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The Journal of Neuroscience, February 15, 1999, 19(4):1263-1272
Rapid, Activation-Induced Redistribution of Ionotropic Glutamate
Receptors in Cultured Hippocampal Neurons
Dmitri V.
Lissin1,
Reed
C.
Carroll1,
Roger A.
Nicoll2, 3,
Robert C.
Malenka1, 3, and
Mark von
Zastrow1, 2
Departments of 1 Psychiatry, 2 Cellular and
Molecular Pharmacology, and 3 Physiology, University of
California at San Francisco, San Francisco, California 94143
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ABSTRACT |
We have examined the membrane localization of an AMPA
receptor subunit (GluR1) and an NMDA receptor subunit (NR1)
endogenously expressed in primary cultures of rat hippocampal neurons.
In unstimulated cultures, both GluR1 and NR1 subunits were concentrated
in SV2-positive synaptic clusters associated with dendritic
shafts and spines. Within 5 min after the addition of 100 µM glutamate to the culture medium, a rapid and selective
redistribution of GluR1 subunits away from a subset of synaptic sites
was observed. This redistribution of GluR1 subunits was also induced by
AMPA, did not require NMDA receptor activation, did not result from
ligand-induced neurotoxicity, and was reversible after the removal of
agonist. The activation-induced redistribution of GluR1 subunits was
associated with a pronounced (~50%) decrease in the frequency of
miniature EPSCs, consistent with a role of GluR1 subunit
redistribution in mediating rapid regulation of synaptic efficacy. We
conclude that ionotropic glutamate receptors are regulated in native
neurons by rapid, subtype-specific membrane trafficking, which may
modulate synaptic transmission in response to physiological or
pathophysiological activation.
Key words:
glutamate; synaptic transmission; synaptic plasticity; receptor regulation; redistribution; immunocytochemistry
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INTRODUCTION |
AMPA- and NMDA-type glutamate
receptors are ligand-gated ion channels that play a critical role in
mediating excitatory neurotransmission and synaptic plasticity. It is
well established that AMPA and NMDA receptors are concentrated in the
postsynaptic membrane, but immunocytochemical and biochemical studies
suggest that this may occur via distinct molecular mechanisms (Dong et
al., 1997 ; Kornau et al., 1997; Sheng, 1997; O'Brien et al., 1998).
Furthermore, recent studies suggest that the distribution of AMPA
(Lissin et al., 1998; Turrigiano et al., 1998) and NMDA (Rao and Craig,
1997) receptors in the postsynaptic plasma membrane are regulated by synaptic activity, a regulation that may play an important role in
certain forms of synaptic plasticity. Previously, we (Lissin et al.,
1998) and others (Rao and Craig, 1997) have used immunocytochemical techniques to demonstrate subtype-selective, activity-dependent changes
in the number of glutamate receptors associated with individual synapses in cultured neurons. To date, these studies have detected redistribution of glutamate receptors occurring only over a prolonged time scale, ranging from several days to weeks for detectable changes
in receptor number at synapses to be observed. Physiological studies,
on the other hand, suggest that the number of functional AMPA receptors
at the synapse can be regulated much more rapidly (Isaac et al., 1995;
Liao et al., 1995; Durand et al., 1996). However, the functional status
of glutamate receptors can be regulated by posttranslational
modifications, such as phosphorylation, that do not require physical
movement of the receptor protein. Therefore, whether or not glutamate
receptors actually can undergo rapid physical redistribution in neurons
is an important question that has not been directly addressed previously.
In the present study, we have used immunocytochemical methods to
visualize the localization of endogenously expressed GluR1 and NR1,
subunits of AMPA and NMDA receptors, respectively, in cultured
hippocampal neurons. Because ligand activation has been found to cause
rapid internalization of many other types of signal-transducing receptors, including receptor tyrosine kinases and G-protein-coupled receptors (Hausdorff et al., 1990; Vieira et al., 1996; Grady et al.,
1997), we asked whether an analogous mechanism might apply to
ionotropic glutamate receptors. Within several minutes after ligand-induced activation, we find that GluR1-containing receptors associated with synapses redistribute away from these structures and
subsequently accumulate in intracellular membranes located within
dendritic shafts and cell bodies of stimulated neurons. This process
affects GluR1-containing, but not NR1-containing, receptors, is
reversible, and is not initiated by membrane depolarization alone,
suggesting that it may be mediated directly by ligand-induced activation of AMPA receptors. Furthermore, this rapid relocalization of
AMPA receptors is associated with a significant change in synaptic transmission, suggesting that the rapid redistribution of
GluR1-containing receptors may play an important role in modulating
synaptic efficacy.
