal high K1 application (pressure 6 psi). Focal high K1 (23.5 mM) or aCSF was delivered via a pipette to the recorded cell 10 mm away from soma, and 100 mM SR-101 was included in the application solutions to visualize and measure the areas affected by focally applied high K1. Software provided by ALA Scientific Instruments was used for programming of the durations of high K1 application.

Imaging Capture Confocal images were obtained using a Zeiss LSM 510 at the HuntCurtis Imaging Facility. A fluorescent imaging system (Polychrome V system from Till Photonics, Germany) was used for Epifluorescence imaging and high resolution visualization of small glial soma for dual patch recording. This system was also used for identifying astrocyte based on SR101 positive staining (Figs. 1A and 2B), and for simultaneous dual patch.

Statistical Analysis Data are presented as mean 6 SEM. Mean differences between groups were using t test or two-way ANOVAs followed by post hoc testing when main effect was significant at P < 0.05. Data were analyzed using Origin 11 software package (OriginLab, Northampton, MA).

Results Syncytial Coupling Equalizes the Membrane Potential among Astrocytes in the Network To test our hypothesis, we identified astrocytes based on SR101 staining from the hippocampal CA1 region (10) and recorded 4

VM under current clamp mode. In control experiments, we used patch pipette solution containing 140 mM K1 (K1-based solution, [K1]p) (Fig. 1A,B). In cell-attached mode, the gigaohm seal formation led to an anticipated VM shift from 0 mV to 238.1 6 18.5 mV (n 5 13) for the low membrane resistance astrocytes (Supp. Info. Fig. 1). The breakthrough of the membrane patch shifted the VM immediately to a resting membrane potential of 278.1 6 0.7 mV (n 5 11) (Fig. 1B), as expected for cells almost exclusively permeable to K1 (17). When similar recordings were made with patch pipettes in which the [K1]i was reduced from 140 mM to 70 mM by substitution with Na1, the recorded VM was essentially unchanged (278.1 6 1.1 mV, n 5 6) (Fig. 1G) and differed significantly from the GHK predicted 11.1 mV depolarization. Furthermore, the GHK equation predicts a 105 mV depolarization if pipette K1 is totally substituted by Na1 (Na1-based solution, [Na1]p). However, Na1-based solution depolarized the cells only by 4.5 mV (273.5 6 0.9 mV, n 5 10), compared to the control astrocytes (Fig. 1C,G). In all recordings with reduced or K1-free pipette solutions, the recorded VM remained at a steady-state level during a recording time of 10 min. In some recordings, we purposely extended time to over one hour, and the steady-state VM remained unchanged, indicating gap junction coupling provided a sustained VM control over recorded astrocytes. These results support the hypothesis that there exists a powerful gap junction coupling among CA1 astrocytes in situ (Supp. Info. Fig. 2). To determine if a similar VM behavior occurs in other uncoupled cells in this brain region (Xu et al., 2014), we used Na1-based pipette solution to record NG2 glia in hippocampal slices prepared from PDGFa1-driven-GFP transgenic mice (Fig. 1D)(Hesp et al., 2015); NG2 glia are known to also function as K1 electrodes (Maldonado et al., 2013). NG2 glia followed the GHK prediction by depolarizing rapidly to a stable VM level of 7.3 6 1.9 mV (n 5 9, P < 0.01) (Fig. 1E,G). Likewise, uncoupled CA1 interneurons also depolarized to 1.3 6 0.7 mV (n 5 5) when recorded with Na1-based pipette solution (Fig. 1F,G). To ensure that such “anomalous” VM behavior was not specific to Na1 ions, we replaced K1 either partially or fully with NMDG1. Substitution of NMDG1 for K1 resulted in VM changes similar to those observed from Na1 substitution (Fig. 1H). Thus, syncytial coupling suppresses the VM depolarization that is expected for an uncoupled single astrocyte recorded after a full substitution of intracellular K1 by Na1 or NMDG. To confirm that gap junction coupling was responsible for the equalization of VM in astrocytes, we pre-incubated the brain slices with 100 lM meclofenamic acid (MFA) for 1 hour to block the gap junction coupling (Xu et al., 2010). Under this condition, the Na1-based solution depolarized astrocytes to a steady-state VM of 213.9 6 1.8 mV (n 5 10) Volume 00, No. 00

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in MFA (Fig. 1I). The computational model predicts that this corresponds to a 99.3% inhibition of coupling strength (s 5 coupling conductance/membrane conductance of nearest neighbors) with MFA (Supp. Info. Fig. 4). To confirm this prediction, 100 mM Ba21 was applied to inhibit a major astrocyte K1 channel, Kir4.1. This increased the membrane resistance, RM, from the control 6.3 6 0.7 MX to 18.4 6 4.4 MX (n 57), corresponding to a 2.9-fold increase in the coupling strength and a predicted VM hyperpolarization to 223.9 mV (Supp. Info. Fig. 4). Consistent with this prediction, the astrocyte VM hyperpolarized to 223.4 6 2.7 mV (n 5 10) in the presence of 100 lM Ba21 in MFA aCSF (Fig. 1I,J). Thus gap junction coupling is responsible for the mildly altered astrocyte VM recorded with MFA and a Na1based pipette solution in situ. Moreover, the experimental results validated the mathematical model we developed for this study. Larger Syncytia Have Stronger Control Over an Individual Astrocyte’s VM The computational model predicts that if the number of cells that are coupled to the recorded cell with Na1-based electrode solution is increased, then the recorded VM will lie closer to the physiological EM of the nearest neighbors (Supp. Info. Fig. 2). To test this prediction, we used freshly isolated hippocampal tissues that contained single to multiple astrocytes with their spatially distinct domains and functional K1 channels well-maintained (Du et al., 2015) (Fig. 2A). A Na1based pipette solution was used to record astrocytes located in the center of the tissues. As the number of cells in the syncytium that were coupled to the recorded cell increased, so did the deviation of the recorded VM from the GHK predicted value for a Na1-based pipette solution (Fig. 2C–F,K). Thus, the number of neighboring astrocytes has an aggregate effect in suppressing the VM depolarization induced by Na1-loading in the recorded astrocyte (Supp. Info. Fig. 2). In single astrocytes, the same Na1-based pipette solution induced an initial hyperpolarization, reflecting the undisturbed initial [K1]i, followed by a depolarization to 20.02 6 0.74 mV (n 5 12), as predicted by the GHK equation (Fig. 2G). A subsequent bath application of elevated 23.5 mM K1 also induced a predicted VM depolarization (Fig. 2H). When switching to voltage-clamp recordings, the depolarization induced outward currents were almost fully eliminated due to the absence of intracellular K1 ions (Fig. 2I left). Removal of the 3.5 mM K1 in the bath solution ([K1]e) further abolished the inward currents (Fig. 2I middle), and the inward currents reemerged when 3.5 mM [K1]e was restored (Fig. 2I right). In contrast, the breakthrough of the membrane patch with K1-base pipette solution shifted the VM to the anticipated resting VM of 278.4 6 1.9 mV Month 2015

(n 5 11) in single astrocytes (Fig. 2J), which was comparable with astrocytes in slices (Fig. 1G). Thus, independent of which pipette solution was used, single astrocytes behave as a perfect K1 electrode. Contribution of Ionic Coupling in Maintaining the Astrocyte VM In Situ In astrocyte recordings with Na1-based solution in situ, the K1-mediated outward currents remained unchanged (Fig. 3A), suggesting the presence of K1 conducting ions in the recorded cell due to ionic coupling with the associated syncytium. To determine how efficient gap junctions are in permitting ion exchange between coupled astrocytes, we used Na1based pipette solution to compare the time required for the full substitution of intracellular K1 in a freshly dissociated, single astrocyte with the time required in a pair of coupled astrocytes (doublet) (Fig. 3B). In both cases, a complete substitution of the endogenous K1 by Na1 from the pipette was indicated by the depolarization of VM from its resting value to 0 mV, as predicted by the GHK equation. The computational modeling predicts that the high ion exchange efficacy enables a doublet to replace all the endogenous K1 by pipette Na1 with a time course similar to that of a single astrocyte (Fig. 3C, s 5 100). Let T50 be the half time for VM to reach 0 mV. Then the ion exchange efficacy at the gap junctions can be estimated by computing the difference between the T50’s in single and doublet astrocytes (Fig. 3C). The T50 was 12 times slower in doublets (4.67 6 0.36 min, n 5 5) than that of single astrocytes (0.39 6 0.25 min, n 5 3) (Fig. 3D,E). Thus, because of its relatively slow kinetics (see Supp. Info. Fig. 5 for further modeling analysis), ionic coupling is unlikely to be the major determinant in maintaining a constant astrocyte VM in situ (Fig. 1C,G,H). A Strong Electrical Coupling Confers Isopotentiality to Coupled Astrocytes Electrical coupling is able to rapidly equilibrate the VM’s of coupled neurons (Connors and Long, 2004). To examine the strength of electrical coupling and the level of VM equilibration in coupled astrocytes, we considered a doublet in which the EM’s of the two astrocytes were experimentally set-up at sharply different levels. In dual patch recordings, a K1-based pipette (P1) was sealed on one of the astrocytes, while a Na1-based pipette (P2) was sealed on the other astrocyte (Fig. 4A1). The membrane of the astrocyte with the K1-based electrode was ruptured first to measure the resting VM of the doublet (Fig. 4A1); this did not interfere with the intercellular K1 concentration, [K1]i. The subsequent breakthrough of P2 initiated a Na1 influx and a reequilibration of [K1]i and [Na1]i in the recorded doublet. Consistent with the ion exchange T50 at doublet gap junctions (Fig. 3D), the computational model predicts that [K1]i and [Na1]i 5

FIGURE 2: Syncytium dictates the VM of an individual astrocyte inside the network. (A) Images show SR101 staining from one, two and three astrocytes in dissociated tissues with their spatially distinct domains and that the elaborate processes were well preserved. (B) A live single astrocyte captured during recording: SR101 staining (left) and an intact bushy morphology (DIC, right). (C–F) Representative recordings from freshly dissociated hippocampal tissues containing a varied number of astrocytes with [Na1]p. A negative current pulse was periodically injected into the cells to monitor their input resistance (Rin) in all the recordings, and the two representative traces from (F) are shown in expanded scale on the right panel. (G) The VM recording from a single dissociated astrocyte with [Na1]p. The cell showed an initial hyperpolarization and then depolarized to the anticipated 0 mV. (H) The same cell in (G) depolarized in response to 23.5 mM bath K1 application. (I) Whole-cell currents from a dissociated single astrocyte made with [Na1]p, first in aCSF (left) and then after removal (middle) and restoration (right) of 3.5 mM [K1]e in aCSF. (J) The VM recording from an isolated astrocyte with [K1]p. (K) Quantification of VM in [K1]p and [Na1]p pipette solutions (one-way ANOVA with post-hoc F test). *P < 0.05, one way ANOVA test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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FIGURE 3: The ion exchange efficacy at gap junction sites. (A) Representative VM recordings from [K1]p and [Na1]p pipette solutions, as indicated in situ. (B) Schematic illustrations of Na1 dialysis and substitution occurring in a single and a doublet astrocytes with a difference in the presence of gap junctional coupling in the latter. (C) Modeling predictions of the time courses for doublet astrocytes to reach to a full Na1 substitution with different gap junction coupling strengths. The numerical 1 is the prediction for the highest ionic diffusion rate in a doublet; this predicts a time course that is most similar to that of a single cell. (D) The VM recordings from single and doublet astrocytes with [Na1]p to determine their T50’s. (E) The T50 values for Na1 ion substitutions in single and doublet astrocytes (two sample t test). **P < 0.01, one way ANOVA test. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

should reach their new steady-state levels within 120 seconds (Fig. 4A2). There is then an intracellular [K1]i gradient from 140 mM at P1 to 0 mM K1 at P2. This corresponds to EM equal to 274 mV and 210 mV at the respective pipettes (Fig. 4A2). Despite a rapid separation between the battery potentials (EM) of P1 and P2, the breakthrough of P2 resulted in an instantaneous equilibration of the VM’s to comparable levels at both pipettes (left inset, Fig. 4B). A strong electrical coupling was evident by the closely matched VM amplitudes in the two cells as they depolarized in parallel towards their new steady-state levels (middle inset, Fig. 4B). Month 2015

