Bergmann Glia Modulate Cerebellar Purkinje Cell Bistability Via Ca2+-Dependent K+ Uptake

Bergmann glia modulate cerebellar Purkinje cell bistability via Ca2+-dependent K+ uptake

Fushun Wang1, Qiwu Xu, Weishan Wang, Takahiro Takano, and Maiken Nedergaard1

Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, University of Rochester, Rochester, NY 14642

Edited* by Rodolfo R. Llinás, New York University Medical Center, New York, NY, and approved April 4, 2012 (received for review December 12, 2011)

Recent studies have shown that cerebellar Bergmann glia display bistability. We found that selectively induced Ca2+ increases in coordinated Ca2+ transients in live mice. However, the functional Bergmann glia also evoked a transient reduction of extracellular + significance of Bergmann glial Ca2+ signaling remains poorly un- K in cerebellar slices and cerebellum in live adult mice. More- 2+ derstood. Using transgenic mice that allow selective stimulation of over, Bergmann glial Ca signaling consistently triggered a glial cells, we report here that cytosolic Ca2+ regulates uptake of K+ switch to up states of longer duration. All of the effects of Bergmann glial Ca2+ signaling on Purkinje cell bistability could by Bergmann glia, thus providing a powerful mechanism for con- + trol of Purkinje cell-membrane potential. The decline in extracellu- be reproduced by simply reducing K in the bath solution or lar K+ evoked by agonist-induced Ca2+ in Bergmann glia transiently depolarizing a single Bergmann glial cell. Thus, our observations indicate that Bergmann glia can regulate cerebellar network ac- increased spike activity of Purkinje cells in cerebellar slices as well tivity by a pathway involving Ca2+-mediated K+ uptake. Because as in live anesthetized mice. Thus, Bergmann glia play a previously Purkinje cells fire continuously in up state, Ca2+ signaling in unappreciated role in controlling the membrane potential and Bergmann glia is associated with a sharp increase in output from thereby the activity of adjacent Purkinje cells. the cerebellar cortex. two-photon laser scanning microscopy | ion-sensitive microelectrode Results Bergmann Glial Ca2+ Transients Changed Purkinje Cell Bistability. To ergmann glial cells in cerebellum are electrically nonexcitable address a possible role of Bergmann glia in regulation of Pur- cells that in many ways serve the same functions as proto- kinje cell activity, we combined whole-cell recordings of Purkinje B NEUROSCIENCE plasmic astrocytes in forebrain. Bergmann glia are chiefly re- cells with imaging of intracellular Ca2+ signaling in Bergmann sponsible for glutamate uptake and extracellular K+ homeostasis glia loaded with the Ca2+ indicator rhod2/am in cerebellar slices. (1). Their highly negative resting membrane potential of −80 to Whole-cell recordings showed that the membrane potentials of −85 mV combined with a large number of inwardly rectifying K+ almost all recorded Purkinje cells spontaneously fluctuated with channels helps maintain a tight control of extracellular K+ con- a frequency of 0.5 ± 0.1 Hz between two stable states of 63.4 ± centration (2). Bergmann glial cells also display Ca2+ transients in 2.16 mV (down state) and 47.2 ± 3.04 mV (up state), giving rise response to glutamate or stimulation of climbing and parallel to a bimodal time distribution (n = 83; Fig. S1). Similar oscil- fibers in slices and to motor activity in vivo (3). However, although lations were recorded by extracellular electrodes, demonstrating glial Ca2+ signaling has been shown to regulate synaptic trans- that the oscillations are not a result of dialysis of intracellular mission in several brain regions, it is presently not established fluid with the patch electrode (Fig. S1). To selectively stimulate whether Bergmann glia can modulate the activity of Purkinje cells. Bergmann glial Ca2+ transients, cerebellar slices were prepared Membrane potentials of almost all neurons in mammalian brain from mice selectively expressing the MrgA1 receptor under the fluctuate between a hyperpolarized state (down state) and depo- human GFAP promoter (15). The MrgA1 receptor is a member larized state (up state) during sleep/anesthesia and quiet wake- of the Gq-coupled receptor family with expression restricted to a fulness (4, 5). These fluctuations of the membrane potentials are subset of nociceptive sensory neurons outside the CNS. Thus, the synchronized between neighboring cells and detected as slow peptide ligand Phe-Met-Arg-Phe amide (FMRF) does not acti- (∼0.5–1 Hz), large-amplitude delta waves (4). Even