www.sciencemag.org/cgi/content/full/science.

1190721/DC1

Supporting Online Material for

Astrocytes Control Breathing Through pH-Dependent Release of ATP

Alexander V. Gourine,* Vitaliy Kasymov, Nephtali Marina, Feige Tang, Melina F. Figueiredo, Samantha Lane, Anja G. Teschemacher, K. Michael Spyer, Karl Deisseroth, Sergey Kasparov*

*To whom correspondence should be addressed. E-mail: a.gourine@ucl.ac.uk (A.V.G.); sergey.kasparov@bristol.ac.uk (S.K.)

Published 14 July 2010 on Science Express DOI: 10.1126/science.1190721 This PDF file includes: Materials and Methods SOM Text Figs. S1 to S26 Table S1 References Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/science.1190721/DC1) Movies S1 to S5

SUPPORTING ONLINE MATERIAL

Astrocytes Control Breathing Through pH-dependent Release of ATP AV Gourine, V. Kasimov, N. Marina, F. Tang, M.F. Figueiredo, S. Lane, A.G. Teschemacher, K.M. Spyer, K. Deisseroth, S. Kasparov

1. Materials and Methods

All experiments were performed on Sprague-Dawley rats in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986. 1.1 Experimental models In vivo preparations. The rats were anesthetized with pentobarbitone sodium (initial dose 60 mg kg-1, i.p.; then 5-10 mg kg1h1, i.v.) Adequate anesthesia was ensured by maintaining stable levels of arterial blood pressure (ABP), heart and central respiratory rate showing lack of responses to a paw pinch. The femoral artery and vein were cannulated for measurement of ABP and administration of anesthetic, respectively. The trachea was cannulated, the animal was vagotomized and ventilated with a mixture of 50% oxygen and 50% nitrogen using a positive pressure ventilator with a tidal volume of ~2 ml and a ventilator frequency similar to the resting respiratory rate (~60 strokes min 1 ). The animal was then placed in a stereotaxic frame, and the ventral surface of the brainstem (VS) was exposed as described previously (S13). Activity of the phrenic nerve was recorded as an indicator of central respiratory drive. The signal was amplified (x 20,000), filtered (500-1500 Hz), rectified and smoothed ( = 50 ms). For imaging experiments the animal was paralyzed with gallamine triethiodide (50 mg kg -1, i.v.; then 10 mg kg-1h-1, i.v.). PO2, PCO2 and pH of the arterial blood were measured every 1-2 h. End-tidal levels of CO2 were monitored on-line using a fast-response CO2 analyzer and kept at a designated level by altering tidal volume and pump frequency. In all the experiments PO2 in the arterial blood was kept at >100 mmHg to reduce the drive from the peripheral chemoreceptors. The body temperature was maintained at 37.0 0.2C. Acute brainstem slices. Animals were killed by halothane inhalation overdose and the brainstem was quickly removed and placed in chilled (~4C) artificial cerebrospinal fluid (aCSF; 124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgSO4, 10 mM D-glucose saturated with 95% O2/ 5% CO2, pH 7.4) with an additional 10 mM Mg2+. The medulla was isolated and sequences of transverse (250 m) or horizontal (400 m) slices were cut. Once cut, the slices were stored before use in aCSF saturated with 95% O2 and 5% CO2 (pH 7.4) at room temperature. Recordings were made in a flow chamber at 35-37C from the slices placed on an elevated grid to permit access of aCSF from both sides of the slice. Organotypic cultured slices. Rat pups (810 days old) were killed with halothane inhalation overdose and transferred into a laminar flow hood. All procedures were performed aseptically. The brainstem was quickly removed and bathed in ice-cold Hanks Balanced Salt Solution (HBSS) without Ca2+ with added 20 mM glucose (total 25.6 mM),

10 mM MgCl2, 1 mM HEPES, 1 mM kynurenic acid, 0.005% phenol red, 100 U ml-1 penicillin, and 0.1 mg ml-1 streptomycin. The medulla was isolated and a sequence of transverse (250 m) slices was cut. Slices were inspected under a low magnification dissecting microscope and sections (2-3 per animal) containing the facial nucleus (a standard anatomical landmark for RTN area) were selected for plating. For the experiments on C1 neurons 3 sections caudal to the facial nucleus were selected. Millicell-CM organotypic culture membrane inserts were used for culturing which were covered with 1.1 ml plating medium containing 50% Optimem-1, 25% fetal bovine serum (FBS), 21.5% HBSS; 25 mM glucose, 100 U ml-1 penicillin, and 0.1 mg ml-1 streptomycin. After 3 days, the plating medium was removed and DMEM medium containing 10% FBS, 2 mM L-Glutamine, 100 U ml-1 penicillin, and 0.1 mg ml-1 streptomycin was added and subsequently replaced twice a week. Viral vectors (AVV-sGFAP- Case12, AVV-sGFAPChR2(H134R)-Katushka1.3, AVV-PRSx8-DsRed2, AVV-PRSx8-EGFP or AVV-SuperIPRSx8-TN-XXL) were added to the medium at the time of slice culture preparation at 5x1085x1010 transducing units ml-1. Experiments were performed after 7-10 days of incubation. Dissociated astrocyte culture. Primary astrocyte cultures were prepared from cerebral cortices, cerebellum and brainstem tissues of rat pups (P2-3) as described (S31). To prepare cultures of VS astrocytes, 500-m-thick brainstem slices were cut. Slices were inspected under a low magnification dissecting microscope and sections containing the facial nucleus were selected. A scalpel microblade was used to dissect the most ventral surface layer (300 m thick) ~ 0.5-2 mm lateral from the midline. These tissue cuts from 2-3 animals were used for primary astrocyte culture preparation. After isolation the cells were plated on poly-D-lysine-coated coverslips and AVV-sGFAP-Case12 was added to the incubation medium. 1.2 Viral gene transfer in vivo Young adult rats (~200 g) were anesthetized (ketamine, 60 mg kg-1 and medetomidine, 250 mg kg-1; i.m.) and placed in a stereotaxic frame. Ventral regions of the brainstem were targeted either unilaterally or bilaterally with microinjections of viral vectors (AVVsGFAP-ChR2(H134R)-Katushka1.3 or AVV-sGFAP-Case12, respectively) using the following coordinates from bregma: X, 1.8 mm; Y, -10.5 mm; Z, -9 mm. The second microinjection was delivered to each side of the medulla 1 mm caudally (Y, -11.5 mm). After microinjections the wound was sutured and anesthesia was reversed with atipemazole (1 mg kg-1). Immediate postoperative care was given and animals were left to recover for 7-10 days before the experiments to ensure a high level of transgene expression. 1.3 Optical imaging [Ca2+]i responses in VS astrocytes were visualized using a genetically encoded Ca 2+ indicator Case12 (S45) expressed in the brainstem via an adenoviral vector with enhanced glial fibrillatory acidic protein (GFAP) promoter (S26). Specificity of this promoter was confirmed using anti-GFAP immunostaining (fig. S1) and patch clamp recordings of the transduced cells (fig. S19). Simultaneous imaging of pH- and ATPevoked responses in the same VS astrocytes using Case12 and conventional Ca2+ indicator Fura-2 confirmed Ca2+ sensitivity and dynamic range of Case12 (fig. S2). Note that Case12 fluorescence declines towards the end of the pH challenge relative to that of Fura-2. This is a result of transient quenching of Case12 fluorescence caused by

