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The Journal of Neuroscience, January 15, 1999, 19(2):692-704
Neuronal Acetylcholine Receptors with  7 Subunits Are
Concentrated on Somatic Spines for Synaptic Signaling in Embryonic
Chick Ciliary Ganglia
Richard D.
Shoop1,
Maryann E.
Martone2,
Naoko
Yamada2,
Mark H.
Ellisman2, and
Darwin K.
Berg1
1 Department of Biology and the 2 National
Center for Microscopy and Imaging Research, University of California,
San Diego, La Jolla, California 92093-0357
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ABSTRACT |
Nicotinic acetylcholine receptors containing  7 subunits are
widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but
appear to be excluded from postsynaptic densities on the cells. We show
here that 7-containing receptors are concentrated on somatic spines
in close proximity to putative sites of presynaptic transmitter
release. Intermediate voltage electron microscopy on thick sections,
together with tomographic reconstruction, permitted three-dimensional
analysis of finger-like projections emanating from cell bodies. The
projections were identified as spines based on their morphology,
cytoskeletal content, and proximity to presynaptic elements. Both
in situ and after ganglionic dissociation, the spines
were grouped on the cell surface and tightly folded into mats.
Immunogold labeling of receptors containing 7 subunits showed them
to be preferentially concentrated on the somatic spines. Postsynaptic
densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic
vesicle-filled projections from the presynaptic calyx were
interdigitated among the spines. Moreover, the synaptic vesicles often
abutted the membrane and sometimes included  profiles as if
caught in an exocytotic event, even when no postsynaptic densities were
juxtaposed on the spine. The results suggest several mechanisms for
delivering transmitter to 7-containing receptors, and they support
new ideas about synaptic signaling via spines. They also indicate that
neurons must have specific mechanisms for targeting 7-containing
receptors to desired locations.
Key words:
nicotinic; acetylcholine; 7; ciliary ganglion; spines; receptors; neuronal; cholinergic; synaptic; calyx; presynaptic; tomography; immunogold; confocal; electron microscopy
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INTRODUCTION |
Nicotinic acetylcholine receptors
(AChRs) are widely expressed in the vertebrate nervous system where
they function as cation-selective ligand-gated ion channels. One of the
most abundant is a species containing 7 subunits (7-AChRs) that
has a high relative permeability to calcium and binds -bungarotoxin
(-Bgt) with high affinity (Couturier et al., 1990 ; Schoepfer
et al., 1990; Anand et al., 1993; Bertrand et al., 1993; Seguela et
al., 1993; Conroy and Berg, 1998). Increasing evidence indicates that
7-AChRs can act presynaptically to modulate neurotransmitter release
(McGehee et al., 1995; Gray et al., 1996; Coggan et al., 1997; Guo et
al., 1998; Li et al., 1998).
In the chick ciliary ganglion, 7-AChRs play a prominent role
postsynaptically. The ganglion contains ciliary neurons innervating striated muscle in the iris and ciliary body and choroid neurons innervating smooth muscle in the choroid layer (Dryer, 1994). Ciliary
neurons become engulfed by large presynaptic calyces early in
development and express high levels of 7-AChRs. The receptors generate substantial synaptic currents, representing >90% of a 5 nA
whole-cell response in situ (Zhang et al., 1996; Ullian et al., 1997). Although the magnitude of the current is not incompatible with the number of 7-AChRs present [ 106/neuron
(Chiappinelli and Giacobini, 1978; Smith et al., 1983)], the synaptic
response is a surprise given the location of the receptors. Electron
microscopic analysis suggests that 7-AChRs are absent from
postsynaptic densities (PSDs) on the neurons (Jacob and Berg, 1983;
Loring et al., 1985) that instead contain a different species of AChR
(Jacob et al., 1984; Loring and Zigmond, 1987). This latter species
(3*-AChRs) is composed of 3,  4, 5, and occasionally 2
subunits and binds monoclonal antibody mAb 35 (Vernallis et al., 1993;
Conroy and Berg, 1995). Despite their preferred location, 3*-AChRs
generate only a minor portion of the synaptic current in many ciliary
neurons (Zhang et al., 1996; Ullian et al., 1997).
Confocal fluorescence microscopy with derivatized -Bgt suggested
that 7-AChRs are concentrated in perisynaptic clusters on ciliary
ganglion neurons (Wilson Horch and Sargent, 1995). Electron microscopic
analysis using horseradish peroxidase (HRP)-conjugated -Bgt
indicated preferential labeling of extrasynaptic membrane among
finger-like processes emanating from the neurons (Jacob and Berg,
1983). The processes have usually been referred to as pseudodendrites
(Szentagothai, 1964; Hess, 1965; Landmesser and Pilar, 1972). A
subsequent electron microscopic study using 125I--Bgt
and autoradiography supported the conclusion that 7-AChRs are
distributed throughout extrasynaptic membrane but left unanswered the
question as to whether the receptors are preferentially concentrated on
the processes (Loring et al., 1985).
We have used immunogold labeling, together with intermediate voltage
electron microscopy (IVEM) and tomographic analysis, to examine the
distribution of 7-AChRs on chick ciliary neurons late in
embryogenesis. We find the receptors are concentrated on somatic spines
in close proximity to multiple sites of presumed transmitter release.
The receptor distribution and spine configuration suggest a model for
chemical transmission through ciliary neurons that makes economical use
of transmitter while supporting high-frequency signaling and possible
sequestration of calcium entering through activated 7-AChRs.
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MATERIALS AND METHODS |
Sample preparation. Ciliary ganglia were dissected
from embryonic day 15 (E15) chicks and either labeled and processed
directly for confocal and electron microscopy or dissociated into a
cell suspension and allowed to settle on coverslips as described
previously (Zhang et al., 1994) before labeling and processing. After
labeling, samples were prepared for fluorescence visualization by
fixation in 4% paraformaldehyde in phosphate buffer, pH 7.4; samples
prepared for electron microscopy (EM) were fixed in 2%
paraformaldehyde plus 2% glutaraldehyde in cacodylate buffer, pH
7.4.
Immunolabeling of cultured cells and tissue sections. After
allowing 20 min for dissociated cells to attach to the culture substratum, we incubated the cultures for 1 hr at 4°C in 10 mM HEPES, pH 7.4, containing either biotinylated -Bgt
(1:500 dilution; Molecular Probes, Eugene OR) to label 7-AChRs or
the rat monoclonal antibody mAb 35 (1:1000 dilution) to label
3*-AChRs (Wilson Horch and Sargent, 1995; Conroy and Berg, 1998).
