Abstract
Control of the glutamate time course in the synapse is crucial for excitatory transmission. This process is mainly ensured by astrocytic transporters, high expression of which is essential to compensate for their slow transport cycle. Although molecular mechanisms regulating transporter intracellular trafficking have been identified, the relationship between surface transporter dynamics and synaptic function remains unexplored. We found that GLT-1 transporters were highly mobile on rat astrocytes. Surface diffusion of GLT-1 was sensitive to neuronal and glial activities and was strongly reduced in the vicinity of glutamatergic synapses, favoring transporter retention. Notably, glutamate uncaging at synaptic sites increased GLT-1 diffusion, displacing transporters away from this compartment. Functionally, impairing GLT-1 membrane diffusion through cross-linking in vitro and in vivo slowed the kinetics of excitatory postsynaptic currents, indicative of a prolonged time course of synaptic glutamate. These data provide, to the best of our knowledge, the first evidence for a physiological role of GLT-1 surface diffusion in shaping synaptic transmission.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Rothstein, J.D. et al. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725 (1994).
Riveros, N., Fiedler, J., Lagos, N., Munoz, C. & Orrego, F. Glutamate in rat brain cortex synaptic vesicles: influence of the vesicle isolation procedure. Brain Res. 386, 405–408 (1986).
Clements, J.D., Lester, R.A., Tong, G., Jahr, C.E. & Westbrook, G.L. The time course of glutamate in the synaptic cleft. Science 258, 1498–1501 (1992).
Bergles, D.E. & Jahr, C.E. Glial contribution to glutamate uptake at Schaffer collateral-commissural synapses in the hippocampus. J. Neurosci. 18, 7709–7716 (1998).
Wadiche, J.I., Arriza, J.L., Amara, S.G. & Kavanaugh, M.P. Kinetics of a human glutamate transporter. Neuron 14, 1019–1027 (1995).
Lehre, K.P. & Danbolt, N.C. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J. Neurosci. 18, 8751–8757 (1998).
Diamond, J.S. & Jahr, C.E. Transporters buffer synaptically released glutamate on a submillisecond time scale. J. Neurosci. 17, 4672–4687 (1997).
Arriza, J.L. et al. Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex. J. Neurosci. 14, 5559–5569 (1994).
Huang, Y.H. & Bergles, D.E. Glutamate transporters bring competition to the synapse. Curr. Opin. Neurobiol. 14, 346–352 (2004).
Stenovec, M., Kreft, M., Grilc, S., Pangrsic, T. & Zorec, R. EAAT2 density at the astrocyte plasma membrane and Ca2+-regulated exocytosis. Mol. Membr. Biol. 25, 203–215 (2008).
Foran, E., Rosenblum, L., Bogush, A., Pasinelli, P. & Trotti, D. Sumoylation of the astroglial glutamate transporter EAAT2 governs its intracellular compartmentalization. Glia 62, 1241–1253 (2014).
González, M.I. & Robinson, M.B. Neurotransmitter transporters: why dance with so many partners? Curr. Opin. Pharmacol. 4, 30–35 (2004).
Kalandadze, A., Wu, Y. & Robinson, M.B. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486. J. Biol. Chem. 277, 45741–45750 (2002).
Barbour, B., Keller, B.U., Llano, I. & Marty, A. Prolonged presence of glutamate during excitatory synaptic transmission to cerebellar Purkinje cells. Neuron 12, 1331–1343 (1994).
Mennerick, S. & Zorumski, C.F. Glial contributions to excitatory neurotransmission in cultured hippocampal cells. Nature 368, 59–62 (1994).
Tong, G. & Jahr, C.E. Block of glutamate transporters potentiates postsynaptic excitation. Neuron 13, 1195–1203 (1994).
Asztely, F., Erdemli, G. & Kullmann, D.M. Extrasynaptic glutamate spillover in the hippocampus: dependence on temperature and the role of active glutamate uptake. Neuron 18, 281–293 (1997).