acidification of the intracellular milieu. As with all other cyclically permutated GFP-based Ca2+ sensors, Case12 fluorescence quenches when pH decreases (S45). Therefore, in most cases cells imaged with Case12 display a reduction in baseline fluorescence clearly evident at the end of the pH stimulus or when the pH-evoked Ca2+ responses are blocked in zero Ca2+ media or in the presence of apyrase or bafilomycin A (Fig. 2b and 2d; fig. S10). These decreases in Case12 fluorescence are fully reversible. In vivo Ca2+ imaging using Case12 (Fig. 1a) was performed using a Leica fluorescence microscope and MiCam02 high-resolution camera. Brainstem surface transduced with AVV-sGFAP-Case12 was continuously superfused (4 ml min-1) with HEPES-buffered solution (HBS; 137 mM NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2, 1.0 mM MgSO4, 4.2 mM NaHCO3, 10 mM HEPES; pH 7.4-7.45) and bulk [Ca2+]i responses to a 0.2 unit pH decrease were recorded. As expected, a 0.2 unit decrease in pH on the VS of the brainstem always increased central respiratory drive (fig. S20). A large area of the brainstem was imaged at low magnification (Fig. 1a), therefore, ROIs in this case encompass multiple astrocytes which cannot be individually resolved under these imaging conditions. Hence the dynamic responses of single cells such as shown in Fig. 1b or Fig. 1e are not visible. Confocal imaging of pH-evoked Ca2+ responses in individual VS astrocytes transduced with AVV-sGFAP-Case12 was performed in brainstem slices of adult rats (Fig. 1b) and organotypic brainstem slice cultures (Fig. 1e). For imaging, a section of the membrane with the organotypic slice was cut out and placed on an elevated grid in a flow chamber (volume 2 ml). Acute horizontal slices containing ventral brainstem areas were prepared as described above. To visualize VS blood vessels, acute brainstem slices transduced with AVV-sGFAP-Case12 were incubated in aCSF containing TRITC labeled lectin (10 mg ml-1) and 0.7% Pluronic F127 for 30 min at 37C. Recordings were performed at 35-37C in HBS. The rate of perfusion was 4 ml min-1. Images were obtained using an upright confocal microscope with 40x water immersion objective. The 488 nm Argon laser line was used to excite Case12 fluorescence which was measured using a bandpass filter 505550nm. Illumination intensity was kept to a minimum (at 0.50.7% of laser output). For [Ca2+]i monitoring in RTN neurons using a genetically encoded indicator TN-XXL, organotypic slices were transduced with AVV-SuperI-PRSx8-TN-XXL as described above. The mechanism of Ca2+ detection by TN-XXL is based on Frster resonance energy transfer (FRET) between two genetically encoded fluorescent proteins, CFP and YFP. Binding of Ca2+ alters the protein conformation leading to an increase in YFP/CFP fluorescence ratio (S30). To enhance TN-XXL expression we used the recently developed IRES-based two step transcriptional amplification strategy (S2; S26). Cells were imaged using a confocal microscope. For excitation the 456 nm line of argon laser was used, while emission was sampled using 480-520 nm and 535-590 nm filters for CFP and YFP respectively. At the end of the recordings KCl (40 mM) was applied as a positive control to estimate a maximal [Ca2+]i increase during neuronal depolarization. There is a consensus that fluctuations in PCO2 are detected by the medullary chemosensors predominantly indirectly via changes in [H+] following the reaction of CO2 with water (S27; S40). Thus, to elicit chemosensory responses the same HEPES-buffered solution (composition is given above) with pH adjusted to 7.2 or 7.0 was applied. pH measurements on the VS of the brainstem slice in our perfusion system demonstrated a

0.15 unit pH decrease within 30 s from the moment when the solution with lower pH was expected to reach the chamber. For low magnification CCD imaging experiments (fig. S9) transverse medullary slices cut from the brains of 15 day-old rats were incubated in aCSF containing 10 M of the membrane-permeant Ca2+ sensitive dye Rhod-2 AM with the addition of 0.05% Pluronic F127 for 1 h in the dark at 37C. Ca2+ imaging was performed using a fluorescence microscope, equipped with a high-resolution CCD camera. The excitation filter was 520/20 nm, the emission filter was a 590 nm long-pass filter, and the dichroic mirror cutoff at 580 nm. The analogue of respiratory acidosis was induced by perfusing the chamber with aCSF (124 mM NaCl, 3 mM KCl, 2 mM CaCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, 1 mM MgSO4, 10 mM D-glucose) saturated with a gas mixture containing 7-8% CO2 (with the balance being O2) to reduce pH by ~0.2-0.3 units. Fluorescence measurements in dissociated cultures of VS astrocytes (fig S5) were performed using an epifluorescence inverted microscope equipped with a CCD camera, with a 20x oil immersion objective. Case12 fluorescence was excited at 490 nm and emitted fluorescence at 510-530 nm was collected. To visualize influx of Ca2+ ions, astrocytes were loaded with Fura-2 AM and incubated in the medium containing 50 M Mn2+. As the affinity of Fura-2 to Mn2+ is much higher than to Ca2+, and since the Fura-2-Mn2+ complexes are non-fluorescent at all wavelengths, a decrease of Fura-2 fluorescence would indicate an influx of Mn2+ via Ca2+-permeable channels (S32). Data were recorded at excitation wavelength at 360 nm (Ca2+-insensitive isobestic point) and emission signal at 510 nm (3 s time interval). 1.4 Measurement of ATP release ATP release (Fig. 2a) was measured using enzyme-based biosensors as previously described (S13). The sensors were aligned with the pyramidal tracts and placed in direct contact with the VS of the brainstem 0.2-0.5 mm lateral from the pyramidal tracts. The sensitive part of the sensor was ~2 mm in length placed over a significant portion of the brainstem surface. ATP and control sensors were placed in equivalent positions on the slice as judged by the distance from the pyramidal tracts and the remaining nerve roots. A luciferase assay was used to determine whether optogenetic activation of astrocytes induces release of ATP. Organotypic slices of rat pups were prepared and transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 as described above. After 7 days in culture, slices were placed into a tissue chamber and superfused with HBS at 34oC at 0.5 ml min-1. ATP levels in the outflow were measured before and after the stimulation using a luminometer and ATP Bioluminescent assay kit. To activate astrocytes, ventral aspects of the slice were illuminated with blue light (445 nm, 1 min, ~7 mW mm-2). 1.5 Generation and validation of AVV-sGFAP-ChR2(H134R)-Katushka1.3 AVV-sGFAP-ChR2(H134R)-Katushka1.3 was produced by homologous recombination as previously described (S6; S26). Adenoviral vector was chosen because astrocytes are highly susceptible to transduction by these vectors (S6). A compact transcriptionally amplified version of GFAP promoter - GfaABC1D was used to target astrocytes (S26). The layout of the vector is shown on Fig. 3e. Because the functionality of the ChR2 is critically

