Neurochem Res DOI 10.1007/s11064-017-2197-9

ORIGINAL PAPER

Astrocyte Sodium Signalling and Panglial Spread of Sodium Signals in Brain White Matter Behrouz Moshrefi‑Ravasdjani1 · Evelyn L. Hammel1 · Karl W. Kafitz1 · Christine R. Rose1   

Received: 7 December 2016 / Revised: 19 January 2017 / Accepted: 28 January 2017 © Springer Science+Business Media New York 2017

Abstract  In brain grey matter, excitatory synaptic transmission activates glutamate uptake into astrocytes, inducing sodium signals which propagate into neighboring astrocytes through gap junctions. These sodium signals have been suggested to serve an important role in neuro-metabolic coupling. So far, it is unknown if astrocytes in white matter—that is in brain regions devoid of synapses—are also able to undergo such intra- and intercellular sodium signalling. In the present study, we have addressed this question by performing quantitative sodium imaging in acute tissue slices of mouse corpus callosum. Focal application of glutamate induced sodium transients in SR101positive astrocytes. These were largely unaltered in the presence of ionotropic glutamate receptors blockers, but strongly dampened upon pharmacological inhibition of glutamate uptake. Sodium signals induced in individual astrocytes readily spread into neighboring SR101-positive cells with peak amplitudes decaying monoexponentially with distance from the stimulated cell. In addition, spread of sodium was largely unaltered during pharmacological inhibition of purinergic and glutamate receptors, indicating gap junction-mediated, passive diffusion of sodium between astrocytes. Using cell-type-specific, transgenic reporter mice, we found that sodium signals also propagated, albeit less effectively, from astrocytes to neighboring oligodendrocytes and NG2 cells. Again, panglial spread was unaltered with purinergic and glutamate receptors blocked. Taken together, our results demonstrate that activation of * Christine R. Rose rose@uni‑duesseldorf.de 1



Institute of Neurobiology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany

sodium-dependent glutamate transporters induces sodium signals in white matter astrocytes, which spread within the astrocyte syncytium. In addition, we found a panglial passage of sodium signals from astrocytes to NG2 cells and oligodendrocytes, indicating functional coupling between these macroglial cells in white matter. Keywords  Astrocyte · Oligodendrocyte · NG2 cell · Corpus callosum · SBFI · Glutamate Abbreviations AMPA α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid AP5 D-(-)-2-Amino-5-phosphonopentanoic acid ATP Adenosine triphosphate CA1 Cornu ammonis 1 Cx Connexin DAPI 4′,6-diamidino-2-phenylindole EYFP Enhanced yellow fluorescent protein GABA Gamma-aminobutyric acid GFP Green fluorescent protein GLAST Glutamate aspartate transporter GLT1 Glutamate transporter 1 hGFAP Human glial fibrillary acidic protein Iba1 Ionized calcium-binding adapter molecule 1 mGluR Metabotropic glutamate receptor MPEP 2-Methyl-6-(phenylethynyl)pyridine hydro-chloride MRS2179 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt NBQX 2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F) quinoxaline NCX Sodium/calcium exchanger NG2 Neural/glial antigen 2 NMDA (R)-2-(Methylamino)succinic acid

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PBS Phosphate buffered saline PLP Proteolipid protein PPADS Pyridoxalphosphate-6-azophenyl-2′,4′disulfonic acid tetrasodium salt ROI Region of interest SBFI(-AM) Sodium-binding benzofuran isophthalate (-acetoxymethyl ester) SEM Standard error of the mean SR101 Sulforhodamine 101 TFB-TBOA (3 S)-3-[[3-[[4-(Trifluoromethyl)benzoyl] amino]phenyl]methoxy]-L-aspartic acid TTX Tetrodotoxin

Introduction Recent work has firmly established that astrocytes in different grey matter regions of the brain respond to synaptically released glutamate by generating intracellular sodium transients [1, 2]. These sodium signals serve an important role in astrocyte metabolism and neuro-metabolic coupling [3]. In addition, they influence the activity of sodium-dependent transporters. Increases in sodium can, for example, cause a reversal of the sodium/calcium exchanger (NCX) and thereby contribute to astrocyte calcium signalling [4, 5]. Moreover, they promote the release of glutamine, driving the glutamate-glutamine shuttle [6, 7], and may cause reversal of GABA uptake, resulting in a release of GABA from astrocytes [8]. Work from Bruce Ransom’s laboratory on cultured hippocampal astrocytes provided first direct description of astrocyte sodium transients in response to activation of sodium-dependent glutamate transporters [9]. This was confirmed later on astrocytes in vitro and in situ derived from different grey matter regions [6, 10–14]. Other studies showed that additional sodium influx pathways such as GABA transport [15, 16] or plasma membrane ion channels exist [13, 17]. Astrocyte sodium signals can be restricted to sub-cellular domains or be global, comprising the entire cell including perisynaptic processes, somata and endfeet on blood vessels [10, 18]. In addition, sodium easily spreads between neighboring astrocytes. Again, this basic phenomenon was established in Bruce Ransom’s laboratory in primary cultures of hippocampal astrocytes [19]. Using pharmacological tools, they provided evidence that the spread of sodium was mediated by gap junctional coupling, resulting in its equilibration between coupled cells. Diffusive spread of sodium through gap junctions was also demonstrated in cultured cortical astrocytes [20] and in acute brain tissue slices of mouse hippocampus [21] and the lateral superior olive [22]. In the latter brain region, a panglial passage of

