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Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission

Nature Neuroscience volume 18, pages 219–226 (2015)Cite this article

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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.

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Figure 1: Characteristics of GLT-1 surface diffusion.
Figure 2: GLT-1 diffusion is compartmentalized on the surface of astrocytes.
Figure 3: Effect of temperature on GLT-1 diffusion and sEPSC kinetics.
Figure 4: GLT-1 can be immobilized without any consequence on uptake or cell surface localization using X-link.
Figure 5: Surface diffusion of GLT-1 confers synaptic glutamate homeostasis on fast timescale.
Figure 6: X-link of endogenous GLT-1 in hippocampal slice preparation affects both astrocytic GLT-1 currents and neuronal sEPSCs.

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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.

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Author notes

  1. Juan A Varela and Aude Panatier: These authors contributed equally to this work.

  2. Laurent Groc and Stéphane H R Oliet: These authors jointly directed this work.

Authors and Affiliations

  1. Neurocentre Magendie, Inserm U862, Bordeaux, France

    Ciaran Murphy-Royal, Aude Panatier & Stéphane H R Oliet

  2. University of Bordeaux, Bordeaux, France

    Ciaran Murphy-Royal, Julien P Dupuis, Juan A Varela, Aude Panatier, Benoît Pinson, Jérôme Baufreton, Laurent Groc & Stéphane H R Oliet

  3. Interdisciplinary Institute for Neuroscience, CNRS UMR 5297, Bordeaux, France

    Ciaran Murphy-Royal, Julien P Dupuis, Juan A Varela & Laurent Groc

  4. Institute for Biochemistry and Cellular Genetics, CNRS UMR 5095, Bordeaux, France

    Benoît Pinson

  5. Institute of Neurodegenerative Diseases, CNRS UMR 5293, Bordeaux, France

    Jérôme Baufreton

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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.

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Correspondence to Laurent Groc or Stéphane H R Oliet.

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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.

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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

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