dependent on the level of expression we employed a two-step transcriptional amplification strategy (TSTA)(S26). TSTA relies on a positive feedback loop build into the vector which operates by co-expression of a chimeric transcriptional enhancer Gal4NFB. In this construct GfaABC1D operates in a bi-directional manner driving expression of both ChR2(H134R)-Katushka1.3 fusion and the enhancer. This results in a strong enhancement of GFAP activity which is weak otherwise. Transcriptional specificity of this system was demonstrated by previous studies (S24; S26) and further confirmed here (fig. S1 and S19). Katushka1.3 as fluorescent marker was chosen because it can be excited by green or yellow laser light (thus avoiding activation of ChR2) and has an emission peak at ~630 nm (S44; fig. S14b). This allowed identification of the cells expressing the construct (fig S14), monitoring Ca2+ responses with Rhod-2AM (peak emission at ~575 nm) using a yellow 561 nm laser line of a confocal microscope, and activation of ChR2 with an additional beam of blue light (fig S21). Validation and initial characterization of the new vector were performed in primary cultures of the rat astrocytes transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 and loaded with Rhod-2 AM (fig. S14a). The presence of both fluorophores in the same cell was ascertained using the spectral scan with 561 nm excitation (fig. S14b). In order to excite ChR2(H134R) with blue light (470 nm) and simultaneously image Rhod-2, the light path of the SP2 microscope was modified (fig. S21). Flashing blue light (LED 470 nm wavelength; 0.3-0.5 mW/mm2; 20/20 ms cycle) triggered robust increases in [Ca2+]i in all transduced astrocytes (Fig. 3f, fig. S15, S17). The profile of the response depended on the level of ChR2 expression, intensity and regime of stimulation. At low levels of both, increases in [Ca2+]i were readily reversible, while high level of transgene expression in >70% of astrocytes was associated with sustained Ca2+ responses lasting for more than 15 min (not shown). Similar results were obtained in acute slices of the adult rat brainstem transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 (fig. S16). Preliminary experiments indicated that 20/20 ms duty cycle of stimulation induces Ca2+ responses which are more rapid and better reversible in comparison to those induced by a continuous light. 1.6 Patch clamp recording of RTN neurons and astrocytes, optogenetic activation of astrocytes in cultured slices The primary population of central respiratory chemoreceptors is believed to be located near the VS, in particular, in the overlapping retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) (S34; S38). RTN neurons were identified by green or red fluorescence following transduction of organotypic slices with AVV-PRSx8-EGFP (Fig. 3a) or AVVPRSx8-DsRed2 (Fig. 3g), respectively. PRSx8 is active in RTN neurons because they characteristically express Phox2 the key transcriptional activator of the PRSx8 promoter (S1; S20). Organotypic slice cultures containing RTN were prepared as described above. Slices were placed into a recording chamber mounted under an upright confocal microscope and patch clamp recordings were made from fluorescent cells located near the VS (Fig. 3a, 3g). Slices were continuously superfused with HBS as described above. Neuronal activity was sampled and processed using a 1401 interface and CED Spike 2 software.

In the experiments involving light stimulation of astrocytes expressing ChR2, slices were co-transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 and AVV-PRSx8-DsRed2 (Fig. 3g). RTN neurons were visualized via DsRed2 because illumination of slices with blue light to identify EGFP-expressing neurons would inevitably excite astrocytes expressing ChR2. Therefore, we had to rely on cell size and morphology to distinguish between red fluorescent neurons and astrocytes in search of a suitable target for patching while using only yellow 561 nm light for excitation (Fig. 3g). Once patched, cells could be easily identified based on their electrophysiological characteristics. After a stable baseline had been recorded, an area surrounding the recording electrode was stimulated by flashing 470 nm light (20/20 ms duty cycle, 0.5-2 min). Light-activation of astrocytes evoked clear-cut depolarizations of all tested RTN neurons (n = 13; Fig 3h left trace). These astrocyte-evoked depolarizations of RTN neurons in some cases were very long lasting, with recovery time ranging 4-70 min (mean 28 9 min). Therefore, for the experiments with ATP receptor blockade we patched additional 8 RTN neurons where light stimulation was performed twice, first in the presence of MRS2179, and second, after 1 hour recovery and MRS2179 wash-out (Fig. 3h, right trace). To assess whether activation of RTN neurons is a stimulus which may trigger astrocytic Ca2+ excitation, RTN neurons were transduced to express DsRed2 and the adjacent astrocytes were transduced to express Case12 (fig. S7). RTN neurons (n=11) were activated by positive current pulses delivered via a patch pipette to generate spiking at a rate of 510 Hz for 2 min. Fluorescence in 83 adjacent Case12-expressing VS astrocytes from 11 separate experiments was monitored in time-lapse mode (fig. S7). 1.7 Light stimulation of VS astrocytes in vivo Animals in which the VS had been transduced with AVV-sGFAP-ChR2(H134R)Katushka1.3 were anesthetized and instrumented as described above. The VS was exposed using ventral approach and illuminated unilaterally with blue light (445 nm wavelength; 7 mW mm-2, 20/20 ms duty cycle) focused with a miniature objective over a designated area of the VS. To determine whether activation of VS astrocytes can trigger phrenic nerve activity from apnea, the latter was induced by mechanically hyperventilating the animal so that PCO2 in the arterial blood and end-tidal level of CO2 were below the apneic threshold (~30 mmHg and 2%, respectively). At the end of the experiments the rats were intracardially perfused with 4% paraformaldehyde, the brains were removed and stored in the same fixative overnight at 4oC. Medulla oblongata was isolated and a sequence of transverse (30 m) slices was cut and processed for immunohistochemical detection of Phox2b expression. Tissue was incubated in rabbit anti-Phox2b antibody for 24 h at 4C (1:800, generous gift of JF Brunet, Ecole Normale Suprieure, Paris, France) followed by a biotinylated goat anti-rabbit (1:500) antibody for 1 hour. Immunostaining was revealed after incubation with Fluorescein avidin DCS (1:250) for 1 hour. 1.8 Data analysis Built-in analysis software tools were used to analyze the results of the imaging experiments. Changes in Case12 and Rhod-2 fluorescence of individual cells or regions of interest were normalized and expressed as F/F0, where F0 is the initial fluorescence. Records from the patch clamp recordings and in vivo experiments were analyzed off-line using CED Spike 2 software. Astrocytic and neuronal Ca2+ responses, changes in

neuronal membrane potential and respiratory responses evoked by decreases in pH or optogenetic activation of astrocytes in the absence and presence of test drugs were compared using Students paired or unpaired t tests, as appropriate. A value of p < 0.05 was considered to be significant.

