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The Journal of Neuroscience, June 15, 2002, 22(12):5001-5015
7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic
Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses
in Rat Sensory Cortex
Robert B.
Levy and
Chiye
Aoki
New York University Center for Neural Science, New York, New York
10003
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ABSTRACT |
NMDA receptor (NMDAR) activation requires concurrent
membrane depolarization, and glutamatergic synapses lacking AMPA
receptors (AMPARs) are often considered "silent" in the
absence of another source of membrane depolarization. During the second
postnatal week, NMDA currents can be enhanced in rat auditory cortex
through activation of the 
7 nicotinic acetylcholine receptor
(7nAChR). Electrophysiological results support a mainly presynaptic
role for 7nAChR at these synapses. However, immunocytochemical
evidence that 7nAChR is prevalent at postsynaptic sites of
glutamatergic synapses in hippocampus and neocortex, along with
emerging electrophysiological evidence for postsynaptic nicotinic
currents in neocortex and hippocampus, has prompted speculation that
7nAChR allows for activation of NMDAR postsynaptically at synapses
lacking AMPAR. Here we used dual immunolabeling and electron microscopy
to examine the distribution of 7nAChR relative to AMPAR (GluR1,
GluR2, and GluR3 subunits combined) at excitatory synapses in
somatosensory cortex of adult and 1-week-old rats. 7nAChR occurred
discretely over most of the thick postsynaptic densities in all
cortical layers of both age groups. AMPAR immunoreactivity was also
detectable at most synapses; its distribution was independent of that
of 7nAChR. In both age groups, approximately one-quarter of
asymmetrical synapses were 7nAChR positive and AMPAR negative. The
variability of postsynaptic 7nAChR labeling density was greater at
postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a
subset of small AMPA receptor-negative synapses with a high density of 7nAChR immunoreactivity. These observations support the idea that
acetylcholine receptors can aid in activating glutamatergic synapses
and work together with AMPA receptors to mediate postsynaptic excitation throughout life.
Key words:
sensory cortex; receptive field properties; 7
nicotinic acetylcholine receptor; glutamate; AMPA receptor; NMDA
receptor; synaptic plasticity; early postnatal development; postsynaptic density; immunocytochemistry; electron microscopy
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INTRODUCTION |
Plasticity of receptive field
properties in sensory cortex depends on experience-dependent
modification of glutamatergic pathways (Kleinschmidt et al., 1987
; Fox
et al., 1996; Rasmusson, 2000) and cholinergic modulation of these
pathways (Bear and Singer, 1986; Sillito and Murphy, 1987; Müller
and Singer, 1989; Baskerville et al., 1997; Kilgard and Merzenich,
1998). Although developmental studies have focused on muscarinic
acetylcholine receptors, recent knowledge regarding central nicotinic
receptors, particularly the 7 nicotinic acetylcholine receptor
subunit (7nAChR), indicates that these too are important for
synaptic plasticity. Specifically, 7nAChR subunits (1) form
homo-oligomeric channels with greater Ca2+
permeability than NMDA receptor (NMDAR) (McGehee, 1999), (2) are
prevalent during the first postnatal week, before onset of the critical
period for experience-dependent developmental plasticity (Fuchs, 1989;
Bina et al., 1995; Broide et al., 1995), and (3) enhance NMDA currents
specifically at synapses of neonatal cortex lacking AMPA receptor
(AMPAR) activity (Aramakis and Metherate, 1998; Aramakis et al.,
2000).
Despite its prevalence, the role of 7nAChR in modulating excitatory
transmission and long-term synaptic changes is unclear. Much of the
physiological effect of nicotine apparently results from presynaptic
modulation of glutamatergic pathways, i.e., enhanced glutamate release
(McGehee et al., 1995; Role and Berg, 1996; Jones et al., 1999).
However, electrophysiological evidence exists for direct activation of
postsynaptic nAChR in hippocampus (Alkondon et al., 1998; Frazier et
al., 1998a,b; Hefft et al., 1999) and neocortex (Roerig et al., 1997;
Chu et al., 2000). Also, previous immunocytochemical results showed a
primarily somatodendritic distribution of 7nAChR within neocortical
and hippocampal pyramidal neurons (Dominguez del Toro et al., 1994).
Past EM immunocytochemical work by us and others agrees with these
immunocytochemical findings: 7nAChR is concentrated at postsynaptic
sites of asymmetric synapses in cortex (Lubin et al., 1999) and
hippocampus (Fabian-Fine et al., 2001). These synapses are presumably
excitatory (Gray, 1959; Altschuler et al., 1984; Somogyi et al., 1986;
Aoki et al., 1991). Although this suggests that some of the direct or
indirect effects of nicotine occur via postsynaptic glutamate
receptors, morphology alone cannot reveal the glutamate receptor
subtypes at these 7nAChR-immunopositive synapses. Immunocytochemical
studies of hippocampus indicate that NMDARs are present in most
asymmetric synapses throughout postnatal development, whereas AMPARs
are acquired selectively (Nusser et al., 1998; Kharazia and Weinberg,
1999; Liao et al., 1999; Petralia et al., 1999; Shi et al., 1999).
These results complement physiological evidence from hippocampus (Isaac
et al., 1995; Liao et al., 1995; Durand et al., 1996) and neocortex (Wu
et al., 1996; Isaac et al., 1997; Rumpel et al., 1998) that many
NMDAR-containing synapses lack AMPAR function early in development.
These synapses are often considered physiologically "silent"
without the local depolarization required for NMDAR activity (Feldman
et al., 1999). Might 7nAChR contribute to postsynaptic activation of
these synapses? To address this question, we examined the distribution
of 7nAChR relative to AMPARs at asymmetric synapses in neonatal and
adult rat somatosensory cortex. EM immunocytochemistry was used to
resolve the presynaptic versus postsynaptic location of 7nAChR and
to determine whether the presence of 7nAChR at nascent synapses
precedes the arrival of AMPARs.
