00:000–000 (2010)

Astrocyte Calcium Signals at to CA1 Correlate With the Number of Activated Synapses But Not With Synaptic Strength

Silke D. Honsek, Corinna Walz, Karl W. Kafitz, and Christine R. Rose*

ABSTRACT: Glial cells respond to neuronal activity by transient mediate the clearance of glutamate from the increases in their intracellular calcium concentration. At hippocampal extracellular space through high-affinity transporters. Schaffer collateral to CA1 pyramidal cell synapses, such activity-induced This glutamate uptake shapes the time course of syn- calcium transients modulate neuronal excitability, synaptic activ- ity, and LTP induction threshold by calcium-dependent release of gliotrans- aptic conductance, moderates the activation of extrasy- mitters. Despite a significant role of astrocyte calcium signaling in plasti- naptic receptors, and limits spillover of glutamate to city of these synapses, little is known about activity-dependent changes of adjacent synapses (Tzingounis and Wadiche, 2007). In astrocyte calcium signaling itself. In this study, we analyzed calcium transi- addition, astrocytes respond to synaptically released stratum radiatum ents in identified astrocytes and NG2-cells located in the glutamate with calcium transients that are mainly in response to different intensities and patterns of Schaffer collateral stimu- lation. To this end, we employed multiphoton calcium imaging with the caused by the activation of metabotropic glutamate low-affinity indicator dye Fluo-5F in glial cells, combined with extracellu- receptors (mGluR). A wealth of reports has provided lar field potential recordings to monitor postsynaptic responses to the evidence that astrocyte calcium signaling also triggers afferent stimulation. Our results confirm that somata and processes of the release of so-called ‘‘gliotransmitters.’’ While vaso- astrocytes, but not of NG2-cells, exhibit intrinsic calcium signaling inde- active substances released by astrocytes regulate pendent of evoked neuronal activity. Moderate stimulation of Schaffer col- laterals (three pulses at 50 Hz) induced calcium transients in astrocytes microcirculation (Zonta et al., 2003; Metea and New- and NG2-cells. Astrocyte calcium transients upon this three-pulse stimula- man, 2006; Iadecola and Nedergaard, 2007; Gordon tion could be evoked repetitively, increased in amplitude with increasing et al., 2008), neuroactive substances modulate neuro- stimulation intensity and were dependent on activation of metabotropic nal excitability, synaptic activity and plasticity glutamate receptors. Activity-induced transients in NG2-cells, in contrast, (Haydon, 2001; Schipke and Kettenmann, 2004; Vol- showed a rapid run-down upon repeated three-pulse stimulation. Theta burst stimulation and stimulation for 5 min at 1 Hz induced synaptic poten- terra and Meldolesi, 2005; Theodosis et al., 2008). tiation and depression, respectively, as revealed by a lasting increase or Based on the findings that astrocytes not only receive decrease in population spike amplitudes upon three-pulse stimulation. Syn- information from but also signal back to them aptic plasticity was, however, not accompanied by corresponding altera- (Haydon, 2001; Auld and Robitaille, 2003; Perea et al., tions in the amplitude of astrocyte calcium signals. Taken together, our 2009), the concept of the tripartite has evolved. results suggest that the amplitude of astrocyte calcium signals reflects the number of activated synapses but does not correlate with the degree of This model emphasizes that signaling at synapses synaptic potentiation or depression at Schaffer collateral to CA1 pyramidal occurs between all three cellular compartments (pre- cell synapses. VC 2010 Wiley-Liss, Inc. and postsynaptic neurons and surrounding astrocytes) and regards astrocytes as actively involved in synaptic KEY WORDS: NG2; glutamate; - interaction; synaptic transmission and plasticity. It has, however, also been plasticity; Fluo-5F debated because not all astrocyte calcium increases cause gliotransmitter release (Fiacco et al., 2007; Petra- vicz et al., 2008). Moreover, a recent study demon- INTRODUCTION strates that knockout of IP3-receptor-dependent cal- cium signaling in astrocytes fails to influence neuronal plasticity (Agulhon et al., 2010; see also comment by It is widely accepted that astrocytes are actively involved in neural Kirchhoff, 2010). transmission. At glutamatergic synapses in the central , The hippocampal Schaffer collateral to CA1 pyram- idal cell synapse is a widely used model system to Institute for Neurobiology, Heinrich-Heine-University of Duesseldorf, study plasticity of glutamatergic synapses. The expres- Germany sion of long-term potentiation (LTP) as well as of Silke D. Honsek is currently at Department of Neurophysiology, Center for Brain Research, Medical University of Vienna, Austria. long-term depression (LTD) is mainly postsynaptic Grant sponsor: German Research Foundation (DFG); Grant number: and requires postsynaptic calcium signals (Rose and Ro2327/3-2. Konnerth, 2001; Malinow and Malenka, 2002). *Correspondence to: Christine R. Rose, Institute for Neurobiology, Hein- Astrocytes have been suggested to modulate synaptic rich-Heine-University, Universitaetsstrasse 1; 26.02.00, 40225 Duessel- transmission at this synapse by calcium-dependent dorf, Germany. E-mail: [email protected] Accepted for publication 10 June 2010 release of glutamate acting on metabotropic glutamate DOI 10.1002/hipo.20843 receptors (Perea and Araque, 2007). In addition, Published online in Wiley Online Library (wileyonlinelibrary.com). astrocyte calcium signals, induced by high-frequency