Diamond, J.S. & Jahr, C.E. Synaptically released glutamate does not overwhelm transporters on hippocampal astrocytes during high-frequency stimulation. J. Neurophysiol. 83, 2835–2843 (2000).
Heine, M. et al. Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Science 320, 201–205 (2008).
Arizono, M. et al. Receptor-selective diffusion barrier enhances sensitivity of astrocytic processes to metabotropic glutamate receptor stimulation. Sci. Signal. 5, ra27 (2012).
Toulme, E. & Khakh, B.S. Imaging P2X4 receptor lateral mobility in microglia: regulation by calcium and p38 MAPK. J. Biol. Chem. 287, 14734–14748 (2012).
Groc, L. & Choquet, D. Measurement and characteristics of neurotransmitter receptor surface trafficking (Review). Mol. Membr. Biol. 25, 344–352 (2008).
Groc, L. et al. Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors. Nat. Neurosci. 7, 695–696 (2004).
Takeda, H., Inazu, M. & Matsumiya, T. Astroglial dopamine transport is mediated by norepinephrine transporter. Naunyn Schmiedebergs Arch. Pharmacol. 366, 620–623 (2002).
Yang, Y. et al. Presynaptic regulation of astroglial excitatory neurotransmitter transporter GLT1. Neuron 61, 880–894 (2009).
Genoud, C. et al. Plasticity of astrocytic coverage and glutamate transporter expression in adult mouse cortex. PLoS Biol. 4, e343 (2006).
Benediktsson, A.M. et al. Neuronal activity regulates glutamate transporter dynamics in developing astrocytes. Glia 60, 175–188 (2012).
Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656–4665 (2003).
Groc, L., Choquet, D. & Chaouloff, F. The stress hormone corticosterone conditions AMPAR surface trafficking and synaptic potentiation. Nat. Neurosci. 11, 868–870 (2008).
Dupuis, J.P. et al. Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses. EMBO J. 33, 842–861 (2014).
Marcaggi, P., Billups, D. & Attwell, D. The role of glial glutamate transporters in maintaining the independent operation of juvenile mouse cerebellar parallel fibre synapses. J. Physiol. (Lond.) 552, 89–107 (2003).
Danbolt, N.C. Glutamate uptake. Prog. Neurobiol. 65, 1–105 (2001).
Tzingounis, A.V. & Wadiche, J.I. Glutamate transporters: confining runaway excitation by shaping synaptic transmission. Nat. Rev. Neurosci. 8, 935–947 (2007).
Rothstein, J.D. et al. Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686 (1996).
Yernool, D., Boudker, O., Jin, Y. & Gouaux, E. Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature 431, 811–818 (2004).
Oliet, S.H., Piet, R. & Poulain, D.A. Control of glutamate clearance and synaptic efficacy by glial coverage of neurons. Science 292, 923–926 (2001).
Pannasch, U. et al. Connexin 30 sets synaptic strength by controlling astroglial synapse invasion. Nat. Neurosci. 17, 549–558 (2014).
Omrani, A. et al. Up-regulation of GLT-1 severely impairs LTD at mossy fibre–CA3 synapses. J. Physiol. (Lond.) 587, 4575–4588 (2009).
Melone, M., Bellesi, M. & Conti, F. Synaptic localization of GLT-1a in the rat somatic sensory cortex. Glia 57, 108–117 (2009).
Choquet, D. & Triller, A. The dynamic synapse. Neuron 80, 691–703 (2013).
Overstreet, L.S., Kinney, G.A., Liu, Y.B., Billups, D. & Slater, N.T. Glutamate transporters contribute to the time course of synaptic transmission in cerebellar granule cells. J. Neurosci. 19, 9663–9673 (1999).
Ventura, R. & Harris, K.M. Three-dimensional relationships between hippocampal synapses and astrocytes. J. Neurosci. 19, 6897–6906 (1999).