Cultures were then rinsed five times with 10 mM HEPES, pH
7.4, followed by 30 min of fixation and rinsing for 20 min in 0.01 M sodium phosphate, pH 7.5, containing 0.15 M
NaCl (PBS). To detect bound biotinylated -Bgt with
immunofluorescence, we incubated labeled cultures 45 min in either Cy3-
or FITC-conjugated streptavidin (1:1000 in PBS; The Jackson Laboratory,
Bar Harbor, ME). To detect bound mAb 35 with immunofluorescence, we
incubated cultures 45 min in a 1:200 dilution of Cy3-conjugated donkey
anti-rat antibody (The Jackson Laboratory). For immunogold labeling,
cultures were first incubated 45 min in PBS with a 1:200 dilution of
mouse anti-biotin (The Jackson Laboratory), rinsed five times in PBS,
and then incubated 45 min with donkey anti-mouse antibody conjugated to
10 nm immunogold (1:50 dilution; Amersham, Arlington Heights,
IL) in PBS with 5% (v/v) normal donkey serum. Alternatively, neurons
labeled with HRP reaction product were incubated with HRP conjugated to
streptavidin (or to donkey anti-rat antibody in the case of mAb 35) for
45 min, rinsed five times in PBS, and then reacted for 5 min using a
peroxidase-labeling kit (Molecular Probes). Nonspecific (background) labeling with biotinylated -Bgt was assessed by incubating the neurons 20 min with 100 nM -Bgt before the incubation
with biotinylated toxin and then processing as normal. Tissue sections
were labeled for immunofluorescent detection of 7-AChRs as described
above, using slightly longer incubation times and approximately twice as many rinses between steps. In some experiments, 7-AChRs were labeled with indirect immunofluorescence by fixing the cells as described above, permeabilizing with 0.1% (w/v) Triton X-100 in PBS,
and then labeling with anti-7-AChR antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).
Phalloidin labeling was performed on neurons in culture immediately
after they had been labeled for 7-AChRs as described above. Cells
were permeabilized in PBS with 5% (v/v) normal donkey serum and 0.1%
(w/v) Triton X-100 for 20 min and then were incubated for 45 min in a
1:1000 dilution of rhodamine-conjugated phalloidin (Molecular Probes).
Confocal microscopy. Fluorescently labeled material was
visualized with a Noran Instruments Odyssey (Middleton, WI) confocal microscope using a 63×, 1.4 numerical aperture objective lens. Approximately 60 optical sections were taken through each sample, recorded digitally, and assembled to achieve the final volume. National
Institutes of Health Image was used to analyze the confocal images.
Electron microscopy. Material for EM was rinsed in 0.1 M sodium cacodylate buffer several times and treated for 30 min with 2% osmium tetroxide in 0.1 M sodium cacodylate.
Samples were then counterstained with uranyl acetate (except for those
containing HRP-reacted material), dehydrated in an ethanol series, and
infiltrated with Durcupan ACM resin. After polymerization for 24 hr at
60°C, the material was sectioned into either 80 nm (thin-sectioned
material) or 1-2 µm (thick-sectioned material).
Thin-sectioned material was examined using a JEOL 100CX electron
microscope. Thick-sectioned material was examined with a JEOL 4000EX
microscope, operating at 400 keV. Volume content of the thick
sections was revealed by tomographic reconstruction. The
1-2-µm-thick sections were tilted through 120° of rotation, and
electron micrographs were taken at increments of 2°. These 61 tilt
angles were then digitized, aligned, and back projected to generate the
volume information using procedures described by Perkins et al. (1997).
Manual tracing from the computer-generated slices using Xvoxtrace
software (developed by S. Lamont, National Center for Microscopy and
Imaging Research) was used to delineate the plasma membrane, spine
membrane position and label, and calyx structures. This information was
used to construct three-dimensional computer graphic representations
using the Synu software package (Hessler et al., 1992). Colors were
chosen manually. Gold particles were represented one-to-one by gold
spheres in the tomogram.
The number of immunogold particles per unit membrane length was
estimated from electron micrographs of thin-sectioned dissociated neurons labeled with biotinylated -Bgt. Neuronal membrane was divided into two classes: "spiny" and "nonspiny" membrane.
Spiny membrane for purposes of gold particle counts was defined as that representing an extension of at least one putative spine width (usually
0.1-0.2 µm) from the cell body and not abutting other structures
that would limit access of the immunogold label. Nonspiny membrane was
that clearly representing soma surface and did not include distortions
or bumps that might have represented the base of a transected spine.
The total number of gold particles along each type of membrane sample
was summed and divided by the total length of membrane observed.
Membrane length was measured using Analyze software (Mayo Clinic
Foundation, Rochester, MN). No attempt was made to calibrate the
numerical relationship between gold particles bound and 7-AChRs
present because the measured values for gold particle density were in
all cases used only for comparative purposes, i.e., comparing the
relative densities of 7-AChRs on spiny versus nonspiny membrane.
Calculations. The proportion of the cell surface devoted to
7-AChR clusters was measured from confocal micrographs of cells fluorescently labeled with biotinylated -Bgt and Cy3-streptavidin. The threshold for detection in these measurements was adjusted to
reveal only the brightest clusters, thereby ensuring a conservative estimate of the proportion of the surface occupied by them. The extent
of folding or packing of the plasmalemma in a spine clump was estimated
for thin-section electron micrographs of intact ganglia. The total
lineal membrane measured throughout complete spiny regions was divided
by that measured for equivalent (degrees of arc) regions of nonspiny
(smooth soma) membrane. This value for the relative enrichment of
plasma membrane in spiny regions was then multiplied by the total area
of such regions calculated above to obtain a value for the relative
proportion of the total cell surface contributed by spine plasma
membrane. This calculation assumes that the brightest 7-AChR
clusters detected by immunofluorescence are coextensive with spiny
regions, consistent with the immunogold labeling results reported below.
The space constant  was estimated for spine-like projections
according to the formula: = [aRm/2Ri]1/2,
where a is the spine diameter,
Rm is the specific resistance of the spine
membrane, and Ri is the resistance of the
spine interior. Rm and
Ri were taken to be 104
cm2 and 102 cm,
respectively, the values found for mammalian cardiac neurons (Plonsey
and Barr, 1989).
Materials. White Leghorn chick embryos were obtained locally
and maintained at 37°C in a humidified incubator. -Bgt was
purchased from Biotoxins (St. Cloud, FL). All other reagents were
purchased from Sigma (St. Louis, MO) unless otherwise indicated.
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RESULTS |
Clusters of 7-AChRs on dissociated neurons
-Bgt was used as a probe for 7-AChRs on chick ciliary
ganglion neurons because it binds with high affinity to receptors containing 7 subunits (Schoepfer et al., 1990) and the receptors account for the vast majority of -Bgt binding in the ganglion (Vernallis et al., 1993). The only other identified neuronal AChR subunit recognized by -Bgt in chick is 8, but the 8 gene is not expressed in the ganglion (Corriveau and Berg, 1993). Confocal fluorescence microscopy of E15 chick ciliary ganglia labeled with biotinylated -Bgt followed by Cy3-streptavidin shows that
7-AChRs are distributed in large clusters on many of the neurons
(Fig. 1A). Such
clusters have been described previously as perisynaptic in location
because of their proximity to but nonoverlap with presynaptic sites of
transmitter release assessed immunocytochemically (Wilson Horch and
Sargent, 1995).
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Figure 1.
Confocal immunofluorescence comparing the
distribution of 7-AChRs on ciliary ganglion neurons in
situ (A) and in dissociated cell culture
(B). Labeling of 7-AChRs was obtained with
biotinylated -Bgt followed by Cy3-conjugated streptavidin. Large
receptor clusters 1-4 µm in diameter were apparent in both kinds of
preparations. Scale bar, 10 µm.