2. Supporting Text: Extended Discussion

2.1 Central Respiratory Chemosensitivity The nature of the central respiratory CO2 chemosensory mechanism has been under intense scrutiny for the past half century (S10; S15; S27; S40). It is a fundamental homeostatic mechanism because CO2 provides the major drive to breathe in mammals. Indeed, the mammalian central nervous circuitry that generates respiratory activity is silent in the absence of CO2 and requires CO2 to operate (see for example S39). Arterial PCO2 is also monitored by the peripheral chemoreceptors of the carotid and aortic bodies, however, up to 80% of the ventilatory responses to CO2 are preserved after their denervation (S17), indicating that the essential respiratory chemosensors are located within the CNS. The anatomical location of the sensors, their cellular identity and the molecular mechanisms underlying detection of rising levels of PCO2/[H+] remain controversial (S15; S36; S42). The primary population of CO2 chemoreceptors is believed to be located near the VS of the brainstem, in particular, in the overlapping retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG) (S34; S38). In addition, other regions of the brainstem such as the medullary raph nuclei, the pontine locus coeruleus and some others have been proposed to contain respiratory chemoreceptors (S36; S40; S42). Indeed, all these structures contain neurons that are activated by increases in PCO2/[H+] in vitro (S40). However, whether these chemoresponsive neurons are true functional respiratory chemosensors is as yet unresolved. 2.2 Astrocytes as Central Respiratory Chemosensors According to our data, glial cells may play an important role as primary central respiratory chemosensors. Astrocytes which reside at and in close proximity to the VS are capable of sensing fluctuations in arterial PCO2 via changes in pH and impart these changes into a modified pattern of breathing via ATP-mediated activation of chemoreceptor neurons. Acidification-evoked Ca2+ excitation of VS astrocytes and its propagation is highly unlikely to be initiated by local neurons. TTX and muscimol were used to minimize neuronal influences and failed to affect pH-evoked [Ca2+]i responses in VS astrocytes in any of the preparations (Fig. 1d, fig S6). Direct recordings from RTN neurons (the only known chemosensitive neurons in this area, see below) demonstrated, as expected, their effective silencing in the presence of these compounds (complete cessation of action potentials driven by current pulses 2-3 minutes after application of TTX (n=4), hyperpolarisation, decrease in input resistance and disappearance of EPSP after application of muscimol (n=5), data not shown). Thus, astrocytic responses to a decrease in pH must have been abolished or at least strongly reduced in the presence of TTX and muscimol if they were driven by neurons, but they were not. In addition, we directly demonstrate here that selective activation of RTN neurons by current injection via a patch pipette does not trigger significant [Ca2+]i elevations even in the immediately adjacent astrocytes (fig S7). Together these results effectively eliminate neurons, and RTN neurons specifically, as the primary pH sensors responsible for triggering [Ca 2+]i elevations in local astrocytes.

Strong pH-evoked astrocytic [Ca2+]i responses also remain in dissociated primary cell cultures prepared from the ventral regions of the medulla oblongata (fig. S5), but not in cortical astrocytes (fig. S8a). These results, taken together with the data from organotypic brainstem slices (Fig. 1e), also exclude a contribution of micro-vasculature to astrocytic pH-sensitivity because after a few days in vitro vascular elements degenerate completely. High pH-sensitivity appears to be a distinctive feature of astrocytes which reside at and in a close proximity to the VS and is not shared by astrocytes from at least one other region of the medulla oblongata. In our previous studies we failed to detect any significant release of ATP in response to CO2 elsewhere in the brainstem, apart from the VS (S13). In this study astrocytes from the dorsal nucleus of the solitary tract were unresponsive to acidification under the same experimental conditions (fig S8b) which triggered robust Ca2+ responses in VS astrocytes. Cortical astrocytes in primary cultures also appear to be insensitive to this stimulus (fig S8a). 2.3 ATP Release in Response to Chemosensory Stimulation Previously, using ATP biosensors placed in a direct contact with the VS pia mater in anesthetized rats we demonstrated that an increase in inspired CO 2 triggers an immediate release of ATP from the ventral medulla oblongata (S13). We now show that a 0.2 unit decrease in pH from its normal value of 7.4 is a sufficient stimulus to trigger ATP release (Fig 2a) which is largely responsible for propagation of astrocytic Ca2+ excitation. Indeed, the effect of exogenously applied ATP on [Ca2+]i in astrocytes residing in the marginal surface layer and immediately adjacent to it is very similar to the effect of lowering external pH (Fig. 2d, fig. S22). Several putative mechanisms of ATP release have been demonstrated in a variety of glial preparations, including exocytosis and the opening of gap junction hemichannels (S3; S5; S46; S51). Based on our pharmacological analysis we conclude that in response to acidification, VS astrocytes release ATP mainly through exocytosis (from conventional or unconventional compartments, see S16), while functional hemichannels, including connexins and pannexins, do not appear to be involved to a major extent. 2.4 ATP Receptors Responsible for Propagation of Astrocytic Ca2+ excitation In the presence of the ATP-hydrolyzing enzyme apyrase, pH-evoked Ca2+ excitation in VS astrocytes was reversibly abolished (Fig. 2b). Three different ATP receptor antagonists: MRS 2179 (3 M), PPADS (5 M) or TNP-ATP (20 nM) all markedly reduced (by about 80%) acidification-induced [Ca2+]i signals in these astrocytes (Fig. 2e). MRS2179, widely considered as selective antagonist of metabotropic P2Y1 receptors (IC50 = 0.3 M), is also effective at rat P2X1 (IC50 = 1.1 M) and able to inhibit P2X3 (IC50 = 12.9 M) receptors (S4; S21). PPADS is a broad spectrum antagonist of many ionotropic P2X and metabotropic P2Y receptors (S41), while TNP-ATP is a high-affinity (nanomolar range) antagonist of P2X receptors, particularly potent at rat P2X1 (IC50 = 6 nM), P2X3 (IC50 = 0.9 nM) and heteromeric P2X2/3 receptor assemblies (IC50 = 7 nM) (S50). This pharmacological profile suggests the involvement of ionotropic P2X receptors, likely to contain the P2X1 receptor subunit. Indeed, expression of P2X1 receptor subunit in astrocytes has been demonstrated recently (S22).

The involvement of ionotropic P2X receptors is also supported by the fact that acidification- as well as ATP-induced [Ca2+]i responses in astrocytes are absent in Ca2+free medium (with an addition of 2 mM Mg2+ and 1 mM EGTA to chelate any residual Ca2+) (figs. S10, S22). Furthermore, VS astrocytes loaded with Fura-2 AM and incubated in the medium containing 50 M Mn2+ showed an immediate decrease in fluorescence in response to ATP (n=8 slices; fig S23). As the affinity of Fura to Mn2+ is much higher than to Ca2+, and since the Fura-Mn2+ complexes are non-fluorescent, these observations directly demonstrate opening of Ca2+-permeable channels. 2.5 Dynamics of Acidification-Induced [Ca2+]i Responses in VS Astrocytes In the in vitro preparations used in this study (acute slices, organotypic cultured slices and dissociated cell cultures), astrocytes imaged using Case12 responded to acidification with the increases in [Ca2+]i which in many cases were not sustained for the duration of the stimulation. It is well known, however, that activation of chemosensitive brainstem neurons and respiratory activity is maintained during and for a short period after the termination of the hypercapnic stimulus, therefore, such a discrepancy warrants a discussion. In order to confirm Case12 specificity and sensitivity we simultaneously imaged pH- and ATP-evoked Ca2+ responses in the same VS astrocytes using Case12 and conventional Ca2+ indicator Fura-2 and found that changes in Case12 fluorescence faithfully follow changes in Fura-2 fluorescence (fig. S2). However, these experiments also demonstrated that towards the end of the pH stimulation period, Case12 fluorescence decreases. Intrinsic pH-sensitivity of Case12 is responsible for the reduction of Case12 fluorescence in experiments where pH is lowered for lengthy periods of time. Case12, as all other Ca2+ sensors based on cyclically permutated GFP, is sensitive to acidification and this becomes noticeable at pH 7.2 and below (S45). When HEPES buffered solution with lower pH is applied, decrease in intracellular pH is not immediate, thus allowing detection of cellular [Ca2+]i responses. If the stimulus is strong and applied for a longer period of time Case12 fluorescence decreases, as evident from many traces at the end of the pH stimulus or when the pH-evoked Ca2+ responses are blocked in zero Ca2+ media or in the presence of apyrase or bafilomycin A (Fig. 2b and 2d; fig. S10). When a conventional indicator (e.g. Rhod-2) is used, acidification-induced increases in [Ca2+]i on the VS are always sustained for the duration of the pH stimulation episode (fig. S6b). This is further confirmed by the in vivo experiments demonstrating that the lateral aspects of the VS, corresponding to the anatomical location of the RTN, display sustained astrocytic [Ca2+]i responses to changes in pH (Fig. 1a). Moreover, amperometric ATP biosensors detect an increase in ATP release from the VS in response to acidification which is also maintained during the whole period of a decreased pH (Fig. 2a). This suggests that the VS astrocytic network is activated for the duration of pH stimulation and confers this activation onto local chemoreceptor neurons which drive the increase in respiratory activity (see detailed discussion below). It is also conceivable, however, that on a longer time scale, other, possibly Ca2+ independent, mechanisms of ATP release could be engaged (S16)