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sodium was observed, encompassing astrocytes as well as oligodendrocytes [22]. In contrast to grey matter, basically no information is available on sodium signalling in white matter astrocytes. White matter does not contain synapses, but axonal release of glutamate has been reported [23, 24]. While it has been shown that white matter astrocytes express sodiumdependent glutamate transporters [25–27], it is unclear if their activation does induce detectable intracellular sodium transients. In addition, gap junctional coupling between white matter astrocytes and also oligodendrocytes has been described [28–31], but if coupling mediates efficient spread of sodium between these cells, is unknown. In the present study, we have addressed these questions by performing quantitative sodium imaging in the corpus callosum, a major white matter tract of the brain.

Materials and Methods Preparation of Brain Slices and Salines Coronal brain tissue slices (250  µm) were prepared from mouse corpus callosum (mus musculus, BALB/c; postnatal days (P) 15–22; both sexes)  at a vibratome (HM650V, Thermo Fischer Scientific, Oberhausen, Germany). For identification of different cell types, transgenic mice (P1520; both sexes), in which expression of fluorescent proteins is controlled by the promotor activity of defined (“cell-specific”) genes, were used. Acute tissue slices for imaging experiments were sectioned in ice-cold saline composed of (in mM): 125 NaCl, 2.5 KCl, 2 C ­ aCl2, 1 M ­ gCl2, 1.25 N ­ aH2PO4, 26 N ­ aHCO3 and 20 glucose, bubbled with 95% ­O2 and 5% ­CO2, resulting in a pH of 7.4. Afterwards, slices were kept at 34 °C for 20  min in saline to which 0.5–1  µM sulforhodamine 101 (SR 101, Sigma–Aldrich, Munich, Germany) was added for vital staining of astrocytes [32]. Brain slices were then kept at room temperature (20–22 °C); experiments were performed at room temperature as well. Sodium Imaging Experiments Bolus-loading of macroglial cells was achieved by injection of the ester form of the sodium-sensitive fluorescent dye SBFI (SBFI-AM (sodium-binding benzofuran isophthalateacetoxymethyl ester), TEFLabs Inc., Austin, TX, USA) into the corpus callosum [33]. For quantitative, ratiometric imaging of sodium [10], we employed an upright microscope (Nikon Eclipse FN-PT, Nikon, Düsseldorf, Germany) with a 40x/N.A. 0.8 LUMPlanFI (Olympus Deutschland GmbH, Hamburg, Germany) water immersion objective coupled to a digital imaging system (Nikon NIS-Elements