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MATERIALS AND METHODS |
Cultured hippocampal neurons. Hippocampi of newborn
(postnatal day 0) Sprague Dawley rat pups were removed, and the dentate gyri were grossly dissected. Cells derived from the remaining tissue
were plated as described previously (Lester et al., 1989), except that
papain was not used. B27-supplemented (Life Technologies, Grand
Island, NY) primary hippocampal cultures were prepared as described
previously (Brewer et al., 1993), and 50 U/ml streptomycin and
penicillin were added. One-half of the growth medium was exchanged 1 d after plating and weekly thereafter.
Immunocytochemistry. Cells were fixed with methanol for 10 min at  20°C and permeabilized with 0.1% Triton X-100 in PBS. AMPA receptors were detected using rabbit polyclonal anti-GluR1
[recognizing the C-terminal cytoplasmic domain, a generous gift
from Dr. Richard Huganir (Dong et al., 1997)]. NMDA receptors
were detected using mouse monoclonal anti-NMDAR1 (PharMingen, San
Diego, CA) antibodies or rabbit polyclonal anti-NMDAR1
[recognizing the C-terminal cytoplasmic domain, a generous gift from
Dr. Richard Huganir (Johns Hopkins University, Baltimore, MD) (Dong et
al., 1997)]. Synapses were labeled using either rabbit
anti-synaptophysin (PharMingen) or mouse anti-SV2 (a generous
gift from Dr. Peter Sargent, University of California, San Francisco,
CA), depending on the species of the anti-receptor antibody
used. SV2 and synaptophysin immunoreactivity colocalized extensively in
our cultures, so these antibodies were used interchangeably as synaptic
markers. Fluorochrome-conjugated anti-mouse and anti-rabbit secondary
antibodies were obtained from Jackson ImmunoResearch (West Grove,
PA). Identification of glutamate receptor clusters and their
colocalization with synaptic markers was accomplished with dual color
microscopy using a Nikon 60× objective (NA 1.4) and standard
fluorescein and Cy3 filter sets (Omega). Fluorescent images were
acquired using a cooled digital CCD camera (Princeton Instruments,
Inc.). Minimal bleed-through between channels was confirmed by imaging
single-labeled specimens. Twelve-bit images were written linearly to an
eight-bit data set after normalizing images to maximize usable contrast
range using IPLab Spectrum software (Signal Analytics). For display in
figures, monochrome and merged color images were processed using Adobe Photoshop software (Adobe Systems, San Jose, CA). For quantitative analysis, raw data present in the normalized (but unprocessed) eight-bit image were analyzed in a blinded manner. Synaptic structures (identified as synaptophysin- or SV2-positive puncta) were scored as
glutamate receptor-positive if the intensity of receptor
immunoreactivity was more than twofold higher than the background level
in the unprocessed image. Using this criterion, receptor-negative
synaptic sites had uniformly low levels of receptor immunoreactivity
[mean ± SD intensity of 27 ± 20 and 19 ± 18 in a
representative eight-bit (256 gray scale) image of GluR1 and NR1
immunoreactivity, respectively]. In contrast, puncta scored as
receptor-positive by this criterion were typically more than fivefold
higher in their intensity of immunochemical staining (157 ± 60 and 131 ± 55 for GluR1 and NMDAR1 immunoreactivity, respectively,
in the same unprocessed eight-bit images). Fluorescein-conjugated
phalloidin (Molecular Probes, Eugene, OR) was used to label actin
enriched in dendritic spines. Colocalization of receptors to
phalloidin-positive structures was determined by costaining with rabbit
anti-GluR1, followed by Cy3-conjugated secondary antibody.
Epifluorescence imaging of these specimens was performed as described
above. Confocal microscopy was performed using a Leica (Nussloch,
Germany) TCS NT confocal microscope and a Leica 100× (NA 1.4)
objective, using dual laser excitation to minimize bleed-through
between FITC and Cy3 channels and collecting serial optical sections
sampled at 0.5 µm steps. Three-dimensional image reconstruction was
performed using Leica TCS Image software. "End-on" views of
dendritic processes were generated from dual label data sets by
calculating a maximum projection image, representing the maximum of all
intensity values along the direction of the z'-axis.
