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• W1978605718 abstract "Article3 February 2003free access Regulated delivery of AMPA receptor subunits to the presynaptic membrane Ursula Schenk Ursula Schenk CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Claudia Verderio Claudia Verderio CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Fabio Benfenati Fabio Benfenati Department of Experimental Medicine, Section of Physiology, University of Genova, Viale Benedetto XV 3, 16132 Genova, Italy Search for more papers by this author Michela Matteoli Corresponding Author Michela Matteoli CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Ursula Schenk Ursula Schenk CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Claudia Verderio Claudia Verderio CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Fabio Benfenati Fabio Benfenati Department of Experimental Medicine, Section of Physiology, University of Genova, Viale Benedetto XV 3, 16132 Genova, Italy Search for more papers by this author Michela Matteoli Corresponding Author Michela Matteoli CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy Search for more papers by this author Author Information Ursula Schenk1, Claudia Verderio1, Fabio Benfenati2 and Michela Matteoli 1 1CNR Institute of Neuroscience, Cellular and Molecular Pharmacology, Center of Excellence for Neurodegenerative Diseases, Department of Medical Pharmacology, via Vanvitelli 32, 20129 Milano, Italy 2Department of Experimental Medicine, Section of Physiology, University of Genova, Viale Benedetto XV 3, 16132 Genova, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:558-568https://doi.org/10.1093/emboj/cdg059 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with α-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization. Introduction Glutamate is the major excitatory neurotransmitter in the brain. At mature glutamatergic synapses, the response to glutamate is transduced by two different ion-permeable receptors, which are clustered in the portion of the postsynaptic membrane facing the presynaptic active zone: the N-methyl-D-aspartate (NMDA) receptors and the AMPA receptors. The NDMA receptors are largely inactive at resting membrane potential and require membrane depolarization to become activated, and the AMPA receptors open upon glutamate binding and mediate most of the depolarizing effect of this neurotransmitter. Thus AMPA receptors, by directly controlling the majority of excitatory synaptic transmission, play a major role in the storage of information in brain (Wisden and Seeburg, 1993; Hollmann and Heinemann, 1994; Nakanishi et al., 1998; Scannevin and Huganir, 2000; Malinow and Malenka, 2002). Recently, evidence has been accumulating indicating that, in addition to their well-established postsynaptic action, AMPA receptors also mediate presynaptic effects (Nicoll et al., 2000). Presynaptic AMPA receptors have been shown to modulate synaptic transmission, by depressing the release of inhibitory transmitters in the adult cerebellum (Satake et al., 2000). Furthermore, glutamate, by acting on AMPA/kainate receptors during synaptogenesis, regulates the actin-based motility of axonal filopodia implicated in synapse formation (Chang and De Camilli, 2001). Finally, lack of activation of glutamate receptors during synaptogenesis prevents the functional maturation of the presynaptic machinery (Bacci et al., 2001). As a result, a novel role for AMPA receptors as modulators of the presynaptic structure and function at both the developing and the mature synapse is emerging. One of the interesting features of postsynaptic AMPA receptors is their regulated dynamic recycling between the plasma membrane and intracellular pools (Carroll et al., 2001; Sheng and Lee, 2001; Malinow and Malenka, 2002). As the number of surface-exposed postsynaptic AMPA receptors regulates the efficiency of synaptic transmission, this recycling is thought to play a major role in finely tuning synaptic strength during development and synaptic plasticity phenomena. In particular, receptor internalization, occurring in response to various extracellular stimuli, plays a potential role in the rapid attenuation of neurotransmission during long-term depression, whereas receptor insertion into the plasmalemma would represent one mechanism by which synaptic strength is increased during long-term potentiation (Carroll et al., 2001; Sheng and Lee, 2001; Malinow and Malenka, 2002). The molecular processes involved in AMPA receptor internalization have been clearly identified (Carroll et al., 2001). Receptor internalization occurs