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Freshly dissociated E15 ciliary ganglion neurons display similar
patterns of 7-AChR clusters (Fig. 1B). The
clusters were most prominent and distinct on the larger cells that
comprised a little over one-half of the dissociated population. The
smaller cells also showed nonuniform distributions of 7-AChRs, but
the receptor clusters were less pronounced. Similar patterns of
immunofluorescent staining were obtained when the cells were fixed,
permeabilized, and labeled with an anti-7 antibody followed by
Cy3-labeled secondary antibody (data not shown). Measuring the size of
the larger cells with prominent clusters yielded a value of 21.2 ± 0.4 µm (mean ± SEM; n = 29 neurons) for
their mean cell diameter. This value is midway between that reported
for E14 and that for 1 d after-hatching ciliary neurons in
vivo and threefold larger than that reported for E14 choroid
neurons in vivo (Pilar and Tuttle, 1982). Accordingly, the
large dissociated cell population probably contained mostly ciliary
neurons and was selected for subsequent analysis. The smaller cells
seemed likely to include many choroid neurons; they were not
characterized further here.
E15 ganglia were chosen for these experiments because it is a time at
which many of the major developmental events have occurred on ciliary
neurons; all have become innervated, preganglionic calyces have formed,
naturally occurring cell death is complete, and the postganglionic
target has been contacted (Dryer, 1994). Also, preliminary experiments
showed that by E15 the receptor clusters were well formed and
comparable with those reported in vivo for older ganglia
(Wilson Horch and Sargent, 1995).
Localization of 7-AChRs on spine-like structures
To examine the fine structure of 7-AChR clusters, we labeled
freshly dissociated E15 neurons with biotinylated -Bgt followed by
HRP-conjugated streptavidin and prepared these neurons for EM.
Dissociated neurons were chosen in this case because they offered a
relatively simple cellular environment and more reliable access for
receptor labeling. Thick sections were examined by IVEM and were used
to generate tomographic reconstructions of the HRP-labeled membrane
areas. Tomographic analysis was used because it enables quantitative
three-dimensional examination of fine structure within the relevant
domains and thereby facilitates accurate representation of complex structures.
HRP reaction product in the thick sections was primarily associated
with cell surface regions having complex membrane folds (Fig.
2A). Tomographic
analysis of the thick sections was achieved by taking multiple images
by IVEM at different tilt angles through the section and assembling the
images into a composite three-dimensional structure (Soto et al., 1994;
Perkins et al., 1997). A single reconstructed cross section generated
by back projection illustrated the complexity of such labeled regions
(Fig. 2B). Complete three-dimensional images
indicated that the regions were composed of dense clusters or clumps of
small finger-like projections emanating from the cell body and were
heavily labeled with HRP reaction product. Individual projections
within the clumps had the approximate dimensions of somatic spines (see
below) and were usually folded back along the cell surface to form
compact mats (Fig. 2C). The labeled mats ranged in size from
1 to 4 µm in diameter and closely approximated both in size and
number the clusters of 7-AChRs identified previously on ciliary
ganglion neurons with confocal microscopy. No HRP reaction product was
detectable on smooth soma membrane.
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Figure 2.
IVEM tomographic reconstruction of the ciliary
ganglion neuron surface showing spine-like structures matted against
the cell soma. Dissociated cells stained with biotinylated -Bgt
followed by HRP-conjugated streptavidin were thick-sectioned and
examined by IVEM at multiple tilt angles. A, Electron
micrograph of 1-µm-thick section showing HRP labeling of a surface
region containing a tangle of processes difficult to follow.
Black dots at the top and bottom
left are 20 nm gold fiducial markers. B, One of
many computer-generated slices through the tomographic volume showing
the plasma membrane surface clearly resolved in the labeled region.
C, A three-dimensional reconstruction of the surface
plasmalemma illustrating the dense mat of spine-like projections
emanating from the cell body. Scale bars, 500 nm.
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To determine whether 7-AChRs are concentrated on the spine-like
structures, we subjected freshly dissociated neurons to immunogold labeling. Analysis of thin sections with conventional EM indicated prominent gold labeling of presumptive spines (Fig.
3A,B).
The extent of labeling varied along and among the spine-like
structures, but it was very rare to find a putative spine completely
lacking label. Relatively little specific labeling was found on
nonspiny regions of the soma. In both cases the labeling was specific
as judged by the basal levels found on cells coincubated with excess -Bgt to block binding of the biotinylated toxin (Fig.
3C).
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Figure 3.
Conventional electron micrographs of thin sections
showing immunogold labeling of 7-AChRs on dissociated ciliary
ganglion neurons. A, B, Examples of
spine-like structures with immunogold labeling achieved by incubating
cells with biotinylated -Bgt followed by anti-biotin antibodies and
gold-conjugated secondary antibodies. C, Control section
showing minimal nonspecific immunogold labeling on a neuron surface
after the cells were incubated with excess -Bgt before biotinylated
-Bgt in the labeling procedure. Scale bars: A,
C, 500 nm; B, 500 nm.
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Tomographic reconstruction of images collected by IVEM from
immunogold-labeled thick sections confirmed the preferential
association of gold particles with membrane domains rich in the folded
spine-like structures (Fig.
4A). Adjacent nonspiny
membrane on the same cell had lower levels of labeling. Again, the
labeling was judged specific because it could be blocked by competition
with unlabeled -Bgt during the initial binding reaction (Fig.
4B). Stereoscopic images of labeled regions
containing the spine-like structures provided clear three-dimensional
views of the configuration (Fig. 4C).
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Figure 4.
Three-dimensional pattern of 7-AChR immunogold
labeling (yellow spheres) on ciliary ganglion neurons
viewed with IVEM and tomographic reconstruction. A, A
three-dimensional reconstructed view of a patch of membrane-containing
matted spines. The spines are heavily labeled with gold particles
associated with 7-AChRs after immunogold labeling as described in
Figure 3. B, Background levels of immunogold particles
on a neuron surface prepared for nonspecific labeling as described in
Figure 3. C, A stereo pair offering a three-dimensional
view of another reconstruction of folded spines containing
immunogold-labeled 7-AChRs. Scale bars, 500 nm.
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Immunogold-labeled thin-section images were used to quantify the number
of gold particles per unit length of membrane. For purposes of
quantification, nonspiny soma membrane was considered to be those
regions having a consistently smooth contour defining the circumference
of the cell with no deformations or irregularities. Spine-like membrane
was taken to be that extending from the cell body by at least one width
equivalent of the projection itself and having no abutting structures
that might have impeded access of the immunogold label. The specific
labeling associated with putative spines calculated in this way was an
order of magnitude greater in density than that associated with smooth
soma (nonspiny) membrane and was 35-40 times greater than the level of
background labeling found on either type of membrane (Fig.
5). The results indicate a clear
targeting of 7-AChRs specifically to the presumptive spines.
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Figure 5.
Relative levels of 7-AChRs on spine-like
projections and nonspiny soma surfaces. The density of gold particles
on spine-like versus nonspiny membrane segments was determined from
conventional EM of thin sections after immunogold labeling for
7-AChRs as shown in Figure 3. Nonspiny membrane was defined as those
segments having a smooth contour continuous with the cell surface and
free of bumps and irregularities; spine-like membrane was defined as
that involving an extension of at least one putative spine width
(usually 0.1-0.2 µm) from the cell body with no abutting structures
that would limit access of the immunogold label. Specific labeling of
7-AChRs (-Bgt-labeled neurons) and nonspecific
labeling (control neurons) were generated as described in Figure 3. The
values shown represent the mean ± SEM of counts on a total of
31.4 lineal µm of nonspiny membrane from 17 neurons and 26.3 lineal
µm of spine-like membrane from 17 neurons (23 spines).
