RIKEN-MIT Neuroscience Research Center, Center for Learning and Memory, Department of Brain and Cognitive Sciences, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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ABSTRACT |
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Cottrell, Jeffrey R.,
Gilles R. Dubé,
Christophe Egles, and
Guosong Liu.
Distribution, Density, and Clustering of Functional Glutamate
Receptors Before and After Synaptogenesis in Hippocampal Neurons.
J. Neurophysiol. 84: 1573-1587, 2000.
Postsynaptic differentiation during glutamatergic synapse formation is
poorly understood. Using a novel biophysical approach, we have
investigated the distribution and density of functional glutamate
receptors and characterized their clustering during synaptogenesis in
cultured hippocampal neurons. We found that functional
-amino-3-hydroxy-5-methyl-4-isoxazolpropionate (AMPA) and
N-methyl-D-aspartate (NMDA) receptors are evenly
distributed in the dendritic membrane before synaptogenesis with an
estimated density of 3 receptors/µm2. Following
synaptogenesis, functional AMPA and NMDA receptors are clustered at
synapses with a density estimated to be on the order of
104 receptors/µm2, which
corresponds to ~400 receptors/synapse. Meanwhile there is no
reduction in the extrasynaptic receptor density, which indicates that
the aggregation of the existing pool of receptors is not the primary
mechanism of glutamate receptor clustering. Furthermore our data
suggest that the ratio of AMPA to NMDA receptor density may be
regulated to be close to one in all dendritic locations. We also
demonstrate that synaptic AMPA and NMDA receptor clusters form with a
similar time course during synaptogenesis and that functional AMPA
receptors cluster independently of activity and glutamate receptor
activation, including following the deletion of the NMDA receptor NR1
subunit. Thus glutamate receptor activation is not necessary for the
insertion, clustering, and activation of functional AMPA receptors
during synapse formation, and this process is likely controlled by an
activity-independent signal.
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INTRODUCTION |
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Glutamatergic synapses are the
primary source of excitatory synaptic transmission in the CNS. Their
formation is critical in the establishment of synaptic connections and
the refinement of these connections that occurs during development
(Jessell and Kandel 1993) and likely during learning and
memory (Milner et al. 1998
). Despite the importance of
these synapses, the details of their formation, especially regarding
postsynaptic differentiation, remain to be determined. A model system
for understanding postsynaptic differentiation during synaptogenesis is
the cholinergic vertebrate neuromuscular junction (NMJ) (Hall
and Sanes 1993
; Sanes and Lichtman 1999
), where
acetylcholine receptors (AChRs) are inserted at a high-density
(~1,000/µm2) along the muscle membrane before
synapse formation. Following axonal contact and the activation of a
receptor tyrosine kinase on the muscle (Glass et al.
1996
), these AChRs aggregate independently of activity to the
synaptic site. Subsequently, the subjunctional nuclei increase, and
extrajunctional nuclei decrease, expression of AChRs, resulting in a
dramatic difference in AChR density between junctional
(~104/µm2) and
extrajunctional (~10/µm2) regions
(Hall and Sanes 1993
; Sanes and Lichtman
1999
). Because of similarities in their structure and function,
there may be similarities between the formation of the postsynaptic
site of the NMJ and glutamatergic synapses.
To understand postsynaptic differentiation during glutamatergic synapse
formation, we need to determine the distribution and density of
glutamate receptors during synapse formation, the mechanism of
glutamate receptor cluster formation, and the signals that regulate
postsynaptic differentiation and glutamate receptor clustering. These
processes in developing glutamatergic synapses are poorly understood. A
number of studies, based primarily on antibody-labeling experiments,
have shown that the ionotropic glutamate receptor subtypes
-amino-3-hydroxy-5-methyl-4-isoxazolpropionate (AMPA) and
N-methyl-D-aspartate (NMDA) receptors and the
metabotropic glutamate receptors (mGluRs) are clustered at synaptic
sites (Baude et al. 1995
; Craig et al.
1993
; Richmond et al. 1996
). However, the exact
receptor densities before and after synapse formation have not been
determined primarily because the antibody approach does not have
adequate sensitivity or quantitative power. Furthermore although a
number of hypotheses have been proposed (Craig et al. 1994
; Nusser et al. 1998
; Richmond et al.
1996
; Steward 1995
; Tovar and Westbrook
1999
), there is little information regarding the exact
mechanism of receptor clustering. Last, the signals regulating
glutamate receptor clustering during synapse formation remain to be
determined. Antibody-labeling data indicate that glutamate receptor
clustering occurs independently of neuronal activity (Craig et
al. 1994
; Mammen et al. 1997
). In contrast, electrophysiological data suggest that NMDA receptor activation is
required for AMPA receptor insertion, clustering, and/or activation during synaptogenesis (Durand et al. 1996
; Isaac
et al. 1997
; Liao and Malinow 1996
; Wu et
al. 1996
). This hypothesis has been supported by recent
morphological studies (Gomperts et al. 1998
; He
et al. 1998
; Liao et al. 1999
; Petralia
et al. 1999
; Takumi et al. 1999
).
In this study, we have addressed these issues regarding postsynaptic differentiation during glutamatergic synapse formation in cultured hippocampal neurons. Using a novel glutamate receptor map technique, we determined the distribution and density of functional glutamate receptors before and after synapse formation. Furthermore we assessed the time course of functional AMPA and NMDA receptor clustering and determined the role of neuronal activity and glutamate receptor activation in the insertion and clustering of functional glutamate receptors during synapse formation.