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MATERIALS AND METHODS |
Tissue preparation. Adult (200-300 gm) and postnatal
day (PD) 7 Sprague Dawley rats were deeply anesthetized with sodium
pentobarbital (50 mg/kg body weight) and then perfused transcardially
with a mixture of 1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were
postfixed by immersion at room temperature (RT) for 1 hr (for adults)
or overnight (for PD 7) in the same fixative. The prolonged
postfixation for neonatal brains was included to compensate for less
complete perfusion attributable to their relatively undeveloped
vasculature. Variations in the extent of fixation did not have a
measurable effect on subsequent quantitative immunolabeling results
within each age group. Coronal sections, 40 µm thick for adults,
60-80 µm for PD 7, were made with a vibrating microtome (Leica
Microsystems), and fixation was terminated by treatment with 1%
sodium borohydride in 0.1 M PB. Sections were
stored in PBS, pH 7.4 containing 0.02% sodium azide at 4°C for up to
2 months before use in experiments, except for representative sections
that were mounted on gelatin-coated glass slides and Nissl stained
according to standard histological methods.
Pre-embedding immunocytochemistry for AMPA receptor.
Pre-embedding immunocytochemistry using horseradish peroxidase
(HRP)-diaminobenzidine (DAB) for visualization was chosen to ensure
the most sensitive detection of AMPAR subunits. Coronal sections
containing somatosensory (S1) cortex (Paxinos and Watson, 1998) were
subjected to 6-10 rapid freeze-thaw cycles using 20% DMSO as a
cryoprotectant to enhance antibody penetration (Wouterlood and
Jorritsma-Byham, 1993). Sections were rinsed several times in PBS,
incubated for 1 hr at RT in 1% bovine serum albumin (BSA)/PBS to block
unspecific binding sites, and then incubated for 1-2 d at RT in a
mixture of AMPAR subunit antibodies [rabbit GluR1, 1:450, and
rabbit GluR2/3, 1:320; both purchased from Chemicon (Temecula, CA)] diluted in 1% BSA/PBS. Sections were rinsed three times with PBS, incubated for 1 hr at RT in biotinylated anti-rabbit IgG (Vector Laboratories, Burlingame, CA), and then rinsed again three times with PBS. Immunoreactivity was visualized using HRP-DAB
(ABC Elite kit, Vector Laboratories). Sections were then processed
immediately for electron microscopy (see below), apart from one set
that was mounted on glass slides and used for light microscopy.
Tissue processing for electron microscopy. After AMPAR
immunocytochemistry, sections were immersed for 1 hr at RT with 1% OsO4 in 0.1 M PB. Although
many EM studies using postembedding immunocytochemistry have used
osmium-free processing to preserve optimal immunoreactivity, these
methods do not allow detection of HRP-DAB reaction product, and they
also yield poorer ultrastructural preservation than is obtained with
processing using osmium. In our laboratory, osmium treatment was
compatible with optimal subsequent detection of 7nAChR by
postembedding immunocytochemistry (see the following section). After
osmium treatment, sections were dehydrated in stepwise concentrations
of ethanol up to 70% and immersed for 1-3 d at 4°C in 4% uranyl
acetate in 70% ethanol. Sections were then dehydrated and embedded in
EMBED 812 between Aclar sheets (Electron Microscopy Sciences, Fort
Washington, PA). Sections were then drawn under a microscope equipped
with a drawing tube, taking note of prominent features (e.g., large
blood vessels, pial surface, areal density of pyramidal neurons, and
border of the corpus callosum) for use in later identification of
cortical layers under the electron microscope. Portions of each section spanning all layers of S1 cortex were capsule embedded, and 70-90 nm
sections were cut on an MT-7 ultramicrotome (RMC, Tucson, AZ) and
collected on Formvar-coated grids. Grids were then subjected to
postembedding 7nAChR labeling (see below) or viewed directly using a
JEOL 1200 XL electron microscope (JEOL Inc., Tokyo, Japan).
Postembedding immunocytochemistry for 7nAChR.
7nAChR labeling was performed by the postembedding gold (PEG)
procedure of Erisir et al. (1997) modified from that of Phend et al.
(1992). Conditions were adjusted to gain optimal detection of 7nAChR by PEG while maintaining high-quality ultrastructural preservation (particularly of membranes) and preservation of HRP-DAB reaction product corresponding to AMPAR subunit immunoreactivity. Mouse monoclonal antibody 306, directed against a predicted cytoplasmic domain of 7nAChR (Schoepfer et al., 1990; McLane et al., 1992), was
obtained from Research Biochemicals International (Natick, MA) and used
at a dilution of 1:250. This antibody was used previously in a light
microscopic study of 7nAChR distribution in adult rat brain
(Dominguez del Toro et al., 1994) and more recently in an EM study of
adult rat hippocampus CA1 (Fabian-Fine et al., 2001). In the latter
instance, the specificity of synaptic labeling with monoclonal antibody
(mAb) 306 was confirmed by the use of a polyclonal antibody directed
against a region containing the same epitope; mitochondrial labeling
seen with mAb 306 was shown to be a lower-specificity reaction with a
species of 48 kDa, smaller than the native molecular mass of the
7nAChR subunit. Incubation time in primary antibody was 2.5 hr; the
inclusion of 0.1% Triton X-100 in the incubation mixture to remove
osmium and enhance antibody penetration allowed us to avoid etching
steps that cause severe deterioration of ultrastructure (Phend et al.,
1992). Longer incubation times (up to 18 hr) were avoided, because this
did not improve the extent or specificity of 7nAChR labeling and
caused detectable loss of HRP-DAB reaction product. Gold-conjugated
anti-mouse secondary antibody (10 nm) was obtained from Ted Pella, Inc.
(Redding, CA) and used at 1:40 dilution. Samples in which primary
antibody was omitted showed virtually no labeling. After PEG, grids
were postfixed for 10 min in 2% glutaraldehyde and counterstained for
20-30 sec with Reynold's lead citrate to produce acceptable levels of
contrast in the section without reducing the detection of HRP-DAB
reaction product. Grids were then air dried and viewed under the
electron microscope.
Quantitative analysis. In samples from each age group,
cortical layers were identified under the electron microscope by
comparing prominent features with drawings of the flat embedded tissue
(see Tissue processing for electron microscopy above) and by comparison with Nissl-stained sections. In each region, neuropil was sampled only
near the tissue-resin interface where penetration of pre-embedding immunoreagents was thorough, as assessed by the overall extent of
HRP-DAB labeling. Random fields were photographed at 20,000× magnification, excluding those in which cell bodies, blood vessels, or
empty space occupied more than half the field. Approximately 365 µm2 of neuropil was analyzed in each
cortical layer in each animal.
Micrographs were printed at a final magnification of 50,000×.