To examine the similarity between the dynamics of doublets and single astrocytes, we repeated the same experiment in single astrocytes (Fig. 4A3). The breakthrough of the P2 resulted in a pattern of VM equilibration similar to that observed in doublets (Fig. 4C, and right inset). The VM trajectories, in single and doublet astrocytes, all reached steadystate levels with comparable time courses (Fig. 4B,C). To quantify their similarities, we fitted a single exponential to each VM depolarization, starting at the time of breakthrough of P2. The resulting time constants (s) and depolarization amplitudes (A) are comparable in all four pipettes (Fig. 4D), 7

FIGURE 4: Astrocyte doublets show equilibrated VM. (A1) Schematic illustration of the recording procedure to achieve a separation of EM in a doublet. The K1-based electrode, P1, is ruptured first. This does not alter the [K1]i gradient, but enables the reading of VM. (A2) The model prediction of EM separation between P1 and P2 during Na1-dialysis and at the steady-state levels. (A3) Similar to a1 to separate the EM in a single astrocyte at P1 and P2. (B–C) The VM recordings from a doublet (B) and a single astrocyte (C) following the procedure described in a. Each trace is color coded with its corresponding recording pipette (P). In a doublet, the initial VM, just after the breakthrough of P2, is shown in expanded scale to disclose the immediate equilibration of VM in both cells. In both doublet and single astrocytes, the progression of VM to the steady-state levels are shown in expanded scale (middle and right insets, respectively). Each trace was superimposed with a single exponential fit in order to determine a time constant (s) and VM amplitude. (D) The derived s’s and the VM amplitudes among the four pipettes. (E) Illustration of the use of K1 free solutions to maximally reduce the transjunctional voltage and allow for accurate measurement of the CC and gap junction resistance (Rg) (one-way ANOVA with post-hoc F test). (F–G) The respective current clamp (F) and combined voltage and current clamp (G) recording modes for measurement of CC and Rg, as indicated. The access resistance (Ra) was not compensated for the delivered VC in P1(G). The resulting strong CC and low Rg indicate that doublets electrically behave as single astrocytes. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Ma et al.: Electrical Coupling of Astrocytes in a Syncytium

indicating that doublets behave electrically as one single astrocyte. The time constants in doublets were also similar to the T50 of single astrocytes (Fig. 3D,E). This indicates that Na1 influx predominantly replaced the endogenous K1 in the P2 attached cell; moreover, the cross-diffusion of Na1 and K1 across the gap junction was minimal in coupled doublets, as predicted by the computational model (Fig. 4A2 and Supp. Info. Fig. 6). These results demonstrate that a powerful electrical coupling equilibrates the VM’s of coupled astrocytes to a comparable level. To directly estimate the gap junction coupling resistance in doublets, we replaced all K1 by Na1 in the recording solutions (Figs. 2I and 4E). This allows for maximal reduction in current shunting through the low resistance membrane. Under this condition, current injection in one astrocyte induced almost the same VM shift in both astrocytes with a calculated coupling coefficient (CC) of 94.3 6 3.2% (n 5 3) (Fig. 4F) and a coupling resistance (Rg) of 4.2 6 1.5 MX (n 5 3, pair distance 30.5 6 3.1 lm) (Bennett, 1966). As the Rg appeared to be even lower than the RM of single astrocytes (Du et al., 2015), this suggested that, with maximally reduced current shunting, doublets should behave as single astrocytes in voltage clamp recordings (Supp. Info. Fig. 3, Table 2). To test this, a command voltage was delivered to P1 and the VM shift in P2 was measured in current clamp (Fig. 4G). Consistently, the VM shift in P2 followed the command voltages closely, VM/Vc 5 91.4 6 1.0% (n 5 3) (Fig. 4G). These results demonstrate that the physical barrier between coupled astrocytes is almost absent electrically; therefore, equilibration of VM among coupled astrocytes to an isopotential can be readily achieved so that doublets act as a single astrocyte.

To simulate locally elevated ½K 1 e near a subdomain area of an astrocyte or an NG2 glia, we puffer applied 23.5 mM ½K 1 e through a patch pipette to the recorded cells (Fig. 5C). The high ½K 1 e affected area was monitored by SR101 fluorescence included in the solution. A 5 ms, high K1 puff affected an area with a diameter of 26.4 6 0.3 mm (n 5 4) around the patched cells; this was less than a typical 50 mm astrocytic domain (Bushong et al., 2002; Xu et al., 2014) (Fig. 5C). The high ½K 1 e puffer induced depolarization was significantly less in astrocytes (3.86 6 0.48 mV, n 5 4) than in NG2 glia (12.13 6 0.66 mV, n 5 4) (Fig. 5B–D). Puff aCSF alone did not alter the VM (Fig. 5B). The significantly reduced VM depolarization in astrocytes was very close to 5 mV, as predicted by the computational model (Supp. Info. Fig. 7). Thus, syncytial coupling is able to minimize local high ½K 1 e -induced VM depolarization. Because single astrocytes behave as perfect K1 electrodes, one expects that, at steady state, VM lies very close to EM and the K1 uptake driving force (VM 2 EM) is negligible. If a single astrocyte is presented with a local increase in ½K 1 e , then a significant K1 driving force arises only transiently, as VM rapidly depolarizes towards the newly established EM (Fig. 5E). However, individual astrocytes within a syncytium are able to maintain a constant, nonzero driving force. This is because, as we have demonstrated, the VM of an individual astrocyte is held nearly constant by the syncytial isopotential. Therefore, the K1 uptake driving force can be maintained until locally elevated ½K 1 e is completely taken up by the astrocytes (Supp. Info. Fig. 7). Thus, the syncytial isopotential transforms a “restricted local K1 uptake” model in a single astrocyte to a “sustained local K1 uptake” model in coupled astrocytes (Fig. 5E).

Syncytial Isopotentiality Facilitates K1 Uptake Under a Sustained Driving Force The syncytial isopotential suggests that an anticipated regional VM depolarization, in response, for example, to a local increase in extracellular K1 (½K 1 e ) around a subdomain area of a single astrocyte, can be minimized. For a local ½K 1 e increase from 3.5 mM to 23.5 mM, the GHK equation predicts a 43.2 mV depolarization, whereas under isopotential conditions, the numerically predicted depolarization is approximately 5 mV (Supp. Info. Fig. 7). We tested this prediction by comparing the high ½K 1 e responses from astrocytes and uncoupled NG2 glia in situ; both of these glial subtypes function similarly as K1 electrodes (Kuffler et al., 1966; Maldonado et al., 2013). We first bath applied 23.5 mM high K1 to maximally depolarize all the coupled astrocytes and NG2 glia in situ. The astrocytes and NG2 glia responded to high ½K 1 e with comparable time courses and maximum VM amplitudes: astrocytes 43.4 6 1.0 mV (n 5 7) vs. NG2 glia 42.8 6 1.1 mV (n 5 4). These values followed the GHK prediction closely (Fig. 5A,B).

Discussion

Month 2015

Neuronal and glial gap junction coupling was discovered sequentially over 50 years ago (Furshpan and Potter, 1957; Kuffler and Potter, 1964). While the discovery of the former immediately led to the notion of “electrical synapse” underlying one of the neuronal synaptic transmission mechanism, the electrical role of gap junctions in glial cells is still poorly defined (Ransom, 1996). To answer this fundamental question, a novel experimental design has been conceived based a basic electrophysiological property of astrocytes; they behave as K1 electrodes (Kuffler et al., 1966; Ransom and Goldring, 1973). To examine this issue directly from native astrocytes, freshly dissociated hippocampal tissue has been used as a new model (Du et al., 2015). To guide experimental design, predict and explain the experimental results, a computational model has been developed in this study. Astrocyte Gap Junction Coupling Functions to Achieve Syncytial Isopotentiality The following insights into astrocytic syncytium have been revealed. First, a surprisingly low gap junction coupling resistance 9

FIGURE 5: Syncytial coupling attenuates local elevated extracellular K1-induced VM depolarization. (A) Bath 23.5 mM high K1-induced VM depolarization from an astrocyte and an NG2 glia, as indicated. (B) Summary of bath and local high [K1]e induced VM depolarization. (C) A 5 ms local high ½K 1 e was applied to a recorded astrocyte with an affected subdomain area monitored by SR101 included in the high [K1]e solution (two sample t test). (D) A 5 ms local high [K1]e puff induced VM depolarization in an astrocyte and an NG2 glial cell. (E) Schematic illustration of VM response to a high [K1]e puff. In an uncoupled astrocyte, the VM quickly depolarizes to the new elevated [K1]e established EM. Once VM fi EM, the K1 uptake stops. In coupled astrocytes, the VM remains close to a constant. This maintains the driving force, VM – EM, for a “sustained K1 uptake”. *P < 0.05, one way ANOVA test for comparison of the VM depolarization between astrocytes and NG2 glia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

(Rg, 4.2 MX) and high CC (94%) have been found in pairs of coupled astrocytes. From modeling simulation, 2,000 gap junctions are expected in contacts made between two neighboring astrocytes to achieve such low Rg and high CC values (Supp. Info. Tab.2). This CC is 2.1-folds higher than that of cultured astrocytes (44%) (Kettenmann and Ransom, 1988; Ransom and Kettenmann, 1990), and the Rg is even lower than the astrocyte’s membrane resistance (RM,  6.4 MX) (Du et al., 2015). The strong electrical coupling enables any pair of neighboring astro10

cytes to immediately equilibrate their membrane potentials to closely matched levels and thus electrically behave as a single astrocyte. Second, at the syncytial level, strong electrical and ionic coupling equalizes the membrane potentials among coupled astrocytes to achieve syncytial isopotentiality. Astrocytes ensheath neuronal circuits throughout the brain and maintain a homeostatic interstitial environment that is crucial for neuronal signaling and information processing. A new insight into this function is that the homeostatic support is achieved through a coordinated Volume 00, No. 00

Ma et al.: Electrical Coupling of Astrocytes in a Syncytium

action of astrocytes governed by gap junction coupling established syncytial isopotentiality. It has been speculated that a potential role of gap junction is to “clamp” neighboring astrocytes to an identical resting membrane potential, or syncytial isopotentiality, which could be essential for the maintenance of a uniform extracellular ion concentration (Muller, 1996). In this study for the first time, we have experimentally demonstrated that gap junction coupling confers isopotentiality in astrocyte syncytium. This finding is supported by an early in vivo observation from cat cortex: during interictal discharge the recorded glial cell VM remained relatively stable, although the amplitude of extracellular K1 concentration varied dramatically (Futamachi and Pedley, 1976), suggesting that isopotentiality should also function physiologically in vivo. Syncytial Isopotentiality Facilitates K1 Clearance In this study, a demonstrated function of syncytial isopotentiality is to minimize the anticipated high ½K 1 e -induced membrane potential depolarization around a local area in the network, whereby a sustained K1 uptake driving force can be maintained for a higher K1 clearance efficiency (Fig. 5H). This observation has a significant implication for the frequently discussed “K1 spatial buffering” hypothesis. The K1 spatial buffering hyperthesis was proposed by Orkand et al (Orkand et al., 1966). Evidence that glial cells do utilize this mechanism for K1 clearance came from experiments performed in M€ uller cells (Newman, 1984; Newman et al., 1984; Oakley et al., 1992), where a single M€ uller cell, a specialized astrocyte in the retina, spatially transported K1 from the plexiform layers to the vitreous body, blood vessels and subretinal space, a mechanism termed “K1 siphoning”. Now, increasing evidence indicates that this “spatial dependent” K1 clearance mechanism may work as well in syncytial coupled astrocytes (Coles et al., 1986; Gardner-Medwin and Nicholson, 1983; Holthoff and Witte, 2000; Kofuji et al., 2000; Wallraff et al., 2006). In this study, we have demonstrated that syncytial isopotentiality makes K1 uptake more efficient. As demonstrated in this study, syncytial isopotentiality minimizes the local high ½K 1 e -induced VM depolarization, and this maintains a sustained driving force for K1 uptake. By extension, syncytial isopotentiality also increases the driving force for K1 release in distant regions where [K1]e remains at the physiological level. Additionally, increase in both driving forces creates a maximum driving force for intracellular K1 transfer from high [K1]e region to remote regions with normal [K1]e. Therefore, syncytial isopotentiality facilitates all three critical steps in “K1 spatial buffering”: K1 uptake, intercellular transfer and release (Kofuji and Newman, 2004). Month 2015