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Features of somatic spines
By several criteria the finger-like projections appeared to
represent somatic spines. Sometimes individual projections stretched out from the cell body, extending through the full thickness of the
1-2 µm section. Inspection of numerous examples of both folded and
extended projections indicated they were approximately cylindrical in
shape with diameters that varied from 0.1 to 0.5 µm and with lengths
ranging up to 4 µm. Inspection of thin sections by conventional EM
failed to reveal microtubules and instead sometimes showed internal
membrane structures with the shape and size expected for endoplasmic
reticulum. Double labeling with biotinylated -Bgt and phalloidin
followed by confocal fluorescence microscopy showed that the membrane
structures associated with 7-AChR clusters were highly enriched in
bundles of actin filaments, as expected if the structures represented
spine clumps (Fig. 6). These structural features, together with the occasional presence of a PSD (see below),
strongly suggest they are in fact somatic spines.
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Figure 6.
Confocal double-label micrographs showing
codistribution of actin filament bundles and 7-AChRs on neurons.
A, Phalloidin staining (green) of
a dissociated E15 ciliary ganglion neuron to detect bundled actin in
spines. B, Same neuron stained with biotinylated -Bgt
followed by Cy3-streptavidin (red) to visualize
7-AChR clusters. C, Overlay of phalloidin and -Bgt
labeling showing extensive overlap (yellow).
Membrane ruffles that extended from the bottom and
right sides of the cell, presumably serving to anchor
the cell to the substratum, contained actin bundles without
codistributed 7-AChRs. Scale bar, 10 µm.
Figure 7.
Confocal double-label micrographs showing
extensive overlap of 7- and 3*-AChR clusters on dissociated
ciliary ganglion neurons. A, Neuron labeled for
7-AChRs with biotinylated -Bgt followed by FITC-conjugated
streptavidin (green). B, Same
neuron labeled for 3*-AChRs with mAb 35 followed by
Cy3-conjugated secondary antibody (red).
C, Overlay of Cy3 and FITC staining showing extensive
overlap (yellow). Codistribution was most
pronounced for the larger clusters of 7- and 3*-AChRs. Smaller
clusters containing only one of the receptor types were also apparent.
D, Conventional EM of a thin section through a
dissociated neuron stained with mAb 35 and HRP-conjugated secondary
antibodies to label 3*-AChRs. Patches of HRP staining can be
distinguished on the spines and soma membrane (arrows).
A portion of the calyx containing synaptic vesicles remains attached to
the cell in this case. Scale bars: A-C, 5 µm;
D, 500 nm.
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Estimates of the total cell surface membrane devoted to somatic spines
were generated to calculate the proportion of 7-AChRs located on
them. Confocal microscopy indicated that 12.5 ± 1.6% (mean ± SEM; n = 16 neurons) of the surface area on freshly
dissociated E15 neurons was occupied by spine clumps (assuming
7-AChR clusters and spine clumps to be coincident). IVEM and
tomographic analysis of dissociated neurons indicated that the total
surface membrane in spine clumps was 8.4-fold (± 0.8; mean ± SEM; n = 8 regions) more dense per degree of arc than
that along nonspiny portions of the soma. Together these numbers
produced the unexpected result that spines approximately double the
surface area of the neuron.
The mean cell diameter reported above for freshly dissociated E15
neurons would predict a mean total surface area of ~1400 µm2 per cell if the cells were spheres.
Capacitance measurements on dissociated E15 neurons produced a mean
value of 27.3 ± 0.6 pF (Zhang et al., 1994) for cells prepared
and generally size-selected as described here. The capacitance
measurements, therefore, indicate an electrically accessible mean total
surface area of ~2700 µm2 for the cells. This is
nearly twice that calculated from the mean cell diameter and very close
to that predicted for the cells if the spines double the surface area.
A limitation of the analysis is that some of the cells extend membrane
ruffles along the culture substratum, presumably to enhance attachment
(Fig. 6A). It is not clear whether the membrane
ruffles contribute to the capacitance measurement; they are not visible
with phase-contrast optics. Even allowing for them in the capacitance
measurement, however, suggests that a substantial portion of the spine
membrane is likely to be electrically accessible to a recording
electrode positioned on the soma.
Codistribution of AChR subtypes
In addition to 7-AChRs, ciliary ganglion neurons contain
heteromeric 3*-AChRs composed of 3, 4, 5, and occasionally
2 subunits that can be distinguished by binding mAb 35 (Vernallis et
al., 1993; Conroy and Berg, 1995; Wilson Horch and Sargent, 1995). mAb
35 recognizes the neuronal AChR 3 and 5 gene products but not the
4, 7, 8, 2, or 4 gene products (Conroy and Berg, 1998).
Such receptors were reported previously to be concentrated both at PSDs
on the neurons and on the pseudodendrites (Jacob et al., 1984)
identified here as somatic spines. Confocal analysis suggested
previously that the 3*-AChRs were primarily concentrated in
perisynaptic clusters as reported for 7-AChRs (Wilson Horch and
Sargent, 1995).
When dissociated ciliary ganglion neurons were double labeled for
7-AChRs with biotinylated -Bgt and for 3*-AChRs with mAb 35, confocal fluorescence microscopy showed considerable overlap in the
distribution for the two classes of receptors (Fig.
7A-C). This was most apparent
for the larger clusters of 7- and 3*-AChRs. Small clusters
containing only 3*-AChRs or only 7-AChRs, however, were also
present. The microclusters of 3*-AChRs that lack 7-AChRs and ring
the larger coclusters have the size and position expected for PSDs on
the cells (Jacob and Berg, 1983; Jacob et al., 1984; Wilson Horch and
Sargent, 1995). HRP staining of dissociated neurons labeled with mAb 35 confirmed that 3*-AChRs were associated with spines emanating from
the cells, as seen above with gold labeling for 7-AChRs (Fig.
7D). The HRP reaction product was more patchy, however, and
less widely distributed than that associated with 7-AChRs.
Proximity of spines and synapses in vivo
Previous studies have shown 7-AChRs to be undetectable at PSDs
on ciliary ganglion neurons (Jacob and Berg, 1983), and yet the
receptors contribute importantly to synaptic currents in the neurons
(Zhang et al., 1996; Ullian et al., 1997). These results, coupled with
the finding reported here that 7-AChRs are concentrated on somatic
spines, motivated a re-examination of the relationship between spines
and presynaptic sites of transmitter release in the ganglion.
Tomographic analysis provided an opportunity to obtain a detailed
three-dimensional view of the spines in vivo. This served
two purposes, one being an assessment of spine proximity to presynaptic
specializations such as vesicle-docking sites and the other being an
indication of whether the spine configuration observed on dissociated
cells was a fair representation of that found in situ when
the preganglionic elements remained in place.
E15 ciliary ganglia were thick-sectioned and examined by IVEM.
Tomographic reconstruction of the images indicated a complex mat of
intertwined and folded somatic spines, best seen by stereoscopic projection (Fig. 8A).
