Redistribution and stabilization of cell surface glutamate receptors during synapse formation - PubMed (original) (raw)
Redistribution and stabilization of cell surface glutamate receptors during synapse formation
A L Mammen et al. J Neurosci. 1997.
Abstract
Although the regulation of neurotransmitter receptors during synaptogenesis has been studied extensively at the neuromuscular junction, little is known about the control of excitatory neurotransmitter receptors during synapse formation in central neurons. Using antibodies against extracellular N-terminal (N-GluR1) and intracellular C-terminal (C-GluR1) domains of the AMPA receptor subunit GluR1, combined with surface biotinylation and metabolic labeling studies, we have characterized the redistribution and metabolic stabilization of the AMPA receptor subunit GluR1 during synapse formation in culture. Before synapse formation, GluR1 is distributed widely, both on the surface and within the dendritic cytoplasm of these neurons. The diffuse cell surface pool of receptor appears to be mobile within the membrane and can be induced to cluster by the addition of N-GluR1 to live neurons. As cultures mature and synapses form, there is a redistribution of surface GluR1 into clusters at excitatory synapses where it appears to be immobilized. The change in the distribution of GluR1 is accompanied by an increase in both the half-life of the receptor and the percentage of the total pool of GluR1 that is present on the cell surface. Blockade of postsynaptic AMPA and NMDA receptors had no effect on the redistribution of GluR1. These results begin to characterize the events regulating the distribution of AMPA receptors and demonstrate similarities between synapse formation at the neuromuscular junction and at excitatory synapses in cultured neurons.
Figures
Fig. 1.
Characterization of the N-GluR1 antibody. An antibody was raised against an N-terminal region of GluR1 as described in Materials and Methods. A, Immunoblots of either total (T) or biotinylated- and streptavidin-immunoprecipitated (B) spinal cord cultures were probed with antibodies to either N-GluR1 or C-GluR1. Although N-GluR1 recognized several proteins, in addition to GluR1, in total extracts of spinal cord, there was only a single, appropriately sized protein in the pool of surface molecules. B,C, Live cultures of spinal cord neurons were labeled with N-GluR1 (B) or N-GluR1 plus peptide (0.1 mg/ml) (C) for 1 hr and then fixed and labeled with C-GluR1 as described in Materials and Methods. Surface labeling with N-GluR1 completely overlapped with C-GluR1 and was completely blocked by peptide.
Fig. 2.
Induction of extrasynaptic receptor patches by the N-GluR1 antibody. Live, 3-d-old cultures of spinal cord neurons were incubated with whole N-GluR1 (A–D, I–L) or a Fab fragment of N-GluR1 (E–H) and then fixed and processed as described. Antibody-induced surface patches of N-GluR1 (A), which are not seen with a Fab fragment of N-GluR1 (E), have corresponding clusters of C-terminal GluR1 staining (compare B with_F_). In nonpermeabilized neurons (I) surface N-GluR1 staining is observed, whereas C-terminal staining is not detectable. The nonsynaptic location of this staining is confirmed by the lack of associated synaptophysin stain (D, H, L), and the absence of cell–cell contact at these sites (C, G, K).
Fig. 3.
The distribution of surface GluR1 changes over time in culture. Antibody-induced, nonsynaptic patches of GluR1 are abundant on the dendrites of neurons after 4 d in culture (A–C). On day 11, however (D–F), all live cell staining with N-GluR1 is confined to synapses, defined by the presence of presynaptic synaptophysin stain (F). Note that N-GluR1-induced antibody patching appears to cluster all the surface immunostaining at both day 4 (A) and day 11 (D) but leaves a significant portion of the total GluR1 signal, seen with the C-terminal antibody, unclustered (B, E).
Fig. 4.
Time course of surface GluR1 redistribution_in vitro_. Synaptic clusters and extrasynaptic patches of GluR1 were visualized as described in Figures 2 and 3. GluR1-immunopositive neurons were categorized as either synaptic, extrasynaptic, or transitional on the basis of the following scheme. “Extrasynaptic” neurons averaged more than eight extrasynaptic patches and one or fewer synaptic cluster per major dendrite. “Synaptic” neurons averaged two or more synaptic clusters and two or fewer extrasynaptic patches per major dendrite. “Transitional” neurons fell between these two categories. Neurons were taken from a series of three platings. The total number of neurons at each point is listed above that point. For every neuron, the length of each major dendrite examined was usually 60 μm.