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v4.3) and an Orca FLASH V2 camera (Hamamatsu Photonics Deutschland GmbH, Herrsching, Germany). Excitation light (340/380 nm) was generated by a PolychromeV monochromator (TILL Photonics, Martinsried, Germany) and emission collected >440  nm. Images were obtained at 1–5  Hz. Fluorescence was analyzed from regions of interest (ROIs) positioned around cell bodies of SBFI-labelled cells and background correction was performed as reported earlier [10, 21]. For calculation of the fluorescence ratio F ­ 340/F380 from ROIs, OriginPro Software (OriginLab Corporation, Northampton, MA, USA) was used. Changes in SBFI fluorescence were expressed as changes in sodium concentration based on in situ calibrations as described before [10, 21, 33]. Chemicals were obtained from Sigma–Aldrich (Munich, Germany), except for TTX (tetrodotoxin; Biotrend Chemicals, Cologne, Germany). Substances were generally applied by bath perfusion exept for glutamate which was focally applied by standard micropipettes coupled to a pressure application device (PDES-02D, NPI Electronic GmbH, Tamm, Germany). To induce local sodium influx into an individual cell, a saline-filled micropipette was positioned onto its soma and a 1  ms stimulation pulse delivered using an isolated stimulator (A-M systems, Model 2100, Sequim, USA). This results in the electroporation of the stimulated cell and causes an immediate, transient increase in its intracellular sodium as reported before [18, 20–22]. In a minority of stimulated astrocytes, the electroporation induced a persistent increase in sodium and a strong and rapid loss of fluorescence at 340 nm. Because this indicates cell damage and loss of membrane integrity, these experiments were discarded. Immunohistochemistry Mouse brains (P15-22) were fixed with 0.1  M phosphate buffered saline (PBS) containing 4% paraformaldehyde and 10–25  µm coronal vibratome sections  (HM650V, Thermo Fischer Scientific, Oberhausen, Germany) were prepared. For immuno stainings targeting GFP (green fluorescent protein), EYFP (enhanced YFP) and ionized calcium-binding adapter molecule 1 (Iba1), the sections were incubated in blocking solution comprising normal goat serum (GIBCO/Life Technologies, Darmstadt, Germany; 5% in PBS, 1  h) and TritonX-100 (Sigma–Aldrich Chemical, Munich, Germany, 0.4%). Subsequently, they were incubated with primary antibodies for GFP (chicken anti-GFP, Abcam, Cambridge, UK; ab13970, 1:1000 in blocking solution) and Iba1 (rabbit anti-Iba1, WAKO Chemicals GmbH, Neuss, Germany; 019-19741, 1:4000 in blocking solution) overnight. The final step was an incubation in secondary antibody solutions (PBS with 0.4%

TritonX-100, 1  h) containing goat anti-chicken Alexa 488 (1:500, Invitrogen, Grand Island, NY, USA) and goat antirb 594 (1:500, Invitrogen, Grand Island, NY, USA) for 2 h. For nuclear labelling, slices were additionally incubated in DAPI (20 min). The sections were coverslipped in Mowiol (Calbiochem, Fluka, distributed by Sigma–Aldrich Chemical, Munich, Germany) or Mountant Permafluor (Thermo Fisher Scientific, Schwerte, Germany) mounting medium. In control sections, all or either one of the primary antibodies were omitted from the protocol. Control stainings omitting one of the primary antibodies exhibited identical labeling patterns for the remaining antibody as for the double stainings. Excluding both primary antibodies never resulted in a labeling product. Image z-stacks (10–20 optical sections, 0.3–0.5  µm each) were captured using a motorized confocal laser scanning microscope (Nikon Eclipse C1), equipped with a Nikon PlanApoVC 60x/1.4 oil objective. Appropriate excitation was realized through argon (488  nm), helium–neon (543  nm) and 407  nm lasers (all Melles Griot, Bensheim, Germany). The parameters of image acquisition and processing were kept identical for all stacks. Simultaneous or sequential scanning of both fluorophores revealed no difference in their staining pattern, indicating no cross-excitation or spectral bleedthrough. Maximum Intensity Projections were calculated from z-stacks of optical sections by using ImageJ software (NIH, Bethesda, MD). Images were overlaid employing Adobe Photoshop CS2 (Adobe Systems, San Jose, CA). The PMT settings and the image processing were kept constant. Data Analysis and Presentation Data are presented as means ± S.E.M. (unless stated otherwise) and were statistically analyzed by Wilcoxon paired test. “p” represents error probability, *0.01  ≤  p  <  0.05, **0.001 ≤ p < 0.01, ***p < 0.001. “n” represents the number of individual cells analyzed, while “N” represents the number of individual experiments/slices. Each set of experiments was obtained from slices of at least three different animals. Images were edited in ImageJ (NIH Image, Bethesda, MD, USA) and Adobe Illustrator (Adobe Systems Incorporated, San Jose, CA, USA).

Results Identification of Macroglial Cell Types White matter does not contain neuronal cell bodies, but hosts macroglial as well as microglial cells. For identification of the different types of glial cells in the corpus callosum and to illustrate their distribution and spatial

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arrangement in our preparation, we first performed immunocytochemical stainings and made use of specific reporter mice. Transmission light images were used to localize corpus collosum fiber tracts; DAPI stainings were employed to detect the location of cellular nuclei and thereby identify individual cell bodies (not shown). For astrocytes, reporter mice, in which GFP is expressed under the control of the human GFAP promotor (“hGFAPGFP-mice”; [34]) were stained for GFP to amplify its fluorescence (N = 30 slices; Fig.  1a). These stainings revealed that 15% of DAPI-positive nuclei in our preparation represented astrocytes (Fig. 1d). They also illustrate the characteristic arrangement of white matter astrocyte processes

running in parallel to the fibre tracts [27]. Reporter mice, in which GFP is expressed under the control of the PLP promotor (“PLP-GFP-mice”; [35]), showed that 42% of cells are mature, myelinating oligodendrocytes (N  =  30 slices; Fig. 1b, d). Furthermore, NG2-EYFP mice, in which the encoding sequence for the NG2 protein is replaced by EYFP [36, 37], were employed, demonstrating that about 17% of cells are NG2 cells (N = 30 slices; Fig.  1c, d). Immunohistochemical staining for Iba1 identified a total of 8% of cells as microglial cells (N = 12 slices; Fig.  1d). These numbers are similar to those obtained in an earlier study addressing cellular identity in the postnatal mouse corpus callosum [38] and confirm that the majority of cells