Asymmetric synapses were identified, and postsynaptic elements were
classified as dendritic spines or dendritic shafts, by established morphological criteria (Peters et al., 1991). Both axospinous and axon-shaft synapses were included in the analysis; most of the
identifiable synapses were axospinous (see Results). A fraction of the
postsynaptic profiles were not clearly identifiable as spine or shaft,
particularly in the neonatal group; these profiles were included in the
analysis as long as they did not clearly belong to another category
(e.g., axosomatic or axoaxonal synapses). Symmetric synapses were less
numerous and rarely immunolabeled. Therefore these were excluded from
the quantitative analysis.
Synapses were assessed for AMPAR and 7nAChR labeling on the basis of
examination of the photographs printed for comparable density and
contrast; for details of the criteria used to assign degrees of
labeling in particular figures, see the figure legends. Postsynaptic
labeling for AMPARs was assessed by the local presence of
electron-dense floccular HRP-DAB product directly over the postsynaptic density (PSD). When present, HRP-DAB product was uniformly distributed over the PSD.
Assessment of labeling by 10 nm gold particles representing 7nAChR
staining took into account the distance between the gold particle and
the antigen binding site; i.e., particles falling on the PSD were
considered part of the postsynaptic membrane, whereas those on the
synaptic cleft were assumed to belong to either the presynaptic or
postsynaptic membrane with equal probability (except for data presented
in Fig. 2A, where labeling over the cleft was
categorized separately). Active zone length was determined using a
measuring pen (Precision Technology Devices, Skokie, IL) calibrated to
the scale bar imprinted on each micrograph. To simplify analysis,
perforated synapses (those with discontinuous active zones) were
excluded. Variations between data sets were tested for statistical
significance by ANOVA followed by unpaired two-tailed t
tests (Microsoft EXCEL software); specific details are provided in
Results and figure legends.
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RESULTS |
Subcellular distribution of 7nAChR in adult cortex
In adult cortex, 10 nm gold particles corresponding to 7nAChR
immunoreactivity were concentrated over thick PSDs of asymmetric (typically axospinous) synapses throughout superficial, middle, and
deep cortical layers (Fig.
1A,
Sp1, Sp2,
B,C). In cases where PSDs were
separated across two active zones or perforated within a single active
zone, gold particles clustered specifically over PSDs and were almost
absent along nonjunctional synaptic plasma membranes (data not shown).
Thick PSDs with high and low densities of immunogold occurred
immediately adjacent to one another (Fig. 1B). More than two-thirds of all asymmetric
postsynaptic profiles were immmunoreactive for 7nAChR, and this
value did not vary significantly across layers or between animals
(Table 1, Adult, Total). Asymmetric
synapses also showed labeling at the presynaptic membrane and in the
adjacent cytoplasm (Fig. 1B,C).
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Figure 1.
7nAChR immunoreactivity at asymmetric synapses
of adult somatosensory cortex. A, Electron micrograph
showing two axon terminals (T1,
T2) forming synapses with dendritic
spines (Sp1,
Sp2) in layers II/III. 7nAChR
labeling (10 nm gold particles, indicated by arrowheads)
is seen over the PSD in both synapses. 7nAChR is also seen near
plasma membranes (black arrows) and in the cytoplasm
(open arrows) of adjacent neuronal processes.
B, The PSD of a synapse formed by an axon terminal
(T1) onto a spine (Sp)
in layers II/III, viewed en face, shows gold particles
(arrowheads) distributed at random across its surface.
An adjacent synapse (T2) onto a
dendritic shaft (Sh) is strongly labeled with HRP-DAB
reaction product indicating the presence of AMPAR (curved
arrow; see more examples in Fig. 5). C, An axon
terminal (T) forming a synapse with a spine
(Sp) in layer VI, showing 7nAChR labeling on (no
symbol) and near (arrowhead) the PSD, as well as
vesicle-associated labeling (arrows) in the presynaptic
terminal. D, 7nAChR labeling of rough endoplasmic
reticulum (ER) in a pyramidal cell body of layers II/III. On ER
cisternae cut in section, gold particles are located preferentially on
the cytoplasmic side of the ER membrane (arrowheads;
compare with Fig. 2F). Asterisks
denote the ER lumen. Scale bar (shown in D for
A-D): 200 nm.
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Labeling was distributed more sparsely over nonsynaptic
portions of plasma membranes of dendritic spines and within the
cytoplasm of dendritic spines (Fig. 1C,
arrowhead) and shafts (data not shown). In contrast, a
subcellular compartment other than the PSD that showed a high density
of 7nAChR was the rough endoplasmic reticulum (Fig.
1D), which was highly immunoreactive in both
pyramidal and nonpyramidal cells. The predominance of immunogold
labeling over PSDs, as indicated by these examples, was confirmed by
quantitative analysis of the distribution pattern of colloidal gold
particles within the vicinity of 420 synapses sampled across varying
laminar depths of adult cortices (Fig.
2). This analysis showed that nearly 50%
of the synaptic gold particles aggregated directly on the PSD (Fig.
2A). Moreover, analysis of gold particle distribution along the length of PSDs revealed that 7nAChR immunoreactivity is
distributed evenly, with a slightly higher concentration toward the
center of the active zone captured within single ultrathin sections
(Fig. 2B, left panel).
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Figure 2.
Synaptic 7nAChR labeling is concentrated over
the postsynaptic density. A, The distribution of 10 nm
gold particles relative to the synaptic cleft was tallied for
orthogonally sectioned asymmetric synaptic profiles (see Table 2
footnote) in all cortical layers combined for three animals of each age
group. Both presynaptic and postsynaptic distances are expressed
relative to the thickness of the postsynaptic density; i.e., on
cleft or near+ is within one PSD width of the
synaptic cleft; near and far are in
adjacent zones of equal width. The data are from 420 profiles and 1721 particles for adult and 270 profiles and 1423 particles for PD 7. B, Distribution of 10 nm gold particles along the PSD is
similar between PD 7 and adult tissue. For each age group, a random
subset of relatively long orthogonally cut synaptic profiles (>250
µm for PD 7; >300 µm for adult) was drawn from all animals across
all cortical layers, and the position of each gold particle was
computed as a fraction of the distance from the center to the edge of
the synapse, i.e., center = 0, edge = 1. Data are from 45 synapses and 195 gold particles for adult and 45 synapses and 292 gold
particles for PD 7.
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Subcellular distribution of 7nAChR in PD 7 cortex
7nAChR labeling in PD 7 cortex was qualitatively similar to
labeling in adult cortex, with extensive labeling of dendritic elements
exhibiting asymmetric synapses (Figs. 2,
3A-E) and on the
endoplasmic reticulum of neuronal perikarya (Fig.
