Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca2+-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca2+ elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2. 1. Introduction The morphology and the location of astrocytes place them in a unique position to be able to listen and respond to neuronal activity [1–5]. Astrocytes express a wide variety of functional neurotransmitter receptors essential for sensing neuronal activity [6]. Many of these receptors are G-protein-coupled receptors (GPCRs) that, upon activation, stimulate phospholipase C and form inositol (1,4,5)-triphosphate (IP3) which increases the intracellular calcium (Ca2+) concentration through the release of Ca2+ from intracellular stores [6]. The intracellular cascade resulting in Ca2+ rise in astrocytes is the main mechanism these cells use to transduce synaptic activity. It is well established that the GPCR- mediated Ca2+ variations in astrocytes can trigger release of chemical substances [7, 8] such as excitatory amino acids (D-serine, glutamate) [2, 9, 10], ATP, and related nucleotides and nucleosides [11–13] or proinflammatory mediators such as eicosanoids (prostaglandins or PG) [2, 14] and tumor necrosis
References
[1]
L. Pasti, A. Volterra, T. Pozzan, and G. Carmignoto, “Intracellular calcium oscillations in astrocytes: a highly plastic, bidirectional form of communication between neurons and astrocytes in situ,” The Journal of Neuroscience, vol. 17, no. 20, pp. 7817–7830, 1997.
[2]
P. Bezzi, G. Carmignoto, L. Pasti et al., “Prostaglandins stimulate calcium-dependent glutamate release in astrocytes,” Nature, vol. 391, no. 6664, pp. 281–285, 1998.
[3]
A. Araque, E. D. Martín, G. Perea, J. I. Arellano, and W. Bu？o, “Synaptically released acetylcholine evokes Ca2+ elevations in astrocytes in hippocampal slices,” The Journal of Neuroscience, vol. 22, no. 7, pp. 2443–2450, 2002.
[4]
G. Perea and A. Araque, “Properties of synaptically evoked astrocyte calcium signal reveal synaptic information processing by astrocytes,” The Journal of Neuroscience, vol. 25, no. 9, pp. 2192–2203, 2005.
[5]
M. Santello, P. Bezzi, and A. Volterra, “TNFα controls glutamatergic gliotransmission in the hippocampal dentate gyrus,” Neuron, vol. 69, no. 5, pp. 988–1001, 2011.
[6]
A. Verkhratsky, R. K. Orkand, and H. Kettenmann, “Glial calcium: homeostasis and signaling function,” Physiological Reviews, vol. 78, no. 1, pp. 99–141, 1998.
[7]
P. Bezzi and A. Volterra, “A neuron-glia signalling network in the active brain,” Current Opinion in Neurobiology, vol. 11, no. 3, pp. 387–394, 2001.
[8]
A. Volterra and J. Meldolesi, “Astrocytes, from brain glue to communication elements: the revolution continues,” Nature Reviews Neuroscience, vol. 6, no. 8, pp. 626–640, 2005.
[9]
J. Marchaland, C. Calì, S. M. Voglmaier et al., “Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes,” The Journal of Neuroscience, vol. 28, no. 37, pp. 9122–9132, 2008.
[10]
M. T. Martineau Shi, J. Puyal, A. M. Knolhoff et al., “Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission,” The Journal of Neuroscience, vol. 33, no. 8, article U3605, pp. 3413–3423, 2013.
[11]
T. Liu, L. Sun, Y. F. Xiong et al., “Calcium triggers exocytosis from two types of organelles in a single astrocyte,” The Journal of Neuroscience, vol. 31, no. 29, pp. 10593–10601, 2011.
[12]
C. Verderio and M. Matteoli, “ATP in neuron-glia bidirectional signalling,” Brain Research Reviews, vol. 66, no. 1-2, pp. 106–114, 2011.
[13]
M. Oya, T. Kitaguchi, Y. Yanagihara et al., “Vesicular nucleotide transporter is involved in ATP storage of secretory lysosomes in astrocytes,” Biochemical and Biophysical Research Communications, vol. 438, no. 1, pp. 145–151, 2013.
[14]
Sanzgiri, R. P. A. Araque, and P. G. Haydon, “Prostaglandin stimulates glutamate receptor-dependent astrocyte neuromodulation in cultured hippocampal cells,” Journal of Neurobiology, vol. 41, no. 2, pp. 221–229, 1999.
[15]
P. Bezzi, M. Domercq, L. Brambilla et al., “CXCR4-activated astrocyte glutamate release via TNFa: amplification by microglia triggers neurotoxicity,” Nature Neuroscience, vol. 4, no. 7, pp. 702–710, 2001.
[16]
D. Rossi, L. Brambilla, C. F. Valori et al., “Defective tumor necrosis factor-α-dependent control of astrocyte glutamate release in a transgenic mouse model of Alzheimer disease,” The Journal of Biological Chemistry, vol. 280, no. 51, pp. 42088–42096, 2005.