via clathrin-mediated endocytosis (Carroll et al., 1999; Man et al., 2000; Wang and Linden, 2000), driven by activation of either protein kinase C, calcineurin or protein phosphatase 1, depending on the cell type (reviewed in Carroll et al., 2001). Once internalized, the receptors are either transported for degradation or stabilized in intracellular vesicles through binding to AMPA receptor interacting proteins (Dong et al., 1999; Carroll et al., 2001). The phosphorylation state of AMPA receptor subunits appears to strictly regulate this process (Chung et al., 2000; Malinow and Malenka, 2002). On the other hand, the mechanisms responsible for the delivery of AMPA receptors to the synaptic membrane are much less clear. This process is thought to be mediated by exocytosis of receptor-containing vesicles, regulated by neuronal activity (Lledo et al., 1998; Maletic-Savatic et al., 1998; Broutman and Baudry, 2001; Liao et al., 2001; Passafaro et al., 2001; Shi et al., 2001). In this study, we have investigated the traffic of presynaptic AMPA receptors, taking advantage of the extensive characterization of the vesicular trafficking in the nerve terminal (Greengard et al., 1993; Jahn and Sudhof, 1999; Cremona and De Camilli, 1997; Lin and Scheller, 2000). We demonstrate that, at steady state, a major pool of GluR2 and GluR1 subunits is associated with synaptic vesicle membranes and that AMPA receptors are recruited to the presynaptic membrane in response to depolarization, through clostridial toxin- sensitive exocytosis. Results Expression of AMPA receptor subunits in axonal growth cones of cultured hippocampal neurons A panel of antibodies directed against either the C- or the N-terminus of various AMPA receptor subunits (GluR1, GluR2/3, GluR2 N-ter) was used to investigate the expression of these proteins in primary cultures of hippocampal neurons. When tested on homogenates of adult rat brain as well as on cell extracts of mature hippocampal cultures, all the antibodies recognized a single band with the expected molecular mass (see Supplementary data, available at The EMBO Journal Online). Immunofluorescence of mature neuronal cultures with the anti-GluR1 and -GluR2/3 antibodies resulted in a typical staining of the somatodendritic compartment and of dendritic spines (Wyszynski et al., 2002). Application of antibodies directed against the N-terminal domain of GluR2 to living neurons, followed by immunofluorescence for the bound antibodies, specifically stained neuronal cell bodies and dendrites, consistent with the in vivo exposure of this AMPA receptor subunit on the somatodendritic plasma membrane (not shown). Antibodies directed against the AMPA receptor subunit GluR4 produced a faint staining in cell extracts of mature hippocampal cultures by western blot analysis and did not produce any detectable signal by immunofluorescence. Once the specificity of the antibodies had been assessed, we used them to investigate the possible presence of AMPA receptor subunits in the axonal compartment of cultured hippocampal neurons. Five to seven days in vitro (DIV) old cultures were double stained with the antibodies to AMPA receptor subunits GluR2/3 and with antibodies directed against the synaptic vesicle protein synaptobrevin/VAMP2. GluR2/3 antibodies labeled with variable intensity, axonal growth cones (Figure 1), which could be identified by the lack of staining with antibodies against the somatodendritic marker MAP2 (Figure 1A) or by immunoreactivity for the axonal protein tau-1 (insert). Axonal growth cones were also characterized by the presence of small synaptic vesicle clusters (Figure 2A and D), as previously described (Mundigl et al., 1993). The percentage of GluR2/3-positive growth cones was 77 ± 14%. GluR2/3 immunoreactivity was still present in the growth cone after it has contacted a target dendrite (Figure 2G and H, arrow). GluR2/3 immunoreactivity was also detectable in packages of synaptic vesicles which travel along the axon (Figure 2I and L; Kraszewski et al., 1995; Ahmari et al., 2000). A percentage of axonal growth cones (42 ± 7.3%) was also found to be positive upon staining with antibodies to GluR1 (Figure 2M–O) (see also Craig et al., 1993). Figure 1.Immunocytochemical localization of GluR2/3 in axonal growth cones of cultured hippocampal neurons. Seven day old neurons double labeled for GluR2/3 (B) and for the somatodendritic marker MAP2 (A). GluR2/3 immunoreactivity is detectable in a MAP2-negative axonal process (arrowheads). Inserts show a GluR2/3-positive process immunoreactive for the axonal protein tau-1. Scale