Electrophysiology in cultured hippocampal neurons. Cultures
(12- to 15-d-old) were prepared exactly as for the
immunocytochemistry experiments. Whole-cell recordings were made with
an Axopatch-1D amplifier using low resistance patch pipettes (2-5
M ). Pipette solutions contained (in mM): 116 K-gluconate, 6 KCl, 2 NaCl, 20 HEPES, 0.5 EGTA, 2 MgATP, and 0.3 NaGTP,
adjusted to pH 7.2 with KOH. The extracellular solution contained (in
mM): 115 NaCl, 5 KCl, 5 HEPES, 20 glucose, 1.8 CaCl2, and 1 MgCl2, adjusted to pH 7.3 with NaOH (maintained at 35°C). For recording miniature EPSCs
(mEPSCs), cells were held at 60 mV in extracellular solution containing 10 mM lidocaine. mEPSCs were acquired for 10 min, after which the lidocaine was washed out and cells were placed in
current clamp during the addition of AMPA. After a 15 min treatment
with AMPA, lidocaine was added back to the extracellular solution, and
the cells were voltage clamped at 60 mV for further acquisition. mEPSCs were acquired using Fetchex (Axon Instruments, Foster City, CA)
and were analyzed using MiniTM (J. Steinbach, Washington University, St. Louis, MO). Threshold mEPSC amplitude was set at 3 pA, and 300-1000 events were collected and averaged to calculate the mean mEPSC amplitude and frequency for each culture preparation examined.
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RESULTS |
The localization of AMPA and NMDA receptors in primary cultures of
rat hippocampal neurons was examined using antibodies recognizing endogenous GluR1 (an AMPA receptor subunit) and NR1 (an NMDA receptor subunit). All studies were performed in 2-week-old cultures in which
glutamate receptors have been demonstrated previously to be localized
at postsynaptic sites in dendritic spines (Craig et al., 1993; O'Brien
et al., 1998).
Accordingly, in control cultures, GluR1 subunits (detected using an
antibody recognizing the C-terminal cytoplasmic domain) were visualized
in numerous puncta associated with synaptic sites identified by SV2
immunoreactivity (Fig.
1A, left
panels, arrow, example of a colocalized synaptic
puncta). Quantitation of this colocalization indicated that, in control
cultures, the vast majority (~85%) of GluR1-containing puncta
colocalized with synapses (Fig. 1C, filled
bar), and most synapses were associated with detectable GluR1 immunoreactivity (Fig. 1D, filled
bar). To determine whether ligand-induced activation causes a
physical redistribution of glutamate receptors, as is the case for
certain other classes of signal-transducing receptors [such as
G-protein-coupled receptors and receptor tyrosine kinases (Vieira et
al., 1996; Grady et al., 1997)], we applied 100 µM
glutamate and then examined the localization of glutamate receptor
subunits. This manipulation caused a rapid and pronounced
redistribution of GluR1-containing receptors, as indicated by visual
inspection of fluorescence micrographs (Fig. 1B,
arrows, example of a GluR1-negative synapse). Quantitation of these observations, visualized in multiple fields (n = 20 fields acquired blindly from a total of three separate
experiments), confirmed a substantial decrease in the proportion of
GluR1-positive puncta associated with synapses (Fig. 1C,
open bar) occurring within 15 min after application of
glutamate and a concomitant decrease in the number of synapses
containing detectable GluR1 immunoreactivity (Fig.
1D, open bar). This glutamate-induced
redistribution of GluR1 subunits was not associated with any detectable
change in the number of SV2 puncta visualized in the cultured neurons (data not shown), indicating that glutamate caused a rapid
redistribution of GluR1 subunits from synaptic to nonsynaptic sites
without changing the overall number of synapses.
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Figure 1.
Glutamate causes a rapid redistribution of GluR1
subunits away from synapses in cultured hippocampal neurons.
A, Immunocytochemical localization of GluR1 and SV2 in
cultured hippocampal neurons incubated in the absence of added
glutamate. A higher magnification image of the region indicated by the
box is displayed in the bottom panels.
Arrows in these panels point out an example of a GluR1
puncta colocalized with SV2 immunoreactivity. B,
Immunocytochemical localization of GluR1 subunits and SV2 in neurons
incubated in the presence of 100 µM glutamate for 15 min.