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METHODS |
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Cell culture
Procedures for culturing hippocampal neurons from neonatal rats
and mice were as previously described (Liu and Tsien
1995). All pharmacological treatments were added to the culture
medium at 0 days in vitro (DIV) and refreshed every 3 days. The
glutamate receptor antagonist cocktail included 10 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX; RBI), 5 µM
3-((R)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid
(D-CPP; RBI), 500 µM RS-1-aminoindan-1,5-dicarboxylic
acid (AIDA; Tocris), 500 µM 2S-alpha-ethylglutamic acid (EGLU;
Tocris), and 500 µM RS-alpha-cyclopropyl-4-phosphonophenylglycine
(CPPG; Tocris) to block AMPA receptors, NMDA receptors, and group
I-III mGluRs, respectively (Jane et al. 1996
;
Pellicciari et al. 1995
).
FM1-43 labeling and synaptic density measurements
Cultures were stained with 10 µM N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide (FM1-43; Molecular Probes) in (in mM): 39 NaCl, 90 KCl, 30 glucose, and 25 HEPES, pH adjusted to 7.4 with NaOH for 1 min. They were then washed for >5 min in Tyrode solution (in mM): 128 NaCl, 5 KCl, 30 glucose, 25 HEPES, 1 MgCl2, 2 CaCl2, plus 0.5 µM tetrodotoxin (TTX; Oretek), 5 µM glycine (Sigma), and 50 µM picrotoxin (Sigma), pH adjusted to 7.4 with NaOH. During electrophysiological experiments, the FM1-43 solution contained 5 µM D-CPP and 10 µM DNQX to block NMDA and AMPA receptors, respectively. Therefore we would expect no significant changes in the number and properties of these receptors due to acute receptor activation during FM1-43 loading. Although possible, we know of no cases in which simple depolarization without simultaneous receptor activation changed the function of synaptic receptors. Imaging was performed with an inverted Olympus Fluoview Personal Confocal Microscope, using an Olympus 40× planapochromat water immersion lens (1.15 NA). The number of FM1-43 punctae per 20 µm was determined by manually counting on 400 dendritic segments taken from 10 different regions from at least two different cultures. Values are the mean of 10 regions ± the standard error of the mean (SE).
Electrophysiology
Whole cell patch-clamp recordings
(Vm = 60 mV) were made from
presumptive pyramidal cells at room temperature using an Axon Instruments model 200B Integrating Patch Clamp amplifier with a 1- kHz
(8-pole Bessel) low-pass filter. Data were digitized at 10 kHz by a
Digidata 1200B A/D converter. Patch pipettes (2-4 M
) contained (in
mM): 125 CsMeSO3, 10 HEPES, 8 NaCl, 1 CaCl2, 10 EGTA, 2 Mg-ATP, 0.3 GTP, pH adjusted to
7.25 with CsOH. Extracellular solution was Tyrode solution as described
above. Access resistance was monitored on-line and was typically <10
M
. Glutamate iontophoresis (MVCS 02C microiontophoresis controller,
NPI Electronics) was performed as previously described (Liu et
al. 1999
).
Glutamate receptor map
Patch-clamp recordings were performed from the cell body, and
the glutamate iontophoresis electrode was visually guided to the
dendritic location where the map was to begin. A confocal image of the
dendrite and iontophoresis electrode was taken and transferred into
home-designed software. The area to be mapped was selected by
indicating to the software the starting and ending dendritic locations
and the desired interval between each point, typically 0.5-1 µm.
During each step, glutamate was applied to the dendrite via 100 nA
iontophoretic current, and the resulting glutamate-evoked response was
recorded at the cell body. This iontophoretic current saturates the
glutamate receptors of single synapses (see Fig. 1E). Each
successive glutamate application was delayed by 1.5 s.
Microelectrode movements were controlled by micromanipulators (MP-285;
Sutter Instrument). Custom-written software was used to control
glutamate iontophoresis, acquisition of current signals with whole cell
recordings, and off-line data analysis.
Receptor density measurements
Receptor density was estimated from the glutamate-evoked
responses according to the following relationship (assuming a
homogenous population of channels with a constant ,
Vr, and
po)
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The surface area of neurons at 8 DIV was roughly estimated by determining the surface area of a model cell based on confocal images of cells filled with the intracellular dye Alexa Flour 568 hydrazide (5 µM; Molecular Probes). This model neuron assumed a spherical cell body with a diameter of 10 µm; three major dendrites with a length of 50 µm and diameter of 3 µm; 15 second-tier dendrites with a length of 50 µm and diameter of 1 µm; and 15 third-tier dendrites with a length of 30 µm and a diameter of 0.5 µm.
NMDA receptor po calculation
To reduce glutamate spillover to extrasynaptic locations (see RESULTS), a suction electrode (tip diameter ~ 2 µm) was placed opposite to the iontophoresis electrode. Multiple pulses of glutamate were applied to the synapse in the absence of extracellular Mg2+ or at +40 mV and in the presence of 5 or 20 µM (5R,10S)-(+)5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine (MK-801; Tocris), and suction (2 PSI; Picospritzer IID, General Valve) was applied for 200 ms at 1 ms following the iontophoretic pulse.
The NMDA receptor po at any time
t was calculated from the following equation whose
derivation is described in Rosenmund et al. (1995)
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RESULTS |
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Characterization of the glutamate receptor map
To determine the distribution and density of functional glutamate
receptors in cultured hippocampal neurons, we combined a rapid and
localized glutamate iontophoresis technique (Liu et al.
1999) (see APPENDIX) with a computer-assisted
positioning system to move the electrode along the dendrite (Fig.
1A).
First, presynaptic boutons were labeled with FM1-43 (Ryan et al.
1993
). Then, whole cell patch-clamp recordings were made from
the soma, and brief, saturating pulses of glutamate were
iontophoretically applied (
100 nA; 1 ms; 150 mM Na-glutamate) to the
dendrite at 0.5 to 1 µm steps. The resulting glutamate-evoked
currents were recorded at the cell body in the presence of TTX to block
action potentials.