though the vate endogenous brain receptors (15). As previously reported, bath application of FMRF peptide induced robust Ca2+ in- existence of bistability of Purkinje cells in awake mice has been + questioned, it is generally agreed that bistability is a common creases in EGFP Bergmann glia (Fig. 1A) concomitant with a phenomenon in both anesthetized and sleeping animals (5). The sharp increase in the duration of Purkinje cell up state (Fig. 1 B bistability of the resting membrane potential in Purkinje cells was and C). The FMRF-induced changes in Purkinje cell-membrane first discovered by Llinás and Sugimori in cerebellar slices (6, 7). potential were transient, lasting 20–120 s. The regular pattern of bistability returned with a delay of ∼10–60 s after normalization The current model predicts that bistability of Purkinje cells, similar 2+ to thalamic neurons, relies on the balance between noninactivat- of Ca in surrounding Bergmann glial cells. Of note, FMRF did + 2+ not evoke Ca2+ increases in Bergmann glia and did not reduce ing inward currents (persistent Na currents and/or T-type Ca + currents) (4, 8, 9) and an outward K+ leak current (5–7, 10). extracellular K in slices prepared from WT mice (Fig. S2). We However, Purkinje cell bistability is not simply an expression of next assessed the effect of ATP because it has previously been shown that ATP mediates Ca2+ increases in Bergmann glia after intrinsic membrane properties because complex spikes generated fi by climbing fiber activation can toggle the membrane potential parallel ber stimulation. Bath application of ATP also triggered elevations in Bergmann glial Ca2+ accompanied by a transient between up and down states in vivo (5). Recently, glutamate + uncaging on distal dendrites of striate spiny neurons was shown to increase in Purkinje cell up state in both MrgA1 mice and WT induce up states that lasted hundreds of milliseconds (11), and small hyperpolarization currents or interneuronal activity can toggle Purkinje cells between up and down states (12). Moreover, Author contributions: F.W. and M.N. designed research; F.W., Q.X., and W.W. performed stimulating a single astrocyte can trigger up-state synchronizations research; F.W. and T.T. analyzed data; and T.T. and M.N. wrote the paper. of neighboring neurons in cortical slices, in a mechanism that The authors declare no conflict of interest. depends on purinergic and glutamatergic signaling (13). *This Direct Submission article had a prearranged editor. Our earlier studies have shown that astrocytes in hippocampus Freely available online through the PNAS open access option. + 2+ and cortex increase K uptake in response to agonist-induced Ca 1To whom correspondence may be addressed. E-mail: [email protected] + signaling (14). Because the extracellular concentration of K is or [email protected]. a critical determinant of Purkinje cell-membrane potential, we This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. here asked whether Bergmann glia can modulate Purkinje cell 1073/pnas.1120380109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1120380109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 A Rhod-2 2+ GFP Rhod-2 Merge 0 s 30 s 120 s Fig. 1. Agonist-induced Ca signaling in Berg- mann glia modiﬁes Purkinje cell bistability and increases their spiking activity. (A) Two-photon imaging of freshly prepared vibratome sections from a MrgA1+ mouse expressing EGFP under the GFAP promoter (green; Left) loaded with the Ca2+ indicator rhod2/am (red). EGFP and rhod2 signal FMRF BCoverlap, indicating that rhod2 primarily is taken up –35 by Bergmann glia. Right three images display rhod2 –40 emission changes in response to exposure to the –45 MrgA1 receptor agonist FMRF (15 μM). (B) Whole- –50 cell recording of a Purkinje cell (PC) combined with –55 PC imaging rhod2 in Bergmann glia in a slice from –65 mV –60 a MrgA1+ mouse. The durations of up states in- Rhod-2 –65 2+ Membrane potential (mV) crease concurrently with a Ca increase in Berg- 20 mV 0.00 0.05 0.10 0.15 0.20 0.25 100% Relative frequency mann glia evoked by FMRF. (C) Histogram depicting DEATP 6s the bimodal distribution of Purkinje cell-membrane –35 potential before (gray) and after (red) FMRF expo- –40 sure (10 s were analyzed, bin = 1 mV). (D) ATP (100 –45 μM) increased Purkinje cell spiking and rhod2 signal –50 in neighboring Bergmann glial cells. (E) Histogram –55 depicting the bimodal distribution of Purkinje cell- PC –60 membrane potential before (gray) and after (pink) –65 mV-- –65 ATP exposure (10 s were analyzed, bin = 1 mV). (F)