VC 2010 WILEY-LISS, INC. 2 HONSEK ET AL. stimulation (HFS) of Schaffer collaterals, cause release of D-ser- NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, ine from astrocytes, thereby saturating postsynaptic NMDA 6 MgCl2, and 20 glucose, bubbled with 95% O2/5% CO2 receptors and enabling LTP-induction (Yang et al., 2003; Hen- resulting in a pH of 7.4. Slices were kept for 30 min at 348C neberger et al., 2010). in ACSF containing 2 mM CaCl2 and 1 mM MgCl2.For Neuronal LTP is characterized by a change in postsynaptic some experiments, sulforhodamine 101 (SR101) was added to receptor complement (Malinow and Malenka, 2002), and a the ACSF to selectively label astrocytes as described previously similar mechanism has recently been suggested in NG2-cells. (Kafitz et al., 2008). Subsequently, slices were kept at room These glial cells, also known as precursor cells, temperature until they were used for experiments, which were have been shown to be synaptically innervated by neurons (Ber- also performed at room temperature. gles et al., 2000), and to exhibit changes in the expression of calcium-permeable AMPA-receptors upon activation (Ge et al., Calcium Imaging 2006). Similarly, calcium signaling in perisynaptic Schwann cells at the amphibian is modulated in For part of the measurements of spontaneous calcium sig- an activity-dependent manner, likely due to receptor modula- nals, the calcium indicator dye Oregon-Green-BAPTA-1-AM tion or alteration in calcium handling (Belair et al., 2010). (OGB1-AM, 200 lM) was pressure injected into the stratum Despite the suggested relevant role of astrocyte calcium signal- radiatum of the CA1 region using a glass micropipette (5 s, ing in synaptic function and plasticity, only few studies have 8 psi, tip diameter 1 lm), resulting in the bulk-loading of addressed the question if astrocyte calcium transients might be numerous cells in the field of view (Kafitz et al., 2008). Alter- subject to activity-dependent alterations as well. In hippocampal natively, individual glial cells were loaded with the indicator astrocytes, it was shown that neuronal activity dynamically con- Fluo-5F via patch-pipettes. Patch-pipettes were pulled from trols the frequency of calcium oscillations, an effect presumably borosilicate glass (Hilgenberg, Waldkappel, Germany) and filled due to intrinsic modification of mGluR signaling pathways (Pasti with intracellular solution containing (in mM): 120 K-metha- et al., 1995; Pasti et al., 1997). It has also been demonstrated nesulfonate, 32 KCl, 10 HEPES, 4 NaCl, 0.16 EGTA, 4 Mg- that astrocytes can discriminate and preferably respond to the ac- ATP, 0.4 Na-GTP, 500 lM Fluo-5F, pH 7.3 (KOH), resulting tivity of distinct excitatory pathways (Perea and Araque, 2005; in a resistance of 4–5 MX. For multiphoton imaging experi- Schipke et al., 2008). Furthermore, evoked calcium signals are ments, 200 lM AlexaFluor594 was added to the intracellular activated by specific input patterns in a nonlinear manner, a phe- solution to enable ratio calculation (see below). After obtaining nomenon which was ascribed to intrinsic properties of astrocytes the whole-cell mode, basic electrophysiological properties were as well (Araque et al., 2002; Perea and Araque, 2005). Astrocyte determined using an EPC10 double- or an Axopatch 200B-am- processes show a high degree of rapid motility at synapses (Hirr- plifier (HEKA electronic, Lambrecht, Germany and Molecular linger et al., 2004; Haber et al., 2006), and LTP induction or Devices, Sunnyvale, CA, respectively). Two minutes after break- prolonged stimulation have been shown to give rise to enhanced through, patch-pipettes were withdrawn carefully and cells were synaptic ensheathment by astrocyte processes (Wenzel et al., allowed to rest for at least five minutes before starting imaging 1991; Genoud et al., 2006; Lushnikova et al., 2009). Fast struc- experiments. Cells that did not reseal to a resistance of at least X tural rearrangements at synapses following induction of synaptic 0.5 G were not included in the analysis. potentiation or depression could influence the activation of Widefield calcium imaging was performed using an epifluores- metabotropic glutamate receptors and calcium signaling in peri- cence system (TILL photonics, Martinsried, Germany) attached synaptic glial processes (Deitmer and Rose, 2010). In this study, to an upright microscope (Axioskop: Zeiss, Oberkochen, Ger- we therefore analyzed calcium transients in astrocytes located in many; 40x water immersion objective: N.A. 0.8, Olympus stratum radiatum Europe, Hamburg, Germany). Excitation light was generated by the in response to moderate stimulation of > Schaffer collaterals before and after induction of potentiation or a monochromator (Polychrome 5); fluorescence emission ( 515 depression at Schaffer collateral to CA1 pyramidal cell synapses. nm) was detected by a CCD-camera (TILL imago super-VGA). Multiphoton laser-scanning microscopy was performed with a Our results show that the amplitude of astrocyte calcium transi- custom-build system based on a FluoView300 (Olympus ents correlates with the number of activated synapses, but is not Europe, Hamburg, Germany). A femtopulsed infrared laser significantly altered within a short time frame after induction of (MaiTai; Spectra Physics, Darmstadt, Germany) at 810 nm was synaptic plasticity. used for multiphoton excitation. Fluo-5F emission was detected between 510 and 540 nm and AF594 emission between 585 and 640 nm. Images were acquired at 2 Hz in multiphoton MATERIALS AND METHODS measurements and at 5 Hz in widefield measurements. Data analysis was performed offline using TILL Vision, FluoView 5.0 and IgorPro Software (Wavemetrics, Lake Preparation and Salines Oswego, OR). Fluoview TIFF files from multiphoton experi- Acute hippocampal slices (thickness 250 lm) were obtained ments were imported in IgorPro 6.0, where intensity values in from Balb/c mice at postnatal Days 13 to 16 (P13-P16). Dis- selected regions of interest in the green (Fluo-5F) and red section was performed in ACSF consisting of (in mM): 125 (AF594) channel were measured over time and the ratio green/