10

2.6 The Initial Chemosensory Event Together the data reported and discussed here suggest very strongly that (i) VS astrocytes release ATP in response to a decrease in pH and (ii) [Ca2+]i responses detected in VS astrocytes are largely mediated by prior release of ATP. The most logical conclusion from these observations is that astrocytes release ATP in response to acidification and ATP amplifies and propagates this signal by acting on the same and adjacent astrocytes as well as local chemoreceptor neurons to produce an enhancement in the respiratory activity. Indeed, essentially the same sequence of events can be mimicked by optogenetic activation of astrocytes using ChR2 which brings about the key physiological outcome of central chemoreception an increase in respiratory activity. However, the key initial mechanism linking a decrease in pH with [Ca2+]i elevation and (presumably) exocytotic ATP release as yet remains obscure. In search of this primary mechanism we tested pharmacologically a possible involvement of several plausible pHsensitive mechanisms, including pH-sensitive K+, TRPV and TRPP channels (Table S1). None of the agents used and known to interfere with a range of these channels had any effect on pH-evoked [Ca2+]i responses in VS astrocytes (fig. S24 and Table S1). Our data also do not support the idea that membrane depolarization of astrocytes is the key initial event, since depolarization of VS astrocytes by >20 mV (significantly more in comparison to depolarizations reported to be evoked by a pH decrease (~5 mV) (S43) via a patch pipette had no significant effect on [Ca2+]i in the depolarized cell or neighboring astrocytes (fig. S25). Thus, the key initial pH chemosensory transduction event remains unknown and requires a wider screening of other potential targets. 2.7 ATP mediates pH-evoked Responses of Brainstem Respiratory Neurons and RTN Chemoreceptor Neurons ATP release is responsible not only for the spread of Ca2+ excitation among VS astrocytes it also imparts this astrocytic activation to the respiratory network, leading to a modified pattern of breathing. Indeed, robust astrocytic Ca2+ responses were detected in the areas located just beneath, and in a close proximity to, the brainstem respiratory rhythm and pattern generator. The stimulatory effects of ATP on the activity of the rhythm-generating circuits and individual medullary respiratory neurons have been demonstrated in several studies from our laboratory and others (S12; S13; S19; S28; S29; S49). Blockade of ATP receptors following microinjections of P2 antagonists into the ventral medulla or by direct application of the antagonists onto the VS markedly reduces CO2-evoked increases in breathing (S13; S48). At a single cell level, blockade of ATP receptors abolishes CO2-evoked excitation of individual medullary inspiratory neurons (S49). Interestingly, a recent study has demonstrated involvement of astroglia in purinergic modulation of the activity of the preBtzinger complex, an area directly involved in respiratory rhythmogenesis (S18). In this study we have also demonstrated that pH-evoked responses of Phox2bexpressing chemoreceptor neurons located in the RTN are largely mediated by prior release of ATP. RTN, which is adjacent to the VS, has been advocated to play a crucial role in central respiratory CO2/pH chemosensitivity (S7; S15). It contains a distinct population of Phox2b-expressing pH-sensitive neurons which either reside within the marginal glial layer or have extensive projections to it (Mulkey et al., 2004). Phox2bexpressing RTN neurons project to the brainstem respiratory network and their activation

11

leads to an increase in respiratory activity (S1). In this study we specifically targeted these neurons using AVV-PRSx8-EGFP, ADD-PRSx8-DsRed2 (to identify RTN neurons for patch clamp recordings) or AVV-SuperI-PRSx8-TN-XXL (to monitor their [Ca 2+]i). Although MRS2179 had no effect on acidification-induced responses of 2 cells (which were also insensitive to applied ATP; not shown), in the majority (85%) of Phox2bexpressing neurons located near the VS pH-evoked depolarizations were inhibited when ATP actions were blocked in the presence of MRS2179 or apyrase (Fig. 3a, 3b; fig. S13). MRS2179 also effectively abolished [Ca2+]i responses to decreases in pH in 9 additional RTN neurons imaged using TN-XXL (Fig. 3c and 3d). A previous study (S33) has failed to reveal such a role for ATP and, therefore, such a discrepancy warrants a detailed discussion. Mulkey et al (S33) did not specifically target Phox2b-expressing cells recordings were made from a sample of neurons located within the anatomical region of RTN in acute transverse slices prepared from neonatal rats. In our study RTN neurons were identified using adenoviral vectors AVV-PRSx8-EGFP or AVV-SuperI-PRSx8-TN-XXL (Phox2b is the key transcriptional activator of the PRSx8 promoter) in organotypic brainstem slices cut specifically at the level of RTN. Patch clamp or [Ca2+]i recordings were made from fluorescent cells located near the ventral edge of the slice immediately ventral to the facial nucleus. In our sample ventrally located neurons expressing EGFP or TN-XXL displayed profound pH-evoked responses. In electrophysiological experiments four neurons were successfully loaded with Rhod-2 via the patch pipette and two of these cells were found to contain galanin immunoreactivity (fig. S26) a peptide found in about 50% of all RTN chemosensitive neurons (S47). In addition, in a separate experiment we targeted EGFP-positive neurons in an anatomical location caudal to the facial nucleus (corresponding to the C1 group of Phox-2b-positive cells) and found that they are insensitive to changes in pH (n = 9; fig. S12) confirming results of a previous report (S23). Thus, we conclude with a high degree of confidence that the units recorded in our experiments are representative of the population of VS chemoreceptor Phox-2bexpressing RTN neurons. Electrical properties of units recorded in our experiments could be slightly different from those reported by Mulkey et al (S33; S34), due to differences in preparation, perfusion media and recording configurations. Mulkey et al (S33; S34) utilized a loose patch mode, which is semi-extracellular, i.e. without full dialysis of the cell with the pipette solution. Reported RTN neurons displayed slow spontaneous discharge (S33; S34) which in neurons recorded in our experiments often (although not always, see fig. S7) ceased after a few minutes as the cells became dialyzed and slightly hyperpolarized (quite common for whole cell recordings). However, depolarizations of RTN neurons in response to changes in pH in our experiments are very similar in magnitude to those reported by Ritucci et al (S43) from RTN neurons in acute slices of neonatal rats. There is another methodological reason to explain why the results of Mulkey et al. (S33) are not directly comparable with the data presented in our study. That study was conducted at room temperature where sensitivity of glial ATP-releasing mechanism to changes in pH would be expected to be significantly reduced. As in the Mulkey et al study (S33) ATP release was not demonstrated in conjunction with the effect of an antagonist (or the lack of such an effect) these data remain inconclusive. In support of the data presented here, Mulkey et al (S33) demonstrated that the RTN neurons are directly excited by ATP through P2Y receptors (MRS2179 is a potent antagonist of P2Y 1 receptors,

