Redistribution and Stabilization of Cell Surface Glutamate Receptors During Synapse Formation

The Journal of Neuroscience, October 1, 1997, 17(19):7351–7358 Redistribution and Stabilization of Cell Surface Glutamate Receptors during Synapse Formation Andrew L. Mammen,1,2 Richard L. Huganir,1,2 and Richard J. O’Brien1,2,3 1Howard Hughes Medical Institute, and Departments of 2Neuroscience and 3Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Although the regulation of neurotransmitter receptors during addition of N-GluR1 to live neurons. As cultures mature and synaptogenesis has been studied extensively at the neuromus- synapses form, there is a redistribution of surface GluR1 into cular junction, little is known about the control of excitatory clusters at excitatory synapses where it appears to be immo- neurotransmitter receptors during synapse formation in central bilized. The change in the distribution of GluR1 is accompanied neurons. Using antibodies against extracellular N-terminal (N- by an increase in both the half-life of the receptor and the GluR1) and intracellular C-terminal (C-GluR1) domains of the percentage of the total pool of GluR1 that is present on the cell AMPA receptor subunit GluR1, combined with surface biotiny- surface. Blockade of postsynaptic AMPA and NMDA receptors lation and metabolic labeling studies, we have characterized had no effect on the redistribution of GluR1. These results begin the redistribution and metabolic stabilization of the AMPA re- to characterize the events regulating the distribution of AMPA ceptor subunit GluR1 during synapse formation in culture. Be- receptors and demonstrate similarities between synapse for- fore synapse formation, GluR1 is distributed widely, both on the mation at the neuromuscular junction and at excitatory syn- surface and within the dendritic cytoplasm of these neurons. apses in cultured neurons. The diffuse cell surface pool of receptor appears to be mobile Key words: GluR1; synaptogenesis; spinal cord; metabolism; within the membrane and can be induced to cluster by the glutamate receptors; rat; tissue culture The processes involved in the generation and maintenance of phoretic mapping, immunocytochemistry, and immunoelectron synapses are critical for neuronal development and synaptic plas- microscopy have demonstrated that these receptors are localized ticity. To date, information regarding the regulation of neuro- at excitatory synapses (for review, see Ehlers et al., 1996). Little transmitter receptors during synapse formation has largely been is known, however, about the mechanisms regulating clustering derived from studies of the developing vertebrate neuromuscular and maintenance of GluRs at central synapses. junction (Hall and Sanes, 1993). On the surface of embryonic We have previously characterized the development and subunit myotubes before synapse formation, acetylcholine receptors composition of GluR clusters in cultured spinal neurons (O’Brien (AChRs) are distributed diffusely at a density of 10 2/mm 2. Under et al., 1997). Over a period of 10 d, the AMPA receptor subunit the influence of molecules secreted by the motor nerve terminal, GluR1, visualized with an antibody that recognizes the intracel- AChRs become highly enriched at sites of nerve–muscle contact lular C terminus, changes its distribution from diffuse to highly (;10 4 AChRs/mm 2) and depleted at extrajunctional areas clustered at sites of synaptic contact, similar to the pattern seen (Anderson and Cohen, 1977; Frank and Fischbach, 1979; Salpeter with GABAA (Killisch et al., 1991) and glycine receptors and Harris, 1983; Salpeter and Marchaterre, 1988). The synaptic (Bechade et al., 1996). Here, we use an extracellular antibody to enrichment of AChRs is attributable to the clustering of preex- GluR1 to study the distribution of surface receptors during syn- isting extrasynaptic receptors and the directed insertion of newly aptogenesis in the same system. When applied to live neurons, the synthesized receptors (Role et al., 1985). In addition to the bivalent antibody induces the aggregation of extrasynaptic recep- dramatic changes in receptor localization that accompany synap- tors, rendering them easily visualizable, while simultaneously togenesis at the neuromuscular junction, the metabolic half-life of staining preexisting synaptic clusters. We demonstrate that a pool receptors increases from 1 to 10 d (Berg and Hall, 1975b; Reiness of mobile, extrasynaptic receptors is expressed on the surface of and Weinberg, 1981; Steinbach, 1981). immature dendrites, which diminishes as GluR1 redistributes to Ionotropic glutamate receptors (GluRs) mediate excitatory synapses. Coincident with the redistribution of GluR1 to syn- synaptic transmission between neurons in the CNS. These recep- apses, there is a metabolic stabilization of AMPA receptors, tors have been cloned and divided into AMPA, kainate, and evidenced by an increase in their half-life, determined both by NMDA subclasses on the basis of their electrophysiological and surface biotinylation and metabolic labeling. In