The dimensions and configuration of the spines were similar to those
present on dissociated cells. PSDs could be distinguished on somatic
membrane in the immediate vicinity of the spine clumps. Individual
reconstructed sections generated by the tomographic back projection
illustrated the proximity of spines, calyx, and synaptic vesicles (Fig.
8B). When the locations of individual synaptic
vesicles were scored and added to the tomographic reconstruction, it
became clear that the vesicles were concentrated not only over the PSDs
but also over the folded spines even though no PSDs were apparent on
the spines in the field of view (Fig. 8B,C). Adding the calyx profile to
the image illustrated how it engulfed the folded spines and extended
small projections among the spines (Fig. 8C).
View larger version (92K):
[in this window]
[in a new window]
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Figure 8.
Tomographic reconstruction of ciliary ganglion
neuron surface in situ showing complexes of matted
spines, overlaid synaptic vesicles, and nearby PSDs. A,
Stereo pair of images showing a three-dimensional view of a membrane
patch containing matted spines on a neuron in situ. Note
the locations of nearby PSDs (green).
B, One of many computer-generated slices through the
reconstructed volume. A vesicle-filled calyx extension underlies the
mat of spines (arrowhead). A synapse adjacent to the
spines is indicated by the arrow. C, Same
image as in A (75% scale) with overlaid synaptic
vesicles (red) on the right and the
accompanying calyx structure (gray) on the
left containing the same vesicles and rotated 70°
out from the plane of the neuron surface. For clarity,
only a representative 20% of the vesicles are indicated. The calyx
image shows thin projections that extend into the mat of spines;
synaptic vesicles are densely packed over the spines even though PSDs
are located only at the perimeter. C, Calyx;
Gl, glial cell; N, neuronal soma;
Sp, spine. Scale bars, 500 nm.
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Conventional EM on thin sections provided additional detail. Spines
were often closely packed to form a clump overlaid on the cell soma but
were interdigitated with calyx projections filled with synaptic
vesicles (Fig. 9A). PSDs could
be found both on the soma membrane close to the base of the spines and
on the spines themselves in some cases (Fig.
9A,B). Synaptic vesicles were
packed close to the calyx membrane ensheathing the spines, often being in such proximity as to suggest that some of the vesicles may have been
docked at release sites (Fig.
9B,C). Occasionally the spines
projected deep into the calyx where they were surrounded with synaptic
vesicles. In some instances a spine was caught in cross section
emerging from the soma near a PSD in the same field of view; synaptic
vesicles could be seen tightly packed along its length (Fig.
9C). In the example shown, an profile can also be
distinguished in the calyx membrane, possibly representing a synaptic
vesicle caught in midexocytosis onto the spine surface (Fig.
9C). Higher magnification tomographic sections of other regions provided additional examples of profiles opposed to spines
even though no PSD was present in the entire volume sampled (Fig.
9D,E). The absence of clathrin
coats suggested that the structures represented exocytotic events
rather than endocytotic ones. Clathrin-coated pits were occasionally
observed both on calyx and spine membrane, confirming that the
structures could be distinguished when present.
View larger version (167K):
[in this window]
[in a new window]
|
Figure 9.
The relationship of calyx membrane, spines,
synaptic vesicles, and PSDs as shown by conventional electron
micrographs of the intact ganglion in thin sections
(A-C) and by high-magnification
tomographic-reconstructed views from thick sections (D,
E). A, A mat of folded spines invested
with a calyx projection (arrow) filled with synaptic
vesicles. A PSD can be distinguished both on the soma membrane near the
spines (double arrowhead) and on a spine itself
(arrowhead). B, A mat of folded spines
overlaid with calyx densely packed with synaptic vesicles. Two PSDs can
be distinguished on the spines (arrowheads), and
synaptic vesicles are tightly packed near the spine membrane in other
regions as well that lack PSDs. C, A spine in longitudinal section
showing the base (asterisk) with a PSD on the adjacent
soma membrane (arrow) and synaptic vesicles tightly
lining the calyx membrane along the spine as if some may have been
docked for release. An profile (arrowhead) can be
distinguished in the calyx membrane as if a vesicle had been trapped in
midexocytosis onto the spine surface. D,
E, Two high-magnification tomographically reconstructed
images ~40 nm apart from the same thick section. Multiple spines are
seen in cross section (one of which is labeled) stacked up along the
vesicle-containing calyx. Several profiles are indicated
(arrowheads). The absence of clathrin coats in these
cases supports the interpretation that they represent exocytotic rather
than endocytotic events. Examination of serial reconstructed images
confirmed the absence of PSDs from the entire 1-µm-thick volume
sampled. c, Calyx; n, neuron;
sp, spine. Scale bars: A,
B, 500 nm; C, 500 nm; D,
E, 200 nm.
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|
| |
DISCUSSION |
The principal findings reported here are that 7-AChRs are
concentrated on ciliary ganglion somatic spines. The spines are arranged in clumps on the ciliary neuron surface and are folded into
tightly matted arrays. The matted spines effectively double the total
surface area of the cell and position much of it close to presynaptic
calyx membrane. Although 7-AChRs may be excluded from PSDs on the
neurons, the receptors are in close proximity to numerous potential
sites of transmitter release by virtue of their distribution along
spines. The elaborate spine configuration implies a specialized
synaptic function, and the accompanying distribution of 7-AChRs
indicates that neurons have specific mechanisms for targeting
7-AChRs to desired locations.
Spine-like projections emanating from neuron cell bodies have been
described in several kinds of autonomic ganglia (Piezzi and
Rodriquez-Echandia, 1968; Watanabe, 1971; Smolen, 1988; Robertson and
Jackson, 1996). Identification of the projections as somatic spines in
the present experiments depended on several criteria. Chief among these
were their dimensions, their cytoskeletal composition (presence of
actin filaments and absence of microtubules), and their proximity to
presynaptic elements. Usually another defining feature of both
dendritic and somatic spines is the presence of a PSD (Harris and
Kater, 1994). Analysis of ciliary ganglion sections revealed PSDs on
some somatic spines but not on others. It is unclear whether this
represents a structural heterogeneity among the spines or whether PSDs
were often missed because the spines rarely were captured in their
entirety within the section being examined. A previous report described
rare somatic spines with opposing PSDs on both sides of the spine neck
on ciliary ganglion neurons; the structures did not appear until 2 weeks after hatching and were found in only a few of many ganglia
examined (Takahashi, 1967). Serial-section EM analysis of ciliary
neurons in situ may be necessary to define the full extent
of spines and to quantify PSDs on them.