Membrane potential (mV) potential Membrane –70 Rhod-2 0.00 0.05 0.10 0.15 0.20 0.25 Percentage of time Purkinje cells spent in up state 100% 20 mV Relative frequency before, during, and after exposure to FMRF or ATP 6 s (**P < 0.01, n =6–7). (G) Action potential frequency FHG Ibefore, during, and after exposure to FMRF and 100 18 ** 100 < – ** ** 16 ** 0.5 ATP (**P 0.01, n =6 7). (H) Comparison of ISIs of 80 14 80 12 0.4 action potentials before, during, and after exposure 60 60 10 0.3 to FMRF or ATP. ISI was analyzed during up state

8 CV 40 40 0.2 – 6 ISI (ms) only (P = 0.13 for FMRF, P = 0.9 for ATP, n =6 7). (I) in upstate 20 4 20 0.1 Percent of time Frequency (Hz) 2 CVs for action potentials before, during, and after 0 0 0 0.0 application of FMRF and ATP (P = 0.6 for FMRF, P = beforeFMRFafter beforeATPafter beforeFMRFafter beforeATPafter beforeFMRFafter beforeATPafter beforeFMRFafter beforeATPafter 0.3 for ATP, n =6–7).

mice (Fig. 1 D and E and Fig. S2). Of note, Purkinje cells only Overall, the analysis showed that agonist-induced Ca2+ fired action potentials during up state. As a consequence, Ca2+ increases in Bergmann glia were linked to a sharp increase in the signaling in Bergmann glia led to a significant increase in Pur- duration of Purkinje cell up state and thereby in spiking activity. kinje cell output detected as an increased frequency of spiking The increase in up state was transient because normalization of (Fig. 1 F and G). Interspike interval (ISI) and coefficient of Ca2+ in Bergmann glia after a delay of 10–60 s was followed by variance (CV) of action potential firing during up state did not a return to normal periodic oscillations of the membrane po- change significantly after application of either FMRF or ATP tential in Purkinje cells. (Fig. 1 H and I and Fig. S2), suggesting that neither FMRF nor ATP directly modified the properties of the voltage-gated Ca2+ Signaling in Bergmann Glia Reduced Extracellular K+ Concentration. channels generating Purkinje cells action potentials. Because buffering extracellular K+ is a well-known function of Because it cannot be excluded that ATP activates Purkinje cell astrocytes, and Ca2+ signaling in hippocampal astrocytes has + P2Y1 receptors, the P2Y2 and P2Y4 receptor-specific agonist been shown to increase their uptake of K (14, 17, 18), we next UTP was also evaluated. UTP-induced Ca2+ increases in Berg- tested whether agonist-induced Ca2+ signaling in Bergmann glia mann glia were directly comparable to those of ATP and FMRF triggered a transient decrease in extracellular K+ in cerebellar and were also associated with a transient increase in the duration slices. We combined two-photon imaging of Ca2+ and whole-cell of Purkinje cell up states (Fig. 2 A and B). To address whether recording of Bergmann glia with measurements of extracellular agonist-induced increases in cytosolic Ca2+ of Bergmann glia was K+ by using ion-sensitive microelectrodes. required for receptor-mediated changes in Purkinje cell output, Bath application of FMRF in MrgA1+ mouse cerebellar slices UTP was next applied to slices prepared from inositol 1,4,5-tri- triggered a robust increase in Bergmann glial Ca2+ accompanied −/− + sphosphate (IP3) receptor subtype 2 knockout mice (IP3R2 ). by a transient reduction of extracellular K and a minor hyper- In contrast, Bergmann glia did not exhibit Ca2+ transients in polarization of Bergmann glial membrane potential (Fig. 3A). −/− 2+ response to UTP in slices prepared from IP3R2 mice (Fig. Similarly, application of ATP consistently triggered Ca increases 2C), consistent with the report that Bergmann glia only expresses and hyperpolarization of Bergmann glia concomitant with a sig- fi + IP3R2 and not subtypes 1 or 3 (16). UTP also failed to trigger ni cant drop in extracellular K (Fig. 3B), providing strong sup- significant changes in Purkinje cell bistability in slices prepared port for a role of Bergmann glia in active K+ uptake. To address −/− 2+ from IP3R2 mice (P = 0.8, n = 6; Fig. 2 C–E). Similarly, whether Bergmann glial Ca was required for receptor-mediated +/+ + −/− whereas the spiking frequency increased in IP3R2 mice (P < K uptake, UTP was applied to slices prepared from IP3R2 −/− +/+ 2+ 0.01), no significant change was observed in IP3R2 mice (P = mice and WT littermates (IP3R2 ). UTP-induced Ca in- 0.437; Fig. 2F). Purkinje cell up state ISI and CV were un- creases in Bergmann glia were also linked to a transient decrease affected by the UTP exposure in both genotypes, suggesting that in extracellular K+ and hyperpolarization of Bergmann glia in +/+ UTP did not alter the membrane properties of Purkinje cells IP3R2 mice (Fig. 3C). Consistent with the notion that agonist- (Fig. 2 G and H). Similarly, ATP increased bistability in Purkinje induced K+ uptake in Bergmann glia is Ca2+-dependent, slices −/− −/− cells from WT mice but failed to induced changes in IP3R2 prepared from IP3R2 mice failed to exhibit UTP-mediated mice too (Fig. S2). increases in Ca2+ and reduction in extracellular K+ concentration