bar: 12 μm. Download figure Download PowerPoint Figure 2.Immunocytochemical localization of GluR2/3 and GluR1 in axonal compartments of hippocampal neurons. (A–F) Seven day old neurons double labeled for GluR2/3 (B and E) and for the synaptic vesicle protein synaptobrevin/VAMP2 (A and D). GluR2/3 immunoreactivity is detectable along the axon and in the growth cone, identified by the presence of synaptic vesicle clusters. (G–L) Ten day old neurons double labeled for GluR2/3 (H and L) and for synaptobrevin/VAMP2 (G and I). GluR2/3 is still detectable in the axonal growth cone (G and H, arrow) contacting the postsynaptic dendrite. Note the presence of GluR2/3 immunostaining in the dendritic shaft of the postsynaptic neuron. GluR2/3 immunoreactivity is also detectable in corresponding packages of synaptic vesicles which travel along the axon (I and L). (M–O) Five day old neurons double labeled for GluR1 (N) and for synaptobrevin/VAMP2 (M). Scale bars: A–C, 15 μm; D–F, 6 μm; G–L, 1.8 μm; M–O, 6.6 μm. Download figure Download PowerPoint Functional activation of AMPA receptors in axonal growth cones To investigate whether the presence of AMPA receptor subunits in the growth cone is accompanied by the expression of functional AMPA receptors on the plasma membrane, 5–7 DIV old neurons were loaded with the sodium-sensitive dye SBFI and stimulated with 100 μM AMPA, applied in the presence of 50 μM cyclothiazide. Exogenous application of the agonist resulted in a [Na+]i transient both in the somatodendritic region and in the axonal growth cone (Figure 3A). The identification of the growth cone was carried out by morphological criteria and by retrospective immunocytochemical labeling with antibodies aginst tau-1 or MAP2 at the end of the recording session (see Figure 1). The growth cone response to AMPA was specifically blocked by the AMPA receptor antagonist CNQX (data not shown). A CNQX-sensitive response to AMPA was also recorded in growth cones of neurons loaded with the calcium-sensitive dye FURA-2 (Figure 3B), consistent with the activation of voltage-gated calcium channels upon AMPA receptor activation, as previously described (Fischer et al., 2000). The percentage of axonal growth cones responding to AMPA was found to be 75 ± 4.2%. These data indicate that functional glutamate receptors are present in the membrane of axonal growth cones. Figure 3.Functional AMPA receptors in axonal growth cones of hippocampal neurons. Temporal analysis of [Na+]i (A) or [Ca2+]i (B) changes (measured as variations in the 340/380 fluorescence ratio) in single neurons loaded with either SBFI (A) or Fura-2 (B), following stimulation with 100 μM AMPA in the presence of 50 μM cyclothiazide. Recordings are carried out from the cell body and from the axonal growth cone, as indicated. (C and D) Time-lapse recordings of the peripheral portion of the axon, carried out under control conditions (C) or after the application of 100 μM AMPA (D). Note that application of AMPA almost completely inhibits the growth cone motility. Arrowheads point to the leading edge of the growth cone. Both in panels C and D, the timing between consecutive images is 4 s (images 1–3) and 40 s (images 3 and 4). (E–H) Immunocytochemical staining of an axonal growth cone (E and F) and of giant growth cone (G and H) stained for synaptobrevin/VAMP2 (green) and for F-actin (red, TRITC–phalloidin). (E and G) Control conditions; (F and H) neurons treated with 100 μM AMPA for 5 min. Note that the vesicle-rich core of the growth cones becomes disorganized upon AMPA treatment. Scale bars: C and D, 6 μm; E–H, 9 μm. Download figure Download PowerPoint It has been recently described that glutamate receptor activation regulates the actin-based motility of both dendritic spines and axonal filopodia (Fischer et al., 2000; Chang and De Camilli, 2001). To test whether the activation of AMPA receptors affects the motility of the axonal growth cone, time-lapse recordings of the peripheral portion of the axon were carried out under control conditions or after the application of 100 μM AMPA. The series of images shown in Figure 3C confirms previous studies indicating that axonal growth cones are highly motile structures, endowed with filopodia which repeatedly extend and retract (Zheng et al., 1996). Application of AMPA almost completely inhibited motility (Figure 3D), as previously reported in growth cones of mouse spinal cord neurons (Owen and Bird, 1997), dendritic growth cones (Mattson et al., 1988) and filopodia of hippocampal neurons (Chang and De Camilli, 2001). Removal of AMPA reversed the inhibition, arguing