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 3 red was computed (Yasuda et al., 2003; Yasuda et al., 2004) chemistry were documented at a Nikon 90i microscope or Calcium transients are expressed as changes in the ratio of taken as maximum projections of images at an Olympus confo- Fluo-5F and AF594 signals (DR) divided by the baseline ratio cal laser scanning microscope (FV 300, Olympus). (R)(DR/R in arbitrary units, a.u.). Traces were smoothed (5 point box smoothing); elevations of more than three standard Chemicals deviations above baseline were defined as calcium transients. Statistical analyses were performed with the t-test implemented Standard chemicals were purchased from Sigma (Tauf- in IgorPro 6.0 and data are presented as mean 6 S.D. kirchen, Germany). 2-Methyl-6-(phenylethynyl)pyridine hydro- chloride (MPEP), 6-Amino-N-cyclohexyl-N,3-dimethylthia- Synaptic Stimulation and Field zolo[3,2-a]benzimi dazole-2-carboxamide hydrochloride (YM2 Potential Recordings 98198), and (2S)-3-[[(1S)-1-(3,4-Dichlorophenyl)ethyl]amino-2 hydroxypropyl] (phenyl-methyl) phosphinic acid (CGP55845) Schaffer collaterals were stimulated with glass micropipettes were purchased from Tocris (Eching, Germany). TTX was pur- (Hilgenberg, Waldkappel, Germany) placed into the stratum chased from Alomone Labs (Jerusalem, Israel) or from Biotrend radiatum at a distance of 5–30 lm from a given cell. Current Chemicals (Cologne, Germany). Fluorescent indicators were pulses (0.1-ls duration) were delivered by an isolated stimula- purchased from Invitrogen. tor (World Precision Instruments; A365), driven by a pulse generator (MAX 21-Stimulator, Werner Zeitz Instruments, Augsburg, Germany). Basal stimulation consisted of three pulses, delivered at 50 Hz. Evoked calcium transients were not RESULTS included in the analysis if spontaneous calcium transients were obscuring the evoked responses. For analysis of the influence of Glial Cells in the CA1 area of the stratum radiatum were dis- LTP/LTD induction paradigms on glial calcium transients, only tinguished by staining with the fluorescent indicator dye sulfo- experiments, in which synaptic potentiation or depression was rhodamine 101 (SR101; Nimmerjahn et al., 2004; Kafitz et al., confirmed by field potential recordings (see below) were 2008; Meier et al., 2008), and/or based on their morphological included in the analysis. Receptor antagonists were delivered by and electrophysiological properties. I/V-curves were obtained in pressure application (5 s, 8 psi) close a cell of interest using a the voltage-clamp mode during dye-loading of individual cells Picospritzer II (General Valve/Parker Hanifin, Flein/Heilbronn, with patch-pipettes. As described previously, astrocytes did not Germany) coupled to standard micropipettes. display voltage-gated conductances (Fig. 1A), and were charac- Field potentials were recorded with ACSF-filled glass micro- terized by a membrane resistance below 5 MX, a high capaci- pipettes placed in the pyramidal cell layer of the CA1 region tance and a membrane potential close to 285 mV. Morpholog- using an Axopatch 200B amplifier (Molecular Devices, Sunny- ically, astrocytes were characterized by small, ramified processes vale, CA) or a HEKA EPC10 double amplifier (HEKA Elek- that often formed endfeet on blood vessels. Cellular processes tronik, Lambrecht, Germany). The pipette potential was zeroed were visible up to a distance of 20–30 lm from the , and data were acquired in current clamp at 10–20 kHz and fil- indicating an overall cellular diameter of roughly 60–70 lm. tered at 1 kHz, using PClamp 8.2 software (Molecular Devices) Dye-diffusion into adjacent cells, indicating gap-junction cou- or PatchMaster (HEKA electronics). pling, was observed regularly. SR101-negative cells with small somata located in the stra- Immunohistochemistry tum radiatum displayed no signs of dye-coupling and exhibited Immunohistochemistry was performed in hippocampal slices numerous fine processes that did not terminate on blood ves- according to the protocol published by Zhou and coworkers sels. They exhibited slowly desensitizing voltage-dependent out- (Zhou et al., 2006). In brief, acute slices, in which calcium sig- ward currents reflected by their nonlinear I/V-curves (Fig. 1B; nals of single, Fluo-5F/AF594-filled, cells had been analyzed, n 5 37/37 cells). About 70% of these nonlinear cells also were transferred immediately after physiological experiments in showed fast desensitizing inward currents, likely representing 4% paraformaldehyde (PFA). After over night fixation in PFA, voltage-gated sodium currents (Fig. 1B). Every nonlinear cell slices were washed 4x in PBS, blocked and permeabilized in that underwent immunostaining after electrophysiological char- 3% normal goat serum (NGS)/0.1%Triton-X-100 (TX) in acterization was positive for the proteoglycan NG2 (n 5 6/6 PBS. The primary antibody recognizing NG2 (rabbit, Chemi- cells; Fig. 1B). Based on this, nonlinear glial cells with the mor- kon) was incubated for 19 h at room temperature. After three phology described above will generally be termed ‘‘NG2-cells’’ washes in PBS, the secondary antibody (AF488 anti rabbit; in the following. Molecular Probes/Invitrogen) was incubated for 90 min at room temperature and the tissue was washed again three times Glial Calcium Signals in the Absence of Evoked and finally was coverslipped in MOVIOL. With every staining, Neuronal Activity additional negative controls, in which primary antibodies were omitted, were performed. These never resulted in labeled tis- Glial cells exhibit oscillations in their intracellular calcium sues. AlexaFluor594 labeling and images of the immunohisto- concentration in situ and in vivo, that are independent of

Hippocampus 4 HONSEK ET AL.

positive astrocytes during a recording period of 50–400 s (n 5 93 cells, Fig. 2A). In contrast to this, spontaneous calcium transients were observed in only one out of 29 SR101-negative cells (Fig. 2 A). To analyze the spatial distribution of spontaneous calcium signals in individual glial cells, astrocytes, and NG2-cells were filled via patch-pipettes with the low-affinity calcium indicator K in vitro l dye Fluo-5F ( d : 2.3 M, Fig. 2A) and the red indica- tor Alexa Fluor 594 (AF594), allowing for visualization of small cellular processes. To reduce washout of intracellular sig- naling components, patch-pipettes were carefully withdrawn two minutes after obtaining the whole-cell mode and cells were allowed to recover for at least 5 min before commencing cal- cium imaging experiments. Again, within recording periods of up to 400 s, spontaneous calcium transients were observed in the vast majority of astrocytes (11/12 cells), while no spontane- ous events were detected in the somata or processes of NG2- cells (n 5 10 cells; Figs. 2A,C,D). These findings indicate that the absence of spontaneous calcium signals in NG2-cells seen with OGB-1 was not due to its high calcium binding affinity. Further, our results show that basic intracellular calcium signal- ing differs significantly between astrocytes and NG2-cells. Typically, spontaneous astrocyte calcium transients did not occur in the entire cell. Instead, we found that global calcium signals, occurring synchronously in the soma and all cellular processes in the focal plane, were extremely rare (1 out of 77 events; Figs. 2 B,C). About 50% of recorded events included the soma as well as part of the visible processes (n 5 36/76 events), while the other half was restricted to part of the proc- FIGURE 1. Identification of glial cells. A: Left: Whole-cell cur- esses, but did not occur in the soma (40/76; Figs. 2B,C). rents (not leak-subtracted) recorded in an SR101-positive astrocyte challenged with hyper- and depolarizing voltage steps from a hold- The mean amplitude of spontaneous calcium transients was ing potential of -85 mV (12 steps from -110 mV to 150 mV). not significantly different (P > 0.06) between somata and proc- Right: Average intensity z-projection (stack of 123 slices with spac- esses of astrocytes (DR/R 5 1.6 6 1.0 a.u.; range, 0.4–4.5 a.u. l ing of 0.5 m) of the AF594-filled astrocyte; SR101-stained astro- for n 5 69 spontaneous events in 18 cell somata; and DR/R 5 cytes are visible in the background. B: Left: Whole-cell currents 1.4 6 1.2 a.u.; range, 0.3–10.3 a.u. for 111 spontaneous events recorded in an identified NG2-positive cell challenged with hyper- and depolarizing voltage steps from a holding potential of 285 in 38 processes of 11 cells). Interestingly, calcium transients in mV (12 steps from 2110 mV to 150 mV). Top: nonleak-sub- regions of interest in which signals were detected first did not tracted traces. Center: P/4 leak-subtraction revealed voltage-gated always display the largest amplitude. These results support the conductances. Bottom: same recording at different scaling to visu- existence of cellular microdomains in astrocytes, in which local alize small inward currents. Right, Top: Average intensity z-projec- calcium signaling occurs independent of adjacent cellular tion (stack of 149 slices with spacing of 0.5 lm) of the AF594- filled glial cell (same animal as the astrocyte image in A). SR101- domains. Alternatively, our findings indicate the existence of a stained astrocytes are visible in the background. C: Image of a cell regenerative component of astrocyte calcium transients. filled with AF594 during a patch-clamp recording (left) and subse- quently immunostained for the NG2-proteoglycan (right). All scale bars 20 lm. Calcium Signals Evoked by Three-Pulse Synaptic Stimulation To analyze calcium signals evoked by synaptic activity, a saline- neuronal activity (Aguado et al., 2002; Nett et al., 2002; Zur filled glass microelectrode for Schaffer collateral stimulation was Nieden and Deitmer, 2005). To evaluate spontaneous calcium placed into the stratum radiatum at a distance of 5–30 lmfroma signals in identified astrocytes and NG2-cells, we costained sli- cellular process of a Fluo-5F/AF594-loaded cell. At the same time, ces with the AM-ester of the high-affinity calcium indicator postsynaptic responses were monitored by recording extracellular K in vitro OregonGreen-BAPTA1 (OGB1-AM; d : 170 nM), field potentials in the CA1 pyramidal cell layer (Fig. 3A). and the astrocyte-specific indicator SR101 (Meier et al., 2008). Schaffer collaterals were electrically stimulated with three cur- Multiphoton imaging of cell somata and primary processes rent pulses delivered at 50 Hz, a stimulation paradigm resembling revealed spontaneous calcium transients occurring in the ab- the intrinsic firing pattern of hippocampal neurons (Albensi et al., sence of evoked synaptic transmission in about 90% of SR101- 2007). At a stimulation intensity of 75 lA, field potentials were