3F). Where fortuitous planes of section revealed
extensions of synaptic specializations away from the postsynaptic
membrane or accentuated curvatures of the PSD (Fig. 3B,
black arrow), immunogold particles followed these irregular
PSD profiles.
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Figure 3.
7nAChR immunoreactivity at asymmetric synapses
of PD 7 somatosensory cortex. A, Two axon terminals
(T) forming synapses with spines
(Sp1,
Sp2) in layer I, showing 7nAChR
labeling on the postsynaptic density (compare with Fig. 1). The top
synapse also shows gold particles on the synaptic cleft and at the
presynaptic membrane. Also note the cluster of 7nAChR labeling in
the cytoplasm of an unidentified neurite (N).
B, En face view of a large irregular 7nAChR-positive
postsynaptic density in layers II/III. Black arrow
points to the curvature of the PSD. The axon terminal, identifiable by
synaptic vesicles (open arrows), converges on the PSD
from two directions and appears to form another synapse below with a
second dendritic compartment (Den), also 7nAChR
positive (arrowheads). C,
D, Axospinous synapses in layers II/III; compare the
exclusively postsynaptic labeling in C
(arrowheads) with the discrete clusters of presynaptic
(arrow) and postsynaptic (arrowhead)
labeling in D. The sparse,
pleiomorphic vesicles seen in the axon terminal
(T) in C are characteristic of
immature synapses. E, An 7nAChR-positive synapse in
layer VI. As in A, a nonsynaptic cluster of 7 label
is seen in an adjacent neurite (N); also present
in the postsynaptic compartment are patches of HRP-DAB reaction
product indicating AMPAR labeling (curved arrows;
compare with Fig. 1B; see also Fig. 6).
A, D, and especially E
show terminals with large cytoplasmic volumes devoid of vesicles. This
is a recurrent feature of PD 7 neuropil. F, 7nAChR
labeling of rough endoplasmic reticulum (ER) in a pyramidal cell body
of layers II/III. Note the predominantly cytoplasmic location of gold
particles (arrowheads; compare with Fig.
1C). Asterisks denote the ER lumen. Scale
bar (shown in F for
A-F): 200 nm.
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Occasionally, gold particles clustered near plasma membranes lacking
PSDs. These sites appeared to be nascent postsynaptic specializations,
based on the alignment of the plasma membrane with that of the axon
terminal (Fig. 3E, profile N). Axonal
labeling at or near the presynaptic membrane was also seen (note Fig.
3D in particular) but was not as prevalent as postsynaptic labeling.
The overall incidence of 7nAChR immunoreactivity at asymmetric
synapses in neonates was comparable to that seen in adults in that
slightly more than two-thirds of all synapses were labeled, and
immunoreactivity likewise did not vary significantly across layers
(Table 1, PD 7, Total). Quantitative analysis of colloidal gold
particles across presynaptic to postsynaptic distances from the cleft
and along the lengths of PSDs of 270 representative synapses revealed a
striking resemblance to the pattern seen for adult synapses (Fig.
2A,B).
Of the 7nAChR-positive asymmetric synapses in PD 7 cortex, 66.2%
were axospinous, and they most likely represent synapses onto
excitatory (pyramidal or spiny stellate) neurons; 10.3% were axon-shaft, and the remainder of postsynaptic profiles was not clearly
identifiable (Table 2, PD 7). These
figures closely reflect the overall distribution of synapses (Table 2,
PD 7, Total, 7nAChR positive plus 7nAChR negative). The
proportion of 7nAChR-positive synapses that are axospinous is
slightly higher in adulthood than in PD 7 cortex (Table 2, Adult and PD
7, 7(+)). The difference, which is not statistically significant,
parallels an overall increase in the proportion of synapses (labeled
plus unlabeled) that are axospinous (Table 2, Adult and PD 7, Total).
This trend probably reflects the greater differentiation of adult
cortex.
AMPA receptor distribution by light microscopy
The presence of 7nAChR at the majority of postsynaptic sites of
asymmetric, presumably excitatory synapses in neonatal as well as adult
cortex [see also Lubin et al. (1999)], suggested that a subset of the
silent synapses (i.e., those lacking AMPAR) reported previously might
indeed be subject to postsynaptic nicotinic stimulation. We examined
the distribution of AMPAR in relation to 7nAChR using a combination
of previously characterized antibodies against peptides corresponding
to the C termini of GluR1 and GluR2/3 subunits to get an inclusive
measure of the presence of AMPAR.
First, light microscopy was performed to confirm that GluR1/2/3
immunocytochemistry was adequate using tissue fixation conditions compatible with 7nAChR immunolabeling. Light microscopy of
AMPAR-stained adult cortex, visualized by HRP-DAB (Fig.
4B), showed
somatodendritic labeling of pyramidal and nonpyramidal neurons
throughout the cortex, with large pyramidal cell bodies in layer V
being especially prominent (Fig. 4B, thick
bracket). Staining was relatively intense in layer I, because of
nearly complete labeling of the neuropil, and relatively light in layer
IV (Fig. 4B, thin bracket) and in the
superficial part of layer VI. This laminar distribution pattern agreed
with our expectations, on the basis of earlier reports on the laminar
distribution of GluR1 and GluR2/3 examined separately (Petralia and
Wenthold, 1992; Kharazia et al., 1996; Arai et al., 1997; Brennan et
al., 1997; Petralia et al., 1997).
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Figure 4.
Light micrographs of AMPAR immunoreactivity
(GluR1/2/3 subunits) in adult (A, B) and PD 7 (C-G) somatosensory cortex. A,
Nissl stain of adult cortex shows location of layers. B,
HRP-DAB staining for AMPAR: cell bodies and apical dendrites of layer
V pyramidal neurons are most prominent (thick bracket),
but staining is appreciable in all neuronal cell types and across all
layers. Staining of layer I neuropil is relatively dense, whereas
staining is relatively light in layer IV (thin bracket).
C, Nissl stain of PD 7 cortex. Note that the cell bodies
are more densely packed and the laminar boundaries less well defined
than in the adult. SP, Subplate region.
D, AMPAR distribution at PD 7: note diffuse neuropil
labeling in layers I and II/III and relatively light staining of
layer IV (thin bracket), staining of layer V pyramidal
cell bodies (thick bracket), and darkly stained cell
bodies in the subplate layer (open arrow).