against a non-specific effect due to neuronal damage (data not shown). Application of AMPA to cultured neurons also affected the distribution of synaptic vesicles within the growth cone. Whereas in control cultures synaptic vesicles, labeled by antibodies against synaptobrevin/VAMP2, were most frequently retained in the core of the axonal growth cone (Figure 3E, 72% of growth cones; Mundigl et al., 1993, 1998), a vesicle relocation up to the tips of growth cone filopodia could be detected upon AMPA application (Figure 3F, 81% of growth cones). The AMPA-induced relocation of synaptic vesicles was even more clearly detectable in the giant growth cones that are occasionally found in hippocampal cultures. The large flat geometry of these growth cones makes them especially suitable for high-resolution light microscopy (Mundigl et al., 1993, 1998). Figure 3H shows the disorganization of the organelle-rich core of the growth cone consequent to AMPA stimulation (compare with control, Figure 3G). Synaptic vesicle markers become detectable at the periphery of the growth cone, coinciding with patches of filamentous actin (Figure 3H). Quantification of the growth cone area immunoreactive for synaptobrevin/VAMP2, normalized to the total growth cone area, revealed a two-fold increase (197 ± 15%) in AMPA-treated cultures compared with controls (P < 0.01). Regulated recruitment of AMPA receptors to the plasma membrane of growth cones In neurons loaded with either FURA-2 (Figure 4A) or SBFI (not shown), a second stimulation with 100 μM AMPA, carried out in the presence of cyclothiazide, 10–15 min after the first AMPA application, resulted in a significantly smaller growth cone response (see right panel for quantitation). On the other hand, equivalent [Ca2+]i responses were recorded from neurons subjected to two subsequent challenges with 50 mM KCl (Figure 4B and relative quantitation). These results indicate that a specific reduction of functional AMPA receptors on the growth cone surface occurs in response to receptor activation, suggesting that the exposure of AMPA receptors on the axonal plasma membrane may be dynamically regulated. Figure 4.Regulated delivery of functional AMPA receptors into the axonal plasma membrane. (A) Subsequent stimulations with 100 μM AMPA in the presence of 50 μM cyclothiazide lead to a reduced [Ca2+]i responsivity of the growth cone in Fura-2-loaded cultures. (B) Equivalent [Ca2+]i responses in a growth cone repetitively stimulated with 50 mM KCl. (C) Two subsequent stimulations with 50 mM KCl, carried out in 320 mM sucrose, lead to an enhancement of the growth cone response to 100 μM AMPA. (D) No increase in the growth cone response to AMPA takes place, upon repetitive KCl stimulation in high sucrose, in cultures intoxicated with 80 nM BoNTE. (E) Subsequent stimulations with 100 μM AMPA lead to a reduced growth cone responsivity in BoNTE-intoxicated cultures. Download figure Download PowerPoint We then tested the possibility that AMPA receptors could be recruited to the axonal growth cone plasma membrane upon stimulation, as previously indicated for postsynaptic AMPA receptors (Shi et al., 1999; Liao et al., 2001). To this aim, FURA-2-loaded neurons were repetitively stimulated with 50 mM KCl in the presence of high concentrations of extracellular sucrose, a condition known to inhibit clathrin-mediated endocytosis (Daukas and Zigmond, 1985; Heuser and Anderson, 1989). As shown in Figure 4C, a significantly larger response to AMPA was detected in the growth cone after KCl stimulation. To investigate whether the enhancement of the response to AMPA could be produced by insertion of new receptors into the plasma membrane from an intracellular vesicular pool, we performed the same experiment after impairing the fusion of intracellular vesicles with the plasma membrane using clostridial toxins. Figure 4D shows that treatment with 80 nM botulinum toxin E (BoNTE), which proteolytically cleaves the t-SNARE SNAP25, completely prevented the enhancement of AMPA response consequent to depolarization. The same treatment did not impair receptor internalization (Figure 4E), thus arguing against a generalized neurotoxic effect of BoNTE (see right panels for quantitation). Similar results were obtained after intoxicating mature neurons with tetanus toxin (TeNT), which proteolytically cleaves the v-SNARE synaptobrevin/VAMP2. The extent of the second response to AMPA, normalized to the first one, was: 2.19 ± 0.62, n = 7, P < 0.05, controls; 0.86 ± 0.27, n = 5, P > 0.1, BoNTE-treated; 1.06 ± 0.7, n = 5, P > 0.1, TeNT-treated. These