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Figure 1B illustrates an AMPA receptor map
(Vm = 60 mV) at an FM1-43 labeled
presynaptic terminal. The peak glutamate-evoked response rose as the
electrode neared the synapse and declined as it moved away, resulting
in a Gaussian-shaped peak amplitude distribution around the FM1-43
labeled bouton. This distribution is consistent with functional AMPA
receptors being clustered beneath the presynaptic terminal
(Baude et al. 1995
; Craig et al. 1993
). Since the width of this distribution at the points where the AMPA receptor response approaches the baseline is typically between 3 and 4 µm, the synaptic AMPA receptor clusters were affected by glutamate
applications when the electrode was within 2 µm of either side of the
synapse. Knowing that the width of the postsynaptic density in which
the receptor cluster is located is comparatively small (<0.2 µm), we
conclude that the glutamate applications affect the AMPA receptors
along a <4 µm length of the dendrite by the time the peak AMPA
receptor current is measured (<3 ms). We used calculations of
glutamate diffusion and of the glutamate concentration dependency of
AMPA receptor probability of opening
(po) to estimate the length of a
hypothetical cylindrical dendrite whose AMPA receptors would be
activated by our glutamate applications. In line with our experimental
data, the predicted length of affected dendrite was 3 µm (see
APPENDIX).
Since NMDA receptors frequently colocalize with AMPA receptors at
individual synapses (Bekkers and Stevens 1989;
Jones and Baughman 1991
), both receptor components can
be detected by glutamate application to single synapses under
conditions in which the voltage-dependent NMDA receptor
Mg2+ block is removed. This is evident in the
current-voltage (I-V) relationship of the glutamate-evoked
responses at an individual synapse (Fig. 1C) in which the
NMDA receptor component is distinguished by its J-shaped I-V
relationship and long decay time (Mayer and Westbrook
1987
). Thus we can determine the distribution of functional NMDA receptors and isolate individual receptor clusters with an NMDA
receptor map (Fig. 1D; Vm = +40 mV, 10 µM DNQX). As the NMDA receptor response was reduced to
baseline ~4 µm from the synapse, we estimated that the NMDA
receptor-mediated evoked response is derived from an 8-µm segment of
the dendrite by the time the peak NMDA receptor-mediated current is
measured (10 ms). The lower spatial resolution of the NMDA receptor map
is largely due to the slower NMDA receptor gating kinetics
(Dzubay and Jahr 1996
; Gibb and Colquhoun
1992
; Hestrin et al. 1990
; Lester and
Jahr 1992
; Lester et al. 1990
), which allow the
glutamate to diffuse away by the time the peak NMDA receptor current is
measured. This knowledge of the spatial resolution of the glutamate
receptor map is critical for our estimation of the distribution and
density of functional glutamate receptors at localized dendritic regions.
For our analysis, it is important that we saturate the glutamate
receptors at single postsynaptic sites with the glutamate applications.
Therefore we determined the dose-response relationship between
iontophoretic current and evoked current at a single synapse in the
absence of extracellular Mg2+. As evident from
the dose-response curve of Fig. 1E, the strength of the
iontophoretic pulse we used in the glutamate receptor map (100 nA) is
sufficient to saturate both AMPA and NMDA receptors in single
postsynaptic sites. The similar apparent affinities of AMPA and NMDA
receptors for glutamate evident in Fig. 1E
(EC50 ~
50 nA for both) are due to the rapid
time course of the glutamate application (Dubé and Liu
1999
). Furthermore the addition of cyclothiazide to block AMPA
receptor desensitization did not significantly affect the peak evoked
AMPA receptor response, suggesting that the glutamate iontophoresis
results in little desensitization. Therefore the glutamate-evoked
response can provide a good estimation of the number of receptors at
the glutamate application site (data not shown).
The estimation of the number and density of receptors at a synaptic
site depends on the assumption that an FM1-43 fluorescent puncta
represents a single synaptic release site as opposed to a cluster of
synapses or a synapse with multiple active zones. Analysis of serial
electron microscopy sections in hippocampal cultures has shown that the
majority of synaptic boutons contain single active zones, with
estimates ranging from 69% (Schikorski and Stevens
1997) to 97% (Forti et al. 1997
). Furthermore
morphological analysis from our lab suggests that ~80% of FM1-43
punctae represent single synapses (Liu et al. 1999
).
Here, we provide physiological evidence that each FM1-43 puncta spot
contains only one functional synapse. When we mapped multiple synapses
from single cells, we found that there was little variability in the
peak AMPA receptor response from synapse to synapse within individual
neurons (Fig. 1F). In fact, the variation within the neurons
(25 ± 5% of mean) was much smaller than the variation between
neurons (190%). This suggests that most of the mapped synapses within
individual neurons contained similar numbers of AMPA receptors and,
therefore, that each FM1-43 puncta contains a similar number of
postsynaptic sites. Since it is unlikely that all of these FM1-43
punctae contained multiple synapses, we propose that the vast majority
of FM1-43 punctae from which we collected data contained only one
active zone and one single synapse. However, in one case, the recorded AMPA receptor response was approximately double the size of the response from other synapses on the same neuron (Fig. 1F). A
logical interpretation of this response is that this synapse was one of the minority of FM1-43 punctae that contained either multiple active
zones or clusters of multiple synapses.
Distribution and density of functional AMPA receptors during synaptogenesis
Using the glutamate receptor map, we characterized the
distribution of functional AMPA receptors in the dendritic membrane before and after synaptogenesis. Following plating, hippocampal neurons
develop neurites and begin to form synapses after 4 DIV (Basarsky et al. 1994; Fletcher et al.
1994
). However, until after 7 DIV, the number of synapses as
labeled by FM1-43 remains very low. Between 8 and 10 DIV, there is a
dramatic increase in the density of FM1-43 labeled boutons (Fig.
