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. 2018 Jan 25;9:370. doi: 10.1038/s41467-017-02723-6

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Activation of G_q-mediated signaling pathways in preBötC astrocytes facilitates the respiratory rhythm. a Schematic of AVV-sGFAP-DREADD_Gq-EGFP vector layout. b CNO-induced [Ca²⁺]_i responses in cultured astrocytes transduced to express DREADD_Gq. c The effect of CNO is blocked by the PLC inhibitor U73122. d Summary data illustrating PLC activity in cultured brainstem astrocytes expressing DREADD_Gq and the effects of CNO on PLC activity as assessed by measuring [³H]-inositol phosphate (InsP) production relative to the total inositol lipid pool in naive astrocytes and in astrocytes transduced to express DREADD_Gq or CatCh. Higher resting InsP level in brainstem astrocytes expressing DREADD_Gq indicate constitutive activity of the receptor. e Summary data illustrating the rate of spontaneous and CNO-induced fusion of quinacrine-labeled vesicular compartments in astrocytes transduced to express CatCh or DREADD_Gq. DREADD_Gq expression in astrocytes is associated with a significantly higher rate of spontaneous vesicular fusion events. In d and e, numbers of individual tests performed in three different cultures prepared from different animals are indicated. f–h Confocal images illustrating DREADD_Gq-EGFP expression in a subset of preBötC astrocytes (scale bars: 100 μm). DREADD_Gq expression is limited to astrocytes as no neurons (identified by NeuN immunoreactivity) expressed the transgene (see high-magnification inset images. Inset scale bars: 25 µm). i Representative example of changes in ATP biosensor current after biosensor placement on the surface of the brainstem slice transduced to express CatCh and DREADD_Gq in astrocytes residing in opposite preBötC areas. Asterisk (*) marks the movement deflection due to removing of the biosensor from the slice surface. j Summary data illustrating facilitated tonic release of ATP in acute brainstem slices of adult rats transduced to express DREADD_Gq by the preBötC astrocytes; k Group data showing the effect of DREADD_Gq expression in preBötC astrocytes on frequency distribution of all respiratory-related events and resting ƒ_R in conscious rats. Number of animals in each experimental group is indicated in parentheses. p values—Mann–Whitney U rank test (d, e, k) or Wilcoxon matched-pairs signed-rank test (j)