Fig. 1  Immunohistochemical analysis of corpus callosum microanatomy. a–c Images of indirect immunolabelling in corpus callosum targeting hGFAP-GFP (a), PLP-GFP (b), and NG2-EYFP (c) showing localization of resident macroglia. Dashed lines illustrate the borders

of the corpus callosum, which is situated between CA1 stratum oriens (ventral) and cortex (dorsal). Scale bar: 25  µm. d Relative distribution of DAPI-positive corpus callosum cells including microglia

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in this preparation represents cells of the oligodendrocyte lineage (including NG2 cells) (see also [39]). Glutamate‑Induced Sodium Signals To study sodium signals in the corpus callosum, the AMester form of the sodium indicator SBFI was injected into the tissue. While microglial cells do not stain upon bolusloading with the AM-ester form of ion-selective fluorescent indicators [40], macroglial cells, namely astrocytes, oligodendrocytes as well as NG2 cells, take up these dyes. For identification of astrocytes in acute tissue slices, SR101 was employed. This fluorescent marker labels virtually the entire astrocyte population in the cortex and hippocampus grey matter [32, 41]. In the ventrolateral medulla, in contrast, astrocytes are only weakly labelled with SR101 [42]. In corpus callosum of hGFAP-GFPmice, we found that ~75% of GFP-labelled cells were also brightly labelled with SR101 (n = 589, N = 16; Fig. 2a, b). On the other hand side, virtually all SR101-positive cells were also GFP-positive (n = 775, N = 16; Fig. 2a, b), demonstrating that SR101 labels the majority of astrocytes in this preparation. In SR101-positive astrocytes, focal pressure application of glutamate (1  mM, 250  ms) induced a transient sodium increase by 5.4 ± 0.4  mM that recovered to baseline following a monoexponential decay (n = 38, N = 8; Fig.  2c). Glutamate-induced astrocytic sodium transients were unaltered by application of NBQX (2,3-Dihydroxy6-nitro-7-sulfamoyl-benzo(F) quinoxaline, 10  µM) to block ionotropic AMPA (α-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid) receptor channels (Fig.  2 c, d) as well as by additional application of AP5 (2-amino5-phosphonopentanoic acid, 50 µM), which blocks NMDA ((R)-2-(Methylamino)succinic acid) receptors (Fig.  2b, c). Inhibition of sodium-dependent glutamate uptake by TFBTBOA ((3  S)-3-[[3-[[4-(Trifluoromethyl)benzoyl]amino] phenyl] methoxy]-L-aspartic acid, 1  µM), in contrast, reduced sodium transients by more than 80% (Fig. 2c, d). Glutamate-induced sodium signals were also observed in the majority of (68% of a total of 68 cells analyzed) SR101negative cells. In responding cells, a minority (19% of all SR101-negative cells) showed sodium transients similar to SR101-positive astrocytes, indicating that at least part of the 25% of SR101-negative hGFAP-GFP-expressing cells (cf. Fig. 2a, b) are in fact false SR101-negative astrocytes. The remainder (49% of all SR101-negative cells) showed a rather small sodium increase (1.8 ± 0.1  mM; Fig.  2c). Although the amplitude of the glutamate-evoked signals was much smaller in this latter group, they appeared to be mediated primarily by glutamate uptake and there was little pharmacological difference to astrocytes, showing a

