Astrocytes control breathing through pH-dependent release of ATP

Abstract

Astrocytes provide structural and metabolic support for neuronal networks, but direct evidence demonstrating their active role in complex behaviors is limited. Central respiratory chemosensitivity is an essential mechanism that, via regulation of breathing, maintains constant levels of blood and brain pH and partial pressure of CO2. We found that astrocytes of the brainstem chemoreceptor areas are highly chemosensitive. They responded to physiological decreases in pH with vigorous elevations in intracellular Ca2+ and release of adenosine triphosphate (ATP). ATP propagated astrocytic Ca2+ excitation, activated chemoreceptor neurons, and induced adaptive increases in breathing. Mimicking pH-evoked Ca2+ responses by means of optogenetic stimulation of astrocytes expressing channelrhodopsin-2 activated chemoreceptor neurons via an ATP-dependent mechanism and triggered robust respiratory responses in vivo. This demonstrates a potentially crucial role for brain glial cells in mediating a fundamental physiological reflex.

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. Right traces: changes in VS astrocytic [Ca²⁺]_i in response to a decrease in pH. Pseudocolored images (left) were taken at times indicated by blue 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. Dashed line outlines approximate boundary of the RTN. py – pyramidal tract. (b) VS astrocytes identified by 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. Two fluorescent images obtained before and at the peak of [Ca²⁺]_i response. Circle indicates an astrocyte responding first to pH change in the field of view. Yellow arrow shows the direction of the flow in the chamber. (c) Zoomed in Ca²⁺ transients 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 3-5 separate experiments are given in brackets. (e) Acidification-evoked [Ca²⁺]_i responses in VS astrocytes of organotypic brainstem slice transduced with AVV-sGFAP-Case12. (f) VS vasculature visualized with lectin in a horizontal slice prepared from an AVV-sGFAP-Case12–transduced rat. Arrows point at pH-responsive astrocytes.

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. “net ATP” 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). Note the dip in fluorescence 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, P2 receptor antagonists, mGlu_1a 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 3-5 separate experiments are given in brackets (*p < 0.05).

Fig. 3. ATP mediates responses of chemoreceptor neurons to decreases in pH or following 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 absence (left), presence (middle) or after washout (right) of MRS2179. (i) Effects of MRS2179 on depolarizations of RTN neurones evoked by optogenetic activation of neighboring astrocytes (*p<0.05).

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 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. (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. Note spontaneous recovery of the response over time. (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). Coronal brainstem section. (g) Phox2b-expressing chemoreceptor RTN neurons (red nuclei) embedded in the astrocytic network (astrocytes are transduced with Case12 in this example to reveal their morphology).