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 5

FIGURE 2. Spontaneous calcium signaling in glial cells. A: Bar only a subset of analyzed regions. About half of all local transients chart illustrating the percentage of astrocytes and NG2-cells exhibit- included the soma (black part of the bar), whereas the rest (gray ing spontaneous calcium transients in cell somata or primary proc- part of the bar) only occurred in the processes and did not invade esses as determined with bolus-loading of the calcium-indicator dye the soma. The number of cells showing global or local calcium OGB1-AM (left) or with intracellular loading of Fluo-5F (right). transients vs. total number of analyzed cells is indicated below the The number of cells showing calcium transients vs. the total num- bars. C,D: Left: Spontaneous calcium signaling in different regions ber of analyzed cells is indicated below the bars, the percentage of of interest (r1-r7 and r1-r9) in the focal plane: local and global cal- responding cells is indicated in/above the bars. B: Bar chart illus- cium oscillations occur the astrocyte (C) and are absent in the trating the spatial profile of spontaneous calcium transients in NG2-cell (D). Fluorescence changes are shown as DR/R in arbitrary astrocytes. Global calcium signals involved all cellular regions ana- units (see methods). Right: Inverted AF495 fluorescence image illus- lyzed during a recording, whereas local calcium events involved trating the analyzed regions of interest. Scale bars: 10 lm.

reliably evoked, and the presence of population spikes indicated (1 lM) to block voltage-gated sodium channels together with 1 suprathreshold activation of postsynaptic CA1 pyramidal neurons Cd2 (15–30 lM) to block voltage-gated calcium channels. In in the majority of experiments (Fig. 3A). In addition to postsy- the presence of these blockers, electrical stimulation did not naptic activity, this relatively moderate stimulation paradigm eli- result in astrocyte calcium signals (n 5 4, not shown), confirm- cited calcium transients in both astrocytes and NG2-cells. ing their dependence on generation and neuro- To ensure that astrocyte calcium signals were not due nal transmitter release. to direct electrical stimulation, but induced by synaptic trans- Astrocytes typically responded to the three-pulse stimulation mission, we performed experiments in which we applied TTX with a delay of 1–2 s (n 5 17; Fig. 3B). Processes closest to

Hippocampus 6 HONSEK ET AL.

the stimulating pipette often responded first, but did not neces- sarily exhibit the highest amplitude (Fig. 3B). Mean amplitudes of evoked responses in astrocytes were in a similar range as those observed in spontaneous events (DR/R 5 2.1 6 1.7 a.u.; range, 0.3–4.4 for n 5 22 somata; and DR/R 5 1.1 6 0.5 a.u.; range, 0.3–2.7 a.u. for 53 processes; Fig. 3C). As observed in astrocytes, NG2-cells responded to the three- pulse stimulation with calcium transients (Fig. 3D). These cal- cium transients often had a delay of less than 1 s. Similar to astrocytes, processes closest to the stimulation pipette typically responded first, but did not necessarily exhibit the highest amplitudes. The mean amplitudes of calcium transients evoked in NG2-cells were DR/R 5 3.9 6 2.3 a.u.; range, 0.4–6.6 a.u., for n 5 6 somata and DR/R 5 1.6 6.1 a.u., range, 0.2–4.2 a.u., for 35 processes (Fig. 3E).

Calcium Signals Evoked by Repetitive Three-Pulse Stimulation To study glial cell calcium transients in response to repeated afferent stimulation, we applied the three-pulse stimulation ev- ery 2 min while continuously recording calcium signals. With this stimulation paradigm, the average population spike ampli- tude slightly increased with consecutive stimulations (n  17; Fig. 4A). In individual astrocytes, time course and amplitude of synap- tically induced calcium signals were relatively variable with repeated stimulation in a given region of interest (n 5 12–21 cell somata and 17–53 processes; Fig. 4B). Normalized mean amplitudes of astrocyte calcium transients upon repetitive stim- ulation had a high standard deviation (SD), reflecting the high variability of the responses (Fig. 4B). In addition, and in con- trast to the extracellular field potentials, run-down was observed to some extend in astrocytes, with the sixth response being reduced by about 40% as compared with the first response (n 5 12–21 cell somata and 17–53 processes; Fig. 4B). In NG2-cells, in contrast, we observed an unexpected strong run-down of synaptically evoked calcium signals. On average, calcium transients were observed in response to the first two or three stimulations (Fig. 4C). Some cells, however, showed small FIGURE 3. Responses to a single 3-pulse stimulation. A: Left: responses at the beginning of the stimulation paradigm, and Image of a hippocampal slice preparation. The positions of the elec- exhibited a larger responses at a later time (not shown). None trical stimulation and field potential electrodes are indicated sche- matically. The black box illustrates the typical recording area for of the NG2-cells reliably responded upon repetitive synaptic imaging experiments. Right: Typical field potential as recorded in the stimulation, and normalized mean amplitudes were reduced by CA1 pyramidal cell layer upon basal stimulation with three pulses at 80% at the third stimulation (Fig. 4C; n 5 3–35). 50 Hz. B, D: Top: Intracellular calcium transients measured in several Having established that astrocytes responded repetitively to regions of interest in an astrocyte (B) and a NG2-cell (D) upon three- the three-pulse stimulation paradigm, we next performed phar- pulse stimulation. The time point of synaptic stimulation is indicated macological experiments using widefield imaging to elucidate by the vertical line and fluorescence changes are shown as DR/R in ar- bitrary units. The position of the stimulating electrode and the ana- the underlying mechanisms of astrocyte calcium transients. To lyzed regions of interest of each cell are illustrated in the respective this end, we first recorded a control response in an individual false-colored AF594 image at the bottom (note that only the focal astrocyte and then pressure-injected receptor antagonists (three plane in which the measurement was performed is shown). Scale times for 5 s) with a glass micropipette in the proximity of the l 6 bars: 10 m. C, E: Bar charts illustrating the mean amplitudes S. analyzed cell (Fig. 5A). Injection of saline did not significantly D. of calcium transients evoked in somata and processes of astrocytes n (C) and NG2-cells (E). The number of analyzed regions of interest is alter astrocyte responses to synaptic stimulation (Figs. 5A–C; indicated below the bars. 5 7). The metabotropic 5 (mGluR5) antag- onist MPEP (100 lM) reduced the amplitude of stimulation-