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1120380109 Wang et al. Downloaded by guest on October 2, 2021 2+ UTP in IP R2+/+ Fig. 2. Ca dependency of Berg- AB3 –40 mann glial modulation of Purkinje cell

–45 bistability. (A) The P2Y2 and P2Y4 receptor agonist UTP (100 μM) induced –50 increases in cytosolic Ca2+ in Berg- –55 mann glia and an increase in up state of Purkinje cells (PC) in a IP R2+/+ (WT) –60 3 PC mouse. (B) Histogram plots the mem- –65 mV--- Membrane potential (mV) –65 brane potential distributions before 0.00 0.05 0.10 0.15 0.20 0.25 (gray) and after (pink) UTP exposure Rhod-2 Relative frequency (10 s, bin = 1 mV). (C) UTP failed to 100% 20 mV 2+ −/− 5 s induce increases in Ca in an IP3R2 –/– UTP in IP3R2 mouse and failed to change the reg- CD–40 ular pattern of bistability in Purkinje –45 cells. (D) Histogram shows the mem- –50 brane potential distributions before (gray) and after (green) UTP exposure –55 −/− PC in the IP3R2 recording in C (10 s, –60 bin = 1 mV). (E) Time spent in up state –65 mV --- –65 before, during, and after exposure to Rhod-2 Membrane potential (mV) +/+ −/− 20 mV UTP in IP3R2 and IP3R2 littermates 100% 0.00 0.05 0.10 0.15 0.20 0.25 5 s (**P < 0.01, n =6–7). (F) The spiking Relative frequency frequency before, during, and after Before +/+ −/− +/+ exposure to UTP in IP R2 and IP R2 EFUTP in IP3R2 G H 3 3 UTP in IP R2–/– < – 100 3 16 80 0.5 littermates (**P 0.01, n =67). (G) After ** ** 14 Comparison of ISIs of action potentials 80 0.4 12 60 during up states before, during, and after exposure to UTP in IP R2+/+ and 60 10 0.3 3 −/− +/+

8 40 CV IP3R2 littermates (P = 0.14 for IP3R2 , −/−

ISI (ms) – 40 6 0.2 P = 0.29 for IP3R2 , n =6 7). (H) CVs

4 20 before, during, and after exposure to NEUROSCIENCE 20 0.1 − −

Time at up-state (%) +/+ /

Spiking frequency (Hz) 2 UTP in IP3R2 and IP3R2 litter- +/+ 0 0 0 0.0 mates (P = 0.65 for IP3R2 , P = 0.81 −/− for IP3R2 , n =6–7).

(Fig. 3D). A comparison of FMRF, ATP, and UTP showed com- cells. As shown in Fig. S3 B and C, the relative time Purkinje parable increases in Ca2+, extracellular K+, and hyperpolarization cells spent in up state increased from 39.1% ± 8.5% to 68.2% ± of Bergmann glia (Fig. 3 E–G). In addition, UTP had no significant 11.4% (n = 6) when an adjacent Bergmann glial cell was effect on extracellular K+ or Bergmann glial membrane potential hyperpolarized by injecting a negative current (−100 pA). De- −/− in slices prepared from IP3R2 mice (Fig. 3 F and G). UTP also polarization of Bergmann glial cells (+100-pA current) had the failed to alter bistability and thereby the output of Purkinje cells in opposite effect (Fig. S3 D–F). Concomitantly, the gap in mem- −/− 2+ IP3R2 mice (Fig. 2). Importantly, the Ca -mediated change in brane potential between the two states narrowed when Berg- extracellular K+ was positively linked to hyperpolarization of mann glial cells were hyperpolarized and widened when Bergmann glia (R2 =0.486,n = 24; Fig. 3H). Thus, these Bergmann glial cells were depolarized (Fig. S3G). The ISI and experiments showed that agonist-induced Ca2+ signaling in CV of action potentials did not change significantly in response Bergmann glia is linked to a transient reduction of extracellular to either hyperpolarization or depolarization of Bergmann glia K+. Of note, the relative large tip of the K+-selective micro- cells (Fig. S3H). electrode (∼3 μm) creates an artificially large extracellular space, and the actual K+ decreases might be of a larger amplitude than Extracellular K+ Directly Modulated Purkinje Cell Bistability. The those recorded, as suggested in refs. 19 and 20. analysis above indicates that Ca2+ signaling in Bergmann glia induced both a decrease in extracellular K+ and an increase in Hyper- and Depolarization of Bergmann Glia Modulate Purkinje Neu- the duration of Purkinje cell up states. To critically evaluate ron Bistability. The passive membrane permeability of Bergmann whether a reduction in extracellular K+, in the absence of glial cells is predominantly attributable to K+. Hyper- and de- Bergmann glial Ca2+ signaling, is sufficient to trigger an increase polarization currents are almost exclusively carried by K+ influx or in the duration of up states, we next systematically changed ex- efflux and are accompanied by changes of extracellular K+ in a tracellular K+ while continuously recording from Purkinje cells. symmetrical manner. Previous analysis shows that clamping the Whole-cell recording from Purkinje cells showed that, by de- resting membrane potential of Bergmann glial cells offers an al- creasing bath K+ from 2.5 mM to 1 mM, the duration of up state ternative approach to assessing the role of glial K+ buffering on increased (P < 0.01, n = 6; Fig. 4 A and B). The increase in Purkinje cell bistability (2). We repeated the original observations duration of up state was linked to a twofold increase in the by Hounsgaard and Nicholson (2) and confirmed that injecting frequency of simple spikes (Fig. 4 C and D) without affecting ISI a hyperpolarizing current (−100 pA) in Bergmann glial cells cau- or CV of action potentials during up state (Fig. 4 E and F). The sed a drop in the resting membrane potential, averaging −7.04 ± normal pattern of bistability returned with a delay of 10–30 s 0.69 mV (n = 6), which was paralleled by a decrease in extracel- after bath K+ was returned to 2.5 mM (Fig. 4A). Testing a range lular K+ of 0.288 ± 0.017 mM (n =6;Fig. S3A). Conversely, of physiological K+ concentrations (1–5 mM) revealed that up injecting a depolarizing current (+100 pA) triggered a symmetric state was prevalent when K+ concentration was reduced in the increase in membrane potential, −7.00 ± 0.68 mV (n = 6), and an bath solution (Fig. 4G). The prevalence of up state with lower increase in extracellular K+, averaging 0.290 ± 0.021 mM (n =6; K+ was associated with a narrowed gap between up and down Fig. S3A). states of Purkinje cell-membrane potentials (Fig. 4H). Of note, To assess the effect of Bergmann glial cell-membrane poten- K+-sensitive microelectrodes inserted ∼80–100 μm below the tial on Purkinje cell up and down states, we next used dual whole- surface of the slice showed that changes in extracellular K+ cell recordings of neighboring Purkinje cells and Bergmann glial within cerebellar slices were of much smaller amplitude than the

