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Science 329 (5991): 571-575

Copyright © 2010 by the American Association for the Advancement of Science

Astrocytes Control Breathing Through pH-Dependent Release of ATP

Alexander V. Gourine1,*, Vitaliy Kasymov1, Nephtali Marina1, Feige Tang2, Melina F. Figueiredo2, Samantha Lane2, Anja G. Teschemacher2, K. Michael Spyer1, Karl Deisseroth3, and Sergey Kasparov2,*

1 Neuroscience, Physiology, and Pharmacology, University College London, London WC1E 6BT, UK.
2 Department of Physiology and Pharmacology, University of Bristol, Bristol BS8 1TD, UK.
3 Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.

Figure 1
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Fig. 1. Astrocytes residing near the VS are exquisitely pH-sensitive. (A) In vivo imaging of pH-evoked astrocytic [Ca2+]i responses in the ventrolateral area of the brainstem surface transduced with AVV-sGFAP-Case12 in an anesthetized adult rat. (Far right) Changes in VS astrocytic [Ca2+]i in response to a decrease in pH. (Left) Pseudocolored images were taken at times indicated by arrows. Squares indicate regions of interest. Here and elsewhere, the pH bar shows when the solution with lower pH is reaching and starts leaving the preparation. The dashed line outlines the approximate boundary of the RTN. py, pyramidal tract. (B) VS astrocytes identified by means of Case12 fluorescence in a horizontal slice from an adult rat in which the ventral medulla was transduced with AVV-sGFAP-Case12. Acidification induces rapid increases in [Ca2+]i as determined by changes in Case12 fluorescence. The two fluorescent images were obtained (left) before and (right) at the peak of [Ca2+]i response. The circle indicates an astrocyte responding first to pH change in the field of view. The yellow arrow shows the direction of the flow in the chamber. (C) Zoomed-in Ca2+ transients in order to emphasize the latency differences between responses of individual astrocytes shown in (B). (D) No effect of TTX or muscimol on acidification-induced [Ca2+]i responses in VS astrocytes expressed as percentage of the peak initial response. Numbers of individual astrocytes sampled from three to five separate experiments are given in brackets. (E) Acidification-evoked [Ca2+]i responses in VS astrocytes of organotypic brainstem slice transduced with AVV-sGFAP-Case12. The yellow arrow shows the direction of the flow in the chamber. (F) VS vasculature visualized with lectin in a horizontal slice prepared from an AVV-sGFAP-Case12–transduced rat. Arrows point at pH-responsive astrocytes.


Figure 2
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Fig. 2. Exocytotic release of ATP propagates pH-induced Ca2+ excitation among VS astrocytes. (A) A 0.2-unit decrease in pH induces sustained ATP release from the VS as detected with biosensors placed on the pia mater in horizontal slices prepared from adult rats. "netATP" trace represents the difference in signal between ATP and null (control) sensor currents. (B) Apyrase abolishes pH-evoked [Ca2+]i responses in VS astrocytes. Traces illustrate the effects of apyrase on pH-induced changes in Case12 fluorescence of six individual astrocytes (adult rat slice preparation). The decrease in signal is due to acidification-induced quenching of Case12 fluorescence. (C) The effect of MRS2179 on acidification-induced [Ca2+]i responses of eight individual VS astrocytes (organotypic brainstem slice). (D) Bafilomycin A abolishes pH-evoked Ca2+ excitation of VS astrocytes (five individual astrocytes in slice preparation of an adult rat). (E) The effects of apyrase, ATP receptor antagonists, mGlu1a and mGlu5 receptor antagonists (LY367385 and MPEP, 100 µM each), blockers of pannexin/connexin hemichannels and gap junctions lanthanum (100 µM) and carbenoxolone (CBX), or inhibitors of exocytotic mechanisms on acidification-induced [Ca2+]i responses in VS astrocytes expressed as the percentage of the initial response. Numbers of individual astrocytes sampled from three to five separate experiments are given in brackets (*P < 0.05).


Figure 3
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Fig. 3. ATP mediates responses of chemoreceptor neurons to decreases in pH or evoked by selective light-induced Ca2+ excitation of adjacent astrocytes. (A) (Left) Image of the ventral aspect of an organotypic brainstem slice showing EGFP-labeled Phox2b-expressing RTN neurons, one of which is patch clamped. (Right) Time-condensed record of the membrane potential of an RTN neuron responding to acidification in the absence and presence of MRS2179. AP, action potentials (truncated); R, resistance tests using current pulses. (B) Summary of MRS2179 effect on pH-evoked depolarizations in RTN neurons. (C) Effect of MRS2179 on acidification-induced [Ca2+]i responses of RTN neurons from two different experiments (ratiometric imaging using TN-XXL). (Inset) RTN neurons expressing TN-XXL. (D) Summary data showing significant effect of MRS2179 on pH-evoked [Ca2+]i responses of RTN neurons. (E) Layout of AVV-sGFAP-ChR2(H134R)-Katushka1.3. (F) Primary astrocytes displaying increases in [Ca2+]i in response to 470 nm light. (G) Ventral aspect of the organotypic slice showing a recorded DsRed2-labeled RTN neuron surrounded by ChR2(H134R)-Katushka1.3–expressing astrocytes. (H) Membrane potential of two different RTN neurons illustrating their responses to light activation of adjacent ChR2(H134R)-expressing astrocytes in the (left) absence, (middle) presence, or (right) after washout of MRS2179. (I) Effects of MRS2179 on depolarizations of RTN neurons evoked by optogenetic activation of neighboring astrocytes (*P < 0.05).


Figure 4
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Fig. 4. Optogenetic activation of VS astrocytes stimulates breathing in vivo. (A) Unilateral photostimulation of VS astrocytes expressing ChR2(H134R)-Katushka1.3 is sufficient to trigger respiratory activity from hypocapnic apnea in an anesthetized rat. Hypocapnic apnea was induced by means of mechanical hyperventilation to reduce arterial levels of PCO2/[H+] below the apneic threshold. IPNA, integrated phrenic nerve activity; TP, tracheal pressure; ABP, arterial blood pressure. (B) Lasting effect of light activation of VS astrocytes in an animal breathing normally. RR, respiratory rate. (1) and (2) indicate expanded traces of phrenic nerve activity before and after photostimulation of VS astrocytes. (C) Time-condensed record illustrating effects of repeated stimulations of VS astrocytes on phrenic nerve activity before and after a single application of MRS2179 (100 µM, 20 µl) on the VS. Spontaneous recovery of the response over time can be seen. (D) Summary data of MRS2179 effect on the increases in neural minute respiration (the product of phrenic frequency and amplitude) evoked by light activation of VS astrocytes (*P < 0.05). (E) Rostro-caudal distribution of astrocytes expressing ChR2(H134R)-Katushka1.3 in the brainstem of the rat from the experiment shown in (A). 7, facial nucleus; RTN, retrotrapezoid nucleus; C1, catecholaminergic cell group. (F) ChR2(H134R)-Katushka1.3 (Kat 1.3) expression in astrocytes is identified by red fluorescence distributed near the VS in close association with Phox2b-immunoreactive neurons (green nuclei). Shown is the coronal brainstem section. (G) Phox2b-expressing chemoreceptor RTN neurons (red nuclei) embedded in the astrocytic network (astrocytes were transduced with Case12 in this example so as to reveal their morphology).


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