The immunogold labeling with biotinylated -Bgt permitted a
quantitative demonstration for the first time that 7-AChRs are preferentially concentrated on somatic spines rather than being randomly distributed throughout the surface membrane. Because spines
contribute approximately one-half of the total surface area of E15
ciliary neurons and contain an order of magnitude greater density of
-Bgt-binding sites than does the soma membrane, they account for
~90% of the total 7-AChRs on the neurons. The few -Bgt-binding
sites in ciliary ganglia not associated with 7 protein ( 5%) are
unlikely to have contributed significantly because of their scarcity
(Pugh et al., 1995) and because anti-7 antibodies and -Bgt
produced the same labeling patterns. If one-half of the ~20 fmol of
-Bgt-binding sites in E15 ciliary ganglia (Chiappinelli and
Giacobini, 1978; Corriveau and Berg, 1994) are on the estimated 1600 ciliary neurons present (Landmesser and Pilar, 1974), the 1400 µm2 calculated here for the aggregate spine
surface per E15 neuron should have a mean binding-site density of
~2 × 103 per µm2. This
value is intermediate between the ~0.6 × 103
and 5 × 103 neuronal bungarotoxin-binding
sites per µm2 estimated by EM autoradiography for
AChRs (non-7) at PSDs on chick ciliary ganglion neurons in
situ and on rat sympathetic neurons in culture, respectively
(Loring and Zigmond et al., 1987; Loring et al., 1988). If 7-AChRs
have one to two -Bgt-binding sites (Schoepfer et al., 1990; Chen and
Patrick, 1997; but see Palma et al., 1996), the inferred receptor
density would be approximately an order of magnitude below the 2 × 104 AChRs per µm2 at the
mammalian neuromuscular junction (Fertuck and Salpeter, 1976). Because
7-AChRs are widely expressed in the nervous system, it will be
important to determine whether they are commonly associated with spines.
The HRP-labeling studies with mAb 35 indicate that 3*-AChRs are
distributed along the spines as well. The receptors are less abundant
than are 7-AChRs (Chiappinelli and Giacobini, 1978; Smith et al.,
1985; Corriveau and Berg, 1994) but may be intermingled with them.
Alternatively, they may be segregated to different regions on the
spines or to different spines within a clump. The results corroborate
and extend those obtained with conventional EM and HRP labeling of
neurons in situ (Jacob and Berg, 1983; Jacob et al., 1984).
Those results also showed, however, as did EM autoradiographic analysis
(Loring and Zigmond, 1987), that a portion of the 3*-AChRs is
uniquely concentrated at PSDs. Such receptors may comprise a small
fraction of the total 3*-AChRs and appear in confocal microscopy as
the microclusters of mAb 35-binding sites seen near but not overlapping
with the larger 7- and 3*-AChR coclusters both here on
dissociated cells and previously in situ (Wilson Horch and
Sargent, 1995).
How do 7-AChRs become synaptically activated if they are excluded
from PSDs? The present results suggest two kinds of mechanisms. One
arises from the proximity of spines to PSDs. Transmitter released onto
PSDs could spread rapidly by diffusion to adjacent spine surfaces
(Clements, 1996) and produce the ~1 msec rise times for synaptic
currents caused by 7-AChRs (Zhang et al., 1996; Ullian et al.,
1997). A different possibility is that vesicular release of transmitter
may occur directly onto spine membrane even without PSDs. Such might be
the purpose of having both large amounts of spine surface and vast
numbers of synaptic vesicles packed tightly along the calyx membrane
juxtaposed to spines. The occasional membrane profiles seen at such
locations could represent examples of vesicular release; they lack the
clathrin coats expected for endocytotic events. The multiple spines and
the abundance of synaptic vesicles along them could ensure reliable
high-frequency transmission even if release occurred as a
low-probability event per unit area of membrane. This mode of synaptic
transmission may be more widespread in the nervous system than
previously imagined.
Previous confocal microscopic analysis using immunofluorescence
suggested that synaptic vesicle protein did not overlap in distribution
with 7-AChR clusters on ciliary ganglion neurons (Wilson Horch and
Sargent, 1995). This led to the suggestion that the receptor clusters
were perisynaptic. Confocal imaging of dissociated neurons displayed
the same kinds of 7-AChR clusters in the present experiments, but
double labeling with phalloidin for bundled actin and with biotinylated
-Bgt for 7-AChRs indicated the clusters were coextensive with
somatic spine clumps. (The spines presumably survived the dissociation
procedure because they were folded and packed on the cell surface; the
folding appeared only slightly less compact than on cells in
vivo.) Immunogold labeling of dissociated cells showed that most,
if not all, of the spines were heavily endowed with 7-AChRs, as
indicated above. The EM analysis performed here on sectioned ganglia
indicated a close alignment between synaptic vesicles in the calyx and
juxtaposed spines on the cell body. As a result, 7-AChRs must be in
close apposition to synaptic vesicles. It is not clear why an
association of vesicles and 7-AChRs was not detected previously;
possibly the high density of vesicles throughout the calyx core
necessitated an attenuation of the fluorescence monitoring such that
vesicles positioned peripherally near spines were obscured.
Nearly all of the current generated by 7-AChRs on the spines should
reach the soma. This follows from the observation that the spines are
only a few micrometers in length and have a diameter of ~0.2 µm
that predicts a space constant in excess of 200 µm (Plonsey and Barr,
1989). The EM images revealed no physical restrictions at the base of
the spines that might impede current flow. Many of the 7-AChRs are
probably exposed to transmitter during each synaptic trial because of
the anatomical features described above and because of studies with an
esterase inhibitor suggesting that the 7-AChR synaptic response is
limited by receptor desensitization rather than by transmitter
availability during a stimulus (Zhang et al., 1996). Given these
considerations, it is surprising that estimates of 7-AChR
single-channel properties suggested that only a few percent of the
7-AChRs might be sufficient to account for the synaptic currents
observed (Ullian et al., 1997). If the assumptions about 7-AChR
properties were correct, the results imply that many of the receptors
either may not be functionally available during a given trial because
of some regulatory constraint or may quickly desensitize during initial
exposure to agonist.
What is the function of somatic spines, and why are 7-AChRs
concentrated on them? One reason may be that the enlarged surface area
contributed by spines increases the safety factor for reliable transmission in an economical manner. A second reason is that spines
may compartmentalize some consequence of synaptic signaling (Koch and
Zador, 1993). A prime candidate is calcium influx. The relative calcium
permeability of 7-AChRs is as high as that of NMDA receptors
(Bertrand et al., 1993; Seguela et al., 1993). Given the high-frequency
firing possible in the ciliary ganglion (Dryer, 1994), substantial
calcium influx may occur through 7-AChRs. Localizing the receptors
on spines could sequester the calcium and protect the cell body from
activity-driven cytotoxicity (Harris and Kater, 1994). Sequestering
calcium in spines may also enable higher levels to be sustained for
long-term regulatory effects.
| |
FOOTNOTES |
Received July 15, 1998; revised Oct. 14, 1998; accepted Oct. 23, 1998.
This research was supported by National Institutes of Health Grants NS
12601 and 35469 and Tobacco-Related Disease Research Program Grant
RT65-0050 to D.K.B. and by the National Institutes of Health Grant
RR04050 to M.H.E.
Correspondence should be addressed to Dr. Darwin K. Berg, Department of
Biology, 0357, University of California, San Diego, 9500 Gilman Drive,
La Jolla, CA 92093-0357.
| |
REFERENCES |
-
Anand R,
Peng X,
Lindstrom J
(1993)
Homomeric and native 7 acetylcholine receptors exhibit remarkably similar but non-identical pharmacological properties, suggesting that the native receptor is a heteromeric protein complex.
FEBS Lett
327:241-246[Web of Science][Medline].
-
Bertrand D,
Galzi JL,
Devillers-Thiery A,
Bertrand S,
Changeux JP
(1993)
Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal 7 nicotinic receptor.
Proc Natl Acad Sci USA
90:6971-6975[Abstract/Free Full Text].