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 7

FIGURE 4. Responses to repeated three-pulse stimulation. A: stimulation (1–6). Calcium transients are normalized to the mean Left: Field potentials evoked by a three-pulse/50 Hz stimulation, of six consecutive stimulations. C: Left: Calcium transients six repetitions are shown. Right: Bar chart illustrating the average observed in the soma and three different processes of an NG2-cell population spike amplitude 6 S. D. evoked by the three pulses, upon repetitive three-pulse synaptic stimulation. Corresponding normalized to the average of the six repetitions. B: Left: Calcium regions of interest and the position of the stimulating pipette are transients detected in the soma (gray trace) and a process (black illustrated in the inset. The time of stimulation is indicated by the trace) of an astrocyte upon repeated three-pulse synaptic stimula- arrowheads. Right: Bar chart illustrating average amplitudes of tion. The time point of stimulation is indicated by the arrow- calcium transients 6 S. D. recorded in the somata (gray bars) and heads. Inset shows an inverted AF594 fluorescence image of the processes (black bars) of NG2-cells in response to six consecutive analyzed regions of interest. Right: Bar chart illustrating average stimulations. Calcium transients are normalized to the average of amplitudes of calcium transients 6 S. D. recorded in the somata six consecutive stimulations. Note the strong rundown of the (gray bars) and processes (black bars) of astrocytes upon repetitive response. Scale bars: 10 lm. induced calcium transients in astrocytes to 5.7% 6 2.8% of tensity of a repeated three-pulse stimulation arbitrarily between control (Figs. 5B,C, n 5 5). In contrast, astrocyte responses 25, 50, 75, and 100 lA. Increasing the intensity of the three- were not altered by injection of the mGluR1 antagonist pulse stimulation increased the amplitude of the averaged l n 5 n 5 YM298198 (10 M; 3; Figs. 5B,C) nor by the GABAB- population spike ( 6–11; Figs. 6A,C), indicating a stronger receptor antagonist CGP55845 (5 lM; n 5 3; Figs. 5B,C). postsynaptic activation due to the recruitment of additional fibers. These results thus confirm earlier reports that synaptically The mean amplitude of astrocyte calcium transients increased induced calcium transients in astrocytes are mainly due to acti- proportionally to the stimulation intensity and the population vation of mGluR5 (e.g., Pasti et al., 1997; Porter and McCar- spike amplitude in both somata and processes (n 5 6–35, Figs. thy, 1996; Latour et al., 2001). 6B,C). Taken together, these findings demonstrate that—within To analyze the dependence of astrocyte calcium transients on the tested range—the amplitudes of astrocyte calcium transients the number of stimulated afferents, we varied the stimulation in- linearly correlated with the overall number of activated synapses.

Hippocampus 8 HONSEK ET AL.

paradigms that are typically used for inducing long-term poten- tiation or long-term depression. To induce an increase in synaptic strength, Schaffer collaterals were subjected to a theta-burst-stimulation (TBS) paradigm (Albensi et al., 2007), which consisted of four current pulses at a frequency of 100 Hz, delivered five times at an interval of 200 ls, resulting in a total of 20 pulses. As observed with a sin- gle three-pulse stimulation, TBS reliably induced calcium transi- ents in astrocytes. However, despite a nearly sevenfold increase in

FIGURE 5. Pharmacological profile of synaptically-induced cal- cium transients in astrocytes. A: Image of a Fluo5-filled astrocyte taken with the widefield imaging system. On the top left, the position of a pipette, through which test substances were delivered by pressure application (three times for 5 s) is indicated schematically. On the top right, the position of the pipette for electrical stimulation is indicated. Scale bar: 20 lm. B: Calcium transients in four individual astrocytes in response to a three-pulse stimulation in the control and after injec- tion of saline (first trace), 100 lMMPEP(secondtrace),1lM YM298198 (YM, third trace), or 5 lM CGP55845 (CGP, fourth trace). Stimulation is indicated by the arrowheads and the vertical line. C: Bar chart illustrating normalized amplitudes 6 S. D: Of cal- FIGURE 6. Dependence of astrocyte calcium signals on num- cium transients in the control, and following injection of saline or ber of stimulated afferents. A: Field potentials evoked by three- the different pharmacological antagonists. pulse 50 Hz stimulations at stimulation intensities from 25 to 75 lA. B: Inverted AF594 fluorescence image of the analyzed astro- cyte, illustrating the position of the stimulating electrode, and the analyzed regions of interest in the focal plane. Scale bar: 10 lm. Dependence of Astrocyte Calcium Signals on C: Calcium transients detected in the soma (region 1, gray trace) Synaptic Strength and in a process (Region 2, black trace) of the astrocyte upon three-pulse stimulation with stimulation intensities from 25 to 75 The Schaffer collateral to CA1 pyramidal cell synapse is a lA. The times of stimulation are indicated by the vertical lines. D: widely used model system to investigate mechanisms underly- Bar chart illustrating mean amplitudes of calcium transients in somata (gray bars) and processes of astrocytes (black bars) as well ing activity-induced synaptic plasticity (Malinow and Malenka, as amplitudes of population spikes (open bars) 6 S. D. upon stim- 2002). To study the effect of changes in synaptic strength on ulation with intensities from 25 to 100 lA. Amplitudes are nor- astrocyte calcium signals, we employed different stimulation malized to values obtained at 75 lA.