Number of Nearest Neighbors and Coupling Strength Collectively Determine the Capacity of K1 Clearance Previously, we have shown that each hippocampal astrocyte couples directly with 11 of its nearest neighbors (Xu et al., 2014), Here we show that a strong coupling mediated syncytial isopotentiality facilitates the capacity of K1 uptake to a level that could not be appreciated in the past. These observations, however, also raise an important physiological question: how does the overall capacity of K1 clearance depend on the number of nearest neighbors and the coupling strength? This fundamental question is answered from our modeling simulations (Supp. Info. Fig. 7). First, when coupling number keeps as a constant, the coupling strength (s) determines the slope, or how fast, the high [K1]e can be brought back to the physiological levels. Second, when the coupling strength (s) keeps as a constant, the number of coupling cells determines how close the final [K1]e can approach to the physiological [K1]e level. Third, when the coupling strength (s) is set at 1, as demonstrated in this study, the total capacity of K1 uptake can no longer increase with further increased number of nearest neighbors over 11. Thus the combination of these anatomic and physiological features is likely achieved in astrocytes over the course of evolution for optimal brain function.

Future Studies The precise role of astrocytes in the adult brain remains to be defined (Nedergaard et al., 2003). An emerging view from this study is that astrocytes work as a team and the basic physiological function is achieved at syncytial network levels. This sheds new lights on future astrocyte study and urges more study to examine the functions achieved at syncytial (or system) levels. Astrocyte coupling is important for equalization of intracellular Na1 concentration (Langer et al., 2012; Rose and Ransom, 1997), calcium signaling and wave propagation (Khakh and McCarthy, 2015; Kuga et al., 2011), and regulatory redistribution of glucose toward active neurons in demand (Rouach et al., 2008). Meanwhile, there is an increasing awareness of astrocyte diversity (Giaume and Liu, 2012; Zhang and Barres, 2010). Thus, the experimental approach demonstrated in this study provides a powerful functional measurement to determine in the future the extent to which the syncytial isopotentiality exists in other brain regions and whether and how this crucial network function could be altered in various pathological disorders. Additionally, the numerously expressed Na1-dependent uptake systems (Cahoy et al., 2008; Kimelberg, 2010) should be appreciated and reappraised with the consideration of syncytial 11

isopotential. This study also emphasizes the importance of homeostatic support as a basic astrocyte function in the brain.

Acknowledgment Grant sponsor: National Institute of Neurological Disorders and Stroke; Grant numbers: RO1NS062784 (to M.Z.), RO1NS043246 (to D.D.M.); Grant sponsor: The Ohio State University College of Medicine (to M.Z.).; Grant sponsor: National Science Foundation; Grant numbers: DMS 1410935 (to D.T.) and DMS 0931642 (to the Mathematical Biosciences Institute). The authors thank Drs. Harold K. Kimelberg and Maiken Nedergaard for critical comments on the manuscript. They thank Dr. Bruce R. Ransom for providing insightful comments and suggestions for manuscript improvement during revision.

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Giaume C, Liu X. 2012. From a glial syncytium to a more restricted and specific glial networking. J Physiol Paris 106:34–39. Hesp ZC, Goldstein EA, Miranda CJ, Kaspar BK, McTigue DM. 2015. Chronic oligodendrogenesis and remyelination after spinal cord injury in mice and rats. J Neurosci 35:1274–1290. Holthoff K, Witte OW. 2000. Directed spatial potassium redistribution in rat neocortex. Glia 29:288–292. Kettenmann H, Ransom BR. 1988. Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures. Glia 1:64–73. Khakh BS, McCarthy KD. 2015. Astrocyte calcium signaling: From observations to functions and the challenges therein. Cold Spring Harb Perspect Biol 7:a020404Kimelberg HK. 2010. Functions of mature mammalian astrocytes: A current view. Neuroscientist 16:79–106. Kofuji P, Ceelen P, Zahs KR, Surbeck LW, Lester HA, Newman EA. 2000. Genetic inactivation of an inwardly rectifying potassium channel (Kir4.1 subunit) in mice: Phenotypic impact in retina. J Neurosci 20:5733–5740. Kofuji P, Newman EA. 2004. Potassium buffering in the central nervous system. Neuroscience 129:1045–1056. Kuffler SW, Nicholls JG, Orkand RK. 1966. Physiological properties of glial cells in the central nervous system of amphibia. J Neurophysiol 29:768–787. Kuffler SW, Potter DD. 1964. Glia in the leech central nervous system: physiological properties and neuron-glia relationship. J Neurophysiol 27:290–320. Kuga N, Sasaki T, Takahara Y, Matsuki N, Ikegaya Y. 2011. Large-scale calcium waves traveling through astrocytic networks in vivo. J Neurosci 31: 2607–2614. Langer J, Stephan J, Theis M, Rose CR. 2012. Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ. Glia 60:239–252. Lin JH, Weigel H, Cotrina ML, Liu S, Bueno E, Hansen AJ, Hansen TW, Goldman S, Nedergaard M. 1998. Gap-junction-mediated propagation and amplification of cell injury. Nat Neurosci 1:494–500. Ma B, Xu G, Wang W, Enyeart JJ, Zhou M. 2014. Dual patch voltage clamp study of low membrane resistance astrocytes in situ. Mol Brain 7:18Maldonado PP, Velez-Fort M, Levavasseur F, Angulo MC. 2013. Oligodendrocyte precursor cells are accurate sensors of local k1 in mature gray matter. J Neurosci 33:2432–2442. Meme W, Vandecasteele M, Giaume C, Venance L. 2009. Electrical coupling between hippocampal astrocytes in rat brain slices. Neurosci Res 63:236–243. Muller CM. 1996. Gap-junctional communication in mammalian cortical astrocytes: Development, modifiability and possible functions. In: Spary DC, Dermietzel R. Austin. Gap junctions in the nervous system Editors, Chapter 12. TX: RG Landes Company. pp 203–212. Muller T, Moller T, Neuhaus J, Kettenmann H. 1996. Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation. Glia 17:274–284. Nedergaard M, Ransom B, Goldman SA. 2003. New roles for astrocytes: Redefining the functional architecture of the brain. Trends Neurosci 26:523–530. Newman EA. 1984. Regional specialization of retinal glial cell membrane. Nature 309:155–157. Newman EA. 2001. Propagation of intercellular calcium waves in retinal astrocytes and Muller cells. J Neurosci 21:2215–2223. Newman EA, Frambach DA, Odette LL. 1984. Control of extracellular potassium levels by retinal glial cell K1 siphoning. Science 225:1174–1175. Nimmerjahn A, Kirchhoff F, Kerr JN, Helmchen F. 2004. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat Methods 1:31–37. Oakley B, 2nd Katz BJ, Xu Z Zheng J. 1992. Spatial buffering of extracellular potassium by Muller (glial) cells in the toad retina. Exp Eye Res 55:539–550. Orkand RK, Nicholls JG, Kuffler SW. 1966. Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia. J Neurophysiol 29:788–806.

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Ma et al.: Electrical Coupling of Astrocytes in a Syncytium Ransom BR. 1996. Do glial gap junctions play a role in extracellular ion homeostasis? In: Spary DC, Austin DR. Gap junctions in the nervous system Editors. TX: RG Landes Company. pp 159–173.

Wallraff A, Kohling R, Heinemann U, Theis M, Willecke K, Steinhauser C. 2006. The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus. J Neurosci 26:5438–5447.

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Ransom BR, Kettenmann H. 1990. Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture. Glia 3: 258–266.

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Month 2015

Zhou M, Schools GP, Kimelberg HK. 2006. Development of GLAST(1) astrocytes and NG2(1) glia in rat hippocampus CA1: Mature astrocytes are electrophysiologically passive. J Neurophysiol 95:134–143.

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1

Title: Gap junction coupling confers isopotentiality on astrocyte syncytium

2

Authors: Baofeng Ma1, Richard Buckalew2, Yixing Du1, Conrad M. Kiyoshi1, Catherine C.

3

Alford1, Wei Wang1, #, Dana D. McTigue1, John J. Enyeart1, David Terman3, Min Zhou1,*

4 5 6 7 8 9

Affiliations: 1

Department of Neuroscience, The Ohio State University Wexner Medical Center, Columbus,

OH 43210, USA. 2

Mathematical Biosciences Institute, The Ohio State University, Columbus, OH 43210, USA.

3

Department of Mathematics, The Ohio State University, Columbus, OH 43210, USA.

#

Present address: Department of Physiology, Tongji Medical College, Huazhong University of

10

Science and Technology, Wuhan, Hubei 430030, China.

11

Running title: Electrical coupling of astrocytes in a syncytium.

12

Word counts: Abstract:157, Introduction: 527, Methods: 1,154, Results: 2456, Discussion:

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1130, References: 1162; Figure legend: 1014, Total word: 7,928

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Number of figures: 5

15

Number of tables: 0.

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*Correspondence: Min Zhou, 4066C Graves Hall, 333 West 10th Avenue, Columbus, OH

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43210, Phone: (614) 366-9406, Fax: (614) 688-8742, Email: [email protected]

19 20 21

1

Main Points

2



Astrocytes are electrically coupled strongly with a coupling coefficient of 94%.

3



The strong coupling equilibrates the membrane potentials among astrocytes to achieve a

4

syncytial isopotentiality, whereby a constant chemical and quiescent electrical

5

environment can be powerfully maintained.

6 7

Key words: Electrical coupling; Coupling coefficient; Membrane potential; K+ clearance

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2

1

Abstract:

2

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the

3

basic role of this major brain network remains largely unknown. Using electrophysiological and

4

computational modeling methods, we demonstrate that the membrane potential (VM) of an

5

individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell

6

preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or

7

K+ free pipette solutions that alter the K+ equilibrium potential to non-physiological voltages. We

8

show that an astrocyte‟s associated syncytium provides powerful electrical coupling, together

9

with ionic coupling at a lesser extent, that equalizes the astrocyte‟s VM to levels comparable to its

10

neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local

11

extracellular K+ and thereby maintains a sustained driving force for highly efficient K+ uptake.

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Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby

13

a constant extracellular environment can be powerfully maintained for crucial functions of neural

14

circuits.