TFB-TBOA mediated reduction of signals by ~60% (n = 33, N = 8; Fig. 2c, d). Taken together, our data demonstrate that white matter astrocytes experience transient sodium signals in response to glutamate. Glutamate-induced astrocyte sodium signals are mainly due to activation of glutamate uptake, while sodium influx through glutamate-gated ion channels seems to be negligible. The majority of SR101-negative cells, mostly representing oligodendrocytes and NG2 cells, react to glutamate application with a small sodium increase, to which glutamate transport appears to be the main contributor as well. Intercellular Spread of Sodium Signals Between Astrocytes Having established the occurrence of sodium signals in white matter astrocytes, we next investigated if sodium ions are able to rapidly move between individual cells as demonstrated in astrocytes in grey matter [18, 21, 22]. To this end, we performed direct electroporation in the presence of TTX using an aCSF filled (glutamate-free), glass pipette positioned onto the soma of a given SR101-positive cell, a procedure which allows selective stimulation of single astrocytes [18, 20–22]. Direct single-pulse electroporation resulted in a sodium increase in the stimulated astrocyte, which slowly recovered to baseline (ΔNai: 12.5 ± 1.8  mM; n = 17, N = 7; Fig.  3a, b). The sodium increase was, however, not restricted to the stimulated cell, but comprised neighboring astrocytes (n = 27, N = 7; Fig. 3a, b) as well as SR101-negative cells (n = 104, N = 7 slices; Fig.  3a, b). The peak amplitude of the sodium increase declined with further distance from the stimulated cell (n = 131, N = 7; Fig.  3c). Within the population of identified (SR101-positive) astrocytes, peak amplitudes of sodium signals decayed mono-exponentially (λ  =  31  µM), and within distances of about 30–40  µm, sodium signals had dropped to about 30% of their initial amplitude (Fig. 3c). Taken together, these results demonstrate that sodium signals induced in individual astrocytes can spread within the astrocyte syncytium as well as to other macroglial cells. This spread is characterized by a monotonic weakening of both peak amplitude and slope with distance, indicating that sodium signals do not regenerate actively, but spread by passive diffusion through gap junctions as shown before for grey matter astrocytes. Involvement of “Gliotransmission” in the Spread of Sodium Signals The results obtained above argue for a passive, diffusionbased spread of sodium through gap junctions between

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Fig. 2  Astrocyte identification and glutamate-induced sodium signals. a GFP fluorescence (left) and SR101 labelling pattern (right) in corpus callosum of hGFAP-GFP mouse brain slices. Arrowheads point to co-localization of SR101 and GFP. Dashed lines illustrate the borders of the corpus callosum. Scale bar: 25  µm. b Histogram showing the percentage of SR101-positive cells that are also GFPpositive as well as that of GFP-positive cells, that are also labelled with SR101, in hGFAP-GFP reporter mice. c Transient changes in

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intracellular sodium in SR101-positive (upper traces) and SR101negative cells (lower traces) induced by glutamate (1  mM, 250  ms, arrowheads) and effect of blockers of ionotropic glutamate receptors (NBQX/AP5) as well as glutamate transporters (TFB-TBOA). d Histograms showing the effect of antagonists on the peak amplitude of glutamate-induced sodium transients, normalized to first glutamate application (control) in SR101-positive (left) and negative cells (right)

Neurochem Res Fig. 3  Interglial spread of sodium. a Image of an SBFIloaded slice (top) and the identical image inverted (bottom) showing stimulated SR101positive cell (arrowhead) and regions of interest (ROIs) chosen for analysis of sodium transients as illustrated in b. Dashed lines indicate the borders of the corpus callosum. Scale bar 25 µm. b Sodium transients obtained in a stimulated SR101positive cell as well as in neighboring SR101-positive (black traces) and SR101-negative (grey traces) cells in response to direct electroporation (indicated by grey line). Distances from the stimulated cell are given at the end of individual traces. c Cumulative data plot showing peak amplitudes of sodium increases in all cells neighbouring a stimulated astrocyte, normalized to the amplitude of the latter (left). Right: plot showing data points from SR101-positive astrocytes only. Lines represent a best fit monoexponential decay function (left: ­r2 = 0.85, right: ­r2 = 0.89). Lambda (λ) represents the distance where fit reaches 1/e of maximum. d Same as c, but with blockers of gliotransmission (MPEP, PPADS, MRS) added. Lines represent best fit (left: ­r2 = 0.90, right: ­r2 = 0.94)

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macroglial cells as reported before from grey matter [18, 20–22]. On the other hand, many studies, including a report on corpus callosum [40], suggested the involvement of gliotransmitter release in the propagation of ion signals between astrocytes [20, 43, 44]. We, therefore, studied the effect of inhibitors of metabotropic glutamate receptors (mGluR1, 5) on the intercellular spread of sodium signals by employing MPEP (2-Methyl6-(phenylethynyl)pyridine hydro-chloride, 25  μM; N = 6). Moreover, we applied the purinergic receptor blocker PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid tetrasodium salt; 20 μM; 6 experiments), and the P2Y blocker MRS2179 (2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt; 30  μM; N = 6). Inhibiton of both types of receptors for potential gliotransmitters did not influence the amplitude of sodium signals in neighboring cells in response to direct stimulation of an astrocyte (Fig. 3d). In summary our results show that activation of metabotropic glutamate receptors does not contribute to the spread of sodium between macroglial cells. In addition, they demonstrate that purinergic receptors are not significantly involved in their spread either. This suggests that the major pathway for intercellular passage of sodium is indeed represented by gap junctions.