Fig. 5.
The diffuse dendritic staining for GluR1 is specific. For A and B, an 11 d spinal cord culture was stained with Cy3-labeled C-GluR1, revealing two neurons, one of which is GluR1 immunopositive and one of which is GluR1 immunonegative. Note the differences in diffuse dendritic stain between the immunopositive (closed arrows) and immunonegative (open arrows) neurons. The categorization of these cultured neurons as immunopositive and immunonegative is based on cell body and synaptic stain, and correlates well with in situ hybridization signal for GluR1 mRNA. In C_and D, a GluR1-immunopositive neuron from a 5 d culture, identified by live N-GluR1 stain (C), shows minimal dendritic stain with Cy3-labeled C-GluR1 when the antibody has been preincubated with peptide (for comparison, see Fig.3_B,E). In all control experiments, neuronal cell bodies showed low-level, nonspecific staining, which did not extend into the dendrites.
Fig. 6.
Lack of suppression of extrasynaptic GluR1 in island cultures. Island cultures containing isolated spinal cord neurons were grown for 10 d. Under these conditions all synaptic contacts are autaptic. In A, the staining pattern of N-GluR1 continues to show extrasynaptic patches of GluR1 (arrowheads) despite autaptic clusters of GluR1 (arrows).
Fig. 7.
Time course and efficiency of biotinylation of GluR1 in cultured spinal cord neurons. Cultures of spinal cord neurons were biotinylated at 4°C with 1 mg/ml NHS-SS-biotin. In_A_, cultures were exposed to the biotinylating reagent for different times, and the detergent soluble fraction of each culture was added to streptavidin-conjugated beads and incubated for 2 hr. The supernatant recovered from the precipitation of the 15 min-treated neurons was reprecipitated with streptavidin beads, and the additional streptavidin-precipitated material was loaded as well. All samples were loaded such that each lane represents 1% of the total material per plate. The gel was transferred to immobilon and probed with both C-GluR1 and anti-tubulin antibodies. In B, one plate of neurons was scraped into the biotinylating reagent, sonicated, spun at 14,000 × g for 15 min, and resuspended in precipitation buffer including 0.2% SDS and 1% Triton X-100. A fraction of this was saved and loaded as total extract. The remainder was incubated for 2 hr with streptavidin-conjugated beads. The supernatant, streptavidin-precipitated, and total extracts were loaded such that each lane represents 1% of the material from the plate. The gel was transferred and probed with the C-GluR1 antibody.
Fig. 8.
Half-life of cell surface GluR1 in spinal cord neuronal cultures at day 4 and day 11 in vitro. Plates of spinal cord neurons were biotinylated at day 4 and day 11 and recultured for 0–24 hr, at which time cell extracts were harvested, sonicated, and frozen. Subsequently, these samples were thawed and incubated with streptavidin-linked beads, and the streptavidin-precipitated material was loaded onto gels (A). A standard curve including serial dilutions of the t = 0 streptavidin-precipitated material was included on each gel for purposes of quantitation. After transfer, gels were probed with the C-GluR1 antibody. In B, the natural log of the percent of remaining surface GluR1 was plotted against time, and half-lives were calculated from the regression slopes of the resulting lines. The results of the experiment in A are shown.
Fig. 9.
Determination of the fraction of GluR1 on the cell surface at day 4 and day 11. Spinal cord neuronal cultures were biotinylated, harvested, and incubated with streptavidin beads. Samples of the total extract, streptavidin-supernatant (intracellular), streptavidin-precipitate (surface), and washes were saved and loaded, such that each lane represents protein from 1% of the plate. Serial dilutions of the total extract were also loaded for purposes of quantitation. The gels were transferred, probed with C-GluR1, stripped, and reprobed with anti-tubulin antibody. In each of five experiments, <1% of the total tubulin was precipitated as a “surface” molecule.
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