data point to a regulated insertion of AMPA receptors into the axonal plasma membrane. The regulated, vesicular recruitment of AMPA receptors to the plasma membrane of growth cones was further supported by immunofluorescence experiments carried out in the presence of α-latrotoxin, a potent stimulator of synaptic vesicle exocytosis (Ceccarelli et al., 1988; Schiavo et al., 2000; Sudhof, 2001). When applied to cultured hippocampal neurons in the absence of extracellular calcium, α-latrotoxin not only induces synaptic vesicle exocytosis, but also prevents synaptic vesicle endocytosis (Pennuto et al., 2002). To determine whether the α-latrotoxin-induced exocytosis of synaptic vesicles was paralleled by an increase in the number of AMPA receptor subunits recruited to the surface of growth cones, antibodies directed against the extracellular portion of the GluR2 subunits were added to living neurons, either under control conditions or after stimulation with the toxin. Neurons were then fixed and double stained for the synaptic vesicle protein synaptobrevin/VAMP2 (Figure 5). Under control conditions, virtually no staining for the extracellular portion of GluR2 could be detected (Figure 5B), possibly due to the low amount of receptor subunits present on the cell surface at steady state. Upon α-latrotoxin treatment, a specific staining of growth cones could be detected (Figure 5D), indicating that the massive fusion of synaptic vesicles was accompanied by the insertion of AMPA receptor subunits into the plasma membrane. A significant staining for the extracellular portion of GluR2 subunits was also detected, upon α-latrotoxin treatment, corresponding to synaptic vesicle packages traveling along the axon (Figure 5H). To detect the possible internalization of GluR subunits, cultures were incubated with antibodies directed against the extracellular portion of the GluR2 during stimulation with α-latrotoxin in the presence of extracellular calcium, a condition which enhances synaptic vesicle fusion and membrane retrieval (Ceccarelli et al., 1988). After removal of surface-bound antibodies by acid-strip protocol (Figure 5L; Passafaro et al., 2001), antibodies to GluR2 could be detected upon fixation and permeabilization of the neurons (Figure 5N), further confirming that GluR subunits can be internalized from the growth cone surface upon membrane recycling. A surface GluR2 staining was also detectable in cultures stimulated by repetitive application of 50 mM KCl in the presence of high concentrations of extracellular sucrose (data not shown), i.e. the same experimental treatment which enhances AMPA responsivity. These data further support the existence of a vesicle-mediated recruitment of receptor subunits to the growth cone plasma membrane and open up the possibility that synaptic vesicles may represent the organelles mediating this delivery. Figure 5.Exposure of GluR2 on the growth cone surface upon neuronal stimulation. (A and B) Five day old control neurons were incubated at 4°C with antibodies against the extracellular portion of GluR2 (B), fixed and double stained for synaptobrevin/VAMP2 (A). Virtually no surface GluR2 staining is detectable on the growth cone. (C and D) Culture incubated at 4°C with antibodies against the extracellular portion of GluR2 (D) upon stimulation with 0.1 nM α-latrotoxin (αltx), applied in the absence of extracellular calcium, fixed and double stained for synaptobrevin/VAMP2 (C). A surface GluR2 staining is detectable. (E–H) Ten day old neurons were incubated at 4°C with antibodies against the extracellular portion of GluR2 (F and H) and double labeled for synaptobrevin/VAMP2 (E and G) either in the presence (G and H) or in the absence (E and F) of α-latrotoxin. Note the selective surface staining for GluR2, specifically upon α-latrotoxin treatment. (I and L) Acid-strip protocol applied to cultures treated as in panel D, allows the removal of membrane-bound antibodies (L). (M and N) Cultures stimulated with αltx in the presence of extracellular calcium, subjected to acid-strip protocol, permeabilized and stained for internalized antibodies (N). Note the labeling of growth cones, revealing internalization of surface GluR2 subunits. I and M: double staining of growth cones with FITC–phalloidin. Scale bars: A–D, 7.1 μm; E–H, 4.2 μm; I–N, 7.1 μm. Download figure Download PowerPoint Regulated recruitment of AMPA receptors to synaptosomal plasma membrane To determine whether stimuli which promote the insertion of AMPA receptor subunits into the surface of axonal growth cones are also efficient in enhancing the delivery