-
Chen D,
Patrick JW
(1997)
The -bungarotoxin-binding nicotinic acetylcholine receptor from rat brain contains only the 7 subunit.
J Biol Chem
272:24024-24029[Abstract/Free Full Text].
-
Chiappinelli VA,
Giacobini E
(1978)
Time course of appearance of -bungarotoxin binding sites during development of chick ciliary ganglion and iris.
Neurochem Res
3:465-478[Web of Science][Medline].
-
Clements JD
(1996)
Transmitter timecourse in the synaptic cleft: its role in central synaptic function.
Trends Neurosci
19:163-171[Web of Science][Medline].
-
Coggan JS,
Paysan J,
Conroy WG,
Berg DK
(1997)
Direct recording of nicotinic responses in presynaptic nerve terminals.
J Neurosci
17:5798-5806[Abstract/Free Full Text].
-
Conroy WG,
Berg DK
(1995)
Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions.
J Biol Chem
270:4424-4431[Abstract/Free Full Text].
-
Conroy WG,
Berg DK
(1998)
Nicotinic receptor subtypes in the developing chick brain: appearance of a species containing the 4, 2, and 5 gene products.
Mol Pharmacol
53:392-401[Abstract/Free Full Text].
-
Corriveau RA,
Berg DK
(1993)
Co-expression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development.
J Neurosci
13:2662-2671[Abstract].
-
Corriveau RA,
Berg DK
(1994)
Neurons in culture maintain acetylcholine receptor levels with far fewer transcripts than in vivo.
J Neurobiol
25:1579-1592[Web of Science][Medline].
-
Couturier S,
Bertrand D,
Matter J-M,
Hernandez M-C,
Bertrand S,
Millar N,
Valera S,
Barkas T,
Ballivet M
(1990)
A neuronal nicotinic acetylcholine receptor subunit (7) is developmentally regulated and forms a homo-oligomeric channel blocked by -Btx.
Neuron
5:847-856[Web of Science][Medline].
-
Dryer S
(1994)
Functional development of the parasympathetic neurons of the avian ciliary ganglion: a classic model system for the study of neuronal differentiation and development.
Prog Neurobiol
43:281-322[Web of Science][Medline].
-
Fertuck HC,
Salpeter MM
(1976)
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after I-125--bungarotoxin binding at mouse neuromuscular junctions.
J Cell Biol
69:144-158[Abstract/Free Full Text].
-
Gray R,
Rajan AS,
Radcliffe KA,
Yakehiro M,
Dani JA
(1996)
Hippocampal synaptic transmission enhanced by low concentrations of nicotine.
Nature
383:713-716[Medline].
-
Guo J-Z,
Tredway TL,
Chiappinelli VA
(1998)
Glutamate and GABA release are enhanced by different subtypes of presynaptic nicotinic receptors in the lateral geniculate nucleus.
J Neurosci
18:1963-1969[Abstract/Free Full Text].
-
Harris KM,
Kater SB
(1994)
Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function.
Annu Rev Neurosci
17:341-371[Web of Science][Medline].
-
Hess A
(1965)
Developmental changes in the structure of the synapse on the myelinated cell bodies of the chicken ciliary ganglion.
J Cell Biol
25:1-19[Abstract/Free Full Text].
-
Hessler D,
Young SJ,
Carragher BO,
Martone M,
Lamont S,
Whittaker M,
Milligan RA,
Masliah E,
Hinshaw JE,
Ellisman MH
(1992)
Programs for visualization in 3-dimensional microscopy.
NeuroImage
1:55-67[Medline].
-
Jacob MH,
Berg DK
(1983)
The ultrastructural localization of -bungarotoxin binding sites in relation to synapses on chick ciliary ganglion neurons.
J Neurosci
3:260-271[Abstract].
-
Jacob MH,
Berg DK,
Lindstrom JM
(1984)
Shared antigenic determinant between the Electrophorus acetylcholine receptor and a synaptic component on chicken ciliary ganglion neurons.
Proc Natl Acad Sci USA
81:3223-3227[Abstract/Free Full Text].
-
Koch C,
Zador A
(1993)
The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization.
J Neurosci
13:413-422[Web of Science][Medline].
-
Landmesser L,
Pilar G
(1972)
The onset and development of transmission in the chick ciliary ganglion.
J Physiol (Lond)
222:691-713[Abstract/Free Full Text].
-
Landmesser L,
Pilar G
(1974)
Synapse formation during embryogenesis on ganglion cells lacking a periphery.
J Physiol (Lond)
241:715-736[Abstract/Free Full Text].
-
Li X,
Rainnie DG,
McCarley RW,
Greene RW
(1998)
Presynaptic nicotinic receptors facilitate monoaminergic transmission.
J Neurosci
18:1904-1912[Abstract/Free Full Text].
-
Loring RH,
Zigmond RE
(1987)
Ultrastructural distribution of 125I-toxin F binding sites on chick ciliary neurons: synaptic localization of a toxin that blocks ganglionic nicotinic receptors.
J Neurosci
7:2153-2162[Abstract].
-
Loring RH,
Dahm LM,
Zigmond RE
(1985)
Localization of -bungarotoxin binding sites in the ciliary ganglion of the embryonic chick: an autoradiographic study at the light and electron microscopic level.
Neuroscience
14:645-660[Web of Science][Medline].
-
Loring RH,
Sah DWY,
Landis SC,
Zigmond RE
(1988)
The ultrastructural distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion.
Neuroscience
24:1071-1080[Web of Science][Medline].
-
McGehee D,
Heath M,
Gelber S,
Role LW
(1995)
Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors.
Science
269:1692-1697[Abstract/Free Full Text].
-
Palma E,
Bertrand S,
Binzoni T,
Bertrand D
(1996)
Neuronal nicotinic 7 receptor expressed in Xenopus oocytes presents five putative binding sites for methyllycaconitine.
J Physiol (Lond)
491:151-161[Abstract/Free Full Text].
-
Perkins GA,
Renken CW,
Song JY,
Frey TG,
Young SJ,
Lanont S,
Martone ME,
Lindsey S,
Ellisman MH
(1997)
Electron tomography of large multicomponent biological structures
-
Piezzi RS,
Rodriguez-Echandia EL
(1968)
Studies on the pararenal ganglion of the Bufo arenarum Hensel. I. Its normal fine structure and histochemical characteristics.
Z Zellforsch Mikrosk Anat
88:180-186[Web of Science][Medline].
-
Pilar G,
Tuttle JB
(1982)
A simple neuronal system with a range of uses: the avian ciliary ganglion.
In: Progress in cholinergic biology (Hanin I,
Goldberg AM,
eds), pp 213-247. New York: Raven.
-
Plonsey R,
Barr R
(1989)
In: Bioelectricity. New York: Plenum.
-
Pugh PC,
Corriveau RA,
Conroy WG,
Berg DK
(1995)
A novel subpopulation of neuronal acetylcholine receptors among those binding -bungarotoxin.
Mol Pharmacol
47:717-725[Abstract].
-
Robertson GN,
Jackson PC
(1996)
Axosomatic synapses of the rat ciliary ganglion.
Synapse
22:269-280[Web of Science][Medline].
-
Schoepfer R,
Conroy WG,
Whiting P,
Gore M,
Lindstrom J
(1990)
Brain -bungarotoxin binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.