[17]
M. Domercq, L. Brambilla, E. Pilati, J. Marchaland, A. Volterra, and P. Bezzi, “P2Y1 receptor-evoked glutamate exocytosis from astrocytes—control by tumor necrosis factor-α and prostaglandins,” The Journal of Biological Chemistry, vol. 281, no. 41, pp. 30684–30696, 2006.
[18]
D. Rossi, F. Martorana, and L. Brambilla, “Implications of gliotransmission for the pharmacotherapy of CNS disorders,” CNS Drugs, vol. 25, no. 8, pp. 641–658, 2011.
[19]
D. Stellwagen and R. C. Malenka, “Synaptic scaling mediated by glial TNF-α,” Nature, vol. 440, no. 7087, pp. 1054–1059, 2006.
[20]
G. G. Turrigiano, “The self-tuning neuron: synaptic scaling of excitatory synapses,” Cell, vol. 135, no. 3, pp. 422–435, 2008.
[21]
E. C. Beattie, D. Stellwagen, W. Morishita et al., “Control of synaptic strength by glial TNFα,” Science, vol. 295, no. 5563, pp. 2282–2285, 2002.
[22]
D. Stellwagen, E. C. Beattie, J. Y. Seo, and R. C. Malenka, “Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-α,” The Journal of Neuroscience, vol. 25, no. 12, pp. 3219–3228, 2005.
[23]
D. Zenisek, J. A. Steyer, M. E. Feldman, and W. Almers, “A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells,” Neuron, vol. 35, no. 6, pp. 1085–1097, 2002.
[24]
S. M. Voglmaier, K. Kam, H. Yang, et al., “Distinct endocytic pathways control the rate and extent of synaptic vesicle protein recycling,” Neuron, vol. 51, no. 1, pp. 71–84, 2006.
[25]
S. Chevlet, P. Bezzi, R. Ivarsson et al., “Tomosyn-1 is involved in a post-docking event required for pancreatic β-cell exocytosis,” Journal of Cell Science, vol. 119, no. 14, pp. 2912–2920, 2006.
[26]
C. Calì, J. Marchaland, R. Regazzi, and P. Bezzi, “SDF 1-alpha (CXCL12) triggers glutamate exocytosis from astrocytes on a millisecond time scale: imaging analysis at the single-vesicle level with TIRF microscopy,” Journal of Neuroimmunology, vol. 198, no. 1-2, pp. 82–91, 2008.
[27]
A. Bergeron, P. Bezzi, and R. Regazzi, “Analysis of synaptic-like microvesicle exocytosis of beta-cells using a life imaging technique,” PloS ONE. In press.
[28]
S. M. Voglmaier and R. H. Edwards, “Do different endocytic pathways make different synaptic vesicles?” Current Opinion in Neurobiology, vol. 17, no. 3, pp. 374–380, 2007.
[29]
P. Bezzi, V. Gundersen, J. L. Galbete et al., “Astrocytes contain a vesicular compartment that is competent for regulated exocytosis of glutamate,” Nature Neuroscience, vol. 7, no. 6, pp. 613–620, 2004.
[30]
L. H. Bergersen and V. Gundersen, “Morphological evidence for vesicular glutamate release from astrocytes,” Neuroscience, vol. 158, no. 1, pp. 260–265, 2009.
[31]
S. Coco, F. Calegari, E. Pravettoni et al., “Storage and release of ATP from astrocytes in culture,” The Journal of Biological Chemistry, vol. 278, no. 2, pp. 1354–1362, 2003.
[32]
I. Prada, J. Marchaland, P. Podini et al., “REST/NRSF governs the expression of dense-core vesicle gliosecretion in astrocytes,” The Journal of Cell Biology, vol. 193, no. 3, pp. 537–549, 2011.
[33]
P. Bezzi and A. Volterra, “Monitoring exocytosis in astrocytes with total internal reflection fluorescence microscopy,” in Optical Imaging Neurons: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2011.
[34]
B. Borgonovo, E. Cocucci, G. Racchetti, P. Podini, A. Bachi, and J. Meldolesi, “Regulated exocytosis: a novel, widely expressed system,” Nature Cell Biology, vol. 4, no. 12, pp. 955–962, 2002.
[35]
M. Park, J. M. Salgado, L. Ostroff et al., “Plasticity-induced growth of dendritic spines by exocytic trafficking from recycling endosomes,” Neuron, vol. 52, no. 5, pp. 817–830, 2006.
[36]
V. Ralevic and G. Burnstock, “Receptors for purines and pyrimidines,” Pharmacological Reviews, vol. 50, no. 3, pp. 413–492, 1998.