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BGC –82mV BGC –82mV BGC –82mV BGC –79mV

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Rhod-2 100% 0.4 mMRhod-2 Rhod-2 Rhod-2

EFGH K+ changes (mM) –0.6 –0.4 –0.2 0 UTP UTP UTP UTP 0 1.2 ** FMRF ATP IP R2+/+ IP R2–/– +/+ –/– 3 3 FMRF ATP IP3R2 IP3R2 1.0 0 0 –0.5

0.8 –0.1 –1 –0.5 0.6 –0.2 –1.5 0.4 –1.0 –2 0.2 –0.3 Rhod-2 fluorescence

changes (mM) –2.5 0.0 + –1.5 K –0.4 FMRF ATP UTP UTP Hyperpolarization (mV) –3 * Hyperpolarization (mV) IP R2+/+ IP R2–/– R2 = 0.486 3 3 –0.5 –2.0 * –3.5

Fig. 3. Ca2+ signaling in Bergmann glia is linked to membrane hyperpolarization and a reduction of extracellular K+.(A) Combined whole-cell recordings of Bergmann glia with measurement of extracellular K+ and cytosolic Ca2+ in slices from a MrgA1+ mouse. The MrgA1 agonist FMRF (15 μM) triggered hyperpolarization of the Bergmann glia concomitantly with a decrease in extracellular K+ and an increase in rhod2 signal. (B) Exposure to ATP (100 μM) also triggered Ca2+ signaling and hyperpolarization of Bergmann glia concomitant with a decrease in extracellular K+.(C) UTP evoked increases in cytosolic Ca2+ + +/+ and a hyperpolarization of Bergmann glia as well as a decrease in extracellular K in slices prepared from IP3R2 mice. (D) UTP had little effect in slices −/− + prepared from IP3R2 littermates. (E) Histogram comparing increases in rhod2 signal in slices from MrgA1 mice exposed to FMRF (15 μM, n = 19), slices from +/+ −/− WT mice exposed to ATP (100 μM, n = 16), and slices from either IP3R2 (n = 13) or IP3R2 (n = 13) mice exposed to UTP (100 μM) (**P < 0.01, n = 12). (F) + + +/+ Changes in extracellular K in slices from MrgA1 mice exposed to FMRF (n = 9), slices from WT mice exposed to ATP (n = 9), and slices from either IP3R2 (n = −/− + 9) or IP3R2 (n = 6) mice exposed to UTP (*P < 0.05, n =6–9). (G) Changes in Bergmann glia membrane potential in slices from MrgA1 mice exposed to FMRF, +/+ −/− slices from WT mice exposed to ATP, and slices from either IP3R2 or IP3R2 mice exposed to UTP (*P < 0.05, n =6–9). (H) Scatter histogram displaying Bergmann glia membrane potential as a function of changes in extracellular K+. Data are combined from the agonists mentioned before: ATP, FMRF, and UTP (R2 = 0.486, n = 24).