15

3

1

Introduction:

2

Establishment of a syncytium through gap junction coupling is a prominent feature of

3

astrocytes in the central nervous system (Brightman and Reese 1969; Dermietzel and Spray

4

1993; Giaume et al. 2010; Ransom 1996). Gap junction coupling is known to mediate the

5

exchange of small molecules (< ~1.2 kDa). This facilitates important homeostatic and signaling

6

functions of astrocytes, such as spatial buffering of K+ and Na+ ions and the long-range

7

redistribution of nutrients, metabolites and signaling molecules for the coordination of neuronal

8

activity and brain energy metabolism (Kuga et al. 2011; Langer et al. 2012; Lin et al. 1998;

9

Newman 2001; Orkand et al. 1966; Rose and Ransom 1997; Rouach et al. 2008; Simard et al.

10

2003; Wang et al. 2012).

11

Gap junctions also enable electrical coupling to minimize membrane potential (VM)

12

differences among interconnected cells in a mutual and rapid manner. For pairs of coupled cells,

13

the strength of electrical coupling, termed the coupling coefficient (CC), is commonly evaluated

14

by the ratio of voltage in coupled to that of injected cell (transjunctional voltage to input voltage)

15

using the dual patch recording method. However, the coupling coefficient between any two

16

nearest neighboring astrocytes in the intact brain has been a long-standing but unresolved

17

question. Although the transjunctional voltage analysis has been used to explore this question

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between astroyctes (Ceelen et al. 2001; Kettenmann and Ransom 1988; Meme et al. 2009;

19

Ransom and Kettenmann 1990; Xu et al. 2010), the coupling coefficient cannot be accurately

20

measured due to an extremely low membrane resistance that shunts the experimentally injected

21

currents through astrocyte‟s low resistance membrane (~6 M) (Ma et al. 2014). At the syncytial

22

level, each hippocampal astrocyte is directly coupled with 11 nearest neighbors and several

23

hundreds of astrocytes are coupled in a syncytium (D'Ambrosio et al. 1998; Xu et al. 2010), an 4

1

intriguing question is how the aggregate coupling strength affects the VM and other physiological

2

behavior of any individual astrocyte in a syncytium. No methodology is, as yet, available to

3

tackle these fundamentally important questions.

4

In the present study, we combine electrophysiological and computational modeling

5

methods to investigate the mechanisms by which nearest neighboring astrocytes coordinate the

6

VM‟s in a syncytium. The study is conceived based on a basic feature of astrocytes: they behave

7

as perfect K+ electrodes as a result of their predominant expression of leak type K+ conductances

8

(Kuffler et al. 1966; Ransom and Goldring 1973). Therefore, one can experimentally lower the

9

intracellular K+ concentration ([K+]i) using reduced or K+-free pipette solutions to establish a

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depolarized K+ equilibrium potential (EK) in a recorded astrocyte to levels that can be predicted

11

by the membrane equilibrium potential (EM) from the Goldman-Hodgkin-Katz (GHK) equation.

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The nearest neighbors should then act to hyperpolarize the recorded astrocyte by minimizing the

13

differences in K+ concentrations (ionic coupling) and membrane potential (electrical coupling).

14

Consequently, the deviation of measured VM from the experimentally established EM in the

15

recorded astrocyte can serve as a reliable indicator of the coupling strength among astrocytes in a

16

syncytium (See mathematic modeling). We hypothesized that a strong gap junction coupling

17

among hippocampal CA1 astrocytes suppresses the VM depolarization in recordings made with

18

reduced or free K+ pipette solutions.

19 20

Materials and Methods

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Animals

5

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The C57/BL6 mice of both genders older than P21 (Charles River), and PDGFRA-driven eGFP

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transgenic mice (Hesp et al. 2015) were used in the present study according to the guidelines of

3

the Institutional Animal Care and Use Committee, The Ohio State University.

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Hippocampal slice preparation

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For slice recording, hippocampal slices were prepared as described previously (Ma et al.

6

2014). In brief, after anesthesia with 8% chloral hydrate in 0.9% NaCl, the mouse brain was

7

rapidly removed from skull and submerged into ice-cold oxygenated (95% O2 /5% CO2) cutting

8

solution containing (in mM): 125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 0.1 CaCl2, 3

9

MgCl2, and 10 glucose. Coronal hippocampal slices (250 μm) were cut at 4°C with a Vibratome

10

(Pelco 1500) and transferred to the normal aCSF (in mM): 125 NaCl, 3.5 KCl, 25 NaHCO3, 1.25

11

NaH2PO4, 2 CaCl2, 1 MgCl2, and 10 glucose (osmolality, 295 ± 5 mOsm; pH 7.3–7.4) at room

12

temperature (20-22°C). Slices were kept in aCSF with continuous oxygenation for at least 1 hour

13

before recording.

14

Sulforhodamine 101 Staining

15

For sulforhodamine 101 (SR101, Invitrogen, New York, NY) staining (Nimmerjahn et al.

16

2004), the slices were transferred to a slice-holding basket containing 0.6 M SR101 in aCSF at

17

34 °C for 30 min. Then, the basket was transferred back to the normal aCSF at room temperature

18

before the experiment.

19

Fresh dissociation of hippocampal tissues

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At the animal age older than P21, the spatially distinct domains, electrical coupling, and

21

K+ channel expression have all reached maturity (Bushong et al. 2004; Xu et al. 2010; Zhou et al.

22

2006). To dissociate single and multiple astrocytes with intact domain and functional gap

6

1

junctional coupling, a new hippocampal tissue dissociation method was used (Du et al. 2015).

2

Briefly, after incubation with SR-101 for 30 min, the CA1 regions were dissected out from slices

3

and cut into small pieces (1 mm2), then placed in oxygenated aCSF containing 24U/ml papain

4

and 0.8 mg/ml L-cysteine for 7 min. After papain digestion, a fire polished glass pipette,

5

diameter ~150 μm, was used to triturate the loosened tissues 5-7 times into a tissue suspension,

6

and then transferred into the recording chamber. The dissociated cells were allowed 3-5 min to

7

touch down to the bottom of the chamber before switching on a constant aCSF perfusion at flow

8

rate of 2 ml/min. The viable astrocytes, with their elaborate processes and well-preserved domain

9

shape, are similar to their counterparts in situ (Figures 2A-B).

10

Electrophysiology

11

To record astrocytes in CA1 stratum radium, individual slices were transferred to a

12

recording chamber (RC-22, Warner Instruments, Holliston, MA) mounted on a Olympus

13

BX51WI microscope equipped with infrared differential interference (IR-DIC) and were

14

perfused with oxygenated aCSF (2.5 mM/min) at room temperature.

15

Astrocytes in situ were identified based on SR101 staining; they typically exhibited an

16

irregular soma shape, diameter around 10 m, with several visible primary processes stemming

17

from the soma. The NG2 glial cells were identified by GFP from PDGFRA-driven eGFP

18

transgenic mice (Hesp et al. 2015). The interneurons were identified based on their large soma

19

size and shapes. After establishing whole-cell configuration, the mature astrocytes were

20

unequivocally identified by their expression of passive membrane K+ conductance (Zhou et al.

21

2006). Interneurons expressed large inward Na+ currents in voltage clamp recording and fired

22

action potentials spontaneously when switched to current clamp recording (Figure 1F).

7

1

Recording pipettes were fabricated from borosilicate capillaries (1.5/0.86 mm outer/inner

2

diameter, Warner Instruments, Holliston, MA) using a Flaming/Brown Micropipette Puller

3

(Model P-87, Sutter Instrument). When filled with pipette solution noted below, the pipettes had

4

the open tip resistance of 2.5-3.5 MΩ. The standard pipette solution contained (in mM): 140 KCl

5

or Kgluconate, 0.5 CaCl2, 1 MgCl2, 5 EDTA, 10 HEPES, 3 Mg2+-ATP and 0.3 Na+2-GTP that

6

was titrated with KOH to pH 7.25-7.27. The final osmolality was 280 mOsm. The 140 mM KCl

7

was substituted by NaCl, or NMDGCl either partially or fully in the referred experiments.

8

Whole-cell membrane current or membrane potential was amplified by MultiClamp

9

700A or MultiClamp 700B amplifiers, and the data acquisition was controlled by PClamp 9.2

10

(Molecular Devices, Sunnyvale, CA) installed on Dell personal computers. DIGIDATA 1322A

11

interface was used to convert digital-analog signals between amplifier and computer. A

12

minimum of 2 GΩ seal resistance was required before rupturing the membrane for whole-cell

13

configuration. The total membrane resistance, or input resistance (Rin), was measured by using

14

the "Membrane test" protocol built into the pClampex. The access resistance (Ra) was not

15

compensated for in all the voltage clamp recordings for low RM astrocytes (Ma et al. 2014). The

16

membrane potential (VM) was read either in “I = 0” mode or measured directly in current clamp

17

mode with no holding currents. Astrocytes with a resting membrane potential more positive than

18

-70 mV in both brain slices and freshly isolated single astrocytes were discarded. All the

19

experiments were conducted at room temperature (20 ± 2°C). The liquid junction potential was

20

compensated for before establishment of cell-attached mode in all the recordings and confirmed

21

to be ~ 0 mV after experiments by withdrawal of recording pipette.

8

1

SR101 positive staining was used to confirm the astrocytic identity and determine the

2

number of astrocytes in a freshly dissociated tissue block. The same patch clamp set-up and

3

procedure, for slice astrocyte recording, was used for freshly dissociated astrocyte recording.

4

In the experiment for manipulating coupling strength, brain slices were pretreated with

5

aCSF containing 100 µM meclofenamic acid (MFA) for one hour before recording and perfused

6

with the same solution during recording. When VM was stable, 100 µM MFA plus 100 µM Ba2+

7

was bath applied. In the experiment for determining effect of 100 M MFA + 100 M Ba2+ on

8

the RM, dual patch recording was used as we previous reported (Ma et al. 2014) .

9

Local high K+ application

10

We used VC34 Controller (ALA Scientific Instruments) for focal high K+ application

11

(pressure 6 psi). Focal high K+ (23.5 mM) or aCSF was delivered via a pipette to the recorded

12

cell ~10 µm away from soma, and 100 µM SR-101 was included in the application solutions to

13

visualize and measure the areas affected by focally applied high K+. Software provided by ALA

14

Scientific Instruments was used for programming of the durations of high K+ application.

15

Imaging capture

16

Confocal images were obtained using a Zeiss LSM 510 at the Hunt-Curtis Imaging

17

Facility. A fluorescent imaging system (Polychrome V system from Till Photonics, Germany) is

18

used for Epifluorescence imaging and high resolution visualization of small glial soma and

19

placing of dual patch pipettes on it. This system was used for identifying astrocyte based on

20

SR101 positive staining (Fig. 1A and Fig. 2 B), and for simultaneous dual patch.

21

Statistical analysis

9

1

Data are reported as mean  SEM. Mean differences between groups were using t test or two-

2

way ANOVAs followed by post hoc testing when main effect was significant at p < 0.05. Data

3

were analyzed using Origin 11 software package (OriginLab, Northampton, MA).

4 5

Results

6

Syncytial Coupling Equalizes the Membrane Potential among Astrocytes in the Network

7

To test our hypothesis, we identified astrocytes based on SR101 staining from the

8

hippocampal CA1 region (10) and recorded VM under current clamp mode. In control

9

experiments, we used patch pipette solution containing 140 mM K+ (K+-based solution, [K+]p)

10

(Fig. 1A and B). In cell-attached mode, the gigaohm seal formation led to an anticipated VM shift

11

from 0 mV to -38.1 ± 18.5 mV (n = 13) for the low membrane resistance astrocytes (Supp. Info.

12

Fig. 1). The breakthrough of the membrane patch shifted the VM immediately to a resting

13

membrane potential of -78.1  0.7 mV (n = 11) (Fig. 1B), as expected for cells almost

14

exclusively permeable to K+ (17).