E-G, Higher magnification views of AMPAR staining in
superficial layers (E), layer V
(F), and the subplate layer
(G) of PD 7 somatosensory cortex. Note labeling
of pyramidal cell bodies (large arrows), apical
dendrites (arrowheads), and smaller interneuronal cell
bodies (small arrows); in G, note dark
staining of atypically shaped cell body in the subplate region
(curved arrow). Scale bars:
A-D, 400 µm;
E-G, 40 µm.
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Labeling in PD 7 cortex showed less differentiation across layers (Fig.
4D), corresponding to the relatively undeveloped
laminar pattern seen by Nissl staining (Fig. 4C). However, a
dark band of immunoreactivity was appreciable in layer I (Fig.
4D,E), and strongly stained
neuronal cell bodies were present in layers II/III (Fig.
4E), with less labeling in layer IV (Fig.
4D, thin bracket). Darkly labeled cell
bodies were already apparent in layer V (Fig. 4F) and
in the subplate zone (Fig. 4G) (Herrmann, 1996; Brennan et
al., 1997).
Subcellular distribution of AMPA receptors in adult cortex by
electron microscopy
EM examination (Fig. 5) confirmed
the somatodendritic distribution of AMPARs. Patches of AMPAR labeling
were seen within dendritic shafts, along the plasma membrane or over
microtubules (Fig. 5C). HRP-DAB product occurred within
dendritic spines (Fig. 5A), and relatively less occurred in
the portion of the spine head away from the PSD [Fig. 5A,
arrowhead, C, to the left and
right of the spine apparatus (SA)]. In cases
where spine necks were labeled (Fig. 5C, curved
arrow), labeling was also associated with the membrane of the
dendritic shaft from which the spine emerged.
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Figure 5.
Subcellular distribution of AMPAR immunoreactivity
is mostly independent of 7nAChR distribution in adult cortex.
A, Three axon terminals (T)
forming synapses with spines in layer I demonstrate intense (AMPAR++;
open arrow in Sp1),
moderate (AMPAR+; filled straight arrow in
Sp2), or minimal/absent postsynaptic
AMPAR labeling (AMPAR+/ ; curved arrow in
Sp3), respectively, visualized as
HRP-DAB reaction product. These assignments correspond to the
three-level classification scheme used in this study for statistical
analysis of AMPAR labeling (Table 1). The PSD in Sp1 also
contains three 10 nm gold particles representing 7 AChR labeling;
these are reproduced in the inset
(circled) with greater magnification and reduced
contrast for the sake of visualization. In
Sp2, note non-synapse-associated AMPAR
(arrowhead) in the spine head. B, Two
axon terminals (T) in layers II/III form
asymmetric synapses with a single dendritic shaft [(Sh)
identifiable as such by its size and the presence of a large
mitochondrion (M)]. Note that the
postsynaptic density of the bottom synapse is labeled for AMPAR
(straight arrow), and the top synapse is unlabeled
(curved arrow). Both postsynaptic densities are also
7nAChR-positive (10 nm gold particles). C,
Independent distribution of 7nAChR and AMPAR labeling in a dendritic
shaft (Sh) and several axospinous synapses in layers
II/III. All three show minimal AMPAR labeling but varying degrees of
7nAChR labeling. Patches of AMPAR immunoreactivity are visible over
microtubules running lengthwise in the shaft (straight
arrows) and cut in cross section in another dendrite nearby
(arrow with asterisk). AMPAR is also seen
in the lower spine neck (curved arrow) and along the
spine apparatus (SA) in Sp1 (open
arrows). Note the absence of AMPAR labeling in presynaptic
terminals. 7nAChR labeling (10 nm gold particles) is present both
presynaptically and postsynaptically, and is also seen on the spine
apparatus and within the dendritic shaft. Scale bar (shown in
C): A, 250 nm; inset, 400 nm; B, C, 200 nm.
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The intensity of PSD labeling varied considerably (Fig. 5A,
compare open arrow, curved arrow, and
smaller solid arrow). In some instances, synapses with
obvious AMPAR immunoreactivity, and those below the level of
detectability (referred to as AMPAR negative), could be seen in close
proximity within the same membrane-bound compartment (Fig.
5B), showing that the diffusibility of HRP-DAB product did
not lead to unspecific labeling of all PSDs. Most of the asymmetric
synapses had detectable postsynaptic AMPAR labeling in all cortical
layers examined (Table 1, AMPAR++, AMPAR+), with the proportion
diminishing to 52% in the deepest layer (Table 1, Adult, Total). In
agreement with previous reports (Petralia and Wenthold, 1992),
presynaptic AMPAR labeling was rarely seen.
Localization of 7nAChR relative to AMPA receptors at asymmetric
synapses of adult cortex
There was no significant correlation between the postsynaptic
distribution of AMPAR and 7nAChR; i.e., the incidence of 7nAChR labeling at intensely and moderately AMPAR-positive synapses ranged from two-thirds to three-quarters across the layers but was not significantly different from its incidence at AMPAR-negative synapses (Table 1). Individual synapses varied widely in their 7nAChR and
AMPAR content. For example, in Figure 5A, the most strongly AMPAR-positive synapse (open arrow) is also 7nAChR
positive (inset, circle), whereas the two other
synapses, with less AMPAR immunoreactivity, are 7nAChR negative
(straight and curved arrow, respectively). Compare this with Figure 1B, where an intensely
AMPAR-positive, 7nAChR-negative synapse (curved arrow) is
seen near an AMPAR-negative synapse with abundant 7nAChR labeling
(arrowheads).
Distribution of AMPA receptor relative to 7nAChR at synapses in
PD 7 cortex
The relative distribution of 7nAChR and AMPAR in neonates
resembled that of adult cortex (Fig. 6;
compare with Fig. 5). The overall incidence of postsynaptic AMPAR
labeling was comparable to that seen in adults; i.e., approximately
two-thirds across all layers (Table 1). However, the intensity of AMPAR
labeling in PD 7 synapses never approached that of the most intensely
labeled synapses in the adult (Fig. 6A, compare with
Fig. 5A). Colocalization of AMPAR and 7nAChR was seen
postsynaptically at PSDs of asymmetric synapses (Fig.
6A) and at extrasynaptic sites (Fig.