Scale bars, 5 µm. C, Quantitation of the proportion of
GluR1-immunoreactive puncta associated with SV2 in control (untreated)
and glutamate-treated neurons. D, Quantitation of the
proportion of SV2-positive synaptic puncta associated with detectable
concentrations of GluR1 immunoreactivity. Data are derived from
analysis of a total of 20 independent fields, selected at random and
examined in a blinded manner, from three separate experiments.
GluR1-positive puncta were defined by a GluR1 immunoreactivity at least
twofold greater than background (unclustered regions) in the raw
(unprocessed) fluorescence image, as described in Materials and
Methods. Bars represent the mean proportion, and error
bars represent the SEM from the independent fields
(n = 20).
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In contrast to the rapid redistribution of GluR1 immunoreactivity
observed in the presence of glutamate, no apparent change in the
localization of NR1 subunits (detected using a rabbit antibody recognizing the C-terminal cytoplasmic domain) was observed under the
same conditions (Fig.
2A,B).
Quantitation of these observations in the unprocessed images (collected
from 20 fields acquired blindly from a total of three separate
experiments) confirmed the absence of any detectable change in the
synaptic localization of NR1 subunits (Fig.
2C,D). Similarly, no apparent change in the
synaptic localization of NR1 subunits was detected using a monoclonal
antibody recognizing a different epitope of NR1 compared with the
alternate synaptic marker synaptophysin (data not shown). To further
confirm the subunit specificity of this receptor redistribution, the
localization of NR1 and GluR1 was examined simultaneously in the same
neurons, using rabbit anti-GluR1 and mouse anti-NR1 antibodies.
Clusters of GluR1 and NR1 immunoreactivity were extensively colocalized in unstimulated cultures, consistent with the localization of both AMPA
and NMDA receptors in the same postsynaptic membranes (Fig.
3A, arrows,
examples of colocalized puncta). After the application of glutamate,
however, the localization of GluR1 relative to NR1 subunits changed
rapidly. This was indicated by the appearance of an increased
proportion of puncta selectively containing only GluR1 or NR1
immunoreactivity, but not both, in cultures incubated in the presence
of glutamate for 30 min (Fig. 3B, arrows,
examples of puncta associated with high levels of NR1 immunoreactivity but devoid of detectable GluR1 immunoreactivity; similar results were
observed in four independent experiments).
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Figure 2.
NR1 subunits do not undergo detectable
redistribution under the same conditions. The same experiments as
described in Figure 1 were conducted using anti-NR1 immmunocytochemical
staining in control (untreated) (A) and
glutamate-treated (B) neurons. Scale bars, 5 µm. Arrows in A and B
point out examples of an NR1 puncta colocalized with SV2. Analysis of
the proportion of NR1-positive puncta associated with synapses
(C) and the number of synapses associated with
detectable concentration of NR1 immunoreactivity
(D) were determined as in Figure 1.
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Figure 3.
Dual localization of GluR1 and NR1 subunits in the
same neurons. Cultured hippocampal neurons were incubated in the
absence (A) or presence (B)
of 100 µM glutamate for 15 min and then fixed,
permeabilized, and processed for immunocytochemical analysis using
rabbit anti-GluR1 and mouse anti-NR1, as described in Materials and
Methods. Representative fluorescence micrographs obtained under each
condition are displayed. Arrows in A
indicate examples of puncta associated with both GluR1 and NR1
immunoreactivity. Open arrows in B
indicate examples of NR1-containing puncta devoid of any detectable
GluR1 immunoreactivity, which were observed frequently in
glutamate-treated cultures. Scale bars, 5 µm.
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The selective agonist AMPA also caused a rapid and pronounced
redistribution of GluR1 subunits (Fig.
4A,B).
Redistribution of GluR1 could be observed as early as 5 min after
application of AMPA to the cultures, although the maximum effect
appeared to require 10-15 min incubation in the continuous presence of AMPA. Significantly, this redistribution of GluR1 was observed when
AMPA was applied in the presence of high concentrations of the
selective NMDA antagonist 2-amino-5-phosphonovaleric acid (D-APV) (100 µM), indicating that this
redistribution was independent of NMDA receptor activation as a
consequence of AMPA-induced neuronal activity (Fig. 4C). To
determine whether this effect was a consequence of the membrane
depolarization caused by activation of AMPA receptors, we examined the
effects on GluR1 distribution of replacing media with high potassium
(150 mM) solution for 30 min. This manipulation failed to
cause any apparent redistribution of GluR1 subunits away from synapses.
