Fig. 1.

PreBötC astrocytes modulate the activity of the respiratory rhythm-generating circuits. a Schematic of AVV-sGFAP-dnSNARE-EGFP vector layout. b Summary data illustrating a reduction in the number of juxtamembrane quinacrine-labeled vesicular compartments in cultured brainstem astrocytes expressing dnSNARE. c Plots of TIRF intensity changes showing loss of quinacrine fluorescence from a proportion of labeled organelles in response to application of the Ca²⁺ ionophore ionomycin (1 μM) in two individual cultured astrocytes transduced to express control transgene (black traces) or dnSNARE (red traces). In cultures of astrocytes expressing dnSNARE, digitonin was applied at the end of the recording to permeabilize the membranes, resulting in a rapid loss of quinacrine fluorescence. d Averaged temporal profile of ionomycin-induced vesicular fusion events detected in cultured astrocytes expressing control transgene or dnSNARE. e Total number of ionomycin- and sodium dithionite-induced vesicular fusion events detected in individual cultured astrocytes expressing control transgene or dnSNARE. In b, d, and e, numbers of individual tests performed in three different cultures prepared from different animals are indicated. f Schematic drawings of the rat brain in parasagittal and coronal projections illustrating the location of the preBötC. NAsc, semi-compact division of the nucleus ambiguus; XII, hypoglossal motor nucleus. Representative confocal image of dnSNARE-EGFP expression in preBötC astrocytes is shown on the right (scale bar: 200 μm). High-magnification inset shows expression of dnSNARE-EGFP in GFAP-positive preBötC astrocytes (inset scale bar: 50 μm). NAsc neurons are identified by choline acetyltransferase (ChAT) immunoreactivity. VS, ventral surface of the brainstem. g, h Group data showing the effects of dnSNARE or TeLC expression in preBötC astrocytes on frequency distribution of all respiratory-related events detected in 30-min assay (top) and resting respiratory frequency recorded during periods of calm wakefulness and/or quiet sleep (ƒ_R, bottom) in conscious adult rats. In control animals preBötC astrocytes were transduced to express CatCh-EGFP. i Group data showing the effect of TMPAP expression in preBötC region on frequency distribution of the respiratory-related events and resting f_R in conscious rats. In g, h, and i, number of animals in each experimental group is indicated in parentheses. p values—Mann–Whitney U rank test