15

When similar recordings were made with patch pipettes in which the [K+]i was reduced

16

from 140 mM to 70 mM by substitution with Na+, the recorded VM was essentially unchanged (-

17

78.1  1.1 mV, n = 6) (Fig. 1G) and differed significantly from the GHK predicted 11.1 mV

18

depolarization. Furthermore, the GHK equation predicts a 105 mV depolarization if pipette K+ is

19

totally substituted by Na+ (Na+-based solution, [Na+]p). However, Na+-based solution

20

depolarized the cells only by 4.5 mV (-73.5  0.9 mV, n = 10), compared to the control

21

astrocytes (Fig. 1C and G). In all recordings with reduced or K+-free pipette solutions, the

22

recorded VM remained at a steady-state level during a recording time of ~ 10 min . In some

10

1

recordings, we purposely extended time to over one hour, and the steady-state VM remained

2

unchanged, indicating gap junction coupling provided a sustained VM control over recorded

3

astrocytes. These results support the hypothesis that there exists a powerful gap junction

4

coupling among CA1 astrocytes in situ (Supp. Info. Fig. 2).

5

To determine if a similar VM behavior occurs in other uncoupled cells in this brain region

6

(Xu et al. 2014), we used Na+-based pipette solution to record NG2 glia in hippocampal slices

7

prepared from PDGFα1-driven-GFP transgenic mice (Fig. 1D)(Hesp et al. 2015); NG2 glia are

8

known to also function as K+ electrodes (Maldonado et al. 2013). NG2 glia followed the GHK

9

prediction by depolarizing rapidly to a stable VM level of 7.3  1.9 mV (n = 9, P < 0.01) (Fig. 1E

10

and G). Likewise, uncoupled CA1 interneurons also depolarized to 1.3  0.7 mV (n = 5) when

11

recorded with Na+-based pipette solution (Fig. 1F and G).

12

To ensure that such “anomalous” VM behavior was not specific to Na+ ions, we replaced

13

K+ either partially or fully with NMDG+. Substitution of NMDG+ for K+ resulted in VM changes

14

similar to those observed from Na+ substitution (Fig. 1H). Thus, syncytial coupling suppresses

15

the VM depolarization that is expected for an uncoupled single astrocyte recorded after a full

16

substitution of intracellular K+ by Na+ or NMDG.

17

To confirm that gap junction coupling was responsible for the equalization of VM in

18

astrocytes, we pre-incubated the brain slices with 100 M meclofenamic acid (MFA) for 1 hour

19

to block the gap junction coupling (Xu et al. 2010). Under this condition, the Na+-based solution

20

depolarized astrocytes to a steady-state VM of -13.9 ± 1.8 mV (n = 10) in MFA (Fig. 1I). The

21

computational model predicts that this corresponds to a 99.3% inhibition of coupling strength (s

22

= coupling conductance / membrane conductance of nearest neighbors) with MFA (Supp. Info.

23

Fig. 4). To confirm this prediction, 100 µM Ba2+ was applied to inhibit a major astrocyte K+ 11

1

channel, Kir4.1. This increased the membrane resistance, RM, from the control 6.3 ± 0.7 M to

2

18.4 ± 4.4 M (n =7), corresponding to a 2.9-fold increase in the coupling strength and a

3

predicted VM hyperpolarization to -23.9 mV (Supp. Info. Fig. 4). Consistent with this prediction,

4

the astrocyte VM hyperpolarized to -23.4 ± 2.7 mV (n = 10) in the presence of 100 μM Ba2+ in

5

MFA aCSF (Fig. 1I and J). Thus gap junction coupling is responsible for the mildly altered

6

astrocyte VM recorded with MFA and a Na+-based pipette solution in situ. Moreover, the

7

experimental results validated the mathematical model we have developed for this study .

8

Larger Syncytia have Stronger Control over an individual Astrocyte’s VM

9

The computational model predicts that if the number of cells that are coupled to the

10

recorded cell with Na+-based electrode solution is increased, then the recorded VM will lie closer

11

to the physiological EM of the nearest neighbors (Supp. Info. Fig. 2). To test this prediction, we

12

used freshly isolated hippocampal tissues that contained single to multiple astrocytes with their

13

spatially distinct domains and functional K+ channels well-maintained (Du et al. 2015) (Fig. 2A).

14

A Na+-based pipette solution was used to record astrocytes located in the center of the tissues. As

15

the number of cells in the syncytium that were coupled to the recorded cell increased, so did the

16

deviation of the recorded VM from the GHK predicted value for a Na+-based pipette solution (Fig.

17

2C-F and K). Thus, the number of neighboring astrocytes has an aggregate effect in suppressing

18

the VM depolarization induced by Na+-loading in the recorded astrocyte (Supp. Info. Fig. 2).

19

In single astrocytes, the same Na+-based pipette solution induced an initial

20

hyperpolarization, reflecting the undisturbed initial [K+]i, followed by a depolarization to -0.02 ±

21

0.74 mV (n = 12), as predicted by the GHK equation (Fig. 2G). A subsequent bath application

22

of elevated 23.5 mM K+ also induced a predicted VM depolarization (Fig. 2H). When switching

23

to voltage-clamp recordings, the depolarization induced outward currents were almost fully 12

1

eliminated due to the absence of intracellular K+ ion (Fig. 2I left). Removal of the 3.5 mM K+ in

2

the bath solution ([K+]e) further abolished the inward currents (Fig. 2I middle), and the inward

3

currents reemerged when 3.5 mM [K+]e was restored (Fig. 2I right). In contrast, the breakthrough

4

of the membrane patch with K+-base pipette solution shifted the VM to the anticipated resting VM

5

of -78.4  1.9 mV ( n = 11) in single astrocytes (Fig. 2J), which was comparable with astrocytes

6

in slices (Fig. 1G). Thus, independent of which pipette solution was used, single astrocytes

7

behave as a perfect K+ electrode.

8

Contribution of Ionic Coupling in Maintaining the Astrocyte VM in situ

9

In astrocyte recordings with Na+-based solution in situ, the K+-mediated outward currents

10

remained unchanged (Fig. 3A), suggesting the presence of K+ conducting ions in the recorded

11

cell due to ionic coupling with the associated syncytium.

12

To determine how efficient gap junctions are in permitting ion exchange between coupled

13

astrocytes, we used Na+-based pipette solution to compare the time required for the full

14

substitution of intracellular K+ in a freshly dissociated, single astrocyte with the time required in

15

a pair of coupled astrocytes (doublet) (Fig. 3B). In both cases, a complete substitution of the

16

endogenous K+ by Na+ from the pipette was indicated by the depolarization of VM from its resting

17

value to ~ 0 mV, as predicted by the GHK equation. The computational modeling predicts that

18

the high ion exchange efficacy enables a doublet to replace all the endogenous K+ by pipette Na+

19

with a time course similar to that of a single astrocyte (Fig. 3C, s = 1). Let T50 be the half time

20

for VM to reach 0 mV. Then the ion exchange efficacy at the gap junctions can be estimated by

21

computing the difference between the T50‟s in single and doublet astrocytes (Fig. 3C). The T50

22

was ~12 times slower in doublets (4.67  0.36 min, n = 5) than that of single astrocytes (0.39 

23

0.25 min, n = 3) (Fig. 3D-E). Thus, because of its relatively slow kinetics (see Supp. Info. Fig. 5 13

1

for further modeling analysis), ionic coupling is unlikely to be the major factor in maintaining a

2

constant astrocyte VM in situ (Fig. 1C, G and H).

3

A Strong Electrical Coupling Confers Isopotentiality to Coupled Astrocytes

4

Electrical coupling is able to instantaneously equilibrates the VM‟s of coupled neurons (Connors

5

and Long 2004). To examine the strength of electrical coupling and the level of VM equilibration

6

in coupled astrocytes, we considered a doublet in which the EM‟s of the two astrocytes were

7

experimentally set up at sharply different levels. In dual patch recordings, a K+-based pipette

8

(P1) was sealed on one of the astrocytes, while a Na+-based pipette (P2) was sealed on the other

9

astrocyte (Fig. 4A1). The membrane of the astrocyte with the K+-based electrode was ruptured

10

first to measure the resting VM of the doublet (Fig. 4A1); this did not interfere with the

11

intercellular K+ concentration, [K+]i. The subsequent breakthrough of P2 initiated a Na+ influx

12

and a re-equilibration of [K+]i and [Na+]i in the recorded doublet. Consistent with the ion

13

exchange T50 at doublet gap junctions (Fig. 3D), the computational model predicts that [K+]i and

14

[Na+]i should reach their new steady-state levels within 120 seconds (Fig. 4A2). There is then an

15

intracellular [K+]i gradient from 140 mM at P1 to 0 mM K+ at P2. This corresponds to EM equal

16

to -74 mV and -10 mV at the respective pipettes (Fig. 4A2).

17

Despite a rapid separation between the battery potentials (EM) of P1 and P2, the

18

breakthrough of P2 resulted in an instantaneous equilibration of the VM‟s to comparable levels at

19

both pipettes (left inset, Fig. 4B). A strong electrical coupling was evident by the closely

20

matched VM amplitudes in the two cells as they depolarized in parallel towards their new steady-

21

state levels (middle inset, Fig. 4B).

22

To examine the similarity between the dynamics of doublets and single astrocytes, we

23

repeated the same experiment in single astrocytes (Fig. 4A3). The breakthrough of the P2 14

1

resulted in a pattern of VM equilibration similar to that observed in doublets (Fig. 4C, and right

2

inset). The VM trajectories, in single and doublet astrocytes, all reached steady-state levels with

3

comparable time courses (Fig. 4B-C). To quantify their similarities, we fitted a single

4

exponential to each VM depolarization, starting at the time of breakthrough of P2. The resulting

5

time constants () and depolarization amplitudes (A) are comparable in all four pipettes (Fig. 4D),

6

indicating that doublets behave electrically as one single astrocyte. The time constants in

7

doublets were also similar to the T50 of single astrocytes (Fig. 3D-E). This indicates that Na+

8

influx predominantly replaced the endogenous K+ in the P2 attached cell; moreover, the cross-

9

diffusion of Na+ and K+ across the gap junction was minimal in coupled doublets, as predicted by

10

the computational model (Fig. 4A2 and S6). These results demonstrate that a powerful electrical

11

coupling equilibrates the VM‟s of coupled astrocytes to a comparable level.

12

To directly estimate the gap junction coupling resistance in doublets, we replaced all K+

13

by Na+ in the recording solutions (Fig. 2I and 4E). This allows for maximal reduction in current

14

shunting through the low resistance membrane. Under this condition, current injection in one

15

astrocyte induced almost the same VM

16

coefficient (CC) of 94.3  3.2% (n = 3) (Fig. 4F), and a coupling resistance (Rg) of 4.2  1.5 M

17

(n = 3, pair distance 30.5  3.1 m) (Bennett 1966). As the Rg appeared to be even lower than the

18

RM of single astrocytes (Du et al. 2015), this suggested that, with maximally reduced current

19

shunting, doublets should behave as single astrocytes in voltage clamp recordings (Supp. Info.

20

Fig. 3, Table 2). To test this, a command voltage was delivered to P1 and the VM shift in P2 was

21

measured in current clamp (Fig. 4G). Consistently, the VM shift in P2 followed the command

22

voltages closely, VM/Vc = 91.4  1.0% (n = 3) (Fig. 4G). These results demonstrate that the

23

physical barrier between coupled astrocytes is almost absent electrically; therefore, equilibration

shift in both astrocytes with a calculated coupling

15

1

of VM among coupled astrocytes to an isopotential can be readily achieved so that doublets act as

2

a single astrocyte.