Neuron
5:35-48[Web of Science][Medline].
-
Seguela P,
Wadiche J,
Dineley-Miller K,
Dani JA,
Patrick JW
(1993)
Molecular cloning, functional properties, and distribution of rat brain 7: a nicotinic cation channel highly permeable to calcium.
J Neurosci
13:596-604[Abstract].
-
Smith MA,
Margiotta JF,
Berg DK
(1983)
Differential regulation of acetylcholine sensitivity and -bungarotoxin-binding sites on ciliary ganglion neurons in cell culture.
J Neurosci
3:2395-2402[Abstract].
-
Smith MA,
Stollberg J,
Lindstrom JM,
Berg DK
(1985)
Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor.
J Neurosci
5:2726-2731[Abstract].
-
Smolen A
(1988)
Morphology of synapses in the autonomic nervous system.
J Electron Microsc Tech
10:187-204[Web of Science][Medline].
-
Soto GE,
Young SJ,
Martone ME,
Deerinck TJ,
Lamont S,
Carragher BO,
Hama K,
Ellisman MH
(1994)
Serial section electron tomography: a method for three-dimensional reconstruction of large structures.
NeuroImage
1:230-243[Medline].
-
Szentagothai J
(1964)
The structure of the autonomic interneuronal synapse.
Acta Neurovegetativa
26:338-359.
-
Takahashi K
(1967)
Special somatic spine synapses in the ciliary ganglion of the chick.
Z Zellforsch Mikrosk Anat
83:70-75[Web of Science][Medline].
-
Ullian EM,
McIntosh JM,
Sargent PB
(1997)
Rapid synaptic transmission in the avian CG is mediated by two distinct classes of nicotinic receptors.
J Neurosci
17:7210-7219[Abstract/Free Full Text].
-
Vernallis AB,
Conroy WG,
Berg DK
(1993)
Neurons assemble acetylcholine receptors with as many as three kinds of subunits while maintaining subunit segregation among receptor subtypes.
Neuron
10:451-464[Web of Science][Medline].
-
Watanabe H
(1971)
Adrenergic nerve elements in the hypogastric ganglion of the guinea pig.
Am J Anat
130:305-330[Web of Science][Medline].
-
Wilson Horch HL,
Sargent PB
(1995)
Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion.
J Neurosci
15:7778-7795[Abstract].
-
Zhang Z-w,
Vijayaraghavan S,
Berg DK
(1994)
Neuronal acetylcholine receptors that bind -bungarotoxin with high affinity function as ligand-gated ion channels.
Neuron
12:167-177[Web of Science][Medline].
-
Zhang Z-w,
Coggan JS,
Berg DK
(1996)
Synaptic currents generated by neuronal acetylcholine receptors sensitive to -bungarotoxin.
Neuron
17:1231-1240[Web of Science][Medline].
Copyright © 1999 Society for Neuroscience 0270-6474/99/192692-13$05.00/0
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M. K. Temburni, M. M. Rosenberg, N. Pathak, R. McConnell, and M. H. Jacob
Neuronal Nicotinic Synapse Assembly Requires the Adenomatous Polyposis Coli Tumor Suppressor Protein
J. Neurosci.,
July 28, 2004;
24(30):
6776 - 6784.
[Abstract]
[Full Text]
[PDF]
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N. Wang, A. Orr-Urtreger, J. Chapman, R. Rabinowitz, and A. D. Korczyn
Deficiency of Nicotinic Acetylcholine Receptor beta 4 Subunit Causes Autonomic Cardiac and Intestinal Dysfunction
Mol. Pharmacol.,
March 1, 2003;
63(3):
574 - 580.
[Abstract]
[Full Text]
[PDF]
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C. L. Brumwell, J. L. Johnson, and M. H. Jacob
Extrasynaptic alpha 7-Nicotinic Acetylcholine Receptor Expression in Developing Neurons Is Regulated by Inputs, Targets, and Activity
J. Neurosci.,
September 15, 2002;
22(18):
8101 - 8109.
[Abstract]
[Full Text]
[PDF]
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R. D. Shoop, E. Esquenazi, N. Yamada, M. H. Ellisman, and D. K. Berg
Ultrastructure of a Somatic Spine Mat for Nicotinic Signaling in Neurons
J. Neurosci.,
February 1, 2002;
22(3):
748 - 756.
[Abstract]
[Full Text]
[PDF]
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R. D. Shoop, K. T. Chang, M. H. Ellisman, and D. K. Berg
Synaptically Driven Calcium Transients via Nicotinic Receptors on Somatic Spines
J. Neurosci.,
February 1, 2001;
21(3):
771 - 781.
[Abstract]
[Full Text]
[PDF]
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J. L. Bruses, N. Chauvet, and U. Rutishauser
Membrane Lipid Rafts Are Necessary for the Maintenance of the {alpha}7 Nicotinic Acetylcholine Receptor in Somatic Spines of Ciliary Neurons
J. Neurosci.,
January 15, 2001;
21(2):
504 - 512.
[Abstract]
[Full Text]
[PDF]
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S. Bibevski, Y. Zhou, J. M. McIntosh, R. E. Zigmond, and M. E. Dunlap
Functional Nicotinic Acetylcholine Receptors That Mediate Ganglionic Transmission in Cardiac Parasympathetic Neurons
J. Neurosci.,
July 1, 2000;
20(13):
5076 - 5082.
[Abstract]
[Full Text]
[PDF]
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J. Cuevas, A. L Roth, and D. K Berg
Two distinct classes of functional {alpha}7-containing nicotinic receptor on rat superior cervical ganglion neurons
J. Physiol.,
June 15, 2000;
525(3):
735 - 746.
[Abstract]
[Full Text]
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R. D. Shoop, N. Yamada, and D. K. Berg
Cytoskeletal Links of Neuronal Acetylcholine Receptors Containing alpha 7 Subunits
J. Neurosci.,
June 1, 2000;
20(11):
4021 - 4029.
[Abstract]
[Full Text]
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M. K. Temburni, R. C Blitzblau, and M. H Jacob
Receptor targeting and heterogeneity at interneuronal nicotinic cholinergic synapses in vivo
J. Physiol.,
May 15, 2000;
525(1):
21 - 29.
[Abstract]
[Full Text]
[PDF]
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Q.-s. Liu and D. K. Berg
Actin Filaments and the Opposing Actions of CaM Kinase II and Calcineurin in Regulating alpha 7-Containing Nicotinic Receptors on Chick Ciliary Ganglion Neurons
J. Neurosci.,
December 1, 1999;
19(23):
10280 - 10288.
[Abstract]
[Full Text]
[PDF]
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Q.-S. Liu and D. K. Berg
Extracellular Calcium Regulates Responses of Both alpha 3- and alpha 7-Containing Nicotinic Receptors on Chick Ciliary Ganglion Neurons
J Neurophysiol,
September 1, 1999;
82(3):
1124 - 1132.
[Abstract]
[Full Text]
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K. T. Chang and D. K. Berg
Nicotinic Acetylcholine Receptors Containing alpha 7 Subunits Are Required for Reliable Synaptic Transmission In Situ
J. Neurosci.,
May 15, 1999;
19(10):
3701 - 3710.
[Abstract]
[Full Text]
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Top
Abstract
Introduction