Kullmann, D.M., Erdemli, G. & Asztely, F. LTP of AMPA and NMDA receptor–mediated signals: evidence for presynaptic expression and extrasynaptic glutamate spill-over. Neuron 17, 461–474 (1996).
Krugers, H.J., Hoogenraad, C.C. & Groc, L. Stress hormones and AMPA receptor trafficking in synaptic plasticity and memory. Nat. Rev. Neurosci. 11, 675–681 (2010).
Mikasova, L. et al. Disrupted surface cross-talk between NMDA and Ephrin-B2 receptors in anti-NMDA encephalitis. Brain 135, 1606–1621 (2012).
Rothstein, J.D., Van Kammen, M., Levey, A.I., Martin, L.J. & Kuncl, R.W. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann. Neurol. 38, 73–84 (1995).
Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702 (1997).
Scimemi, A. et al. Amyloid-beta1–42 slows clearance of synaptically released glutamate by mislocalizing astrocytic GLT-1. J. Neurosci. 33, 5312–5318 (2013).
Liévens, J.C. et al. Impaired glutamate uptake in the R6 Huntington's disease transgenic mice. Neurobiol. Dis. 8, 807–821 (2001).
Massie, A. et al. Time-dependent changes in GLT-1 functioning in striatum of hemi-Parkinson rats. Neurochem. Int. 57, 572–578 (2010).
Groc, L. et al. Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors. Nat. Neurosci. 7, 695–696 (2004).
Bard, L. et al. Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins. Proc. Natl. Acad. Sci. USA 107, 19561–19566 (2010).
Peacey, E., Miller, C.C., Dunlop, J. & Rattray, M. The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies. Mol. Pharmacol. 75, 1062–1073 (2009).
Groc, L. et al. Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies. J. Neurosci. 27, 12433–12437 (2007).
Bergles, D.E. & Jahr, C.E. Synaptic activation of glutamate transporters in hippocampal astrocytes. Neuron 19, 1297–1308 (1997).
Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656–4665 (2003).
Tong, G. & Jahr, C.E. Block of glutamate transporters potentiates postsynaptic excitation. Neuron 13, 1195–1203 (1994).
Heine, M. et al. Surface mobility of postsynaptic AMPARs tunes synaptic transmission. Science 320, 201–205 (2008).
Mennerick, S. & Zorumski, C.F. Glial contributions to excitatory neurotransmission in cultured hippocampal cells. Nature 368, 59–62 (1994).
Acknowledgements
We thank M. Rattray (University of Bradford) for the generous gift of GLT-1flag construct, U. Gether (University of Copenhagen) for the generous gift of DATflag construct and M. Goillandeau (CNRS) who developed the software for the detection and analysis of synaptic events. We thank D. Bouchet (IINS CNRS), A. Ledantec (IINS CNRS) and N. Cassagno (INSERM) for cell cultures. We thank the Bordeaux Imaging Center, C. Poujol and D. Choquet for technical support. We thank our laboratory members for critical discussions. This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Conseil Régional d'Aquitaine, Agence National de la Recherche, Fondation de la Recherche Medicale, LABEX BRAIN ANR-10-LABX-43, Marie Curie ‘Edu-GLIA’ (PITN-GA-2009-237, an Initial Training Network) and Marie Curie ‘deepNMDAR’ fellowship funded by the European Commission under the Seventh Framework Programme.