6B,C). As in adults, the incidence
of 7 labeling was not significantly correlated with the degree of
AMPAR labeling (Table 1). Specifically, neither the laminar position
nor AMPAR immunoreactivity was a good predictor of 7nAChR
immunoreactivity. One notable point is that the synapses with intense
AMPAR immunoreactivity were 7nAChR immunoreactive almost without
exception. However, a substantial fraction of all synapses (22% in the
adult group, 25% in PD 7, combined across all layers) were AMPAR
negative but 7nAChR positive and might therefore be candidate sites
for cholinergic modulation of NMDAR-containing, AMPAR-lacking silent
synapses.
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Figure 6.
Subcellular distribution of AMPAR immunoreactivity
relative to 7nAChR immunoreactivity in PD 7 cortex resembles that
seen in adult cortex. A, An axon terminal
(T) forming a synapse with a spine
(Sp) in layer I, exhibiting both AMPAR (HRP-DAB
reaction product) and 7nAChR (10 nm gold) reactivity on the
postsynaptic density (straight arrow). Small patches of
relatively less intense AMPAR staining are seen elsewhere in the
postsynaptic compartment (arrowheads). An unidentified
process nearby (curved arrow) is also positive for both
AMPAR and 7nAChR. B, An example of an
7nAChR-positive, AMPAR-negative PSD (straight arrow)
in layer I; compare the dense AMPAR staining of an adjacent neuronal
process (curved arrows). 7nAChR reactivity is also
seen over electron-dense material located away from the PSD
(arrowheads). C, Axon terminals
(T) forming AMPAR-positive (black
arrow) and AMPAR-negative (open arrow) synapses
in layers II/III; both show 7nAChR reactivity over the postsynaptic
density, whereas the AMPAR-positive synapse shows 7nAChR associated
with the presynaptic membrane as well (curved arrow).
The dendritic elements (Den) are difficult to classify
as spine or shaft. Nearby neuronal processes show clusters of 7nAChR
labeling with (arrowheads) and without
(arrowheads with asterisks) associated
AMPA receptor labeling. D, AMPAR-positive,
7nAChR-positive synapse (arrow) on a dendritic shaft
(Sh) [note mitochondrion
(M)] in layer VI. Scale bar (shown in
D for A-D): 200 nm.
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Quantitative analysis of postsynaptic
7nAChR immunoreactivity
A count of 10 nm gold particles corresponding to 7nAChR
labeling over discrete labeled synapses confirmed our qualitative observation that the density of 7nAChR labeling was higher in neonatal than in adult synapses (Fig. 7).
To simplify the analysis, we included only orthogonally sectioned
synapses and used PSD length, rather than PSD area, as a measure of the
size of each profile. Considerable variability in the density of
labeling (Fig. 7, y-axis) and lengths of PSDs (Fig. 7,
x-axis) was noted among both adult and PD 7 populations of
synapses. A weak positive correlation was seen between synapse length
and the number of 10 nm gold particles per synapse, regardless of the
degree of AMPAR labeling present: AMPAR++, AMPAR+, and AMPAR+/ (Fig.
7, compare panels). Across the three categories of AMPAR
labeling, the density of 7nAChR labeling was consistently greater
for PD 7 synapses than for adult synapses (Fig. 7, compare the slopes
of the linear regression plots). This difference between PD 7 and adult
was statistically significant within each category of AMPAR labeling
(p < 0.05; two-tailed t test) and
likewise within each cortical layer examined (superficial, middle, and
deep, with all categories of AMPAR immunoreactivity combined; data not
shown). To assess the consistency of results between experimental
animals, pairwise t tests were performed among animals of
the same age group and within the same cortical layer. Of 18 possible
comparisons, 17 yielded no significant difference between animals
(p > 0.5), whereas one pair (in PD 7, deep
layers) was significantly different (p = 0.003).
We did not detect significant layer-dependent differences in the
density of 7nAChR labeling, and data from all cortical layers are
combined in Figure 7 and subsequent figures.
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Figure 7.
Scatter plots relating synaptic profile length to
amount of postsynaptic 7nAChR labeling, divided according to the
degree of AMPAR labeling: minimal/absent (AMPAR+/), moderate
(AMPAR+), or intense (AMPAR++). See Figure 5A for
an electron micrograph illustrating these categories. Gray
symbols represent PD 7, and black symbols
represent adult synapses. Synapses across all cortical layers and from
three animals of each group are combined in each plot.
7nAChR-negative profiles and obliquely sectioned profiles were
excluded from the count. 7nAChR labeling was calculated by adding
the number of 10 nm gold particles on the PSD to one-half the number of
particles on the synaptic cleft. Total least-squares regression line is
shown for each data set. Coefficients of correlation
(R2) are 0.088 for AMPAR++ adult
(n = 42), 0.105 for AMPAR++ PD 7 (n = 15), 0.089 for AMPAR+ adult
(n = 209), 0.196 for AMPAR+ PD 7 (n = 129), 0.075 for AMPAR+/ adult
(n = 88), and 0.070 for AMPAR+/ PD 7 (n = 67).
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We examined whether the variation in density of 7nAChR labeling was
the result of random variations in the degree of labeling or whether it
might reflect an underlying heterogeneity of 7nAChR density.
Previous EM immunocytochemical studies have modeled the detection of
antigen in ultrathin sections as a Poisson process (Kharazia and
Weinberg, 1999; Racca et al., 2000). In Figure
8, the density of 7nAChR labeling at
discrete synapses is seen to differ in two ways from a Poisson
distribution around a single mean. (1) In both adult and PD 7 data
sets, there is a disproportionately large number of postsynaptic
profiles without gold particles [Fig. 8, leftmost bin in
each histogram; compare black bar (experimental) with
gray bar (Poisson)], and this population is larger in the PD 7 group than in the adult group. (2) The PD 7 group contains a
statistically distinct subpopulation of 21 synaptic profiles with a
relatively high density of 7nAChR (50 or more particles per
micrometer); these profiles constitute 8.9% of the total in the PD 7 data set versus 2.4% in the adult data set.
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Figure 8.
Histograms showing the density of 7nAChR
labeling on postsynaptic profiles (black bars) in all
adult versus PD 7 synapses. The data are the same as those used to
generate Figure 2, except that unlabeled synapses are included
(leftmost bin of each panel).