Taken together, these results provide evidence for a panglial spread of sodium signals induced in astrocytes to both oligodendrocytes and NG2 cells. Peak amplitudes of sodium signals in the latter two cell populations decay much faster than within the astrocyte syncytium, indicating less effective spread and, consequently, weaker overall functional coupling between these cell types.

Panglial Spread of Sodium Signals

Axons in white matter release neurotransmitters that are sensed by surrounding glial cells including (myelinating) oligodendrocytes [45, 46]. Astrocytes contact myelinated axons at the nodes of Ranvier only, which can also contain processes of NG2 cells [47, 48]. Axonal release of glutamate in both rodent optic nerve and in corpus callosum activates ionotropic and metabotropic glutamate receptors on astrocytes, generating intracellular calcium signals [39, 49, 50]. Expression of glutamate receptors and their activation in response to axonal glutamate release, resulting in intracellular calcium signalling, is not restricted to astrocytes, but includes oligodendrocytes as well as NG2 cells [23, 24, 51–55]. In the present study we now report that glutamate also induces sodium transients in white matter glial cells. In SR101-positive astrocytes, glutamate-evoked sodium signalling was virtually omitted in the presence of TFBTBOA, indicating that it is not related to sodium influx through ionotropic glutamate receptors, but largely caused by activation of glutamate uptake. White matter astrocytes have indeed been shown to exhibit high expression levels of sodium-dependent (TBOA-sensitive) glutamate transporters [25, 26]. This is similar to astrocytes in different grey matter regions, such as the hippocampal CA1 stratum radiatum or cerebellar cortex, where glutamate and excitatory synaptic activity evoke prominent sodium transients due to

The results obtained above showed that the spread of sodium does not include SR101-positive astrocytes only, but also a significant population of SR101-negative cells. To clarify the cellular identity of this population, we made use of specific reporter mice. For identification of oligodendrocytes “PLP-GFP-mice” were employed (Fig. 4a). In slices obtained from these animals, single-pulse electroporation of an (SR101-positive) astrocyte resulted in a spread of the sodium signal within astrocytes as described above (n = 32, N = 14; Fig.  4b, c). In addition, sodium signals also clearly invaded PLP-positive cells (n = 44, N = 14; Fig. 4b, c). This invasion was less effective than that between astrocytes, and peak amplitudes of sodium signals dropped to less than 20% at distances of 20–40 µm from the stimulated cell (Fig. 4c). To unambiguously identify NG2 cells, experiments were performed in tissue slices obtained from NG2-EYFP reporter mice (Fig. 5a). Again, we obtained effective spread within SR101-positive astrocytes (n = 52, N = 12; Fig.  5b, c). Moreover, sodium spread from the stimulated astrocyte to neighboring NG2 cells, albeit less effectively (n = 24, N = 12 mice; Fig. 5b, c). Similar to oligodendrocytes, peak amplitudes had decreased to about 20% within 20–40  µm of their origin.

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Discussion In the present study, we demonstrate that astrocytes in the mouse corpus callosum experience significant sodium transients in response to glutamate, largely mediated by activation of sodium-dependent glutamate transport. Relatively small glutamate-induced sodium increases are also observed in SR101-negative cells, the majority of which represent oligodendrocytes. Astrocyte sodium signalling is not restricted to the stimulated cell, but sodium passively travels to neighboring astrocytes. In addition, spread of sodium includes oligodendrocytes and NG2 cells, demonstrating panglial sodium signalling between the three major classes of macroglial cells. Sodium Signals in White Matter Macroglia

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Fig. 4  Spread of sodium from astrocytes to PLP/GFP-positive glia. a Images of SBFI, SR101 and GFP fluorescence of a corpus callosum slice obtained from a PLP-GFP-mouse. Arrowhead points to a stimulated SR101-positive cell; in addition, regions of interest (ROIs) chosen for analysis of sodium transients shown in b are indicated. Dashed lines illustrate the border of the corpus callosum. Scale bar 25 µm. b Sodium transients obtained in a stimulated SR101-positive cell as well as in a neighboring SR101-positive and a SR101-nega-

tive/GFP-positive cell in response to direct electroporation (indicated by grey line). c Top Cumulative data plot showing peak amplitudes of sodium increases in all astrocytes neighbouring a stimulated astrocyte, normalized to the amplitude of the latter (left). Bottom plot showing data points from neighboring PLP-GFP-positive cells only. Lines represent a best fit monoexponential decay function (top ­r2 = 0.91, bottom ­r2 = 0.96). Lambda (λ) represents the distance where fit reaches 1/e of maximum

activation of glutamate transporters GLAST and/or GLT-1 [10, 12, 13]. In addition to astrocytes, we also detected small glutamate-induced sodium signals in the majority of SR101negative cells. Their peak amplitude was reduced by 60% with TFB-TBOA, indicating that sodium-dependent glutamate uptake represented the main influx pathway for sodium. Because white matter oligodendrocytes, but not NG2 cells, express high levels of glutamate transporters