2C), reflecting a rapid rate
of synapse formation during these days in culture. When we mapped the
dendrites of neurons from 6 to 7 DIV that had no FM1-43 labeled
boutons, we found a small but consistent level of evoked AMPA receptor
current along every mapped dendritic region (Fig. 2A). When
dendrites were mapped after synapse formation (>8 DIV), functional
AMPA receptor clusters were found exclusively colocalized with
presynaptic terminals, while a small AMPA receptor current was evoked
in all mapped extrasynaptic regions (Fig. 2B). These data
indicate that, prior to synaptogenesis, there is a pool of functional
AMPA receptors in the dendrite and that, following synapse formation,
functional AMPA receptors cluster at synaptic sites and a receptor pool
remains in the extrasynaptic membrane.
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To estimate the density of AMPA receptors prior to synapse formation,
we plotted the glutamate-evoked responses from dendrites that contained
no synapses (<8 DIV) against the surface area of the dendritic segment
affected by the glutamate applications, which varies only with the
diameter of the dendrite (Fig. 2D, see METHODS).
These data could be fit linearly (Fig. 2D; slope = 0.88, 95% C.I. = 0.81-0.95, R = 0.72, P < 0.0001), indicating that the pool of AMPA
receptors before synapse formation is evenly distributed throughout the
dendrite. The slope of the linear fit is equal to the AMPA
receptor-mediated current density from these dendritic regions
(0.88 ± 0.04 pA/µm2;
Vm = 60 mV). From this value, we
estimated the density of this AMPA receptor pool to be 2.7 ± 0.1 receptors/µm2. (See METHODS for
details of all density estimates.) Current densities were converted to
conductance densities by normalizing them by the holding potential at
which the recordings were made. All conductance densities and estimated
receptor densities are reported in Table
1.
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To determine if this pool of AMPA receptors aggregates to form synaptic AMPA receptor clusters, we estimated the number and density of AMPA receptors per synapse and predicted the resultant decrease in the density of the AMPA receptor pool following synaptogenesis. Using the mean peak evoked AMPA receptor-mediated current from synapses at 8-10 DIV (125 ± 14 pA, n = 11), we calculated the synaptic AMPA receptor conductance and receptor density (Table 1) and found that each synapse contains ~370 ± 40/synapse. Since neurons at 8 DIV have an estimated surface area of ~5,000 µm2 (see METHODS), each neuron contains on the order of 14,000 functional AMPA receptors in the membrane prior to synapse formation given a baseline receptor density of ~3/µm2. If clusters formed by the aggregation of existing inserted receptors, the formation of as few as 10 synapses would decrease the extrasynaptic receptor density by 25%, assuming that the receptors are freely diffusible.
We analyzed the AMPA receptor current from extrasynaptic regions of
cells that contained functional AMPA receptor clusters by plotting the
glutamate-evoked response from these areas against the stimulated
dendritic surface area. These data could also be fit linearly with the
same line as the data from before synaptogenesis (Fig. 2D;
slope = 0.88, 95% C.I. = 0.73-1.01, R = 0.84, P < 0.0001). Since the AMPA receptor current density
(slope = 0.88 ± 0.08 pA/µm2;
Vm = 60 mV) was exactly the same as
that for before synapse formation, the estimated density of
extrasynaptic AMPA receptors remained constant following synapse
formation (Table 1). These density estimates highlight the tight
clustering of functional AMPA receptors at synaptic sites and suggest
that synaptic AMPA receptor clusters do not form solely by the lateral
aggregation of receptors present in the dendritic membrane prior to
synapse formation (see DISCUSSION).
Estimation of the NMDA receptor probability of opening
As with AMPA receptors, we wanted to assess the density of
functional NMDA receptors in the dendritic membrane. However, this calculation depends critically on the NMDA receptor
po, a value whose estimates have
varied by a factor of 100 and been derived by relatively indirect
methods (Hessler et al. 1993; Huettner and Bean
1988
; Jahr 1992
; Rosenmund et al. 1993
,
1995
). Because of this wide discrepancy, we estimated the
po of NMDA receptors in our system by
perfusing the NMDA receptor open channel blocker MK-801 (5 or 20 µM),
directly activating synaptic NMDA receptors with iontophoretically
applied glutamate, and using the rate of NMDA receptor blockade to
calculate the po (Huettner and
Bean 1988
; Jahr 1992
; Rosenmund et al.
1993
, 1995
). Since glutamate spillover to extrasynaptic
receptors would complicate the interpretation of our data, we placed a
suction pipette on the opposite side of the glutamate electrode, and
suction (2 PSI) was applied for 200 ms beginning at 1 ms following the
end of the glutamate application. This technique significantly reduces
glutamate spillover to extrasynaptic NMDA receptors (Dubé
and Liu 1999
). At a holding potential of +40 mV, repeated
applications of glutamate to a single synapse elicited constant AMPA
and NMDA receptor-mediated currents (Fig. 3A, top). In the
presence of 20 µM MK-801, the NMDA receptor-mediated current
decreased with each pulse of glutamate and approached baseline within
10 applications, while the AMPA receptor-mediated current remained
relatively constant (Fig. 3A, bottom). Since the
MK-801 blockade recovery is markedly faster at positive holding potentials (
= 2 min) (Huettner and Bean 1988
),
performing these experiments at +40 mV may have affected the data
analysis. However, there were no differences in the results of these
experiments (n = 3) and those with negative holding
potentials in zero Mg2+ (n = 2).
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Figure 3B shows the NMDA receptor-mediated charge transfer
(Q) following sequential glutamate applications to a single
synapse in 20 and 5 µM MK-801 normalized against the NMDA
receptor-mediated Q from the first glutamate application.
This value decayed exponentially during the glutamate applications in
both 20 µM (R = 0.97) and 5 µM (R = 0.99) MK-801, with the rate of decay markedly faster in 20 µM MK-801.
These data indicate that equal proportions of NMDA receptors were
blocked with each pulse of glutamate and that the rate of MK-801 block
varied only according to the concentration of the drug. Using the
methods described in Rosenmund et al. (1995) (see
METHODS), we used the rate of MK-801 block to estimate the po of NMDA receptors to be 0.034 ± 0.002 in MK-801 concentrations of 20 and 5 µM (n = 5). Results in both MK-801 concentrations were not different and were
therefore pooled.