Theoretical Poisson distributions (gray bars)
were obtained by fitting the experimental data over a restricted range
[bin range 5-100 for adult; bin range 5-50 for PD 7 by total
least-squares, using a procedure written in MATLAB (Math Works, Natick,
MA)]. The fit therefore excluded unlabeled synapses (leftmost
column of each graph) and neonatal synapses with
>50 gold particles per micrometer. The mean of the Poisson
distribution was 23.5 for PD 7 and 15.0 for adult. Experimental data
did not differ significantly from the theoretical Poisson values over
the included range (p > 0.05;
Kolmogorov-Smirnov test). Experimental data from PD 7 for bin ranges
5-100 (i.e., including synapses with >50 gold particles per
micrometer) differed significantly from a Poisson distribution fit to
that range of bins (p < 0.05;
Kolmogorov-Smirnov test; graph not shown). The percentage of
"truly" negative synapses (measured value minus Poisson value,
left-hand bin of each graph) was 12.0%
for adults and 21.1% for PD 7). n = 420 synapses
for adult; n = 270 synapses for PD 7. The adult
sample included one profile with 179 particles per micrometer that was
excluded from the statistical analysis.
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The data for PD 7 are presented as a scatter plot in Figure
9, subdivided according to the degree of
AMPAR labeling at each synapse. The plot shows that the group of
profiles with 50 or more gold particles per micrometer consisted mostly
of those with relatively short active zones (250 nm or less) and
without intense AMPAR labeling. Eleven of these synapses belonged to
the superficial layers, four belonged to the middle layers, and six
belonged to the deep layers. Fourteen of the synapses were judged to be
axospinous, whereas one (in layer VI) was axon-shaft, and six could not
be classified as one or the other. Of these densely 7nAChR-labeled synapses, 14 were found in animal one, 3 were found in animal two, and
4 were found in animal three.
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Figure 9.
Density of 7nAChR labeling as a function of
synapse length in PD 7 synapses. Data points are grouped according to
the degree of postsynaptic AMPAR labeling: intense (black
squares), moderate (gray squares), or
minimal/absent (white squares). Points
above the dotted line correspond to those to the right
of the 50 particle per micrometer bin (inclusive) in the PD 7 histogram
(Fig. 8). Note the relative absence of larger synaptic profiles and
intensely AMPAR-positive synapses in this group. The data set is the
same one used for PD 7 in Figure 7.
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DISCUSSION |
Prevalence of postsynaptic 7nAChR in both adult and neonatal
somatosensory cortex suggests a widespread role in excitatory
transmission
A large body of work documents the influence of cholinergic
activity on experience-dependent plasticity in cortical receptive field
properties (Sillito and Kemp, 1983; Kilgard and Merzenich, 1998). For
example, acetylcholine can switch the flow of excitatory activity from
columnar to intercolumnar connections through the muscarinic receptors
(Xiang et al., 1998) while also boosting the activity of thalamic
(glutamatergic) afferents through nicotinic receptors (Gil et al.,
1997).
To the best of our knowledge, ours is the first study of the ontogeny
of 7nAChR at the EM level. 7nAChR was found at most postsynaptic
densities of synapses identified as glutamatergic by the presence of
AMPAR (GluR1, GluR2, and GluR3) subunits and by morphology in both
adults and neonates. This implies that the contribution made by
7nAChR in excitatory synaptic transmission in neocortex may be much
greater than was recognized previously. We observed dually
7nAChR-positive and AMPAR-positive synapses not confined to the
thalamo-recipient zone (layer IV) but also present in the supragranular
and infragranular layers. Moreover, most of these
synapses were axospinous. Axospinous junctions are unlikely to be on
interneurons, the class previously identified as the main site of
postsynaptic nicotinic activity (Alkondon et al., 1998; Frazier et al.,
1998a,b) but instead are on excitatory neurons, i.e., pyramidal or
stellate cells. This conclusion is strengthened by similar
findings reported in adult guinea pig prefrontal cortex (Lubin et al.,
1999) and in adult rat hippocampal area CA1 (Fabian-Fine et al., 2001).
In the latter case, 7nAChR was detected postsynaptically at almost
all asymmetric synapses examined. This study used the same antibody as
the present one (mAb 306 from Sigma/RBI), but in conjunction with a
different EM processing protocol (freeze-substitution followed by
embedding in Lowicryl). Despite the differences in EM processing
techniques, both sets of data concur in demonstrating strong
postsynaptic 7nAChR immunoreactivity.
Our results and those of Fabian-Fine et al. (2001) also concur in
visualizing substantial 7nAChR immunoreactivity at extrasynaptic sites within dendritic spines (e.g., in association with the spine apparatus) and on the endoplasmic reticulum within neuronal cell bodies, indicating a large reserve pool and rapid, possibly
activity-dependent turnover and trafficking of 7nAChR. Our results
extend previous light microscopic immunocytochemical data on 7nAChR
localization to neuronal cell bodies and dendrites (Dominguez del Toro
et al., 1994) and radioligand binding data obtained with
-bungarotoxin (Barrantes et al., 1995).
The activity of postsynaptic nicotinic receptors has been well
documented in the peripheral nervous system (cf. Jacob and Berg, 1983;
Zhang et al., 1994), and the existence of 7nAChR-mediated EPSPs was
recently reported in hypothalamus (Hatton and Yang, 2002). In
hindsight, it is surprising that much of the past electrophysiological evidence for postsynaptic nicotinic effects in forebrain was confined to hippocampal GABAergic (aspiny) interneurons (cf. Alkondon et al.,
1998; Frazier et al., 1998a,b). Evidence for postsynaptic nicotinic
activity in excitatory neurons of hippocampus (Hefft et al., 1999) and
neocortex (Roerig et al., 1997; Chu et al., 2000) is comparatively
sparse. A possible explanation for why cortical glutamatergic neurons
have not shown more prominent 7nAChR currents by somatic recordings
is that 7nAChR might act through metabolic pathways triggered by
Ca2+ influx (for review, see Edwards,
1995) rather than through membrane depolarization. Alternatively,
perhaps the depolarizing action of 7nAChR is confined to spines,
especially on the distal, fine branches of dendrites (e.g., in layers I
and II), making these currents relatively more difficult to detect in
whole-cell recordings. In addition, decreases in extracellular
Ca2+ during periods of high neuronal
activity (Vernino et al., 1992; Amador and Dani, 1995), the relatively
low agonist affinity and high desensitization rate of
7nAChR (Albuquerque et al., 1997), and the progressive glial
ensheathment of excitatory synapses in maturing animals (Ling and
Leblond, 1973; Parnavelas et al., 1983) could have obscured the
biophysical detection of 7nAChR.