Author information
Authors and Affiliations
Contributions
C.M.-R. carried out QD imaging, uncaging and immunostaining experiments and analysis. C.M.-R. and B.P. performed radiolabeled-glutamate uptake experiments and analysis. J.P.D. carried out electrophysiological experiments and analysis. J.A.V. and J.P.D. performed stereotaxic injections. A.P. recorded glutamate transporter currents. J.B. provided technical support. S.H.R.O. and L.G. directed the work. C.M.-R., J.P.D., L.G. and S.H.R.O. wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Surface diffusion of GLT-1 and DAT on astrocytes
(a) Schematic representation of GLT-1flag labelled by an anti-flag antibody/QD complex. (b) Plot indicating number of visible QD-tagged GLT-1 following acid wash at 10, 15, 20 and 30 min. Low number of QDs after wash indicates low endocytosis of transporter during this time as QDs visible after wash are those that have been internalized. (c) Representative images of GLT-1-tagged QDs before acid wash and after at 10 and 30 minute time points (scale bar 2 μm). (d) Schematic representation of the dopamine transporter (DAT) with an added N-terminal flag domain to allow surface quantum dot labelling. (e) Representative single trajectory of DATflag surface diffusion (scale bar 0.25 μm). (f) Instantaneous DAT and GLT-1diffusion coefficients. DATflag: median = 0.055 μm2/s; IQR ± 0.021-0.109 μm2/s, n=335 trajectories. GLT-1flag: median = 0.23 μm2/s; IQR ± 0.12-0.36 μm2/s; n=720 trajectories (*** P<0.001). (g) Mean square displacement versus time for DAT and GLT-1. Both curves exhibit negative curvature indicating confined behavior. Note the higher degree of confinement for DATflag.
Supplementary Figure 2 Comparison of endogenous GLT-1 properties to GLT-1flag
(a) Mean square displacement versus time for endogenous GLT-1endo and GLT-1flag. Both curves exhibit negative curvature indicating confined behavior. Note the higher degree of confinement for GLT-1endo. (b) Comparison of radio-labelled glutamate uptake in mixed hippocampal culture. In Na+-free and TBOA conditions transport is very low. Naïve cultures, without antibodies or transfected with GLT-1flag have high transport capacity which is not significantly different from GLT-1endo or GLT-1endo X-link conditions (n=8; P>0.05).
Supplementary Figure 3 No change in total levels of GLT-1 between GLT-1flag-transfected and non-transfected astrocytes in mixed hippocampal culture
(a) Example immunostaining image for GLT-1flag (i.e. transfected), total GLT-1 (using an antibody which recognizes both endogenous and transfected GLT-1) and the overlay of both images (scale bars = 40 μm). (b) No difference in total levels of GLT-1 expression was observed between astrocytes transfected with GLT-1flag and non-transfected astrocytes (non-transfected: 100 ± 23.9%, n=13 astrocytes; transfected: 136 ± 18.7%, n=13 astrocytes; P=0.127; n.s.).
Supplementary Figure 4 Graphic summary
In control condition (left) GLT-1 is highly mobile on the surface of astrocytes with access to the confined membrane opposing the synaptic cleft. However, when GLT-1 surface diffusion is reduced (due to decreased temperature or X-link; right) transporters are no longer permitted to diffuse from synaptic to extrasynaptic areas, which results in a reduced glutamate uptake capacity of GLT-1, evidenced by increased sEPSC kinetics.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–4 and Supplementary Table 1 (PDF 1367 kb)
Rights and permissions
About this article
Cite this article
Murphy-Royal, C., Dupuis, J., Varela, J. et al. Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission. Nat Neurosci 18, 219–226 (2015). https://doi.org/10.1038/nn.3901
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nn.3901
This article is cited by
-
Overview Article Astrocytes as Initiators of Epilepsy
Neurochemical Research (2023)
-
Role of glia and extracellular matrix in controlling neuroplasticity in the central nervous system
Seminars in Immunopathology (2023)
-
Neuronal activity drives pathway-specific depolarization of peripheral astrocyte processes
Nature Neuroscience (2022)
-
Astrocytes in the ventral pallidum extinguish heroin seeking through GAT-3 upregulation and morphological plasticity at D1-MSN terminals
Molecular Psychiatry (2022)
-
Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags
Nature Protocols (2022)