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 9

FIGURE 7. Astrocyte calcium transients in response to a TBS before TBS stimulation. Bottom: Normalized amplitude of astro- stimulation. A: Calcium transients in the soma and two different cyte calcium transients in response to three-pulse stimulation processes of an astrocyte stimulated every two minutes with a 3- before and after TBS. The gray line delineates the average run- pulse or a theta-burst stimulation (TBS) as indicated by the thin down observed in all regions of interest in control conditions. C: and thick lines, respectively. Also note the occurrence of spontane- Bar chart illustrating mean amplitudes of calcium transients 6 S. ous calcium transients. B: Top: Average field potentials recorded D. in somata (light gray) and processes (dark gray) of astrocytes before (gray) and after (black) TBS in response to three-pulse in the course of repeated three-pulse stimulation, during TBS, stimulation (stimulation artifacts are masked). Center: Amplitude and after TBS. The open bar represents the mean population of average population spikes elicited in response to three-pulse spike amplitude after TBS values are normalized to control; num- stimulation before and after TBS, normalized to mean amplitude bers below the bars represent numbers of experiments.

the number of delivered pulses (20 vs. 3), the average amplitude cells: 65 6 33%, 29 regions of interest; P > 0.3). Note that of calcium signals evoked by TBS was increased only to about the response of astrocytes to the control three-pulse stimulation 140% as compared with the three-pulse stimulation (163 6 was not saturated, as increasing the stimulation intensity from 70% in 5 somata; 132 6 40% 12 processes; Figs. 7A,C). 75 to 100 lA resulted in a linear increase in the amplitude of Before and after TBS, calcium transients in response to the astrocyte calcium signals (cf. Fig. 6). repeated, standard three-pulse stimulation were recorded to Next we examined the effect of a 5-min stimulation at 1 reveal activity-dependent changes induced by TBS. Again, field Hz, a protocol well established to induce long-term depression potential recordings served to monitor changes in the postsy- (LTD) at Schaffer collateral to CA1 synapses. After the onset naptic responses. After TBS, the amplitude of population of this stimulation paradigm, an initial increase in astrocyte spikes in response to a three-pulse stimulation was increased to calcium was observed in most regions of interest (c.f. Fig. about 230% of control (n 5 6; Figs. 7B,C). In contrast to 8A), which declined to baseline or transformed into an oscil- this, the mean amplitudes of astrocyte calcium transients in latory response (c.f. Fig. 8A) during the five-minute stimula- response to the three-pulse stimulation recorded after TBS were tion. Within periods of at least 15 min after performing the not different from those obtained in control cells not chal- LTD induction protocol, the average amplitude of population lenged with TBS (Fig. 7; mean amplitude after TBS: 77 6 spikes in response to a three-pulse stimulation was decreased 45%, n 5 19 regions of interest; mean amplitude in control to about 25% of control, confirming depression of postsynap-

Hippocampus 10 HONSEK ET AL.

FIGURE 8. Astrocyte calcium transients in response to a TBS ter a 5 min/1 Hz protocol. Bottom: Normalized amplitude of stimulation. A: Calcium transients in the soma and a process of astrocyte calcium transients in response to three-pulse stimulation an astrocyte stimulated every two minutes with a standard three- before and after a 5 min/1 Hz protocol. The gray line delineates pulse stimulation as indicated by the straight lines. After four the average run-down observed in control conditions. C: Bar chart standard stimulations, a typical LTD induction protocol, consist- illustrating mean amplitudes of calcium transients 6 S. D. in ing of a stimulation for 5 min at 1 Hz, was performed (indicated somata (light gray) and processes (dark gray) of astrocytes, in the by the box). Also note the occurrence of spontaneous calcium course of repeated three-pulse stimulation and after a 5 min/1 Hz transients. B: Top: Average field potentials recorded before (gray) protocol. The open bar represents the mean population spike am- and after (black) a 5 min/1 Hz protocol in response to three-pulse plitude after the 5 min/1 Hz protocol. Numbers below the bars stimulation. Center: Normalized amplitude of averaged population represent numbers of experiments. spikes elicited in response to three-pulse stimulation before and af- tic responses (n 5 8;Figs.8B,C).Again,despitetheevident astrocytes exhibited spontaneous calcium transients, whereas change in synaptic strength, the amplitude of astrocyte cal- NG2-cells displayed a stable intracellular baseline calcium cium transients was not altered significantly as compared to concentration. Moderate synaptic stimulation of afferent control experiments (n 5 15 regions of interest, 107 6 93% Schaffer collaterals evoked calcium transients in astrocytes and vs. 65 6 33%; Figs. 8A–C; P > 0.1). NG2-cells. In astrocytes, these were mediated by mGluR5 In summary, these experiments show that the amplitude of activation and could be evoked repetitively. Evoked calcium astrocyte calcium transients evoked by stimulation of glutama- transients in NG2-cells, in contrast, were subject to rapid tergic synapses is not altered within a time window of up to run-down. While the amplitude of synaptically induced astro- 15 min following an activity-induced potentiation or depres- cyte calcium transients increased with increasing stimulation sion of postsynaptic responses. intensity, it was not altered upon induction of synaptic plasticity.

Spontaneous Calcium Signaling in Glial Cells DISCUSSION We found that astrocytes exhibit spontaneous calcium transi- In this study, we characterized spontaneous and evoked cal- ents in the absence of evoked neuronal activity, confirming cium signaling in identified glial cells of the stratum radiatum earlier observations made in culture, in situ and in vivo (Parri of acute mouse hippocampal tissue slices. We found that, in et al., 2001; Aguado et al., 2002; Nett et al., 2002; Hirase the absence of evoked neuronal activity, the vast majority of et al., 2004; Nimmerjahn et al., 2004; Zur Nieden and