changes in bath K+ (Fig. 4H, compare bottom axes) and the palisading Bergmann glial processes labeled more intensively depth of the recordings affected how much changes in bath K+ with rhod2 than the surrounding neuropil (Fig. 5A). Time-lapse changed extracellular K+ (Fig. S4). The most significant effect imaging documented that FMRF (15 μM) in MrgA1+ mice or on Purkinje cell bistability occurred at 3 mM extracellular K+, ATP (100 μM) in GLT1-EGFP mice triggered robust increases which is within the physiological concentration range (2). in Ca2+. The Ca2+ increases engaged all EGFP+ glial processes To address the alternative hypothesis that Bergmann glia (Fig. 5 A–C). Concurrent with the increase in Ca2+, extracellular modulate Purkinje cell bistability via Ca2+-mediated release of K+ decreased from 3.98 ± 0.05 mM to 3.56 ± 0.07 mM (n =5) gliotransmitters, we analyzed the effect of glutamatergic, in response to FMRF and from 3.91 ± 0.04 mM to 3.45 ± 0.05 GABAergic, and purinergic receptor antagonists on Purkinje cell mM for ATP (n = 7; Fig. 5 B and D). Thus, agonist-induced K+ bistability. Mixtures of glutamate, GABA, and purinergic in the live intact cerebellum was directly comparable to obser- receptors had no detectable effects on Purkinje cell bistability in vations in cerebellar slices (Fig. 3). Recording of cerebellar rat cerebellar slices (P > 0.5; Fig. S5). Moreover, the same ECoG revealed that agonist exposure triggered a transient de- combination of receptor antagonists did not significantly reduce crease in the power of low-frequency activity (≤4-Hz delta FMRF-induced changes in Purkinje cell bistability in slices pre- waves), whereas higher frequencies (>4–32 Hz) increased (Fig. pared from MrgA1+ mice (P > 0.5; Fig. S5), suggesting that 5E, Right) (22). Agonist-induced changes in ECoG were tran- FMRF-induced Ca2+ signaling is not associated with glio- sients as the relative power of slow and high-frequency activity transmitter release. This finding is consistent with a study that before and after the Ca2+ increases did not differ (Fig. 5 B and found that FMRF-induced Ca2+ signaling in hippocampal astro- C; P > 0.01, paired t test, n = 7 for ATP or FMRF). Thus, in vivo cytes did not result in gliotransmitter release (15). observations in adult mice (Fig. 5) replicated all of the major findings in cerebellar slices (Figs. 1–4). Agonist-Induced Bergmann Glial Ca2+ Signaling in Vivo Triggered a Reduction in Extracellular K+ and Increased Neural Activity. Berg- Discussion mann glia exhibit spontaneous Ca2+ events in vivo that are It is generally acknowledged that Ca2+ signaling in many brain expressed as highly localized Ca2+ increases within individual regions can modulate synaptic activity. We here describe that in processes or radially expanding waves encompassing up to ∼40 cerebellum, Ca2+ signaling in Bergmann glia transiently in- cells (3). The functional role of Ca2+ signaling in Bergmann glia creased their K+ uptake, reduced the extracellular concentration has not yet been established, although locomotion is linked to an of K+, and prolonged the duration of Purkinje cell up states that increased frequency of Ca2+ events (3). We here investigated the resulted in an increase in spike activity. By combining meas- physiological significance of agonist-induced Ca2+ increases in urements of extracellular potassium with two-photon imaging in Bergmann glia in vivo, as far as Purkinje cell bistability/slow genetically altered mice, we show that selectively stimulating Ca2+ oscillations are concerned. Acute cranial windows were prepared transients in Bergmann glia expressing MrgA1 receptors lowers over the vermis, and the cerebellar cortex bolus was loaded with extracellular K+ with concomitant increases in Purkinje cell up the Ca2+ indicator rhod2/am. Ca2+ imaging was combined with state (Fig. 1). The process is Ca2+-dependent as the agonist-in- recordings of cerebellar electrocortical activity (ECoG). To duced increase in Purkinje cell up state (Fig. 2) or reduction in identify processes of Bergmann glia, MrgA1 receptor mice extracellular K+ (Fig. 3) was minimal or absent when Bergmann expressing EGFP under the human GFAP promoter (15) or glia cells were unable to mobilize intracellular Ca2+ stores + reporter mice expressing EGFP under the glutamate transporter (IP3R2 deletion). Simply lowering extracellular K (Fig. 4) or 1 (GLT1) promoter (GLT1-EGFP) (21) were used. The EGFP+ depolarizing a single Bergmann glial cell (Fig. S3) replicated all of