Rapid redistribution of GluR1-containing receptors was still observed,
however, when AMPA or glutamate was applied to cultures together with
high potassium.
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Figure 4.
Rapid redistribution of GluR1 subunits is induced
by AMPA and does not require activation of NMDA receptors.
A, In control (untreated) neurons, the majority of GluR1
receptor clusters were localized at the periphery of dendritic shafts,
consistent with synaptic localization. B, Within 15 min
after the addition of 100 µM AMPA to the culture medium,
a pronounced redistribution of GluR1 subunits was observed, which was
qualitatively identical to that induced by glutamate. Note that most
punta are in the center of the dendritic shaft. C,
Coapplication of the NMDA receptor antagonist APV (100 µM) together with AMPA did not block the AMPA-induced
redistribution of GluR1 subunits, confirming that this redistribution
does not require activation of NMDA receptors. Scale bars, 5 µm.
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Because a major consequence of depolarizing neurons is to elicit a rise
in postsynaptic Ca2+, it also was of interest to
determine whether such an increase in intracellular
Ca2+ level is required for the redistribution of
GluR1 caused by application of AMPA. To prevent AMPA-induced changes in
intracellular Ca2+, we applied the
membrane-permeable Ca2+ chelator BAPTA-AM (10 µM) for 1 hr before AMPA application. Although we cannot
rule out some effect of BAPTA-AM, significant redistribution of GluR1
was still observed in cells incubated under these conditions (data not
shown). Furthermore, AMPA-induced redistribution of GluR1 was not
blocked by 1 µM tetrodotoxin (data not shown). Together, the results thus far suggest that the rapid redistribution of GluR1
subunits is mediated specifically by ligand-induced activation of AMPA receptors.
It is well known that glutamate can cause significant excitotoxicity
both in vivo and in cultured neurons (Choi, 1994). Previous studies of cultured hippocampal neurons indicate that concentrations of
glutamate comparable to those used in the present studies are capable
of causing irreversible cell damage (Michaels and Rothman, 1990;
Schinder et al., 1996). To evaluate the possibility that the observed
redistribution of GluR1 subunits is a consequence of cell death,
neuronal viability was determined using the established method of
trypan blue staining (Schinder et al., 1996) 24 hr after incubation of
cultures with glutamate or AMPA under conditions identical to those
used for studies of receptor localization. Application of glutamate for
15 min caused excitotoxicity that was in significant excess to that
observed in untreated neurons. However, consistent with previous
studies establishing a requirement for NMDA receptor activation in
rapid excitotoxicity (Michaels and Rothman, 1990), application of AMPA
caused significantly less excitotoxicity (Fig.
5A). This pronounced
difference in the excitotoxic actions of AMPA versus glutamate
application contrasts markedly with their similar effects on the
redistribution of GluR1 (Fig. 4). In addition, the rapid redistribution
of GluR1 subunits observed in AMPA-treated neurons appeared to be
reversed after washout of AMPA (Fig. 5B), suggesting that
the rapid redistribution of GluR1 subunits is reversible. Thus, we
conclude that the rapid redistribution of GluR1 observed in the present
study represents a specific regulation of the composition of
postsynaptic sites that is not the result of agonist-induced
excitotoxicity.
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Figure 5.
Redistribution of GluR1 subunits can be
dissociated from ligand-induced neurotoxicity and is reversible after
removal of agonist. A, Neurotoxicity in cultured
hippocampal neurons was assayed by trypan blue exclusion 24 hr after
treatment of cultures for 15 min with 100 µM glutamate or
AMPA under conditions identical to those used to induce rapid
redistribution of GluR1 subunits. Whereas glutamate caused substantial
neurotoxicity, AMPA caused significantly less neurotoxicity [although
still significantly more than observed in control (untreated)
cultures]. B, AMPA-induced redistribution of GluR1
subunits was reversed within 6 hr after washout of AMPA. Scale bars, 5 µm.
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Because of its reduced potential to cause neurotoxicity, AMPA was used
to induce GluR1 redistribution for more detailed studies of this
process. GluR1-containing receptors present in control neurons were
associated with a large number of structures resembling dendritic
spines, as indicated by their localization at the periphery of the
dendritic shaft and colocalization with intense staining with
phalloidin (Fig. 6A,
arrow, example of a presumed dendritic spine with
colocalized GluR1 immunoreactivity), which labels F-actin concentrated
in these structures (Allison et al., 1998). In cells treated with AMPA,
however, phalloidin-positive spinous structures were observed that were
not associated with detectable GluR1 immunoreactivity (Fig.