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 11

Deitmer, 2005). Specifically, it was shown that spontaneous ac- Evoked Calcium Signals in Astrocytes tivity occurs independent of neuronal action potentials (Aguado It is firmly established that electrical stimulation of neurons et al., 2002; Nett et al., 2002). While spontaneous calcium evokes astrocyte calcium signaling (Dani et al., 1992; Porter oscillations in rat astrocytes were significantly reduced by antag- and McCarthy, 1996), which can also be induced by physio- onists of metabotropic glutamate receptors (Zur Nieden and logical stimuli (Newman, 2005) and observed in vivo (Takano Deitmer, 2005), spontaneous calcium transients in mouse hip- et al., 2006; Wang et al., 2006; Winship et al., 2007; Nim- pocampal astrocytes in tissue slices were not altered following merjahn, 2009). Whereas earlier studies in slice preparations inhibition of mGluRs (Aguado et al., 2002; Nett et al., 2002). often performed prolonged stimulation to study evoked cal- Furthermore, spontaneous calcium signals were not blocked by cium signaling in astrocytes (e.g., Porter and McCarthy, antagonists of purinergic and/or GABAergic receptors, indicat- 1996; Latour et al., 2001; Fellin et al., 2004; Perea and Ara- ing that in mouse astrocytes their generation is mostly inde- que, 2005), we found that three pulses of Schaffer collateral pendent of ATP signaling and activation of glutamatergic and stimulation were sufficient to induce astrocyte calcium transi- GABAergic receptors (Aguado et al., 2002; Nett et al., 2002). ents. At our standard stimulation intensity of 75 lA, the Generally, spontaneous astrocyte calcium signals could be three-pulse stimulation resulted in calcium transients with blocked in both species by depletion of intracellular stores and amplitudes comparable to spontaneous events. Importantly, were sensitive to interference with phospholipase C signaling this moderate stimulation protocol elicited calcium transients and IP -receptor activation (Aguado et al., 2002; Nett et al., 3 that were not yet saturated, as increasing the stimulation in- 2002; Schipke et al., 2004; Zur Nieden and Deitmer, 2005). tensity also caused the amplitude of astrocyte calcium transi- Thus, while the exact mechanism of their initiation in mouse ents to increase. Thus, with the given stimulation parameters, hippocampal astrocytes is unclear, there is strong evidence that the amplitude of astrocyte calcium transients was a valid mea- they are generated by release of calcium from the endoplasmic sure for assessing the -evoked astrocyte reticulum. activation. As reported earlier (e.g., Aguado et al., 2002), the fre- Astrocyte calcium transients evoked by Schaffer collateral quency and patterns of activity were variable and included stimulation were largely blocked by the mGluR5 antagonist random profiles, but also rhythmic oscillations. Spontaneous MPEP, confirming earlier observations (e.g., Porter and McCar- calcium transients were detected in the vast majority of classi- thy, 1996; Pasti et al., 1997; Latour et al., 2001). Thus, neither cal astrocytes, both using the high-affinity calcium indicator a NMDA-receptor dependent component as described from OGB1-AM as well as the low-affinity indicator Fluo-5F. cortical (Nolte et al., 2001; Lalo et al., 2006) and hippocampal About half of the events detected included the soma and part astrocytes (Serrano et al., 2008), nor activation of other recep- of the processes, while the other half was restricted to the tors expressed by astrocytes (Verkhratsky, 2009) did play an sig- processes. Notably, spontaneous events occurring synchro- nificant role in the generation of calcium transients evoked by nously in all regions of interest were only observed once. Tak- our three-pulse stimulation paradigm. ing into consideration that multiphoton measurements are re- The latency of evoked calcium signals was 1–2 s, which is stricted to one focal plane, these results indicate that sponta- in the range of astrocyte responses reported before (e.g., Wang neous calcium signaling in astrocytes typically is not et al., 2006; Winship et al., 2007; Schipke et al., 2008). Typi- synchronized within the entire cell. This finding is in line cally, astrocyte responses to three-pulse stimulation could be with the existence of cellular microdomains, in which calcium evoked repetitively for at least six consecutive stimulations signaling occurs independent of neighboring regions, similar separated by 2 min. The time course and amplitudes of to what has been observed in Bergmann glia cells of the cere- evoked calcium transients were not only variable between bellum (Grosche et al., 1999). individual astrocytes and different regions of a cell, but also Because of the clear separation of astrocytes and NG2-cells between different trials in a given region, a phenomenon also in the present study, we could show that calcium oscillations described in other studies (Zonta et al., 2003; Winship et al., are restricted to classical astrocytes but do not occur in NG2- 2007). Within the investigated range of stimulations cells. The lack of spontaneous calcium signaling in NG2-cells strengths, mean amplitudes of astrocyte calcium transients lin- was not primarily caused by the specific calcium binding prop- early increased with increasing stimulation strength, indicating erties of the indicators used, as the same results were obtained their dependence on the number of activated fibers and with high (Fluo-5F) and low (OGB1) K indicator dyes. These d synapses. findings are in accordance with previous publications, in which oscillatory calcium activity in NG2 glia has not been reported (Ge et al., 2006; Hamilton et al., 2008; Hamilton et al., 2009; Evoked Calcium Signals in NG2-Cells Tong et al., 2009). Because of the restriction to one focal plane with multiphoton imaging (see above), we may have missed NG2-cells are a distinct type of glial cells that are synapti- local events occurring in single processes. Nevertheless, our cally connected to neurons (De Biase et al., 2010). In NG2- measurements strengthen the view that astrocytes and NG2- cells in the optic nerve, calcium signals are evoked by exoge- cells differ significantly in their basic calcium signaling nouslyappliedATPandglutamateandrobustactivationof properties. purinergic receptors and calcium signaling in NG2-cells was