Distribution and density of functional NMDA receptors during synaptogenesis
We assessed the distribution of NMDA receptors before and after
synaptogenesis. All NMDA receptor recordings were performed at either
+40 mV in 1 mM Mg2+ or at 60 mV in 0 Mg2+. When dendrites were mapped before synapse
formation, there was a consistent level of NMDA receptor-mediated
evoked current along the entire mapped dendritic tree (Fig.
4A). When the amplitude of the
NMDA receptor-mediated evoked response from before synapse formation
was plotted against the surface area of the stimulated dendritic
region, the results could be fit linearly (Fig. 4C; slope = 0.29, 95% C.I. = 0.26-0.31, R = 0.88, P < 0.0001), indicating that, like AMPA receptors,
there is a homogeneously distributed pool of functional NMDA receptors
in the dendritic membrane prior to synapse formation. From the slope of
this fit (0.29 ± 0.04 pA/µm2), we
estimated the density of this pool to be 2.7 ± 0.4/µm2 (Table 1).
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Following synapse formation, NMDA receptor clusters were resolved at synaptic sites, and there remained a baseline level of NMDA receptor-mediated evoked current in extrasynaptic locations (Fig. 4B). As with before synaptogenesis, the amplitude of the NMDA receptor-mediated current varied linearly with the stimulated dendritic surface area (Fig. 4C; slope = 0.31 ± 0.06 pA/µm2; 95% C.I. = 0.27-0.35, R = 0.88, P < 0.0001), indicating that the pool of NMDA receptors in the extrasynaptic membrane remain evenly distributed with an estimated density of 2.9 ± 0.6/µm2 (Table 1). Thus there was no significant change in the NMDA receptor density in extrasynaptic regions following synapse formation (P > 0.3).
To estimate the number of NMDA receptors per synapse, it is necessary to subtract from the peak synaptic response the current resulting from spillover to extrasynaptic receptors by the time the peak NMDA receptor-mediated response is measured. We did not do this for AMPA receptor measurements because spillover is significantly smaller, and the total amount of spillover-induced current was normally <10% of the peak response. Baseline NMDA receptor current was calculated from the diameter of the dendrite from which the NMDA receptor current was sampled and from the slope in Fig. 4C. We subtracted this baseline NMDA receptor current from the peak synaptic NMDA receptor current. Using the corrected peak NMDA receptor amplitude values (28 ± 6 pA, n = 10), we calculated the synaptic NMDA receptor-mediated conductance density and estimated the number and density of synaptic NMDA receptors (Table 1).
Having estimated the densities of AMPA and NMDA receptors, we further calculated the ratio of AMPA to NMDA receptor densities in all dendritic regions. Interestingly, there were no significant differences in any of the densities measurements (Table 1; P > 0.5 for all cases). This suggests that the ratio of AMPA to NMDA receptor densities is close to one in all cases, including at synaptic sites.
Time course of AMPA and NMDA receptor clustering during synaptogenesis
Having established that functional AMPA and NMDA receptors are
homogeneously distributed in the dendritic membrane prior to synaptogenesis and are clustered at individual synapses afterward, we
attempted to determine the order in which these receptors cluster in
developing synapses. If there is a significant discrepancy in the
appearance of one type of receptor cluster, then we should observe
synapses that contain only one or the other type of receptor in newly
formed synapses between 8 and 10 DIV (see Fig. 2C). To determine the colocalization of clustered synaptic AMPA and NMDA receptors, we performed AMPA receptor maps at FM1-43 punctae and subsequently returned the glutamate electrode to the putative synapse
to record the glutamate-evoked I-V response. The AMPA and
NMDA receptor clusters were resolved in the I-V plot by
their kinetics and voltage dependency. Like the example shown in Fig. 5A, the majority of FM1-43
punctae contained both AMPA and NMDA receptor clusters (68%,
n = 43). While four AMPA receptor-only clusters were
detected (9%; Fig. 5B), we did not find any NMDA receptor-only synapses. Thus since most nascent glutamatergic synapses
contained functional AMPA and NMDA receptor clusters, these receptor
clusters likely form at a relatively similar time course during synapse
formation. Additionally, a minority of FM1-43 punctae showed neither
AMPA receptor nor NMDA receptor clusters (23%; Fig. 5C).
The glutamate-evoked currents from these locations were no different
from the baseline-evoked currents. These synapses may have been
GABAergic as a similar percentage of dendritic synapses in hippocampal
cultures were found to be GABAergic (Benson and Cohen
1996).
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Neither activity nor glutamate receptor activation is necessary for AMPA receptor insertion, clustering, and activation
The fact that functional glutamate receptor cluster formation and
presynaptic terminal formation correlate both spatially and temporally
during synaptogenesis raises the possibility that vesicle release from
the newly formed terminal may trigger AMPA receptor insertion,
clustering, and/or activation. In fact, electrophysiological data have
suggested that glutamate release and specifically, NMDA receptor
activation, may be necessary for this process during synaptogenesis
(Durand et al. 1996; Isaac et al. 1997
;
Liao and Malinow 1996
; Wu et al. 1996
).
Therefore we used the glutamate receptor map technique to directly test
the role of neuronal activity and glutamate receptor activation in AMPA
receptor insertion, clustering, and activation during synaptogenesis.
We first examined whether NMDA receptor activation is necessary for this process by blocking NMDA receptors during synaptic development with the NMDA receptor antagonist D-CPP (5 µM) beginning at 0 DIV. Between 8 and 10 DIV following NMDA receptor blockade, functional AMPA receptor clusters were observed colocalized with presynaptic terminals (Fig. 6A; 7 synapses, 3 cells, 3 cultures), suggesting that NMDA receptor activation is not required for AMPA receptor insertion, clustering, and activation during synapse formation.