6B, green, arrow). Furthermore,
numerous GluR1 puncta were visualized in AMPA-treated neurons that were
not associated with phalloidin-positive structures and were located
toward the base of the dendritic shaft (Fig. 6B,
red) or in the cell body (Fig. 4B). These
observations suggest that AMPA caused a rapid and specific redistribution of GluR1-containing receptors away from dendritic spines, although phalloidin-positive structures were observed both in
untreated and AMPA-treated neurons.
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Figure 6.
Top. Rapid, AMPA-induced redistribution of
GluR1 subunits from dendritic spines. A, In control
neurons, many clusters of GluR1 subunits (red)
colocalized with phalloidin staining (green),
consistent with colocalization in dendritic spines.
Arrow indicates an example of such a structure, which
appears yellow in the merged image. B,
After incubation of cultures with 100 µM AMPA for 15 min,
many GluR1-containing puncta were observed to be distinct from
phalloidin-stained extensions of the dendritic membrane, consistent
with a redistribution of GluR1 subunits away from dendritic spines.
Arrow indicates an example of such a phalloidin-positive
dendritic specialization, observed frequently in AMPA-treated neurons,
which was devoid of detectable GluR1 immunoreactivity. Scale bars, 2 µm.
Figure 7. Bottom. Analysis of AMPA-induced
redistribution of GluR1 subunits in individual dendrites using confocal
fluorescence microscopy and three-dimensional image reconstruction.
Schematic view of a control neuron (A) and an
AMPA-treated neuron (B), showing the scanned
region containing an individual dendritic shaft (box).
C, D, Representative three-dimensional
reconstructions displaying the localization of GluR1 immunoreactivity
in dendritic specializations viewed obliquely (as indicated by the
schematic) using the maximum projection technique in a control
(untreated) neuron and an AMPA-treated (100 µM for 15 min) neuron, respectively. E,
F,
Collapsed end-on images of the same three-dimensional image
reconstructions as in C and D, displayed
using the maximum projection technique along the
z'-axis, emphasizing the AMPA-induced redistribution of
GluR1 subunits (red) away from SV2-positive synaptic
sites (green) located at the periphery.
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We further examined the localization of GluR1 in dendritic processes at
higher resolution using laser scanning confocal microscopy. Because of
the complex morphology and small size of these processes, only a small
number of receptor clusters were imaged in an individual optical
section. Therefore, three-dimensional image reconstruction was used to
analyze the localization of GluR1 and SV2 immunoreactivity in serial
optical sections imaged at maximum resolution by scanning dendritic
processes from control and AMPA-treated neurons (Fig. 7A,B).
A representative three-dimensional reconstruction of GluR1 immunoreactivity in a portion of a dendritic process from an untreated neuron is shown in Figure 7C, and an equivalent
reconstruction of a process from an AMPA-treated neuron is shown in
Figure 7D. The AMPA-induced redistribution of GluR1 from
peripheral processes to the base or interior of the dendritic process
is emphasized by comparing Figure 7, E and F,
which represent a collapsed end-on (x'/z') view down the cross section of
reconstructed processes from untreated and AMPA-treated neurons,
respectively. GluR1 and SV2 colocalize extensively at the periphery of
dendritic processes (Fig. 7E, yellow), whereas a
centripetal redistribution of GluR1 immunoreactivity away from the
peripheral synaptic sites (marked by SV2 immunoreactivity) was observed
in dendritic processes from AMPA-treated neurons (Fig. 7F,
red and green, respectively).
The rapid redistribution of GluR1 subunits away from the synapse would
be expected to have significant effects on synaptic transmission. To
examine the functional consequences of the rapid redistribution of
GluR1-containing receptors, synaptic transmission in cultured neurons
was analyzed by recording mEPSCs before and after bath application of
AMPA. As expected, the cell membrane potential depolarized from
approximately 60 to 0 mV after the application of AMPA and
returned to original levels when the drug was washed out. Comparison of
whole-cell recordings before and after treatment showed that
application of AMPA caused a large decrease in the frequency of mEPSCs
without causing a significant decrease in their mean amplitude (Fig.
8). This result is consistent with the
hypothesis that the rapid redistribution of GluR1 subunits influences
the efficacy of synaptic transmission in a subset of synaptic
sites.
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Figure 8.