3

Syncytial Isopotentiality Facilitates K+ Uptake under a Sustained Driving Force

4

The syncytial isopotential suggests that an anticipated regional VM depolarization, in

5

response, for example, to a local increase in extracellular K+ ( [ K  ]e ) around a subdomain area of

6

a single astrocyte, can be minimized. For a local [ K  ]e increase from 3.5 mM to 23.5 mM, the

7

GHK equation predicts a 43.2 mV depolarization, whereas under isopotential conditions, the

8

numerically predicted depolarization is approximately 5 mV (Supp. Info. Fig. 7).

9

We tested this prediction by comparing the high [ K  ]e responses from astrocytes and

10

uncoupled NG2 glia in situ; both of these glial subtypes function similarly as K+ electrodes

11

(Kuffler et al. 1966; Maldonado et al. 2013). We first bath applied 23.5 mM high K+ to

12

maximally depolarize all the coupled astrocytes and NG2 glia in situ. The astrocytes and NG2

13

glia responded to high [ K  ]e with comparable time courses and maximum VM amplitudes:

14

astrocytes 43.4  1.0 mV (n = 7) vs. NG2 glia 42.8  1.1 mV (n = 4). These values followed the

15

GHK prediction closely (Fig. 5A-B).

16

To simulate locally elevated [ K  ]e near a subdomain area of an astrocyte or an NG2 glia,

17

we puffer applied 23.5 mM [ K  ]e through a patch pipette to the recorded cells (Fig. 5C). The

18

high [ K  ]e affected area was monitored by SR101 fluorescence included in the solution. A 5 ms,

19

high K+ puff affected an area with a diameter of 26.4  0.3 µm (n = 4) around the patched cells;

20

this was less than a typical 50 µm astrocytic domain (Bushong et al. 2002; Xu et al. 2014) (Fig.

21

5C). The high [ K  ]e puffer induced depolarization was significantly less in astrocytes (3.86  16

1

0.48 mV, n = 4) than in NG2 glia (12.13  0.66 mV, n = 4) (Fig. 5B-D). Puff aCSF alone did not

2

alter the VM (Fig. 5B). The significantly reduced VM depolarization in astrocytes was very close

3

to 5 mV, as predicted by the computational model (Supp. Info. Fig. 7). Thus, syncytial coupling

4

is able to minimize local high [ K  ]e -induced VM depolarization.

5

Because single astrocytes behave as perfect K+ electrodes, one expects that, at steady

6

state, VM lies very close to EM and the K+ uptake driving force (VM − EM) is negligible. If a

7

single astrocyte is presented with a local increase in [ K  ]e , then a significant K+ driving force

8

arises only transiently, as VM rapidly depolarizes towards the newly established EM (Fig. 5E).

9

However, individual astrocytes within a syncytium are able to maintain a constant, nonzero

10

driving force. This is because, as we have demonstrated, the VM of an individual astrocyte is held

11

nearly constant by the syncytial isopotential. Therefore, the K+ uptake driving force can be

12

maintained until locally elevated [ K  ]e is completely taken up by the astrocytes (Supp. Info. Fig.

13

7). Thus, the syncytial isopotential transforms a “restricted local K+ uptake” model in a single

14

astrocyte to a “sustained local K+ uptake” model in coupled astrocytes (Fig. 5E).

15 16

DISUSSION

17

Neuronal and glial gap junction coupling was discovered sequentially over 50 years ago

18

(Furshpan and Potter 1957; Kuffler and Potter 1964). While the discovery of the former

19

immediately led to the notion of “electrical synapse” underlying one of the neuronal synaptic

20

transmission mechanism, the electrical role of gap junctions in glial cells is still poorly defined

21

(Ransom 1996). To answer this fundamental question, a novel experimental design has been

22

conceived based a basic electrophysiological property of astrocytes; they behave as K+ electrode 17

1

(Kuffler et al. 1966; Ransom and Goldring 1973). To examine this issue directly from native

2

astrocytes, freshly dissociated hippocampal tissue has been used as new model (Du et al. 2015).

3

To guide experimental design, predict and explain the experimental results, a computational

4

modeling has been developed in this study.

5

Astrocyte gap junction coupling functions to achieve syncytial isopotentiality

6

The following insights into astrocytic syncytium have been revealed. First, a surprisingly

7

low gap junction coupling resistance (Rg, 4.2 M) and high coupling coefficient (94%) have

8

been found in pairs of coupled astrocytes. This coupling coefficient is 2.1-folds higher than that

9

of cultured astrocytes (44%) (Kettenmann and Ransom 1988; Ransom and Kettenmann 1990),

10

and the Rg is even lower than astrocyte‟s membrane resistance (RM, ~ 6.4 M) (Du et al. 2015).

11

The strong electrical coupling enables any pair of neighboring astrocytes to immediately

12

equilibrates their membrane potentials to closely matched levels and thus electrically behave as a

13

single astrocyte. Second, at the syncytial level, strong electrical and ionic coupling equalizes the

14

membrane potentials among coupled astrocytes to achieve syncytial isopotentiality. Astrocytes

15

ensheath neuronal circuits throughout the brain, and maintain a homeostatic interstitial

16

environment that is crucial for neuronal signaling and information processing. A new insight into

17

this function is that the homeostatic support is achieved through a coordinated action of

18

astrocytes governed by gap junction coupling established syncytial isopotentiality.

19

It has been speculated that a potential role of gap junction is to “clamp” neighboring

20

astrocytes to an identical resting membrane potential, or syncytial isopotentiality, which could be

21

essential for the maintenance of a uniform extracellular ion concentration (Muller 1996). In the

22

present study, for the first time we have experimentally demonstrated that gap junction coupling

23

confers isopotentiality on astrocyte syncytium. This finding is supported by an early in vivo 18

1

observation from cat cortex: during interictal discharge the recorded glial cell VM remained

2

relatively stable although the amplitude of extracellular K+ concentration varied dramatically

3

(Futamachi and Pedley 1976), suggesting that isopotentiality should also function

4

physiologically in vivo.

5

Syncytial Isopotentiality Facilitates K+ Clearance

6

In this study, a demonstrated function of syncytial isopotentiality is to minimize the

7

anticipated high [ K  ]e -induced membrane potential depolarization around a local area in the

8

network, whereby a sustained K+ uptake driving force can be maintained for a higher K+

9

clearance efficiency (Fig. 5H). This observation has a significant implication for the frequently

10

discussed “K+ spatial buffering” hypothesis.

11

The K+ spatial buffering hyperthesis was proposed by Orkand et al (Orkand et al. 1966).

12

Evidence that glial cells do utilize this mechanism for K+ clearance came from experiments

13

performed in Müller cells (Newman 1984; Newman et al. 1984; Oakley et al. 1992), where a

14

single Müller cell, a specialized astrocyte in retina, spatially transported K+ from the plexiform

15

layers to the vitreous body, blood vessels and subretinal space, a mechanism termed “K+

16

siphoning”. Now increasing evidence indicates that this „spatial dependent” K+ clearance

17

mechanism may work as well in syncytial coupled astrocytes (Coles et al. 1986; Gardner-

18

Medwin and Nicholson 1983; Holthoff and Witte 2000; Kofuji et al. 2000; Wallraff et al. 2006) .

19

In the present study, we have demonstrated that syncytial isopotentiality makes K+ uptake more

20

efficient. As demonstrated in this study, syncytial isopotentiality minimizes the local high [ K  ]e -

21

induced VM depolarization, and this maintains a sustained driving forces for K+ uptake. By

22

extension, syncytial isopotentiality also increases the driving force for K+ release in distant

19

1

regions where [K+]e remains at the physiological level. Additionally, increase in both driving

2

forces creates a maximum driving force for intracellular K+ transfer from high [K+]e region to

3

remote regions with normal [K+]e. Therefore, syncytial isopotentiality facilitates all three critical

4

steps in “K+ spatial buffering”, K+ uptake, intercellular transfer and release (Kofuji and Newman

5

2004).

6

Number of Nearest Neighbors and Coupling Strength Collectively Determine the Capacity

7

of K+ Clearance

8

Previously, we have shown that each hippocampal astrocyte couples directly with 11 the

9

nearest neighbors (Xu et al. 2014), Here we show that a strong coupling mediated syncytial

10

isopotentiality facilitates the capacity of K+ uptake to level that could not be appreciated in the

11

past. These observations, however, also rise an important physiological question. That is,

12

whether and how the capacity of K+ clearance is determined by these two parameters?

13

This fundamental question can now be answered by modeling simulations (Supp. Info.

14

Fig. 7). First, when coupling number keeps as a constant, the coupling strength (s) determines the

15

slope, or how fast, the high [K+]e can be brought back to the physiological levels. Second, when

16

the coupling strength (s) keeps as a constant, the number of coupling cells determines how close

17

the final [K+]e can approach to the physiological [K+]e level. Third, when the coupling strength

18

(s) is sets at 1, as demonstrated in this study, the total capacity of K+ uptake can no longer

19

increase with further increased number of nearest neighbors over 11. Thus the combination of

20

these anatomic and physiological features is likely achieved over the course of evolution for the

21

optimal function of astrocytes in the brain.

22

Future Studies

20

1

The precise role of astrocytes in the adult brain remains to be defined (Nedergaard et al.

2

2003). An emerging view from this study is that astrocytes work as a team and the basic

3

physiological function is achieved at syncytial network levels. This sheds new lights on future

4

astrocyte study and urges more study to examine the functions achieved at syncytial (or system)

5

levels.

6

Astrocyte coupling is important for equalization of intracellular Na+ concentration

7

(Langer et al. 2012; Rose and Ransom 1997), calcium signaling and wave propagation (Khakh

8

and McCarthy 2015; Kuga et al. 2011), and regulatory redistribution of glucose toward to active

9

neuorns in demand (Rouach et al. 2008). Meanwhile, there is an increasing awareness of

10

astrocytes diversity (Giaume and Liu 2012; Zhang and Barres 2010). Thus, the experimental

11

approach demonstrated in this study provides a powerful functional measurement to determine in

12

the future the extent the syncytial isopotentiality exists in other brain regions and whether and

13

how this crucial network function could be altered in various pathological disorders.

14

Additionally, the numerously expressed Na+-dependent uptake systems (Cahoy et al.

15

2008; Kimelberg 2010) should be appreciated and reappraised with the consideration of syncytial

16

isopotential. The present study also emphasizes the importance of homeostatic support as a basic

17

astrocyte function in the brain.

18 19

Acknowledgments:

20

This work was sponsored by grants from National Institute of Neurological Disorders and Stroke

21

(RO1NS062784 to MZ, RO1NS043246 to DDM), a start-up fund from The Ohio State

22

University College of Medicine (to MZ) and grants from the National Science Foundation (DMS

23

1410935 to DT and DMS 0931642 to the Mathematical Biosciences Institute). We thank Drs. 21

1

Harold K. Kimelberg and Maiken Nedergaard for critical comments on the manuscript. We

2

thank Dr. Bruce R. Ransom for providing insightful comments and suggestions for manuscript

3

improvement during revision.

4 5

Author contribution: B.M. and M.Z. designed experiments; B.M. and Y.D. performed

6

experiments; D.D.M. contributed the transgenic mice; B.M., R.B., D.T and J.J.E. performed

7

theoretical analysis; B.M., J.J.E., R.B., D.T. and M.Z. wrote the manuscript. All authors

8

discussed the results and commented on the manuscript.