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To control for nonspecific pharmacological effects of the
D-CPP treatment, we cultured hippocampal neurons from mice
in which the NMDA receptor NR1 subunit has been deleted (Li et
al. 1994). This subunit is critical for the formation of
functional NMDA receptors (Monyer et al. 1992
). Indeed,
the synapses from neurons derived from NR1 knockout mice did not
contain any functional NMDA receptors, as demonstrated by the voltage
dependency of the glutamate-evoked response (Fig. 6C).
Consistent with the results of the pharmacological NMDA receptor
blockade, functional AMPA receptor clusters were observed colocalized
with presynaptic terminals at 10 DIV in NR1 deficient neurons (Fig.
6B; 12 synapses, 5 cells, 2 cultures). Together these data
show that NMDA receptor activation is not necessary for AMPA receptor
insertion, clustering, and activation during synaptogenesis.
We next attempted to determine if any type of neuronal activity or
glutamate receptor activation is required for the insertion and
clustering of functional AMPA receptors during synaptogenesis. We added
TTX (1 µM) to the culture medium at the time of plating to block
activity-dependent vesicle release. Between 8 and 10 DIV following TTX
treatment, functional AMPA receptor clusters were resolved colocalized
with FM1-43-stained terminals (Fig. 6D; 8 synapses, 4 cells,
3 cultures), indicating that activity-dependent vesicle release is not
necessary for AMPA receptor insertion, clustering, and activation.
These data do not rule out the possibility that activity-independent
vesicle release may trigger this process. Therefore we added a cocktail
of antagonists to every known glutamate receptor subtype to the culture
medium beginning at 0 DIV. The cocktail included 10 µM DNQX, 1 µM
D-CPP, 500 µM AIDA, 500 µM EGLU, and 500 µM CPPG to
block AMPA receptors, NMDA receptors, and group I-III mGluRs,
respectively (Jane et al. 1996; Pellicciari et
al. 1995
). Following this treatment, functional AMPA receptor clusters were detected colocalized with FM1-43-stained terminals at
8-10 DIV (Fig. 6E; 8 synapses, 5 cells, 5 cultures),
suggesting that glutamate receptor activation is unnecessary for the
insertion and clustering of functional AMPA receptors during synapse formation.
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DISCUSSION |
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Distribution and density of glutamate receptors before and after synapse formation
Since the glutamate receptor map sampled only functional
receptors, there may have been nonfunctional receptors present in the
dendritic membrane that avoided detection and contaminated our density
estimates. According to our data, it is possible that there is an equal
density of receptors in the membrane following synapse formation but
that a much larger proportion of receptors at the synapse are
functional. However, this is an unlikely scenario as antibody labeling
of both AMPA and NMDA receptors have found that there is strong
punctate staining at synaptic sites, suggesting that these proteins
have a markedly higher density at synaptic sites (Craig et al.
1993; Rao and Craig 1997
; Richmond et al. 1996
). Thus the functional glutamate receptor clustering we
observe is due to the physical clustering of receptors at the synapse. Furthermore in contrast with antibody-labeling studies that reported a
number of extrasynaptic NMDA receptor clusters (Rao and Craig 1997
; Rao et al. 1998
), we found that all
clusters of functional NMDA receptors colocalized with presynaptic
terminals. Although there may be nonfunctional clusters of receptors
outside of synaptic locations, this would require an improbable
mechanism to maintain receptor clusters in a nonfunctional state within
a pool of nonclustered functional receptors. Therefore we propose that
all inserted AMPA and NMDA receptors are normally in a functional state
and that the formation of AMPA and NMDA receptor clusters is regulated to occur only at developing synapses.
Interestingly, the glutamate receptor densities we report here are
similar to the estimated AChR densities in the NMJ (Hall and
Sanes 1993; Sanes and Lichtman 1999
). We found
that there is a pool of functional AMPA and NMDA receptors with a
density of ~3/µm2 in the extrasynaptic
membrane after synaptogenesis, while the estimated density and number
of synaptic AMPA and NMDA receptors were on the order of
104/µm2 and 400/synapse,
respectively (see Table 1). These similarities suggest that ionotropic
neurotransmitter receptors may be regulated to maintain a synaptic
density on the order of
104/µm2
that is optimum for signal transmission. It will be interesting to
determine if other ionotropic receptors are clustered at a similar density.
The only major difference in receptor density estimates is prior to
synapse formation, which likely reflects differences in the mechanism
of receptor clustering. At the NMJ, a relatively high density of AChRs
(~1,000/µm2) are inserted into the muscle
membrane prior to synapse formation, and these receptors aggregate to
form a receptor cluster. Like AChRs in the NMJ, it is possible that the
evenly distributed glutamate receptors prior to synaptogenesis
aggregate to form synaptic clusters. However, unlike the NMJ, there is
a relatively low density of glutamate receptors in the dendritic
membrane prior to synapse formation (~3/µm2),
and there is no decrease in extrasynaptic glutamate receptor density
following synapse formation, including following the time points
sampled in this study (>10 DIV; data not shown). These data suggest
that the lateral aggregation of existing receptors is not
the primary mechanism of glutamate receptor clustering and that
glutamate receptor cluster formation must rely almost entirely on the
insertion of new receptors. Thus the mechanism of glutamate receptor
clustering in hippocampal synapses is different from the mechanism of
AChR clustering at the developing NMJ. The newly inserted glutamate
receptors in the developing clusters can be obtained in two ways:
receptors continue to be inserted into the extrasynaptic membrane and
diffuse into the postsynaptic site (Craig et al. 1994;
Nusser et al. 1998
) or receptors are inserted directly
into the postsynaptic site (Craig et al. 1994
; Richmond et al. 1996
; Steward 1995
;
Tovar and Westbrook 1999
). Monitoring the dendritic
membrane with the glutamate receptor map may allow us to determine
where these receptors are inserted.