12

see above) and, therefore, are fully equipped to sense changes in local levels of ATP released in response to pH change by astrocytic networks in which these neurons are embedded (Fig 4g). At the same time, our results do not exclude the possibility of intrinsic pH-sensitivity of one or more types of neurons located elsewhere in the brainstem. 2.8 Activation of Astrocytes using ChR2: Ca2+ Responses and Physiological Effects In order to determine the functional significance of astrocytic Ca2+ excitation on breathing we have expressed a H134R mutant of ChR2 (which is more potent than the parent ChR2 protein (S14) selectively in astrocytes under the control of an enhanced compact GFAP promoter (S26). Ca2+ permeability of ChR2 expressed in Xenopus oocytes and HEK293 cells was documented previously (S25; S35). In our experiments we recorded precisely timed depolarizations (18.8 2.1 mV, n = 8; fig. S19) and strong Ca2+ signals in astrocytes expressing ChR2 in response to blue light at physiological levels of extracellular Ca2+ (Fig. 3f; fig. S15-S17). For several reasons ChR2-induced Ca2+ signals in astrocytes cannot be considered as an exact replica of the physiological Ca2+ excitation of these cells. Astrocytes do not operate using fast Na+-mediated action potentials and do not possess high density of voltage-gated Ca2+ channels. Indeed, although in astrocytes depolarization of the cell membrane exactly follows the light stimulation (fig. S19), changes in membrane potential per se do not appear to trigger increases in [Ca2+]i because depolarization of patched astrocytes from the ventral regions of the brainstem by >20 mV failed to elicit significant increases in [Ca2+]i in the same and adjacent astrocytes (fig S25). When external Ca2+ is removed, Ca2+ excitation of astrocytes in response to ChR2 activation with light is markedly reduced, indicating an influx of Ca2+ from the extracellular milieu (fig S17). However, even after 30 min in zero Ca2+ media these responses were not completely abolished (fig S17) suggesting that ChR2 activation in these cells also recruits Ca2+ from the internal stores via an as yet unknown mechanism. We demonstrate here directly that activation of astrocytes expressing ChR2 using light also results in the release of ATP (fig. S18). In patch clamp experiments, activation of the adjacent Phox2b-expressing RTN neurons is prevented in conditions of ATP receptor blockade (Fig. 3h, 3i). Importantly, in in vivo experiments we found that selective stimulation of ventral brainstem surface astrocytes using this approach activates breathing in an ATP-dependent manner and is also sufficiently powerful to induce strong respiratory activity even in those animals kept below apneic threshold by mechanical hyperventilation (Fig. 4a, 4c). One distinctive feature of ChR2 activation in astrocytes is the longevity of the evoked Ca2+ responses in many cases [Ca2+]i elevations in astrocytes significantly outlasted the duration of the light stimulation period (Fig. 3f; fig S15, S16). The profile of these Ca 2+ responses clearly depends on a combination of the level of expression of ChR2 and the intensity of light stimulation which is relatively easy to control in vitro but difficult in vivo. With lower level of expression and light intensity the Ca2+ responses following ChR2 stimulation tended to be quickly reversible, while stronger expression typically led to the lengthening of the response so that it could last for many minutes (e.g. fig. S16). It is also possible that ATP-induced ATP release could amplify and prolong these responses

13

under certain circumstances. The exact mechanisms responsible for long Ca2+ responses are currently under investigation and are beyond the scope of this study. Most importantly, we demonstrate here that the duration of light-induced Ca2+ responses in astrocytes expressing ChR2 matches long-lasting responses in the adjacent Phox2bexpressing neurons (see above) and the prolonged episodes of increased respiratory activity in in vivo rats following light stimulation of VS astrocytes (Fig. 4b). However, the use of light of moderate intensity to drive VS astrocytic excitations allows more precise control of respiratory activity as shown on Fig 4c and movie S5. 2.9 Summary More than 30 years ago Fukuda et al (S11) reported depolarization of brainstem surface silent cells in response to a decrease in pH. A few subsequent reports confirmed that surface glial cells do indeed respond to chemosensory challenges (S37; S43) and that pharmacological manipulation of glial function near the ventral medulla oblongata leads to changes in ventilation (S8; S9). The current study capitalizes on the previous evidence and demonstrates that astrocytes which reside at and in close proximity to the VS are highly chemosensitive and respond to increases in [H+] within a physiological range with robust elevations in [Ca2+]i. Mimicking acidification-induced Ca2+ responses by selective light-induced activation of these astrocytes induces depolarization of Phox2b-expressing RTN chemoreceptor neurons in an ATP-dependent manner and enhances central respiratory drive. This illustrates a potentially pivotal role of CNS astrocytes in a fundamental physiological mechanism the central respiratory chemoreceptor reflex and strengthens the case for the active involvement of astrocytes in informationprocessing within the brain. Acknowledgements We gratefully acknowledge B-H. Liu and J. Hewinson for their help with the preparation of viral vectors, J-F. Brunet (Dpartement de Biologie, Ecole Normale Suprieure, Paris, France) for providing us with the Phox2b antibody, O. Griesbeck (Martinsried, Germany) for providing TN-XXL clone, and B. King for expert advice on the pharmacology of P2 receptors.

14

SUPPORTING FIGURES

Fig. S1 Vector used to visualize Ca2+ responses in astrocytes. (A) Layout of the AVVsGFAP-Case12 expression cassette. This vector expresses Case12 under the control of a shortened version of GFAP promoter, GfaABC1D. mCMV operating in the antisense orientation drives expression of a chimeric transcriptional activator Gal4p65 while the specificity of expression in both directions is determined by GfaABC1D. (B) Case12 expression in dissociated culture, organotypic brainstem slices and following transduction of the adult rat ventral brainstem areas with AVV-sGFAP-Case12 in vivo. Note that Case12 concentrates mainly in the somata while GFAP accumulates in the astrocytic processes. Therefore, they mainly co-localize in cell processes and on rare occasions within the cell bodies. Bar = 20 m.

15

Fig. S2 Simultaneous imaging of pH- and ATP-evoked responses in the same ventral surface astrocytes using Case12 and conventional Ca2+ indicator Fura-2 to confirm Ca2+ sensitivity and dynamic range of Case12 (dissociated VS astrocyte culture). Note that following application of the acidic solution Case12 fluorescence is reduced due to its reversible quenching. This is a common feature of all sensors based on cyclically permutated green fluorescent protein.