Many of the 7nAChR-enriched synapses at PD 7 are AMPA
receptor-negative profiles with short active zones
Comparison with a theoretical Poisson distribution [see similar
analyses by Kharazia and Weinberg (1999), Petralia et al. (1999),
Takumi et al. (1999), and Racca et al. (2000)] indicates that a
fraction of all asymmetric synapses was truly 7nAChR negative in
both adults and neonates, whereas most synapses showed a moderate density of 7nAChR immunoreactivity. A smaller fraction, in neonates only, possessed a distinctly higher density of 7nAChR. The majority of these 7nAChR-enriched profiles appeared to represent synapses with relatively small active zones. The alternative possibility, namely
that these profiles represented sections through the periphery of
larger synapses, with 7nAChR concentrated toward the edge of the PSD
(i.e., in a ring), was ruled out because we did not see this
distribution of 7nAChR either in individual PSDs viewed en face or
in a random sample of larger PSDs viewed in cross section (Fig.
2B).
On the basis of the observation that synaptic AMPAR content increases
steeply with active zone length in adult S1 cortex, other authors have
proposed that synapses with the shortest active zones may be
essentially AMPAR negative (Kharazia and Weinberg, 1999), and
potentiation of these synapses may involve the activity-dependent insertion of AMPAR (Liao et al., 1995; Shi et al., 1999). In the present study, many of the 7nAChR-enriched synapses with short active zones in the neonatal group were not detectably immunoreactive for GluR1, GluR2, or GluR3 and may indeed be functionally AMPAR negative. Such 7nAChR-enriched, AMPAR-negative synapses may rely on
7nAChR-mediated depolarization to relieve the voltage-dependent Mg2+ blockade of NMDAR. Perhaps this is
the same population of synapses shown by Aramakis and colleagues
(Aramakis and Metherate, 1998; Aramakis et al., 2000) to be silent
(AMPAR lacking) immature synapses in which NMDA current is enhanced by
an 7nAChR- or NMDAR-mediated mechanism. These results pointed
clearly to a presynaptic effect (enhancement of glutamate release),
whereas ours show a mostly postsynaptic concentration of 7nAChR and
a lesser presynaptic concentration. This difference might be explained
by a mechanism in which a postsynaptic 7nAChR-mediated influx of
Ca2+ leads to the activation of the
Ca2+/calmodulin-dependent neuronal nitric
oxide synthase (NOS); the product, nitric oxide, is a retrograde
messenger that could enhance presynaptic glutamate release (Cudeiro and
Rivadulla, 1999; for review, see Contestabile, 2000). Our earlier
findings on the subcellular localization of NOS support this idea,
because NOS was found within NMDAR-positive spines forming asymmetric
axospinous junctions (Aoki et al., 1997, 1998).
Possible interactions between 7nAChR and postsynaptic
scaffolding proteins
The clustering of 7nAChR at PSDs of asymmetric synapses implies
that 7nAChR is retained at the PSD through interaction with other
PSD components. Studies of the neuromuscular junction have identified
an extracellular protein, agrin (McMahan, 1990; Sanes and Lichtman,
1999), which is essential for nAChR clustering at the motor endplate
via the transmembrane kinase MuSK (Glass et al., 1996) and the
cytoplasmic protein rapsyn (Apel et al., 1997). Mice deficient in agrin
showed no abnormalities in glutamatergic and GABAergic synapse
formation (Serpinskaya et al., 1999). Likewise, rapsyn deficiency
abolished neuromuscular but not neuronal nAChR clustering (Apel et al.,
1997; Feng et al., 1998). Therefore, 7nAChR may interact with known
or yet unknown scaffolding proteins of glutamatergic CNS synapses. An
interaction was reported recently (Huh and Fuhrer, 2001) between
7nAChR and PICK1, a PDZ domain-containing cytoplasmic protein that
promotes clustering of AMPARs (Dev et al., 1999; Xia et al., 1999). We
found, however, that 7nAChR distribution at asymmetric synapses is
independent of AMPAR distribution. This suggests that 7nAChR and
AMPAR at synapses are not bound to a common postsynaptic scaffolding
protein. 7nAChR and AMPAR may interact through PICK1 or other
interacting proteins at extrasynaptic sites, e.g., during intracellular
assembly and targeting.
AMPAR immunoreactivity in neonatal versus adult cortex
The fact that the most intense AMPAR immunoreactivity was seen
only in adult cortex may reflect the progressive acquisition of AMPAR
subunits over postnatal development (Petralia et al., 1999). In both
adult and neonatal cortices, we saw wide variations in AMPAR
immunoreactivity among thick PSDs, lending support to the idea that
AMPAR content is an important variable in determining the strength of
individual synapses.
Taken together with the existing literature, our results indicate that
the role of 7nAChR in somatosensory cortex, and perhaps in all
cortical areas, is pervasive. Such a role is in keeping with the
widespread distribution of cholinergic afferents through the cortex and
elsewhere in the brain (Woolf, 1991) and their frequent triadic
relationship with glutamatergic terminals and dendritic spines (Aoki
and Kabak, 1992). Mice lacking 7nAChR do not display obvious
anatomical or behavioral abnormalities (Orr-Urtreger et al., 1997;
Paylor et al., 1998), so it is likely that 7nAChR is necessary for
refining, rather than establishing, sensory receptive field properties
and other types of cortical organization dependent on precise synaptic
connectivity. The widely known effects of nicotine on cognitive and
attentional processes suggest that the role of 7nAChR will emerge as
these high-order processes become better understood.
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FOOTNOTES |
Received Dec. 12, 2001; revised March 13, 2002; accepted March 22, 2002.
This research was supported by National Institutes of Health (NIH)
Grants R01 NS41091 and R01 NEI 13145-01 to C.A., and NIH P30
EY13079 core grant, National Institute of Mental Health Training Grant 5T32MH195424-10, and Office of Naval Research Solicitation No. 99-019 to the Center for Neural Science. We thank Claudia Farb, Mona Lubin, and Veeravan Mahadomrongkul for discussion and technical assistance.
The work described in this paper was presented at the Society for
Neuroscience 31st annual meeting, San Diego, CA, November 10-15, 2001 (abstract 362.15).
Correspondence should be addressed to Chiye Aoki, New York University
Center for Neural Science, 4 Washington Place, Room 809, New
York, NY 10003. E-mail: chiye{at}cns.nyu.edu.
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TOP
ABSTRACT
INTRODUCTION