In addition, our data suggest that the ratio of AMPA to NMDA receptor
density is close to one in all dendritic locations, including the
synapse. This raises the possibility that the level of AMPA and NMDA
receptor expression and/or insertion may be coordinated to produce such
a constant ratio. Previous reports estimated the number and density of
synaptic NMDA receptors to be much lower than we report here
(Jahr 1992; Silver et al. 1992
;
Spruston et al. 1995
; Stern et al. 1992
).
However, these calculations all relied on a relatively high value for
the NMDA receptor po (~0.3) (Hessler et al. 1993
; Jahr 1992
).
Critically, a number of other reports have suggested that this value is
significantly lower (po < 0.04)
(Huettner and Bean 1988
; Rosenmund et al. 1993
,
1995
). These discrepant reported
po values may have arisen because of different experimental preparations, each of which has its limitations. Some of these reports used outside-out patches, which removes the
channel from its native environment and requires equilibrating doses of
glutamate, while others used synaptic transmission, which involves
multiple synapses and may include transmission failures. Since an
accurate estimation of receptor density is dependent on this value, we
attempted to overcome the limitations of previous studies by measuring
the NMDA receptor po using direct,
saturating glutamate applications to NMDA receptors in their native
synaptic environment. Under these conditions, we estimated the NMDA
receptor po to be ~0.03. When this
low po is taken into account, the
estimated number and density of synaptic NMDA receptors is much higher
than previously calculated.
Importantly, few glutamate receptor clusters sampled in this study were
on dendritic spines. However, dendritic spines occur infrequently at
the culture ages from which we collected our data as most synapses are
made directly on the dendritic shaft at early stages of culture
development (Boyer et al. 1998; Papa et al. 1995
). A similar situation occurs in the hippocampus in vivo, where the vast majority of synapses are made on dendritic shafts until
spine synapses form after postnatal day 15 (Boyer et al. 1998
; Cotman et al. 1973
; Fiala et al.
1998
; Harris et al. 1992
; Pokorny and
Yamamoto 1981
; Steward and Falk 1991
). In fact,
it has been proposed that during early synaptogenesis, synapses are initially formed on dendritic shafts from which dendritic spines eventually develop (Fiala et al. 1998
). Thus the process
of glutamate receptor clustering that we describe here may be similar
to how it occurs during synaptogenesis in vivo.
Signal for clustering and activation of AMPA receptors at developing synapses
A number of electrophysiological studies have found that there are
more NMDA than AMPA receptor-mediated synaptic events during early
development and that plasticity inducing paradigms causes synapses that
show NMDA receptor-only mediated events to then display both receptor
components (Durand et al. 1996; Isaac et al.
1997
; Liao and Malinow 1996
; Wu et al.
1996
). These data have been interpreted to mean that the
activation of synaptic NMDA receptors is the signal that triggers AMPA
receptor insertion and clustering at that synapse during its formation.
Such a mechanism would require NMDA receptors to cluster prior to AMPA
receptors. As such, immunolabeling has been used to determine the order
of AMPA and NMDA receptor clustering during synapse formation. A number
of reports have found that a proportion of synapses contain only NMDA
receptors, particularly during early development (Gomperts et
al. 1998
; He et al. 1998
; Liao et al.
1999
; Petralia et al. 1999
; Takumi et al.
1999
). This evidence has been used to support the idea that
synaptic NMDA receptor activation is necessary for AMPA receptor
insertion into the synapse.
Surprisingly, with the glutamate receptor map, we did not locate any
NMDA receptor-only synapses during an early stage of synaptogenesis in
cultured hippocampal neurons. Although we did locate several synapses
that contained only AMPA receptor clusters, we found that the majority
of glutamatergic synapses contains both AMPA and NMDA receptor
clusters. Thus functional AMPA and NMDA receptors appear to cluster at
a similar time course in developing glutamatergic synapses. The reason
for the discrepancy between our data and the immunocytochemical data
are unclear, although possibilities include that the glutamate receptor
map technique may detect more AMPA receptor clusters due to its greater
sensitivity than immunolabeling, that there are synapses that contain
only nonfunctional NMDA receptors that avoid detection with our
glutamate receptor map, and that those synapses that contain only NMDA
receptors do not stain with FM1-43 and are therefore both pre- and
postsynaptically "silent" (Malgaroli 1999).
Furthermore functional NMDA receptor-only synapses may exist at this
time, but the proportion of these synapses must be low to have avoided
detection. Regardless, the most direct approach to understanding the
colocalization of functional receptors is to apply glutamate directly
to the synapse and record the resulting response.
Importantly, data regarding the time course of AMPA and NMDA receptor cluster formation are purely correlational, and it is difficult to draw causative information from such results. For example, in our culture system, NMDA receptor-only synapses may be rapidly converted to synapses that contain both AMPA and NMDA receptors. Therefore a direct test to determine whether NMDA receptor activation is necessary for AMPA receptor insertion, clustering, and activation is to study functional AMPA receptor expression in synapses after NMDA receptor blockade during neuronal development.
Following both pharmacological blockade and genetic removal of NMDA
receptor activation, we found that functional AMPA receptors were
clustered at newly formed synapses. In contrast with the electrophysiology studies that suggest that NMDA receptor activation is
essential for functional AMPA receptor clustering (Durand et al.
1996; Isaac et al. 1997
; Liao and Malinow
1996
; Wu et al. 1996
), work with antibody
labeling in cultured neurons indicates that AMPA receptor clustering
can occur independently of activity or glutamate receptor activation
(Craig et al. 1994
; Mammen et al. 1997
).
This discrepancy may be due to the fact that following NMDA receptor
blockade, AMPA receptor clusters are inactive and "silent" on
presynaptic stimulation, as antibody labeling gives no information
regarding the functional status of the receptors. Since the glutamate
receptor map technique sampled functional AMPA receptors, we
ruled out the possibility that AMPA receptor clusters following NMDA
receptor blockade are in an inactive state. Furthermore our results are
not likely attributable to differences between slice and culture
preparations, considering that normal synaptic AMPA receptor current
has been observed in hippocampal slices (Kutsuwada et al.