addition to the pharmacological properties (Seeburg, 1993; Hollman and Heine- surface receptors, a second pool of GluR1 exists within the mann, 1994). Each subclass of ionotropic GluRs includes several cytoplasm of dendrites before and after the period of maximal distinct subunits that exist as heteromeric complexes. Ionto- synaptogenesis. The relative distribution of GluR1 between the surface and cytoplasmic pools of receptors also changes with Received February 28, 1997; revised June 2, 1997; accepted June 10, 1997. time, because of an increase in the number of surface receptors. Correspondence should be addressed to Dr. Richard Huganir, Howard Hughes Medical Institute, PCTB 900, Johns Hopkins Medical School, 725 N. Wolfe Street, MATERIALS AND METHODS Baltimore, MD 21205. Generation of N-terminal GluR1 (N-GluR1). N-GluR1 was made by Copyright © 1997 Society for Neuroscience 0270-6474/97/177351-08$05.00/0 injecting rabbits with the synthetic peptide KQWRTSDSRDHTRVD- 7352 J. Neurosci., October 1, 1997, 17(19):7351–7358 Mammen et al. • Redistribution of Surface Glutamate Receptors WKRPK (Molnar et al., 1994) coupled to albumin. The antisera was centrifuge at 14,000 3 g for 15 min, and the supernatants were processed affinity-purified against the same peptide coupled to thyroglobulin, using as described above for the surface expression experiments. MgCl2 for elution. Fab fragments of N-GluR1 were generated using Quantitation of Western blots. All proteins were visualized by ECL and papain according to the Pierce (Rockford, IL) protocol (no. 44885). Each analyzed using a Molecular Dynamics Personal Densitometer SI. Protein batch of Fab fragments was tested for its ability to detect receptor band intensity was quantitated using ImageQuant. A standard curve was clusters, using C-terminal GluR1 (C-GluR1) as a control. generated for each film, such that band intensity could be expressed as a Patching of surface GluRs. Cultures of embryonic day 19 to postnatal percentage of the biotinylated material at t 5 O (for the half-life day 3 rat spinal cords were prepared as described in O’Brien et al. (1997). experiments) or a percentage of the total extract (for the surface expres- At the appropriate times in culture, the antibody N-GluR1 was added to sion experiments). To calculate half-lives, we plotted the natural loga- growth medium at a concentration of 2 mg/ml for 90 min. Cultures were rithm of the percentage of protein remaining as a function of time and then rinsed in four changes of HBSS and fixed and processed for found the slope of the resulting regression line. Assuming first-order immunohistochemistry as described (O’Brien et al., 1997). Fab fragments decay kinetics, the half-life is equal to ln 2/slope. of N-GluR1 were used at a concentration of 10 mg/ml. In cases in which Metabolic labeling and half-life determination of total GluR1. The C-GluR1 and C-GluR2/3 antibodies were used jointly with N-GluR1, the growth media from spinal cord cultures was removed and replaced with C-terminal antibodies were primarily labeled with Cy3 (CyDye Kit, 1 ml of methionine and cysteine-free media containing 0.8 mCi Tran 35S- Amersham, Arlington Heights, IL). Immunohistochemical control ex- label. Cells were incubated in this media for 30 min, washed with PBS, periments consisted of incubating a 203 concentrate of either the N- or and then returned to the incubator in their original growth media. At C-GluR1 antibodies with appropriate peptide (0.2 mg/ml) for 1 hr at various time points, cell extracts were prepared by adding 1 ml of ice-cold room temperature. This solution was then diluted 1:20 to its final con- lysis buffer (50 mM sodium phosphate, pH 7.5, with 1.0% Triton X-100, centration and added to the coverslips as above. 0.5% deoxycholate, 0.2% SDS, 50 mM NaF, 10 mM sodium pyrophos- Surface half-life experiments. Spinal cord cultures were washed once phate, 5 mM EDTA, 5 mM EGTA, and 10 U/ml aprotinin) to the plates. with PBS/Ca 21/Mg21 (10 mM phosphate buffer, 2.7 mM KCl, 137 mM The cells were scraped off the plates, transferred to a microfuge tube, mixed by inversion, and centrifuged at 4°C for 15 min at 15,000 3 g.The NaCl, 1 mM CaCl2, 0.5 mM MgCl2 , pH 7.4) at 37°C and then cooled gradually to 4°C before they were washed twice with cold PBS/Ca 21/ supernatants were stored at 280°C before use. Once all samples had been Mg 21. Cultures were incubated with 2 ml of biotinylation reagent (1 collected, 100 ml of a 1:1 slurry of protein A-Sepharose CL-4B (Phar- mg/ml NHS-SS-biotin in PBS/Ca 21/Mg21) while being shaken gently at macia, Piscataway, NJ) and lysis buffer with 1% bovine serum albumin 4°C for 12 min, and then washed three times with cold PBS/Ca 21/Mg21 was added to each tube. After rotating for 1 hr at 4°C, the mixtures were including 0.1% BSA. Growth media was then added back, and the plates spun at 2000 3 g for 2 min, and the supernatants were transferred to microfuge tubes containing 150 ml of protein A-Sepharose that had been were returned to the 5% CO2 incubator at 37°C.

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