of receptor subunits to the plasma membrane of mature synapses, synaptosomes purified from adult rat forebrain were stimulated with 0.1 nM α-latrotoxin for 5, 10 or 15 min in the absence of extracellular calcium. A biotinylation labeling of cell surface proteins was then carried out, and biotinylated proteins were separated from non-biotinylated, intracellular proteins by using avidin-coated beads. Western blots prepared from biotinylated and non-biotinylated protein fractions were then probed with antibodies against GluR2/3, the NMDA receptor subunit NR1 or the integral synaptic vesicle protein synaptophysin. As a control, immunolabeling was performed with antibodies to the intracellular protein hsp60 or to TI-VAMP, a marker for vesicles involved in neurite outgrowth (Martinez-Arca et al., 2001). The latter bands, which provide an index of synaptosome permeabilization, are present exclusively in the non-biotinylated fraction, thus excluding synaptosome damage and biotinylation of intracellular proteins as a consequence of the experimental treatments. α-latrotoxin treatment produced an increase in biotinylated (cell surface) synaptophysin, as previously described (Torri-Tarelli et al., 1990), but not in biotinylated TI-VAMP (Figure 6). The lack of TI-VAMP biotinylation is not attributable to the short intravesicular/extracellular tail of the protein, as synaptobrevin/VAMP2, which has an intravesicular/extracellular tail which is shorter than TI-VAMP, is effectively biotinylated (not shown), thus supporting a specific effect of α-latrotoxin on synaptic vesicle exocytosis. An increase in cell surface GluR2/3, similar or even higher compared with synaptophysin, could be detected after α-latrotoxin treatment, in the absence of any significant increase in biotinylated NR1 (Figure 6). These results indicate that a vesicular delivery of GluR2/3, but not of NR1, to the plasma membrane is facilitated by stimuli which are known to promote synaptic vesicle exocytosis. Figure 6.Surface exposure of GluR2 in synaptosome stimulated with α-latrotoxin. Biotinylation labeling of cell surface proteins in control or α-latrotoxin-stimulated synaptosomes, followed by separation of biotinylated (membr) from non-biotinylated (cyto) proteins by avidin-coated beads and analysis by western blotting. α-latrotoxin treatment produces an increase in biotinylated (membrane) synaptophysin and GluR2/3. No increase in surface TI-VAMP or in surface NR1 is detectable, thus indicating that a vesicular delivery of GluR2/3, but not of NR1, to the plasma membrane is induced by α-latrotoxin stimulation. No biotinylation of hsp60 occurs, thus excluding synaptosomal damage. Download figure Download PowerPoint The increase in surface exposure of GluR2/3 subunits could in principle be produced by dendritic vesicular organelles fusing with the postsynaptic membrane. As resealed, metabolically active postsynaptic compartments are also present in the synaptosomal fraction, we carried out a subcellular fractionation of rat forebrain homogenates by differential centrifugation, up to the isolation of a highly purified synaptic vesicle fraction (Huttner et al., 1983). The AMPA receptor subunits GluR2/3 and GluR1, the GluR2/3 binding partner GRIP (GluR2 interacting protein), the NMDA receptor subunit NR1, the plasma membrane marker Na+/K+ ATPase and the synaptic vesicle protein synaptophysin were all clearly detectable in the crude synaptosomal fraction (P2) (Figure 7A). However, only GluR2/3 and, to a lesser extent, GluR1 subunits were found to be present, together with synaptophysin, in a synaptic vesicle fraction purified by continuous sucrose density gradient (Figure 7A). As a further step of purification, an immunoisolation of synaptic vesicles was carried out starting from the latter fraction, using magnetic beads coated with antibodies directed against the synaptic vesicle protein synaptotagmin. Synaptophysin, synaptobrevin/VAMP2 and the AMPA receptor subunits GluR2/3 (Figure 7B) and GluR1 (not shown) co-enriched in the immunoisolated fraction. Finally, the immunolabeling of a highly purified synaptic vesicle fraction prepared via permeation chromatography on controlled-pore glass (CPG-V), revealed that GluR2/3 (not shown) and, more specifically, GluR2 (Figure 7C), copurify with the synaptic vesicle-specific protein synapsin I, indicating that, at steady sta" @default.
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• W1978605718 title "Regulated delivery of AMPA receptor subunits to the presynaptic membrane" @default.
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