16

A
20 m

B
20 m

VS VS
Fig. S3 (A) Ventral medullary astrocytes visualized by Case12 fluorescence. Arrows point at glia limitans. (B) Penetrating arteriole enwrapped by processes of astrocytes expressing Case12. Coronal brainstem sections (50 m) from 2 individual rats used for in vivo imaging experiments (see Fig 1a).

Fig. S4 A representative example of pH-evoked [Ca2+]i oscillations in VS astrocytes of organotypic brainstem slice culture transduced with AVV-sGFAP- Case12. Astrocytes imaged in vitro in some cases (~10%) responded to a decrease in pH with [Ca2+]i oscillations with gradually diminishing amplitude lasting for the whole duration of the stimulus. Traces illustrate pH-evoked changes in Case12 fluorescence of six individual astrocytes.

17

pH 7.4

pH 7.2

50 m

Fig. S5 Primary culture of VS astrocytes transduced with AVV-sGFAP-Case12. A decrease in pH from 7.4 to 7.2 induces rapid transient increases in [Ca2+]i as determined by changes in Case12 fluorescence.

A
Fig. S6 (A) TTX (1 M) does not prevent acidification-induced Ca2+ excitation of VS astrocytes. [Ca2+]i responses were measured as changes in Case12 fluorescence in four individual ventral surface astrocytes (slice preparation of an adult rat). (B) Extracellular acidification (decrease in pH from 7.4 to 7.0), in the presence of TTX (1 M) and muscimol (100 M) induces rapid, biphasic and sustained for the duration of the pH stimulation episode [Ca2+]i signals in Rhod-2 AM loaded VS cells. These cells are likely to be astrocytes as we have directly confirmed TTX- and muscimolinduced neuronal silencing (see text for details). Slice preparation of an adult rat (n=5).

18

Fig. S7 Activation of RTN neurons by injection of positive current fails to trigger significant [Ca2+]i elevations in adjacent astrocytes. For these experiments RTN neurones were targeted with AVV-PRSx8-DsRed2 to express red fluorescence and astrocytes were transduced with AVV-sGFAP-Case12. Using an SP2 confocal microscope, RTN neurones were identified and recorded in whole cell mode. Case12 fluorescence was monitored using the 488 nm laser line. Fluorescence intensities of astrocytes (upper image) adjacent to the patched neuron were monitored while the RTN neuron was induced to fire action potentials at 510 Hz for ~2 minutes by depolarizing current pulses delivered via the patch pipette (lower trace). Activation of the RTN neuron did not produce any substantial [Ca2+]i elevations in adjacent astrocytes. Some cells exhibited spontaneous changes in [Ca2+]i which are not timed with the activity of the neuron. Note that the neuron is not visible on this image because it is expressing DsRed2, while the system is tuned to monitor Case12 (green fluorescence, the image is pseudo-colored).

19

Fig. S8 Astrocytes of the cerebral cortex (A, dissociated cell culture) and the dorsal medullary nucleus of the solitary tract (B, slice preparation of the adult rat brainstem transduced with AVV-sGFAP-Case12 10 days prior to the experiment) are insensitive to acidification. Astrocytes were identified by Case12 fluorescence. Decrease in pH from 7.4 to 7.2 led to a reduction in Case12 signal (Case12 fluorescence quenches when pH decreases) in both preparations. Application of ATP (100 M) evoked profound elevations in [Ca2+]i in cortical and NTS astrocytes, an effect similar to that elicited by ATP in VS astrocytes.

pH 7.4

VS
300 m pH 7.15
Fig. S9 Low magnification Rhod-2 AM imaging in a coronal slice of the rat brainstem illustrating that CO2-evoked decrease in pH at constant [HCO3-] evokes robust [Ca2+]i responses originating and propagating near the ventral surface (see also associated movie S4).

20

Fig. S10 Acidification-evoked [Ca2+]i responses in VS astrocytes depend on extracellular Ca2+. Traces illustrate pH-induced responses of six individual ventral brainstem surface astrocytes in organotypic brainstem slice in control conditions and in the absence of external Ca2+.

Fig. S11 (A, B) Blockade of pannexin and connexin hemichannels with carbenoxolone (CBX, 10 M) and lanthanum (La3+, 100 M) had no effect on [Ca2+]i responses evoked in VS astrocytes by extracellular acidification (slice preparation of an adult rat). CBX is a broad-spectrum gap junction hemichannel blocker which at 10 M concentration inhibits pannexin, but not connexin hemichannels. La3+ is a connexin hemichannel blocker that does not affect gap junctions when applied externally. (C) CBX at concentration 100 M which blocks connexin hemichannels as well as gap junctions, reduces acidification-induced [Ca2+]i elevations in VS astrocytes by 43% (n=18 cells in 4 slices; p=0.0013).

21

Fig. S12 Catecholaminergic C1 neurons are insensitive to extracellular acidification. Since the PRSx8 promoter which was used to target RTN neurons is also active in catecholaminergic neurons located caudally to RTN we tested whether these cells are pHsensitive under the same experimental conditions. Slice cultures were made using sections caudal to RTN and AVV-PRSx8-EGFP-targeted neurons were patched and challenged with the pH stimulus following the same protocol used to study RTN neurons (see Fig 3, main text). R resistance tests using current pulses.

Fig. S13 ATP mediates responses of VS chemoreceptor neurons to decreases in pH. Time-condensed record of pH-evoked changes in the membrane potential of an RTN neuron in the absence and presence of ATP hydrolyzing enzyme apyrase (25 U ml -1). AP action potentials (truncated).

22

20 m

FI (AU)
160 140 120 100 80 60 40 20 0
570

Rhod-2
600

Katushka 1.3
630 660

Wave length (nm)

Fig. S14 (A) Primary astrocytes expressing ChR2(H134R)-Katushka1.3. (B) Spectrum of fluorescence emission of five individual astrocytes transduced with AVV-sGFAPChR2(H134R)-Katushka1.3 and loaded with Ca2+ indicator Rhod-2 AM. 561 nm laser light was used for excitation. Peaks of fluorescence of the two fluorophores are clearly separated allowing Ca2+ imaging using Rhod-2 within the 570-610 nm band.

23

Fig. S15 Repetitive stimulations of astrocytes lead to a build-up of the [Ca2+]i response. The same cells were stimulated 3 times using 20-sec long bursts of blue light (20/20 ms duty cycle). Stronger stimulation and high levels of ChR2(H134R) expression generated [Ca2+]i elevations which lasted for many minutes.

Fig. S16 Lasting Ca2+ responses triggered by flashing blue light in astrocytes transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 and loaded with Ca2+ indicator Rhod-2 AM. In this example rats were microinjected with AVV-sGFAP-ChR2(H134R)-Katushka1.3. Acute brainstem slices were prepared 7 days later, astrocytes were loaded with Rhod-2 and imaged using a confocal microscope.

24

Fig. S17 ChR2-induced [Ca2+]i elevation in astrocytes partially depends on extracellular Ca2+. Cultured astrocytes showing moderate level of ChR2(H134) expression were loaded with Rhod-2 AM. Different areas of the same dish were stimulated. In 2 mM external Ca2+, flashing blue light induced profound [Ca2+]i responses in all the transduced astrocytes. These responses were reduced following 10 min of incubation in zero Ca 2+ media. After 30 min of incubation in zero Ca2+ media, only small [Ca2+]i increases were elicited by light. This suggests that activation of ChR2 in astrocytes could also recruit Ca2+ from the internal stores.

