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. 2013 Jan 9;33(2):435-41.
doi: 10.1523/JNEUROSCI.2813-12.2013.

Differential sensitivity of brainstem versus cortical astrocytes to changes in pH reveals functional regional specialization of astroglia

Vitaliy Kasymov  1 , Olga Larina, Cinzia Castaldo, Nephtali Marina, Maxim Patrushev, Sergey Kasparov, Alexander V Gourine
Affiliations

Differential sensitivity of brainstem versus cortical astrocytes to changes in pH reveals functional regional specialization of astroglia

Vitaliy Kasymov et al. J Neurosci. .

Abstract

Astrocytes might function as brain interoceptors capable of detecting different (chemo)sensory modalities and transmitting sensory information to the relevant neural networks controlling vital functions. For example, astrocytes that reside near the ventral surface of the brainstem (central respiratory chemosensitive area) respond to physiological decreases in pH with vigorous elevations in intracellular Ca(2+) and release of ATP. ATP transmits astroglial excitation to the brainstem respiratory network and contributes to adaptive changes in lung ventilation. Here we show that in terms of pH-sensitivity, ventral brainstem astrocytes are clearly distinct from astrocytes residing in the cerebral cortex. We monitored vesicular fusion in cultured rat brainstem astrocytes using total internal reflection fluorescence microscopy and found that ∼35% of them respond to acidification with an increased rate of exocytosis of ATP-containing vesicular compartments. These fusion events require intracellular Ca(2+) signaling and are independent of autocrine ATP actions. In contrast, the rate of vesicular fusion in cultured cortical astrocytes is not affected by changes in pH. Compared to cortical astrocytes, ventral brainstem astrocytes display higher levels of expression of genes encoding proteins associated with ATP vesicular transport and fusion, including vesicle-associated membrane protein-3 and vesicular nucleotide transporter. These results suggest that astrocytes residing in different parts of the rat brain are functionally specialized. In contrast to cortical astrocytes, astrocytes of the brainstem chemosensitive area(s) possess signaling properties that are functionally relevant-they are able to sense changes in pH and respond to acidification with enhanced vesicular release of ATP.

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Figures

Figure 1.
Figure 1.
Vesicular fusion in cultured astrocytes detected using total internal reflection fluorescence microscopy. A, Incubation of cultured astrocytes with quinacrine (i) and MANT-ATP (ii) labels the same intracellular compartments. Astrocytes were transduced with an adenoviral vector to express DsRed (iii) under the control of a GFAP promoter. Scale bar, 10 μm. B, Cultured ventral brainstem astrocytes display [Ca2+]i oscillations in response to a decrease in pH from 7.4 to 7.0. To monitor Ca2+ responses, astrocytes were transduced to express a Ca2+-sensitive protein, Case12, under the control of GFAP promoter. C, Three-dimensional surface plots illustrating detection of a single vesicular fusion event as evident from a progressive lateral spread and decrease of fluorescence intensity visualized using TIRF microscopy (cultured brainstem astrocyte loaded with MANT-ATP). Images were acquired every 0.5 s.
Figure 2.
Figure 2.
Ventral brainstem astrocytes respond to acidification with an increased rate of exocytosis of putative ATP-containing vesicular compartments. A, B, TIRF images (left) and plots of TIRF intensity changes (middle) showing loss of quinacrine (A) or MANT-ATP (B) fluorescence from a proportion of labeled organelles in cultured ventral brainstem astrocytes in response to a decrease in external pH from 7.4 to 7.0. Plots on the right depict averaged temporal distribution of acidification-evoked fusion events detected in 10 quinacrine loaded (A) and 10 MANT-ATP loaded (B) pH-sensitive brainstem astrocytes. Scale bars, 10 μm; C, Higher basal (at pH 7.4) rate of vesicular fusion events in cultured cortical astrocytes is not affected by acidification. Plots on the bottom depict averaged temporal distribution of fusion events detected in cortical astrocytes exposed to (i) acidification of the external milieu (n = 10 cells) or (ii) application of ATP (10 μm; n = 8 cells). Scale bar, 10 μm. D, Summary data illustrating averaged temporal distributions of acidification-evoked fusion events detected in MANT-ATP-loaded brainstem astrocytes in the absence and presence of MRS2179 (30 μm), ATP degrading enzyme apyrase (25 U ml−1), or after 1 h incubation with a Ca2+-chelator BAPTA-AM (30 μm).
Figure 3.
Figure 3.
Ventral brainstem astrocytes respond to acidification with an enhanced endocytotic recovery of the granules. A, Confocal images of cultured DsRed-expressing brainstem astrocytes in control conditions (pH 7.4) and after stimulation with pH 7.0 (10 min) in the presence of 3 kDa dextran conjugated to Cascade Blue. B, Summary data showing the number of fluorescent puncta in the cytosol of ventral brainstem astrocytes stimulated with ATP or exposed to a decreased pH (7.0) in the absence and presence of brefeldin A (50 μm), MRS2179 (30 μm), or ATP-degrading enzyme apyrase (25 U ml−1). *p < 0.01 compared to the number of fluorescent puncta in control conditions at pH 7.4; #p < 0.01 compared to the number of fluorescent puncta at pH 7.4 and pH 7.0. C, Summary data showing the number of fluorescent puncta in the cytosol of cortical astrocytes exposed to a decreased pH (7.0) or stimulated with ATP. *p < 0.01 compared to the number of fluorescent puncta in control conditions at pH 7.4.
Figure 4.
Figure 4.
Differential expression of vesicular transport- and vesicular fusion-associated proteins in brainstem and cortical astrocytes. A, Immunohistochemical detection of VNUT in cultured ventral brainstem astrocytes. Scale bar, 10 μm. B, Relative transcription level of selected vesicular transport- and vesicular fusion-associated proteins in cultured brainstem versus cortical astrocytes. Inset, Relative mRNA level of VNUT in pH-sensitive versus pH-insensitive ventral brainstem astrocytes. *p < 0.01.
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