[56–61], we conclude that this subset of glutamate-responsive cells most likely represented oligodendrocytes, the most prevalent SR101-negative cell type in our preparation. So far, data on sodium signalling properties of oligodendrocytes is rare. An early work employing ion-selective microelectrodes showed that cultured mouse oligodendrocytes respond to bath application of glutamate with a depolarisation and a rise in intracellular sodium

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Fig. 5  Spread of sodium from astrocytes to NG2/EYFP-positive glia. a Images of SBFI, SR101, and GFP fluorescence in a corpus callosum slice obtained from a NG2-EYFP-mouse. Arrowhead points to a stimulated SR101-positive cell; in addition, regions of interest (ROIs) chosen for analysis of sodium transients shown in b are indicated. Dashed lines illustrate the border of the corpus callosum. Scale bar 25 µm. b Sodium transients obtained in a stimulated SR101-positive cell as well as in a neighboring SR101-positive and a

SR101-negative/EYFP-positive cell in response to direct electroporation (indicated by grey line). cTop Cumulative data plot showing peak amplitudes of sodium increases in all astrocytes neighbouring a stimulated astrocyte, normalized to the amplitude of the latter (left). Bottom plot showing data points from neighboring NG2-EYFP-positive cells only. Lines represent a best fit monoexponential decay function (top: ­r2 = 0.90, bottom: ­r2 = 0.96). Lambda (λ) represents the distance where fit reaches 1/e of maximum

by about 9 mM [62]. Similarly, using fluorescence imaging with SBFI in cultured oligodendrocytes, an increase in sodium upon activation of AMPA receptors was shown [63]. To our knowledge, while numerous reports have demonstrated expression of (calcium-permeable) AMPA receptors and glutamate-evoked calcium signalling in white and grey matter NG2 glia [64–68], sodium signalling in NG2 cells has, so far, not been reported.

Spread of Sodium Between White Matter Macroglia

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The present work demonstrates that sodium signals induced in an individual astrocyte spread monotonically to neighboring astrocytes. Following a monoexponential decay, peak amplitudes decreased to ~37% (1/e) of their initial value at a distance of 31 µm from the stimulated cell. This value is slightly lower than those observed in astrocyte networks in grey matter of hippocampus (~40  µm; [18, 21])

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and lateral superior olive (46  µm; [22]), indicating that sodium signalling domains in the corpus callosum are somewhat less pronounced. In the hippocampal CA1 region, intercellular spread of sodium was completely absent in mice double-deficient for the connexins (Cx) 30 and 43 [18, 21], demonstrating that passive diffusion of sodium through gap junctions provides the major pathway. In addition, it was slightly dampened following inhibition of mGluR1/5, but not altered by blockers of purinergic receptors [21]. A similar observation was made in the present study, indicating that diffusion of sodium through gap junctions also represents the major pathway for intercellular passage of sodium between astrocytes in the corpus callosum. Astrocyte gap junctional coupling in the corpus callosum seems to be less strong as compared to grey matter as earlier work revealed only a weak or even absent dye-coupling [29, 69]. Here we found that sodium ions can apparently diffuse relatively freely between coupled cells and this approach therefore seems to provide a more sensitive measure of coupling than the intercellular spread of dyes (see also [70, 71]). The intercellular spread of sodium differs from that of classical astrocyte calcium waves in the corpus callosum, the propagation of which is independent from gap junctional coupling, but involves release of ATP and activation of purinergic receptors on neighboring astrocytes [40]. Apparently, propagation of calcium waves and spread of sodium signals thus take different routes and employ different mechanisms for their generation. In addition, the speed of signal propagation differs: while classical calcium waves travel at a more or less constant speed of 10–20 µm/s over large distances [43], the velocity of sodium diffusion falls from initial values >120 to <20  µm/s within 50  µm from the stimulated astrocyte [18, 21]. Experiments performed in cell-specific reporter mice demonstrated that, in addition to SR101-positive astrocytes, spread of sodium also involved oligodendrocytes as shown before in the lateral superior olive [22]. In addition, we detected a passage of sodium from astrocytes to NG2 cells. In both cases, and in contrast to the latter study [22], panglial passage of sodium from astrocytes was not as efficient as within the astrocyte syncytium and peak amplitudes decreased about twice as fast with distance. As observed for astrocytes, spread of sodium to SR101 negative cells (representing oligodendrocytes and NG2 cells) was not reduced in the presence of blockers of purinergic and of glutamate receptors, indicating that it did not involve release of gliotransmitters and receptor activation on neighboring cells, but that it was instead mediated by gap junctions. Communication between NG2 cells and other macroglia is, however, considered to be largely mediated by extracellular signalling involving glutamate and/ or ATP [55, 72]. Panglial spread of sodium through gap