Hippocampus 12 HONSEK ET AL. observed during action potential propagation. A glutamatergic Astrocyte Calcium Signals following Changes in component was, however, only visible when desensitization of Synaptic Strength AMPA receptors as well as cellular glutamate uptake was To study the effect of changes in synaptic strength on astro- blocked pharmacologically (Hamilton et al., 2010). In the cyte calcium signals, we employed protocols routinely used for hippocampus, nonlinear glial cells, likely corresponding to induction of LTP and LTD, at Schaffer collateral to pyramidal NG2-cells, express a mosaic of AMPA receptor subunits form- cell synapses. Astrocyte calcium transients in response to these ing glutamate receptors with rapid kinetics and intermediate protocols were nonlinear as compared to the three-pulse stimu- calcium permeability (Matthias et al., 2003). Moreover, they lation protocol. When we performed a theta-burst stimulation, express GABA receptors, and activation of these increases in- A the amplitude of calcium signals was only increased by 40% as tracellular calcium through reversed sodium-calcium exchange compared with our standard, three-pulse stimulation. During (Tong et al., 2009). In addition, intracellular calcium eleva- the 5-min stimulation at 1 Hz, an initial increase in astrocyte tions can be evoked by exogenous glutamate via activation of calcium was observed, which then again declined to baseline or calcium-permeable AMPA receptors and, possibly, mGluRs transformed into an oscillatory response. Such nonlinear behav- (Ge et al., 2006). Synapses between neurons and NG2-cells ior is in line with observations suggesting that astrocytes are undergo LTP following theta burst stimulation which is tuned to specific patterns of neuronal activity and integrate syn- blocked during inhibition of AMPA-receptors and buffering aptic information (Pasti et al., 1995; Pasti et al., 1997; Araque of intracellular calcium increases in NG2-cells (Ge et al., et al., 2002; Perea and Araque, 2005). 2006). The theta burst stimulation induced an increase in the ampli- In our study, we could show that synaptic activation of tude of field potentials evoked by the standard three-pulse stimu- Schaffer collaterals induced calcium transients in NG2-cells. lation, whereas the 5 min/1 Hz stimulation strongly depressed A hitherto unreported observation is that these activity- the amplitude of field potentials. These effects persisted for at induced calcium signals underwent a persistent run-down least 15 min, indicating that synaptic transmission was efficiently when challenged every 2 min with a three-pulse stimulation potentiated or depressed. During this phase of altered synaptic protocol. A similar depression of glial responses upon certain strength, however, we did not observe a significant alteration in stimulation protocols was also reported from other regions in astrocyte calcium signals evoked by three-pulse stimulation. the brain. In the , repetitive stimulation of parallel These experiments were performed with the low affinity cal- fibers at low frequencies that do not affect postsynaptic cur- cium-indicator dye Fluo-5F after withdrawal of patch-pipettes to rents in Purkinje neurons, causes a strong decline of both glu- reduce the risk of calcium buffering and washout-of intracellular tamate uptake and AMPA-receptor-mediated currents in Berg- signaling components, and thus, the likelyhood of masking cal- mann glial cells (Balakrishnan and Bellamy, 2009). While cium-dependent effects on ion channel activity or enzyme phos- NG2-cells do not express glutamate uptake (De Biase et al., phorylation. However, there is a slight possibility that small dif- 2010), the observed activity-dependent run-down might be ferences in intracellular calcium transients, which might be con- caused by a rapid, possibly long-lasting, internalization of fined to a fraction of synapses, might have been missed using AMPA receptors (Ehlers, 2000; Malinow and Malenka, 2002) this indicator. This explanation is rather unlikely, as the or an exchange of calcium-permeable for calcium-imperme- described stimulation paradigms for inducing potentiation or able AMPA receptors (Matthias et al., 2003). AMPA receptors depression resulted in substantial alterations of neuronal signal- of hippocampal NG2-cells undergo a developmental change ing. In addition, calcium transients in astrocytes were observed in subunit composition that results in decreased calcium per- even with very low stimulation intensities such as 25 lA. meability, and it was speculated that activation of calcium- Recent reports have established that astrocytes extend and permeable receptors in immature cells might be necessary to retract processes near dendritic spines over the time course of establish signaling between neurons and glial cells at early minutes (Hirrlinger et al., 2004; Haber et al., 2006). Increased postnatal stages (Matthias et al., 2003). Interestingly, a signifi- coverage of excitatory synapses by glial processes has been dem- cant downregulation of AMPA receptors has also been onstrated as early as 30 min following anoxic LTP (Lushnikova reported as NG2-cells develop into mature et al., 2009), whereas Wenzel and coworkers reported a closer (De Biase et al., 2010). On the other hand, Ge et al. (2006) apposition of astrocyte processes to synapses that peaked eight found evidence for a form of LTP in NG2-cells, which might hours after LTP induction by tetanization (Wenzel et al., 1991). be accompanied by an increased trafficking of calcium-perme- The lack of alterations in astrocyte calcium signaling in response able AMPA receptors to the synapse and increased calcium to synaptic potentiation as well as depression indicates that mor- signaling in response to synaptic activation. While we focused phological changes either do not occur during the induction on astrocyte calcium signaling, and did not investigate the phase of synaptic plasticity or do not lead to substantial altera- run-down of evoked calcium signals in NG2-cells further, tions of astrocyte calcium signals. Whether such changes occur these results demonstrate a significant difference to activity- later on, during the expression phase of synaptic plasticity induced, mGluR5-mediated calcium signaling of astrocytes, in remains to be determined. Considering that astrocytes occupy which calcium transients could be evoked repetitively upon at nonoverlapping domains and can contact up to 100,000 synap- least six stimulations. ses (Bushong et al., 2002), an alternative explanation for the lack

Hippocampus PLASTICITY OF CALCIUM SIGNALS IN HIPPOCAMPAL ASTROCYTES 13 of plasticity-induced changes in the amplitude of astrocyte cal- lates with increased precision of synaptic transmission. Glia cium transients is that heterosynaptic alterations in the strength 57:393–401. In vivo of unstimulated synapses, occurring along with synaptic potentia- Be´lair EL, Valle´e J, Robitaille R. 2010. long-term synaptic plasticity of glial cells. J Physiol 588:1039–1056. tion or depression of stimulated synapses, caused the overall syn- Bergles DE, Roberts JD, Somogyi P, Jahr CE. 2000. Glutamatergic aptic strength in the domain of a given astrocyte to be synapses on oligodendrocyte precursor cells in the hippocampus. unchanged. Because of the observed changes in local field poten- Nature 405:187–191. tial amplitudes, this explanation seems, however, less likely. Bushong EA, Martone ME, Jones YZ, Ellisman MH. 2002. Protoplas- Our findings are in agreement with recordings of astrocyte mic astrocytes in CA1 stratum radiatum occupy separate anatomi- cal domains. J Neurosci 22:183–192. glutamate transporter currents in response to Schaffer collateral Dani JW, Chernjavsky A, Smith SJ. 1992. Neuronal activity triggers stimulation which indicated that astrocyte glutamate uptake— calcium waves in hippocampal astrocyte networks. Neuron 8:429– and presynaptic glutamate release—is unchanged after induc- 440. tion of LTP (Diamond et al., 1998). A recent study confirmed De Biase LM, Nishiyama A, Bergles DE. 2010. Excitability and synap- this finding by showing that the amplitude of glutamate- tic communication within the oligodendrocyte lineage. J Neurosci 30:3600–3611. uptake-mediated astrocyte depolarizations was unchanged after Deitmer JW, Rose CR. 2010. Ion changes and signalling in perisynap- induction of LTP (Ge and Duan, 2007). Yet, a persistent tic glia. Brain Res Rev 63:113–129. increase in the slow component of astrocyte depolarizations was Diamond JS, Bergles DE, Jahr CE. 1998. Glutamate release moni- found, which was suggested to be related to increased accumu- tored with astrocyte transporter currents during LTP. Neuron lation of extracellular potassium following LTP induction (Ge 21:425–433. Ehlers MD. 2000. Reinsertion or degradation of AMPA receptors and Duan, 2007). determined by activity-dependent endocytic sorting. Neuron Taken together, our results imply that synaptically induced 28:511–525. calcium signals in hippocampal astrocytes represent an integral Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG, Carmignoto G. of the overall presynaptic activity and transmitter release, but 2004. Neuronal synchrony mediated by astrocytic glutamate do not reflect changes in the strength of individual synapses through activation of extrasynaptic NMDA receptors. Neuron 43:729–743. contacted by the perisynaptic glial processes. This implies that Fiacco TA, Agulhon C, Taves SR, Petravicz J, Casper KB, Dong X, prospective, calcium-dependent release of gliotransmitters and Chen J, McCarthy KD. 2007. Selective stimulation of astrocyte their actions on neuronal transmission or blood microcircula- calcium in situ does not affect neuronal excitatory synaptic activity. tion will also depend on pre- rather than on postsynaptic activ- Neuron 54:611–626. ity within an astrocyte’s domain. Ge WP, Duan S. 2007. Persistent enhancement of neuron-glia signal- ing mediated by increased extracellular K1 accompanying long- term synaptic potentiation. J Neurophysiol 97:2564–2569. Acknowledgments Ge WP, Yang XJ, Zhang Z, Wang HK, Shen W, Deng QD, Duan S. 2006. Long-term potentiation of neuron-glia synapses mediated by The authors thank Simone Durry and Claudia Roderigo for Ca21-permeable AMPA receptors. Science 312:1533–1537. excellent technical support. Genoud C, Quairiaux C, Steiner P, Hirling H, Welker E, Knott GW. 2006. 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