[37]
S. R. Fam, C. J. Gallagher, and M. W. Salter, “P2 purinoceptor-mediated Ca2+ signaling and Ca2+ wave propagation in dorsal spinal cord astrocytes,” The Journal of Neuroscience, vol. 20, no. 8, pp. 2800–2808, 2000.
[38]
A. Verkhratsky, “Calcium signalling between neurones and glia,” European Journal of Neuroscience, vol. 10, pp. 322–322, 1998.
[39]
N. Stella, M. Tencé, J. Glowinski, and J. Prémont, “Glutamate-evoked release of arachidonic acid from mouse brain astrocytes,” The Journal of Neuroscience, vol. 14, no. 2, pp. 568–575, 1994.
[40]
W. L. Smith and L. J. Marnett, “Prostaglandin endoperoxide synthase: structure and catalysis,” Biochimica et Biophysica Acta, vol. 1083, no. 1, pp. 1–17, 1991.
[41]
V. Parpura, B. J. Baker, M. Jeras, and R. Zorec, “Regulated exocytosis in astrocytic signal integration,” Neurochemistry International, vol. 57, no. 4, pp. 451–459, 2010.
[42]
Q. Zhang, M. Fukuda, E. Van Bockstaele, O. Pascual, and P. G. Haydon, “Synaptotagmin IV regulates glial glutamate release,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 25, pp. 9441–9446, 2004.
[43]
D. Crippa, U. Schenk, M. Francolini et al., “Synaptobrevin2-expressing vesicles in rat astrocytes: insights into molecular characterization, dynamics and exocytosis,” The Journal of Physiology, vol. 570, no. 3, pp. 567–582, 2006.
[44]
T. Pangr？i？, M. Potokar, M. Stenovec et al., “Exocytotic release of ATP from cultured astrocytes,” The Journal of Biological Chemistry, vol. 282, no. 39, pp. 28749–28758, 2007.
[45]
C. M. Anderson and M. Nedergaard, “Astrocyte-mediated control of cerebral microcirculation,” Trends in Neurosciences, vol. 26, no. 7, pp. 340–344, 2003.
[46]
P. G. Haydon and G. Carmignoto, “Astrocyte control of synaptic transmission and neurovascular coupling,” Physiological Reviews, vol. 86, no. 3, pp. 1009–1031, 2006.
[47]
P. Jourdain, L. H. Bergersen, K. Bhaukaurally et al., “Glutamate exocytosis from astrocytes controls synaptic strength,” Nature Neuroscience, vol. 10, no. 3, pp. 331–339, 2007.
[48]
P. Ramamoorthy and M. D. Whim, “Trafficking and fusion of neuropeptide Y-containing dense-core granules in astrocytes,” The Journal of Neuroscience, vol. 28, no. 51, pp. 13815–13827, 2008.
[49]
J. K. Jaiswal, M. Fix, T. Takano, M. Nedergaard, and S. M. Simon, “Resolving vesicle fusion from lysis to monitor calcium-triggered lysosomal exocytosis in astrocytes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 35, pp. 14151–14156, 2007.
[50]
Z. J. Zhang, G. Chen, W. Zhou et al., “Regulated ATP release from astrocytes through lysosome exocytosis,” Nature Cell Biology, vol. 9, no. 8, article U115, pp. 945–953, 2007.
[51]
R. D. Fields and B. Stevens, “ATP: an extracellular signaling molecule between neurons and glia,” Trends in Neurosciences, vol. 23, no. 12, pp. 625–633, 2000.
[52]
M. L. Cotrina, J. H.-C. Lin, A. Alves-Rodrigues et al., “Connexins regulate calcium signaling by controlling ATP release,” Proceedings of the National Academy of Sciences of the United States of America, vol. 95, no. 26, pp. 15735–15740, 1998.
[53]
J. Xu, H. Peng, N. Kang et al., “Glutamate-induced exocytosis of glutamate from astrocytes,” The Journal of Biological Chemistry, vol. 282, no. 33, pp. 24185–24197, 2007.
[54]
N. C. Harata, A. M. Aravanis, and R. W. Tsien, “Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion,” Journal of Neurochemistry, vol. 97, no. 6, pp. 1546–1570, 2006.
[55]
K. Oomagari, B. Buisson, A. Dumuis, J. Bockaert, and J.-P. Pin, “Effect of glutamate and ionomycin on the release of arachidonic acid, prostaglandins and HETEs from cultured neurons and astrocytes,” European Journal of Neuroscience, vol. 3, no. 10, pp. 928–939, 1991.
[56]
J. Kitanaka, K. Takuma, K. Kondo, and A. Baba, “Prostanoid receptor-mediated calcium signaling in cultured rat astrocytes,” Japanese Journal of Pharmacology, vol. 71, no. 1, pp. 85–87, 1996.