9 10

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17 18 19

Figure Legends

20

Figure 1. Physiological VM can be well-maintained in astrocyte recordings with reduced or

21

K+ free pipette solutions in situ. (A) SR101 staining of astrocytes in CA1 region. (B and C)

22

Astrocyte VM recordings, first in cell-attached, then in whole-cell mode after membrane patch

23

breakthrough (arrows). The resting VM‟s were comparable between K+]p and [Na+]p solutions. (D

27

1

and E) An NG2 glia identified from PDGF-driven GFP transgenic mouse CA1 region in situ, and

2

its VM recording with [Na+]p. The VM showed an initial hyperpolarization and a following

3

depolarization. (F) The VM recording from an interneuron with [Na+]p, showing an initially

4

hyperpolarization (-40 mV), followed by a depolarization (~0 mV). A burst of spikes appeared

5

shortly after patch breakthrough (upper inset). (G) Summary of the VM values for the cell types

6

and conditions indicated (one-way ANOVA with post-hoc F test). (H) Summary of the VM

7

values from astrocytes recorded with various pipette NMDG+ concentrations in situ (one-way

8

ANOVA with post-hoc F test). (I) Gap junction inhibition by 100 M MFA depolarized VM and

9

the subsequent Kir4.1 inhibition (100 µM Ba2+) hyperpolarized VM as predicted by the

10

mathematical model (Supp. Info. Fig. 4). (J) Summary of the VM values in MFA, MFA+Ba2+,

11

and a predicted value for the latter from the model (paired sample t test). *P < 0.05.

12 13

Figure 2. Syncytium dictates the VM of an individual astrocyte inside the network. (A)

14

Images show SR101 staining from a single, two and three astrocytes in dissociated tissues with

15

their spatially distinct domains and that the elaborate processes were well preserved. (B) A live

16

single astrocyte captured during recording: SR101 staining (left) and an intact bushy morphology

17

(DIC, right). (C-F) Representative recordings from freshly dissociated hippocampal tissues

18

containing a varied number of astrocytes with [Na+]p. A negative current pulse was periodically

19

injected into the cells to monitor their input resistance (Rin) in all the recordings, and the two

20

representative traces from (F) are shown in expanded scale on the right panel. (G) The VM

21

recording from a single dissociated astrocyte with [Na+]p. The cell showed an initial

22

hyperpolarization and then depolarized to the anticipated 0 mV. (H) The same cell in (G)

23

depolarized in response to 23.5 mM bath K+ application. (I) Whole-cell currents from a 28

1

dissociated single astrocyte made with [Na+]p, first in aCSF (left) and then after removal

2

(middle) and restoration (right) of 3.5 mM [K+]e in aCSF. (J) The VM recording from an isolated

3

astrocyte with [K+]p. (K) Quantification of VM in [K+]p and [Na+]p pipette solutions (one-way

4

ANOVA with post-hoc F test). *P < 0.05, one way ANOVA test.

5 6

Figure 3. The ion exchange efficacy at gap junction sites. (A) Representative VM recordings

7

from [K+]p and [Na+]p pipette solutions, as indicated in situ. (B) Schematic illustrations of Na+

8

dialysis and substitution occurring in a single and a doublet astrocytes with a difference in the

9

presence of gap junctional coupling in the latter. (C) Modeling predictions of the time courses

10

for doublet astrocytes to reach to a full Na+ substitution with different gap junction coupling

11

strengths. The numerical 1 is the prediction for the highest ionic diffusion rate in a doublet; this

12

predicts a time course that is most similar to that of a single cell. (D) The VM recordings from

13

single and doublet astrocytes with [Na+]p to determine their T50‟s. (E) The T50 values for Na+ ion

14

substitutions in single and doublet astrocytes (two sample t test). **P < 0.01, one way ANOVA

15

test.

16 17

Figure 4. Astrocyte doublets show equilibrated VM. (A1) Schematic illustration of the

18

recording procedure to achieve a separation of EM in a doublet. The K+-based electrode, P1, is

19

ruptured first. This does not alter the [K+]i gradient, but enables the reading of VM. (A2) The

20

model prediction of EM separation between P1 and P2 during Na+-dialysis and at the steady-state

21

levels. (A3) Similar to a1 to separate the EM in a single astrocyte at P1 and P2. (B-C) The VM

22

recordings from a doublet (B) and a single astrocyte (C) following the procedure described in a.

23

Each trace is color coded with its corresponding recording pipette (P). In a doublet, the initial 29

1

VM, just after the breakthrough of P2, is shown in expanded scale to disclose the immediate

2

equilibration of VM in both cells. In both doublet and single astrocyte, the progression of VM to

3

the steady-state levels are shown in expanded scale (middle and right insets, respectively). Each

4

trace was superimposed with a single exponential fit in order to determine a time constant (τ) and

5

VM amplitude. (D) The derived τ‟s and the VM amplitudes among the four pipettes. (E)

6

Illustration of the use of K+ free solutions to maximally reduce the transjunctional voltage and

7

allow for accurate measurement of the coupling coefficient (CC) and gap junction resistance (Rg)

8

(one-way ANOVA with post-hoc F test). (F-G) The respective current clamp (F) and combined

9

voltage and current clamp (G) recording modes for measurement of CC and Rg, as indicated. The

10

access resistance (Ra) was not compensated for the delivered VC in P1(G). The resulting strong

11

CC and low Rg indicate that doublets electrically behave as single astrocytes.

12 13

Figure 5. Syncytial coupling attenuates local elevated extracellular K+-induced VM

14

depolarization. (A) Bath 23.5 mM high K+-induced VM depolarization from an astrocyte and an

15

NG2 glia, as indicated. (B) Summary of bath and local high [K+]e induced VM depolarization. (C)

16

A 5 ms local high [ K  ]e was applied to a recorded astrocyte with an affected subdomain area

17

monitored by SR101 included in the high [K+]e solution (two sample t test). (D) A 5 ms local

18

high [K+]e puff induced VM depolarization in an astrocyte and an NG2 glia. (E) Schematic

19

illustration of VM response to a high [K+]e puff. In an uncoupled astrocyte, the VM quickly

20

depolarizes to the new elevated [K+]e established EM. Once VM → EM, the K+ uptake stops. In

21

coupled astrocytes, the VM remains close to a constant. This maintains the driving force, VM – EM,

22

for a “sustained K+ uptake”. *P < 0.05, one way ANOVA test for comparison of the VM

23

depolarization between astrocytes and NG2 glia. 30

B

C Astrocyte in situ



00

VM (mV)

VM (mV)

[K+]p

-40 -40 -60 -60 2 min

D

E



70

VM (mV)

VM (mV) 0

[Na+]p

-60 -60

20 s

H

6

140

140

9

5

1 min

8

-60 -60

0

70

105

140

11

3

3

6

**

**

**

0

10

-40 -40 8

-80 -80

-20 -40 -60 -80

**



I

[Na+]p

-20 -20

[NMDG+]p (mM) Astrocyte

NG2 glia Neuron

VM (mV)

VM (mV)

11

00

-60 -60

00 -20 -20

**

** J

100 M MFA 100 M Ba2+

00

0

MFA

VM (mV)

VM (mV)

6 -20 -20

-40 -40 -60 -60 -80 -80

1 ms

-40

-40 -40

-80 -80

140

40 0

20 20

-40 -40

[Na+]p (mM) Astrocyte 20 20

Neuron



G

GFP

1 min

-60 -60

40 40

-20 -20

10 m

[Na+]p

-40 -40

F

NG2 glia 00

-20 -20

-80 -80

-80 -80

SR101

Astrocyte in situ

00

Patch breakthrough

-20 -20

20 m



A

1 min

6

-10 -20 -30

**

Figure 1, Ma et al., 2015

A

B Patched single astrocyte

Freshly dissociated astrocytes SR101

SR101

10 m

00 t,1

-40 -40

[Na+]p

0.2 s

t,2

-80 -80

t,2

2 min

G





F

00

2 cells

1 min

-20 -40

-40

-60

-60

-80

-80

-60 -60

-40 -40

[Na+]p

-60 -60

I

[Na+]p 2 min

 20 mM [K+]e

20 20

[Na+]p

10 10

1 min

00

-80 -80

2 min

3 cells

H

VM (mV)

[Na+]p

-80 -80

0

-20

30 30

VM (mV)

-40 -40

0

1 cell

-20 -20

-20 -20 VM (mV)

[Na+]p

-60 -60

t,1

-80 -80

00

Vm (mV)

-20 -20

Vm (mV)

-60 -60

E 5 cells



Vm (mV)

D 16 cells

-20 -20 -40 -40

2 cells

3 cells



00

2 cells



C

DIC

1 min

Single astrocyte, [Na+]p 0 mM [K+]e

3.5 mM [K+]e 0 mV

2 nA

2 nA

5 ms

5 ms

5 ms

K Single astrocyte

0

[K+]p

-20

-40 -40

-60 -60 -80 -80

0 mV

2 nA

5 min

Vm (mV)

VM (mV)

-20 -20

0 mV



J

3.5 mM [K+]e

Number of astrocytes in a syncytium >10 7-9 4-6 3 2 1 1 8

8

12

-40

*

-60 -80

-100

12

3

9

11

NS

* NS

* [Na+]p

[K+]p

Figure 2, Ma et al., 2015

A

Astrocyte in situ [K+]p

[Na+]p

-80 mV

-80 mV 2 nA

2 nA 5 ms

5 ms

C

B Single

Doublet K+

[Na+]P

Gap junction

Patch breakthrough Na+ dialysis

Ionic coupling

Na+

K+

Na+

-20 -40 -60

0

Na+

5

10 15 Time (min)

E



0

S=0.01

-80

Full Na+

Na+ substitution

D

S=100 S=1

0

K+

K+

K+

[Na+]P

Voltage (mV)

Seal

20

[Na+]p

5

**

-20

Single

Doublet

-40 [Na+]p

20 m

T50 (min)

VM (mV)

4 3 3

1

-60 2 min

5

2

0

3 3

Single

Doublet

Figure 3, Ma et al., 2015

A1

A2

Doublet ①

EM (mV)

[Na+]P

-40

Single ①





[K+]P

A3

[Na+]p

0

[K+]P

steady-state [K+]P

-80



[Na+]P

60 Time (s)



120





0 [K+ ]

[Na+ ]

B

C [K+]

P

[K+]P





[Na+]P

-20 -20

-40 -40

VM (mV)

VM (mV)

-20 -20

[Na+]P

20 µm

-60 -60 ①

20 s



-80 -80

-60 -60 -80 -80

𝑉𝑚,𝑡 = 𝑉𝑚,0 +

20 µm

-40 -40



20 s

𝐴𝑒 −𝑡/𝜏

𝑉𝑚,𝑡 = 𝑉𝑚,0 +

𝐴𝑒 −𝑡/𝜏

0.2 mV 100 ms

basal VM, [K+]p

5 mV

5 mV

5s

5s

0.3

Amplitude, A (mV)

Time constant,  (min)

D

0.2 8

7

8

7

0.1 0 [K+]p

0 mM [K+]e/ [Na+]P

20 15 8

10

P1

P2

Coupling coefficient =VM/VM

VM

7

[K+]p

G

Iinjection

7

[Na+]p P2

P1

Coupling coefficient = VM/Vc

VM

VC

2 mV 5 ms

transjunctional voltage

8

5 0

[Na+]p

F

E

25

VM 10 mV 5 ms

Vc

Figure 4, Ma et al., 2015

B Depolarized VM (mV)

A Bath high K+, 23.5 mM 4 min

4 min

10 mV

Astrocyte

NG2 glia

50

aCSF

40

GHK prediction

Astrocyte

30 NG2 glia

20

7 4

**

10

3 4 4

0

Subdomain

Bath

D

C

Focal high K+, 23.5 mM, 5 ms 2 mV 1s Astrocyte

26 m

NG2 glia

Subdomain

E Coupled astrocytes

Uncoupled astrocytes

VM→EM

VM→EM

EM (3.5 mM [K]e)



Restricted local K+ uptake

VM

EM (23.5 mM [K]e)

depolarization



Sustained local K+ uptake

Figure 5, Ma et al., 2015

Freshly isolated astrocytes

Astrocytes in a syncytium

Membrane depolarization

: Gap junction

Intracellular [K+]i

0 -20 -40 -60 -80

1 min

Substitution [K+]i with [Na+]i

with [Na+]i Membrane potential (mV)

Membrane potential (mV)

Substitution

[K+]i

Nernstian behavior in single astrocyte membrane potential

0 -20 -40 -60 -80

1 min

Isopotentiality in coupled astrocytes in a syncytium

Original K spatial buffering

1. 2. 3.