25

Fig. S18 Release of ATP following illumination of VS areas in organotypic brainstem slices (n=6) transduced with AVV-sGFAP-ChR2(H134R)-Katushka1.3 as evident from a significant increase in ATP concentration in superfusate (p<0.05). RLU relative luminescence units.

Fig. S19 Patch clamp recording of an astrocyte expressing ChR2(H134R)Katushka1.3 showing immediate depolarization in response to blue light. Note that the membrane potential faithfully follows the light pulses.

Fig. S20 Raw data showing an increase in central respiratory drive in response to 0.2 unit pH decrease on the VS in in vivo imaging experiments illustrated in Fig 1a (anaesthetized and artificially ventilated rat transduced with AVV-sGFAP- Case12). IPNA integrated phrenic nerve activity.

26

Computer
1401 TTL

Scanhead
Detector 560-600 nm Laser 561 nm

5 mW 470 nm Laser diode

Microscope body

Additional dichroic mirror high pass 500 nm

Waterimmersion objective

ChR2 excitation

Emitted Ca2+ related fluorescence

2+

Rhod-2filled cell

Fig. S21 Optical path for confocal Ca2+ imaging and light activation of ChR2. Leica SP2 upright confocal microscope was modified as following: (i) the standard Hg lamp was replaced by a LED-based illumination system; (ii) an additional dichroic mirror was placed into the light path with the cut-off at 495 nm. This mirror reflected the light from the 470 nm LED onto the specimen while being transparent for both, the yellow 561 nm laser and the emitted red fluorescence. Thus, activation of ChR2 was possible while sampling Rhod-2 fluorescence with minimal interference.

27

Fig. S22 (A) Application of ATP mimics the effect of acidification on [Ca2+]i in VS astrocytes. Traces illustrate changes in Case12 fluorescence of four individual VS astrocytes in organotypic brainstem slice culture transduced with AVV-sGFAP- Case12. Two images illustrate ATP-evoked increases in Case12 fluorescence in VS astrocytes. (B) The dependence of ATP-evoked [Ca2+]i responses in VS astrocytes on extracellular Ca2+ (organotypic brainstem slice culture).

28

Fig. S23 ATP opens Ca2+-permeable channels in VS astrocytes as measured by Mn2+induced quenching of Fura-2 fluorescence. Experiments were performed in the presence of 2 mM Ca2+. Addition of Mn2+ (50 M) resulted in a minimal basal quenching of Fura-2 fluorescence signal. Application of ATP (100 M) evoked an immediate opening of Ca2+permeable channels and rapid quench of Fura-2 fluorescence. Digitonin (10 M) was added at the end of the recording to permeabilize cell membranes and obtain a maximum response.

Fig. S24 Summary data showing lack of an effect of various inhibitors of K + and TRP channels on acidification-induced Ca2+ responses in VS astrocytes expressed as percentage of the initial response (horizontal brainstem slice preparations of adult rats transduced with AVV-sGFAP-Case12). Numbers of individual astrocytes sampled from 3-5 separate experiments are given in parentheses. 4-AP, 4-Aminopyridine; TEA, tetraethylammonium. Concentrations of individual drugs are given in Table S1.

29

Fig. S25 Depolarization of VS astrocytes by positive current injections fails to trigger significant [Ca2+]i elevations in the recorded and adjacent astrocytes. For these experiments astrocytes were targeted with AVV-sGFAP-Case12, some of them (arrow) were patched and recorded in whole cell mode. Case12 fluorescence in the same and adjacent astrocytes was monitored using the 488 nm laser line (image on the left, pseudocolor). Fluorescence intensities of astrocytes (upper traces) adjacent to the patched astrocyte (arrow) were monitored while the cell was depolarized by as much as 100 mV by positive current injection via a patch pipette (not visible on this image as it is not fluorescent, position indicated by white arrow). Even with this non-physiological depolarization only a few adjacent astrocytes responded with mild increases in [Ca 2+ ]i. When a weaker stimulus was used (~20 mV) no Ca2+ responses were observed (5 patched astrocytes, responses were monitored in 42 adjacent cells, p>0.1, data not shown).

Fig. S26 Galanin-immunoreactivity of a pH-sensitive RTN neuron identified by EGFP-expression in the organotypic slice culture and loaded with Rhod-2.

30

Supporting Table S1
Effect on resting [Ca2+]i in VS astrocytes Increases [Ca2+]i no no no Effect on pH-evoked [Ca2+]i responses no no no no

Compound

Action

Concentration

4-Aminopyridine AMG9810 Amiloride Halothane

Isoflurane

Ruthenium Red Tetraethylammonium

Blocker of voltagedependent K+channels Potent TRPV1 receptor antagonist Na+ channel blocker, TRPP3 channel blocker Volatile anesthetic; inhibits NMDA receptors, Activates GABAA receptors and select 2-pore K+ channels Volatile anesthetic; inhibits NMDA receptors, Activates GABAA receptors and select 2-pore K+ channels Blocker of TRPV channels Non-selective K+ channel blocker

0.5-1 mM 30 nM 0.5 mM 2 mM

2 mM

induces [Ca2+]i oscillations

no

30 M 25 mM

no no

no no

31

Supporting References

S1.

Abbott SB, Stornetta RL, Fortuna MG, Depuy SD, West GH, Harris TE and Guyenet PG. Photostimulation of retrotrapezoid nucleus Phox2b-expressing neurons in vivo produces long-lasting activation of breathing in rats. J Neurosci 29: 5806-5819, 2009. Benzekhroufa K, Liu BH, Teschemacher AG and Kasparov S. Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy. Gene Ther 16: 681-688, 2009. Bowser DN and Khakh BS. Vesicular ATP is the predominant cause of intercellular calcium waves in astrocytes. J Gen Physiol 129: 485-491, 2007. Brown SG, King BF, Kim YC, Jang SY, Burnstock G and Jacobson KA. Activity of novel adenine nucleotide derivatives as agonists and antagonists at recombinant rat P2X receptors. Drug Development Research 49: 253-259, 2000. Coco S, Calegari F, Pravettoni E, Pozzi D, Taverna E, Rosa P, Matteoli M and Verderio C. Storage and release of ATP from astrocytes in culture. J. Biol. Chem. 278: 1354-1362, 2003. Duale H, Kasparov S, Paton JFR and Teschemacher AG. Differences in transductional tropism of adenoviral and lentiviral vectors in the rat brainstem. Exp Physiol 90: 71-78, 2005. Dubreuil V, Ramanantsoa N, Trochet D, Vaubourg V, Amiel J, Gallego J, Brunet JF and Goridis C. A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons. Proc Natl Acad Sci U S A 105: 1067-1072, 2008. Erlichman JS, Hewitt A, Damon TL, Hart M, Kurascz J, Li A and Leiter JC. Inhibition of monocarboxylate transporter 2 in the retrotrapezoid nucleus in rats: a test of the astrocyte-neuron lactate-shuttle hypothesis. J Neurosci 28: 4888-4896, 2008. Erlichman JS, Li A and Nattie EE. Ventilatory effects of glial dysfunction in a rat brain stem chemoreceptor region. J Appl Physiol 85: 1599-1604, 1998.

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S3. S4.

S5.

S6.

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S8.

S9.

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