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5 NEUROSCIENCE Time in upstate (%) Time Bath K+ (mM) Bath K+ (mM) 0 0 0 0 2.5 1 2.5 1 2.5 1 2.5 1 + + 3.0 3.5 4.0 3.0 3.5 4.0 4.5 (mM K ) (mM K+) (mM K ) (mM K+) 4.5 Slice K+ (mM) Slice K+ (mM)

Fig. 4. Purkinje cell bistability is modulated by extracellular K+ concentration. (A) Whole-cell recording of a Purkinje cell (PC) superfused with 2.5 mM or 1mMK+.(B) Distribution of membrane potential when superfused with 2.5 mM or 1 mM K. (C and D) Time spent in up state (C) and the Purkinje cell spiking frequency (D) in 2.5 and 1 mM K+ (**P < 0.01, n = 5). (E) ISI of action potential ﬁring analyzed during up state. (F) CV of action potential frequency. (G) Time in up state plotted as a function of increasing concentration of extracellular K+. The two x axes show K+ in the bath solution and the actual changes in extracellular K+ detected by a K+-sensitive microelectrode at 100 μm below the slice surface (n = 9). (Inset) Calibration of a double-barreled K+-sensitive microelectrode. (H) Membrane potential of up and down states in Purkinje cells plotted as a function of bath (upper trace) and extracellular K+ (lower trace) (n = 9). The Purkinje cell recordings were similar to all other recordings in this study obtained in neurons located 80–100 μm below the surface of the slice.

the effects of agonist-induced Ca2+ signaling, supporting the standing of how these electrically nonexcitable cells participate in notion that Bergmann glial K+ uptake provides a simple, yet cerebellar network activity. A recent publication observed, similar powerful, mechanism for modulation of Purkinje cell output. In to ﬁndings in this study, that stimulation of astrocytic Ca2+ sig- vivo experiments conﬁrmed that this glia–neuronal signaling naling in cortical slices triggered an increase in up states (13). pathway functions in the intact adult cerebellum because agonist- However, cortical astrocytes modulated bistability by release of induced Ca2+ signaling in Bergmann glia triggered a transient gliotransmitters, as P2 receptors antagonists increased, whereas drop in extracellular K+ concentration concomitant with a de- P1 (adenosine A1) receptor antagonists decreased up states. It is crease in low-frequency power (1–4 Hz) of ECoG (Fig. 5, Left). not surprising that the observations in cortex and cerebellum The ability of Bergmann glia to dynamically regulate extracellular differ (13). Although bistability in cerebellar Purkinje cells is gen- K+ in a Ca2+-dependent manner may be central to our under- erated by intrinsic membrane properties and depends on persis-

Fig. 5. Agonist-induced Ca2+ signaling in Berg- + A eGFP Rhod-2 B C mann glia reduces extracellular K and alters FMRF Before FMRF After 400 cerebellar ECoG in vivo. (A) Two-photon imaging ** ** 300 of EGFP and rhod2 in vermis of an adult MrgA1+ ECoG 200 mouse. FMRF exposure transiently increases 100 rhod2 signal in EGFP+ Bergmann glia processes. K+ 0 Single optical sections at 100 μmbelowthepial 1 min 0.5 mM Rhod-2 fluorescence Rhod-2 0.3 mV μ 100% After ATPAfter surface are shown. (Scale bar: 30 m.) (B)Cere- BeforeFMRF Before bellar ECoG combined with imaging of rhod2 + + D E signal and recordings of K with K -sensitive 1 μ 4.4 4.4 4 Hz 4-32 Hz microelectrodes at a depth of 100 m. (C) rhod2 0.8 0.8 4.2 4.2 ** ** ﬂuorescence emission before, during, and after 0.6 * 0.6 * + 4 4

exposure to FMRF (in MrgA1 mice) or to ATP. (D) (mM) (mM) ** 0.4 0.4 + 3.8 ** + 3.8 K Changes in extracellular K+ before, during, and K 0.2 0.2 3.6 3.6 after exposure to FMRF or ATP (P < 0.01, n =5–7). 0 0

Relative power spectra After ATP After Relative power spectra After ATP After After ATP After (E) Power spectrum analysis of ECoG. The rela- BeforeFMRF Before BeforeFMRF Before BeforeFMRF Before tive power of slow activity (≤4 Hz) decreased, whereas the relative power of higher frequencies (<4–32 Hz) increased during agonist-induced Ca2+ signaling (*P < 0.05, **P < 0.01, n =5–7).