Extracellular Na transfer was proposed to maintain extracellular electoneutrality. At any given time, the K influx and efflux has to be equal, that keeps the intracellular electoneutrality. During K redistribution, the cell volume is not changed, or cell volume regulation is not involved.

Three revisions on the K spatial buffering

EK, high EK, low

Isopotentiality

1. The K is taken up under a sustained and larger driving force. More efficient !!! 2. So is the K release (right side of the drawing). More efficient!!! 3. For intercellular K transfer, the Vm is off the equation in driving force calculation. The transfer driving force now is determined by EK, high-EK, low . More efficient !!!

OASIS - Session Summary Summary Information  

 

Minisymposium Information

Title

Emerging insights into the critical role of astrocyte ion channels in neuron-glia signaling and pathogenesis of neurological disorders

Primary Theme

Theme B: Neural Excitability, Synapses, and Glia: Cellular Mechanisms

Cross-Cut:

Theme C: Disorders of the Nervous System

Description

The importance of astrocyte potassium channels in support of neuronal circuit function is reinforced from several recent studies conducted in animal disease models. Increasing number of astrocyte ion channels, such as Best1, hemichannels and two-pore domain channels have been uncovered. These channels primarily function as signaling channels meditating neuron-astrocyte crosstalk. Development and future perspective in these active research fronts will be the focus of this minisymposium.

Objectives

We have gathered 6 speakers who are active investigators in astrocyte membrane ion channel study. The session is conceived to critically examine the state of field by delivering the latest discoveries to the Society. This session aims at the following objectives. First, speakers will revisit the homeostatic support function of astrocyte potassium channels, Kir4.1 in specific, from study of several disease models, where dysfunctional Kir4.1 is causative for Huntington’s disease, Rett Syndrome, Epilepsy, etc. Second, speakers will present the newly identified neuron-glia communication mechanisms mediated from astrocyte Best1, hemichannels and twopore domain channels. There is an increasing appreciation that, in addition to those traditionally recognized potassium channels functioning as “housekeeping channels”, the ion channels falling into the latter category should be considered as “signaling channels” in astrocytes. The number of fascinating “signaling channels” is still growing. These channels are poorly selective in ion species; therefore their operation depends critically on the physiological membrane potential established by “housekeeping channels”. An issue to be highlighted for the future study is the mechanism by which “housekeeping” and “signaling” channels are functionally interrelated so that the critical homeostatic and signaling function could be achieved in astrocytes coordinately.

Timeliness

Exciting new results have been generated in recent years and more discoveries are expecting. This session will be a timely update of research in this area and guidance towards to future study. Novel notions to the Society are 1) the function of "housekeeping" potassium channels undergoes dynamic regulation, and deficits in these channels are causative to neurological disorders; 2) the variously expressed "signaling channels" shed lights on novel mechanisms initiating neuron-glia communication.

Appeal

The session covers a broad range of issues centering on astrocyte ion channels. These issues range from in-depth understanding of homeostatic support function of astrocytes, novel channel-medicated mechanism underlying

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

bidirectional neuron-glia communication, and causative role of dysfunctional ion channels in several demonstrated pathological disorders. Therefore this minisymposium should be appealing to our society broadly. Relevance

This session will show that dysfunction of Kir4.1 channels is causative to a broad range of neurological disorders. Importantly, the new findings highlighted in this session will reiterate the notion that the operation of neuronal circuit requires the homeostatic support from astrocytes, and urge future study to uncover the entire gene profile of housekeeping potassium channels in astrocytes, an issue yet not fully understood.

Diversity

This proposal consists of six speakers selected worldwide in this areas. Specifically, speakers are from France (1), Korea (1), Puerto Rico (1) and United States (3). Among them, two speakers are female leading investigators in this research area.

Number of Female Speakers

2

Number of Underrepresented Minority Speakers

0

Other

none

Journal Minireview

Yes

 

Chair Information

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Min

2nd Initial: 3rd Initial: Last Name:

Zhou

Suffix (Jr., Sr., III): Degree(s):

MD, PhD

Institution:

Ohio State University

Department:

Neuroscience

City:

Coulumbus

State:

OH

Country:

United States

Zip/Postal Code:

43210

Address Line 1:

4066C Graves Hall

Address Line 2:

333 West 10th Avenue

City:

Columbus

State:

OH

Country:

United States

Zip/Postal Code:

43210

Phone:

614 366 9406

Fax: E-mail:

[email protected]

Disclosure Block :

M. Zhou, None.

 

Co-Chair Information

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Michelle

2nd Initial:

L

3rd Initial: Last Name:

Olsen

Suffix (Jr., Sr., III): Degree(s):

PhD

Institution:

University of Alabama At Birmingham

Department:

Cell, Developmental and Integrative Biology

City:

BIRMINGHAM

State:

AL

Country:

United States

Zip/Postal Code:

35294

Address Line 1:

1918 University Blvd.

Address Line 2:

MCLM 958

City:

BIRMINGHAM

State:

AL

Country:

United States

Zip/Postal Code:

35294

Phone:

(205)975-2715

Fax:

(205)975-9028

E-mail:

[email protected]

Disclosure Block :

M.L. Olsen, None.

 

Speaker(s) Information

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Baljit

2nd Initial: 3rd Initial: Last Name:

Khakh

Suffix (Jr., Sr., III): Degree(s):

PhD

Speaker Status:

Professor or Equivalent Position

Institution:

University of California, Los Angeles

Department:

Physiology

City:

Los Angles

State:

CA

Country:

United States

Zip/Postal Code:

90095

Address Line 1:

10833 Le Conte Avenue

Address Line 2: City:

Los Angles

State:

CA

Country:

United States

Zip/Postal Code:

90095

Phone:

310 825 6258

Fax:

310 206 5661

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

E-mail:

[email protected]

Presentation Title

Astrocyte dysfunction in Huntington’s disease

References:

Tong X, Ao Y, Faas GC, Nwaobi SE, Xu J, Haustein MD, Anderson MA, Mody I, Olsen ML, Sofroniew MV, Khakh BS (2014) Astrocyte Kir4.1 ion channel deficits contribute to neuronal dysfunction in Huntington's disease model mice. Nat Neurosci 17:694-703.

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

B. Khakh, None.

 

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Michelle

2nd Initial:

L

3rd Initial: Last Name:

Olsen

Suffix (Jr., Sr., III): Degree(s):

PhD

Speaker Status:

Assistant Professor or Equivalent Position

Institution:

University of Alabama At Birmingham

Department:

Cell, Developmental and Integrative Biology

City:

BIRMINGHAM

State:

AL

Country:

United States

Zip/Postal Code:

35294

Address Line 1:

1918 University Blvd.

Address Line 2:

MCLM 958

City:

BIRMINGHAM

State:

AL

Country:

United States

Zip/Postal Code:

35294

Phone:

(205)975-2715

Fax:

(205)975-9028

E-mail:

[email protected]

Presentation Title

Deficits in astrocyte mediate potassium homeostasis contribute to Rett Syndrome disease pathogenesis

References:

Nwaobi SE, Lin E, Peramsetty SR, Olsen ML (2014) DNA methylation functions as a critical regulator of Kir4.1 expression during CNS development. Glia 62:411-427.

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

M.L. Olsen, None.

 

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Serguei

2nd Initial:

N

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

3rd Initial: Last Name:

Skatchkov

Suffix (Jr., Sr., III): Degree(s):

PhD

Speaker Status:

Professor or Equivalent Position

Institution:

Univ Central Del Caribe

Department:

Biochem & Physiology

City:

Bayamon

State:

PR

Country:

United States

Zip/Postal Code:

00960

Address Line 1:

Sch Med

Address Line 2:

Call Box 60-327

City:

Bayamon

State:

PR

Country:

United States

Zip/Postal Code:

00960

Phone:

78 787-786-

Fax:

78 787-786-

E-mail:

[email protected]

Presentation Title

Glial face of EAST/SeSAME-Syndrome, Epilepsy, Autism and MS: critical role of polyamine and sodium in Kir4.1 and GLT1/GLAST interactions

References:

Skatchkov SN, Woodbury-Farina MA, Eaton M. (2014) The Role of Glia in Stress: Polyamines and Brain Disorders, Psychiatr Clin North Am. 37(4):653-678 (doi:10.1016/j.psc.2014.08.008) Review.

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

S.N. Skatchkov, None.

 

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Min

2nd Initial: 3rd Initial: Last Name:

Zhou

Suffix (Jr., Sr., III): Degree(s):

MD, PhD

Speaker Status:

Assistant Professor or Equivalent Position

Institution:

Ohio State University

Department:

Department of Neuroscience

City:

Columbus

State:

OH

Country:

United States

Zip/Postal Code:

43210

Address Line 1:

4066C Graves Hall

Address Line 2:

333 W 10th Ave

City:

Columbus

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

State:

OH

Country:

United States

Zip/Postal Code:

43210

Phone:

(614)366-9406

Fax:

(614)688-8742

E-mail:

[email protected]

Presentation Title

mGluR3-mediated TWIK-1 membrane expression and ammonium homeostasis in astrocytes

References:

Wang W, Putra A, Schools GP, Ma B, Chen H, Kaczmarek LK, Barhanin J, Lesage F, Zhou M (2013) The contribution of TWIK-1 channels to astrocyte K(+) current is limited by retention in intracellular compartments. Front Cell Neurosci 7:246.

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

M. Zhou, None.

 

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Justin

2nd Initial:

C

3rd Initial: Last Name:

Lee

Suffix (Jr., Sr., III): Degree(s):

PhD

Speaker Status:

Professor or Equivalent Position

Institution:

Korea Advanced Institute of Science and Technology

Department:

Center for Neural Science and Center for Functional Connectomics

City:

Seoul

State: Country:

Korea, Republic of

Zip/Postal Code:

136-130

Address Line 1:

39-1 Hawolgok-dong, Seongbuk-gu

Address Line 2: City:

Seoul

State: Country:

Korea, Republic of

Zip/Postal Code:

136-130

Phone:

82-2-958-6940

Fax:

82-2-958-6937

E-mail:

[email protected]

Presentation Title

DBT

References:

DBT

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

J.C. Lee, None.

 

file:///C|/Users/zhou16/Desktop/OASIS - Session Summary.html[12/22/2014 9:49:00 AM]

OASIS - Session Summary

Salutation: (Dr./Mr./Ms./Mrs.)

Dr.

First Name:

Nathalie

2nd Initial: 3rd Initial: Last Name:

Rouach

Suffix (Jr., Sr., III): Degree(s):

PhD

Speaker Status:

Professor or Equivalent Position

Institution:

College De France CIRB

Department:

Neuroglial Interactions in Cerebral Physiopathology

City:

Paris

State: Country:

France

Zip/Postal Code:

75005

Address Line 1:

Inserm U1050/CNRS UMR 7241

Address Line 2:

11 place Marcelin Berthelot

City:

Paris

State: Country:

France

Zip/Postal Code:

75005

Phone:

33 0033144271242

Fax:

33 0033144271260

E-mail:

[email protected]

Presentation Title

Astroglial connexin hemichannels tune glutamatergic synaptic transmission through ATP signaling

References:

O. Chever, C.Y. Lee, N. Rouach. 2014. Astroglial connexin 43 hemichannels tune basal excitatory synaptic transmission. The Journal of Neuroscience. 34:11228-32.

Unlabeled/Unapproved:

No

1. Product Name: 2. Product Name Disclosure Block :

N. Rouach, None.

   

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