The rapid redistribution of GluR1 subunits is
associated with decreased frequency of mEPSCs. A, Traces
of miniature synaptic events recorded from cultured hippocampal neurons
in the presence of lidocaine (10 µM) before
(Pre) and after (Post) application of 100 µM AMPA to cells for 15 min, followed by rapid washout.
B, Averaged mEPSC (250 events) pretreatment and
posttreatment with AMPA from experiment in A show only a
slight reduction in amplitude. C, Summary of the effects
of AMPA treatment on the mean amplitude and frequency of mEPSCs. Error
bars represent SEM (n = 9 cells).
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DISCUSSION |
We have demonstrated a rapid, activation-induced redistribution of
GluR1-containing receptors from a subset of synaptic sites. This
regulatory process appears to be highly specific for GluR1-containing AMPA receptors relative to NR1-containing NMDA receptors and occurs without a gross change in neuronal cytoarchitecture, as indicated by
the apparent preservation of synaptic sites in agonist-treated cells.
This redistribution of GluR1 subunits occurs much more rapidly than the
previously reported processes of activity-dependent redistribution of
AMPA and NMDA receptors, which require several days to be detected
experimentally (Rao and Craig, 1997; Lissin et al., 1998). Furthermore,
in contrast to these previously reported changes in the subcellular
distribution of glutamate receptor subunits, the rapid redistribution
of GluR1 observed in the present study does not require activation of
NMDA receptors. Thus, we have identified a novel mechanism of receptor
regulation, which can rapidly and specifically control the number of
GluR1-containing receptors present at synapses.
The observation that this redistribution of GluR1 subunits is
induced specifically by AMPA receptor activation, is not induced by
depolarization or NMDA receptor-mediated neuronal activity, and is not
blocked by the Ca2+ chelator BAPTA-AM suggests the
intriguing possibility that the molecular machinery mediating this
rapid regulation may be controlled directly by an activated
conformation of the AMPA receptor complex itself. In this case,
regulated protein interactions with individual receptor subunits
located at synaptic sites may play a critical role in mediating the
selectivity of subunit redistribution, perhaps independently of
downstream signal transduction. Thus, the regulation of ionotropic
glutamate receptors may share unanticipated similarities with other
classes of receptor protein, such as G-protein-coupled receptors, which
are regulated by conformation-dependent interaction of receptors with
cytoplasmic regulatory proteins (such as receptor kinases and
arrestins) that can occur in the absence of downstream (G-protein-linked) signal transduction (Freedman and Lefkowitz, 1996;
Grady et al., 1997).
Rapid, activation-induced regulation of G-protein-coupled receptors is
often associated with lateral redistribution of receptors in the plasma
membrane (von Zastrow and Kobilka, 1994; Goodman et al., 1996),
followed by rapid endocytosis (Freedman and Lefkowitz, 1996; Okamoto et
al., 1998). Although these mechanisms obviously regulate the
subcellular localization of receptors, they are also thought to play a
fundamental role in modulating the efficacy of signal transduction to
distinct effectors (Vieira et al., 1996; Grady et al., 1997). To our
knowledge, the present results provide the first direct evidence that a
similar process of receptor redistribution may regulate ionotropic
glutamate receptors. Although the precise mechanisms mediating
glutamate receptor redistribution remain to be elucidated, the present
results indicate that GluR1-containing and NR1-containing receptors
differ greatly in their ability to undergo rapid redistribution in
neurons, although these receptors are frequently concentrated in the
same postsynaptic sites. This selectivity of regulation may be
conferred by the highly specific interaction of AMPA and NMDA receptors
with distinct cytoplasmic proteins associated with postsynaptic
densities (Kornau et al., 1995, 1997; Dong et al., 1997; Srivastava et
al., 1998). Indeed, previous studies indicate that AMPA receptors can
be dissociated from the neuronal cytoskeleton much more readily than
NMDA receptors (Allison et al., 1998).