Wang et al. PNAS Early Edition | 5of6 Downloaded by guest on October 2, 2021 tent currents (6, 7), neocortex is extensively interconnected by Implication of Ca2+-Dependent Glial K+ Buffering. Our results show recurrent excitatory collaterals, and bistability operates through that Bergmann glia cells have the ability to transiently boost their a balance between excitation and inhibition (8). Moreover, cer- K+ uptake capacity via receptor-mediated increases in cytosolic ebellar Purkinje cells are inhibitory neurons that do not express Ca2+. We have shown in a previous study that the Na+, adenosine A1 receptors (1, 23). It is, however, of interest that K+ATPase activity is increased during Ca2+ signaling in hippo- both studies concluded that glia Ca2+ signaling triggers an in- campal glia cells, resulting in a reduction in extracellular K+ crease in up states and may reflect that glia use different strategies (14). We speculate that a similar mechanism exists in cerebellum to modulate neural circuits depending on their location in CNS. and that the transient reduction of extracellular K+ hyper- Of note, our study excludes that gliotransmitter played a role in 2+ polarized Purkinje cells, which paradoxically prolonged up states. the increase in up states during Ca signaling in Bergmann glia An in vivo analysis in anesthetized mice confirmed that agonist- cells, because a broad range of receptor antagonists applied either induced Ca2+ signaling in Bergmann glia was linked to a transient individually or as a mixture failed to affect Bergmann glia mod- reduction in extracellular K+ and an increased duration of time ulation of bistability (Fig. S5). spent in up state (Fig. 5). We speculate that the increased spon- 2+ + taneous Ca signaling in nonanesthetized mice may cause a Extracellular K Modulates Purkinje Cell Bistability. During sleep and + persistent reduction in extracellular K that contributes to the anesthesia, the membrane potentials of Purkinje cells oscillate between up and down states (5), whereas Purkinje cells in awake lack of bistability in awake animals (24). If these observations can be generalized to other brain regions, our report provides a animals operate primarily in the up state (24). The bistability 2+ + persists without synaptic input and is also present in cerebellar framework for dissecting the role of Ca -dependent K uptake slices, suggesting that it is driven by intrinsic membrane prop- on synaptic transmission and neural network activity. Neuro- erties (6, 7). Similar to other neurons, it is thought that Purkinje modulators are released diffusely across most of the forebrain during arousal, and neuromodulators directly induce Ca2+ sig- cell bistability is generated by the balance between inward cur- 2+ + rents, consisting of T-type Ca2+ currents and/or persistent Na+ naling in cortical astrocytes (25). Ca -dependent K uptake in currents in combination with K+ leak currents, and hyperpolar- glia may therefore support the tonic up state characteristic of the ization-activated cation channels (6, 7, 12). awake state and affect functional networks on a global scale. The Nernst equation predicts that reducing extracellular K+ will result in membrane hyperpolarization by increasing the Materials and Methods + driving force for K leaking out of the cell. Why then, does a Full details of all methods are available in SI Materials and Methods. reduction in extracellular K+ lead to an increase in up states in Purkinje cells in both cerebellar slices and in vivo preparations? Animals. All slice experiments used cerebellum from 14- to 23-d-old Sprague- A recent study showed that hyperpolarization of Purkinje cells Dawley rat pups or C57BL/6 mouse pups (Charles River). paradoxically increased the duration of up states by activating Ih channels (12). However, it is unlikely that this mechanism is Two-Photon Laser Scanning Microscopy. A custom-built microscope attached responsible for the observations reported here because Ca2+ to a Tsunami/Millenium laser (10 W; Spectra-Physics) and scan box (FV300; signaling in Bergmann glia was linked to hyperpolarization of up Olympus) using FluoView software was used for two-photon laser scanning states and depolarization in down states (Figs. 1 and 2). A more microscopy. likely explanation is that the hyperpolarization of up states (Fig. 4H) decreased the activity of the Ca2+-dependent K+ channels, K+-Sensitive Microelectrodes. Ion-sensitive microelectrodes were fabricated which are necessary for driving the membrane potential back to from double-barreled pipette glass (PB150F-6; WPI). the down state. Conversely, the depolarization of down states increased T-type Ca2+ and Na+ channel activation and thereby ACKNOWLEDGMENTS. We thank Charles Nicholson and Sabina Hrabetova destabilized, and shortened, the duration of down state (10). In for technical assistance with fabrication of K+-sensitive microelectrodes; all, although a reduction in extracellular K+ results in membrane J. Chen, K. McCarthy, and J. Rothstein for generously sharing genetically modified mice; and Lane Bekar for valuable comments on the manuscript. hyperpolarization at up states and depolarization at down states, This study was supported by National Institutes of Health, National Institute the net effect is a stabilization and prolongation of up state of Neurological Disorders and Stroke Grants NS075177 and NS078304; the combined with depolarization and destabilization of down state. W. M. Keck Foundation; and the Dana Foundation.

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