junctions might thus mainly be relevant for astrocytes and oligodendrocytes. The demonstration of a passive spread of sodium between different types of macroglial cells is in line with their demonstrated panglial coupling through gap junctions. Moreover, while in the present study, we have only performed direct electroporation of SR101-positive astrocytes, we assume that sodium signals induced in oligodendrocytes (or NG2 cells) will also spread to astrocytes if functional panglial coupling through gap junctions exists. Such coupling was already reported nearly 30 years ago based on electrophysiological recordings obtained from astrocytes and oligodendrocytes in culture [70, 73]. While the grade of coupling between astrocytes and oligodendrocytes differs between different brain regions, it is mainly based on the formation of heterotypic gap junction channels composed of Cx43 (astrocyte) and Cx47 (oligodendrocyte) or of Cx30/Cx32, respectively [28–31]. In corpus callosum [29], but not thalamus [30], panglial gap junction networks include NG2-positive cells, an observation indicating that spread of sodium from astrocytes to NG2 cells as observed in the present study might also involve gap junctions. Functional Relevance of Sodium Signalling in White Matter The data presented here demonstrate that glutamate induces prominent sodium transients in white matter macroglia. Previous work has shown that NCX operates close to the resting membrane potential and an elevation of intracellular sodium by ~9  mM only, in conjunciton with intracellular calcium signalling, is sufficient to reduce its reversal potential to values around −30 mV [5]. Thus, increases in sodium as determined in our study are most likely sufficient to drive reversal of NCX and induce calcium loading in both astrocytes [4, 74–76] as well as in oligodendrocytes/ oligodendrocyte precursor cells in culture [63, 77]. In the latter, this has been suggested to influence myelin synthesis [77] and is likely involved in cellular maturation [57]. Moreover, reverse NCX activity following influx of sodium has been implicated in GABA-induced NG2 cell migration [78]. Under pathophysiological conditions, strong sodium influx mediates excitotoxic damage to oligodendrocytes [46, 79]. A more direct influence of sodium signalling on oligodendrocyte development was suggested earlier: opening of AMPA receptors and concomittant sodium influx was shown to induce block of ­K+ channels and to inhibit proliferation [52, 80–82]. Our work also shows that sodium signals can efficiently spread between astrocytes and also -albeit to a smaller extent- from astocytes into both oligodendrocytes as well as NG2 glia. In grey matter, activity-related increases in intracellular sodium have been implicated to

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drive astrocyte breakdown of glycogen, glycolysis and the production and release of lactate, thereby playing a prominent role in neuro-metabolic coupling [3, 83]. While in white matter, astrocytes contact myelinated axons at nodes of Ranvier only, astrocyte glycogen stores have been shown to strongly support axonal functionality and viability [84, 85]. Recent work from several laboratories has put forward the idea that axons are not directly fed by astrocytes, but by lactate derived from myelinating oligodendrocytes [86–88]. How astrocyte glycogen is involved in this phenomenon is, so far, not completely understood. The proposed passage of signalling molecules and metabolites within astrocytes and between astrocytes and oligodendrocytes through gap junctions [87, 89, 90], however seems an attractive hypothesis. Acknowledgements  This work was supported by the Deutsche Forschungsgemeinschaft (SPP1757 “Glial Heterogeneity”, Ro2327/8  −  1). We thank Simone Durry for excellent technical assistance. The authors thank Dr. Gerald Seifert and Prof. Christian Steinhäuser, University of Bonn, Germany, as well as Prof. Nikolaj Klöcker, Heinrich Heine University Duesseldorf, Germany, for providing transgenic reporter animals (hGFAP-GFP, PLP-GFP, NG2-EYFP). Compliance with Ethical Standards  Conflict of interest  The authors declare that they have no conflict of interest Ethical Approval  This study was carried out in strict accordance with the institutional guidelines of the Heinrich Heine University Düsseldorf, as well as the European Community Council Directive (86/609/EEC). All experiments were communicated to and approved by the Animal Welfare Office at the Animal Care and Use Facility of the Heinrich Heine University Düsseldorf (institutional act number: O52/05), following the recommendation of the European Commission (published in: Euthanasia of experimental animals, Luxembourg: Office for Official Publications of the European Communities, 1997; ISBN 92–827–9694-9).

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