The ligand-induced removal of GluR1-containing receptors from the
synaptic plasma membrane would be expected to depress synaptic transmission. Consistent with this prediction, prolonged agonist exposure caused a reduction in the frequency of mEPSCs. Thus, we
believe that we have identified a heretofore unknown mechanism for the
functional regulation of synaptic AMPA receptors. For example, it is
possible that the rapid and reversible redistribution of
GluR1-containing AMPA receptors observed in the present studies may
play a role in the physiological phenomenon of silent synapses in which
the efficacy of individual synapses is regulated by a dramatic change
in the number of functional AMPA-type receptors present in the
postsynaptic membrane (Isaac et al., 1995; Liao et al., 1995; Durand et
al., 1996). However, because multiple mechanisms contribute to the
physiological regulation of signal transduction in the postsynaptic
plasma membrane, the precise role of this specific mechanism of
receptor regulation remains to be determined. Furthermore, because the
experimental conditions used to induce the rapid redistribution of
GluR1-containing receptors are not achieved under normal physiological
conditions (i.e., bath application of agonist), it remains to be
determined whether rapid redistribution of AMPA receptor subunits is
induced by physiological synaptic activity.
Our immunocytochemical and electrophysiological data are consistent
with the hypothesis that AMPA causes an "all-or-none" removal of
AMPA receptors from a subset of synaptic sites. In this regard, the
rapid regulation of GluR1 receptors may differ significantly from
rapid, ligand-induced redistribution of certain G-protein-coupled
receptors, which is characterized by a partial decrease in the number
of surface receptors observed at steady state attributable to
continuous endocytosis and recycling in the presence of agonist.
However, caution is required in interpreting the present data because
of the limited sensitivity of our analyses. Indeed, it is unlikely that
our immunochemical methods are sufficiently sensitive to detect
receptor subunits in synapses that contain very small (but perhaps
functionally sufficient) amounts of receptor protein. Similarly, we
cannot state with certainty whether the decrease in mEPSC frequency
observed in the present study represents complete "silencing" of a
restricted subset of synapses or a graded reduction in a larger number
of synapses, leading to a limited subset of synapses falling below our
threshold for detection by whole-cell electrophysiology. Nevertheless,
our observations that the redistribution of AMPA receptors is
associated with a relatively small change in mEPSC amplitude compared
with the more pronounced effect on frequency does suggest some
heterogeneity in the response of individual synapses to activation.
Further studies will be necessary to elucidate specific mechanisms
underlying the observed redistribution of synaptic glutamate receptors
and to determine their contribution to graded versus all-or-none
regulation of synaptic efficacy.
The experimental conditions used to elicit the rapid redistribution of
AMPA receptors are similar to those used to induce excitotoxicity in
hippocampal neurons. Importantly, however, the rapid redistribution of
GluR1 could be induced under conditions that caused relatively little
neuronal death (i.e., AMPA stimulation in the presence of APV).
Furthermore, this redistribution of receptor subunits was reversible
after removal of agonist. Thus, the rapid, activation-induced
redistribution of GluR1-containing receptors observed in the present
study is unlikely to be a direct consequence of excitotoxicity. It is
possible, however, that rapid redistribution of GluR1-containing AMPA
receptors functions physiologically in the context of excitotoxicity,
perhaps as a mechanism to protect neurons from excessive stimulation by
high concentrations of glutamate. If this is true, elucidating
mechanisms that mediate the rapid redistribution of receptor components
from the synaptic plasma membrane may be of fundamental importance to
understanding mechanisms of neural injury and may therefore define
novel therapeutic targets for the development of neuroprotective agents.
In conclusion, we have identified a novel type of regulation of the
subcellular localization of ionotropic glutamate receptors, which
differs substantially from previously described mechanisms of glutamate
receptor regulation. These observations establish that receptor
trafficking mechanisms mediate rapid regulation of ionotropic glutamate
receptors, and they suggest that these mechanisms of receptor
regulation may play an important role in modulating postsynaptic
responses to physiological or pathophysiological stimuli.
| |
FOOTNOTES |
Received July 22, 1998; revised Nov. 13, 1998; accepted Nov. 25, 1998.
These studies were supported by grants from the National Institutes of
Health (to R.C.M., R.A.N., and M.v.Z.), the Human Frontier Science
Program (to R.C.M.), and the McKnight Endowment Fund for Neuroscience
(to R.C.M.). R.C.C. was supported by a National Research Service Award
from the National Institutes of Health. We thank Chris Billante and
Stephen Gomperts for preparing hippocampal cultures, Richard Huganir
and Peter Sargent for providing antibodies, and Heather W. Deacon for
expert advice and assistance with confocal microscopy.
Correspondence should be addressed to Mark von Zastrow, University of
California at San Francisco, Box 0984IRE/UCSF, Room LP-A104 LPPI, 401 Parnassus Avenue, San Francisco, CA 94143-0984.
| |
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Top
Abstract
Introduction