1996
) and in brain stem trigeminal complex slices (Li et
al. 1994
) following the genetic removal of functional NMDA
receptors. Thus NMDA receptor activation is not necessary for AMPA
receptor insertion, clustering, and activation during synapse formation.
These results appear to contradict studies that found synapses that
show NMDA receptor-only synaptic events during early development and
that can be activated to demonstrate both AMPA and NMDA receptor responses (Durand et al. 1996; Isaac et al.
1997
; Liao and Malinow 1996
; Wu et al.
1996
). However, it is theoretically possible that individual
synapses containing both AMPA and NMDA receptors could show NMDA
receptor-only responses due to the presynaptic release profile of
glutamate (Choi et al. 2000
) and the different
activation kinetics of AMPA and NMDA receptors (Dubé and
Liu 1999
) and not to the absence of functional AMPA receptors.
Such proposed synapses have been observed in hippocampal slices
(Choi et al. 2000
). Therefore we speculate that during
synaptogenesis, functional AMPA receptors do indeed cluster
independently of NMDA receptor activation and that NMDA receptor-only
events may result from presynaptic effects and the differential
responses of AMPA and NMDA receptors to relatively prolonged release
profiles of glutamate (Dubé and Liu 1999
).
Our data further suggest that neither neuronal activity nor activation of any known subtype of glutamate receptor is necessary for AMPA receptor insertion, clustering, and activation during synapse formation. Although we have not ruled out the possibility of an activity-independent, vesicularly released molecule that may trigger this process, we hypothesize that an activity-independent, contact-mediated surface signal may be sufficient for AMPA receptor clustering and activation during synapse formation.
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APPENDIX |
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To estimate accurately the number and density of glutamate receptors, it is necessary to determine the dendritic surface area affected by each iontophoretic glutamate application. This calculation requires estimations of the spread of glutamate and of the glutamate concentration dependency of channel opening. The spread of glutamate can be estimated by solving the diffusion equation with the appropriate boundary conditions. Since the distance between the tip of the electrode and the dendritic membrane is significantly smaller than the distance between the tip of the electrode and the surface boundaries of the recording chamber, the diffusion process is equivalent to the release of glutamate from a point source into an infinite space. The solution of the diffusion equation with the boundary conditions described in the preceding text gives the concentration of glutamate (C) at any time (t) and position relative to the electrode tip (r).
For an iontophoretic source continuously applied from 0 to n
ms
![]() |
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|
The concentration dependency of AMPA receptor opening can be estimated
by performing a dose-response paradigm at a single synapse (Fig.
A1A). We converted iontophoretic current to glutamate concentration by assuming that the concentration of glutamate required
to activate 50% of AMPA receptors (EC50) in our
system is similar to that in previous studies (480 µM) (Jonas
et al. 1993; Patneau and Mayer 1990
). Therefore
we assumed that the iontophoretic current that resulted in the
half-maximal response (about
60 nA) produced a glutamate
concentration of 480 µM in the synaptic cleft at the time the peak
AMPA receptor-mediated current was recorded (3 ms). We further
calculated the concentration and time course of glutamate diffusion 0.5 µm away from the tip of the iontophoresis electrode following
increasing iontophoretic pulses from
12 to
200 nA (Fig.
A1A, left inset). Following these applications, the peak glutamate concentration under the tip of the iontophoresis electrode was estimated to range from 100 to 1,600 µM, respectively, and to return to baseline within 2 ms. These simulations indicate that
the glutamate application is rapid and approaches the time course of
synaptic transmission (Clements et al. 1992
). Since the
maximum AMPA receptor po has been
estimated to be 0.58 (Diamond and Jahr 1997
) and the
response evoked by
200 nA current was the maximum, we further
converted the original dose-response curve into probability of channel
opening as a function of glutamate concentration (Fig. A1A, main).
We used the estimated concentration-dependency of AMPA receptor opening
(Fig. A1A) and the glutamate concentration profile (Fig.
A1B, ) to estimate the AMPA receptor
po along the dendrite 3 ms following
initiation of glutamate application (Fig. A1B, - - -). The
po decreased to baseline at ~1.5
µm from the electrode tip, indicating that AMPA receptors along a 3 µm dendritic segment are activated within 3 ms after the release of
glutamate. Thus the peak glutamate-evoked response is derived from AMPA
receptors within a 3 µm segment of the dendrite. This theoretical
calculation was supported by experimental observation (see Fig.
1B).
In principle, similar calculations can be carried out to determine the
length of dendrite in which NMDA receptors are activated by glutamate
applications. However, since the NMDA receptor has a much longer first
latency (the time from receptor binding to its first opening) than the
AMPA receptor (Dzubay and Jahr 1996), glutamate diffuses
away by the time the NMDA receptor response reach its maximum (~10
ms). Therefore it is not practical to predict the dendritic length
whose NMDA receptors will be affected by the spread of glutamate by
these methods. Since the results of the AMPA receptor map matched the
results of the theoretical calculations, we used the width of the
distribution of NMDA receptor-mediated peak in the NMDA receptor map
(Fig. 1D) to approximate this value to be 8 µm.
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ACKNOWLEDGMENTS |
---|
We thank A. Nashat for insightful discussions regarding glutamate diffusion and for providing the solution to the diffusion equation in our experimental conditions; A. M. Craig, I. Bezprozvanny, S. Waddell, J. Murnick, and J. Renger for comments on the manuscript; and S. Tonegawa and T. Iwasato for the generous gift of the NR1 mutant mouse.
This work was supported by grants from the RIKEN-MIT Neuroscience Research Center and the National Institutes of Health.
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FOOTNOTES |
---|
Address for reprint requests: G. Liu, E25-435, Dept. of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (E-mail: liu{at}mit.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 March 2000; accepted in final form 1 June 2000.
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REFERENCES |
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