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The Journal of Neuroscience, December 15, 2002, 22(24):10567-10579
Three-Dimensional Reconstruction of a Calyx of Held and Its
Postsynaptic Principal Neuron in the Medial Nucleus of the Trapezoid
Body
Kurt
Sätzler1, 2, *,
Leander F.
Söhl3, *,
Johann H.
Bollmann1,
J.
Gerard G.
Borst4,
Michael
Frotscher3,
Bert
Sakmann1, and
Joachim H. R.
Lübke3
1 Department of Cell Physiology, Max Planck Institute
for Medical Research, D-69120 Heidelberg, Germany,
2 Interdisciplinary Center of Scientific Computing,
University of Heidelberg, D-69120 Heidelberg, Germany,
3 Anatomical Institute, University of Freiburg, D-79104
Freiburg, Germany, and 4 Department of Neuroscience,
Erasmus University Rotterdam, 3015 GE Rotterdam, The Netherlands
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ABSTRACT |
The three-dimensional morphology of the axosomatic synaptic
structures between a calyx of Held and a principal neuron in the medial
nucleus of the trapezoid body (MNTB) in the brainstem of young
postnatal day 9 rats was reconstructed from serial ultrathin sections.
In the apposition zone between the calyx and the principal neuron two
types of membrane specializations were identified: synaptic contacts
(SCs) with active zones (AZs) and their associated postsynaptic
densities (PSDs) constituted ~35% (n = 554) of
the specializations; the remaining 65% (n = 1010)
were puncta adherentia (PA). Synaptic contacts comprised ~5% of the
apposition area of presynaptic and postsynaptic membranes. A SC had an
average area of 0.100 µm2, and the nearest
neighbors were separated, on average, by 0.59 µm. Approximately
one-half of the synaptic vesicles in the calyx were clustered within a
distance of 200 nm of the AZ membrane area, a cluster consisting of
~60 synaptic vesicles (n = 52 SCs). Approximately
two synaptic vesicles per SC were "anatomically docked."
Comparing the geometry of the synaptic structure with its previously
studied functional properties, we find that during a single presynaptic
action potential (AP) (1) ~35% of the AZs release a
transmitter quantum, (2) the number of SCs and anatomically docked
vesicles is comparable with the low estimates of the readily releasable
pool (RRP) of quanta, and (3) the broad distribution of PSD areas
[coefficient of variation (CV) = 0.9] is likely to contribute to
the large variability of miniature EPSC peaks. The geometry of the reconstructed synapse suggests that each of the hundreds of SCs is likely to contribute independently to the size and
rising phase of the EPSC during a single AP.
Key words:
electron microscopy; three-dimensional reconstruction; synapse; active zones; puncta adherentia; synaptic vesicles
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INTRODUCTION |
A quantitative description of the signal cascade underlying synaptic
transmission requires that the temporal and spatial dependence of those
steps that couple a presynaptic action potential (AP) to a postsynaptic
potential must be known. Important steps are (1) the diffusion of
Ca2+ in the presynaptic terminal driving
the exocytosis of transmitter-containing vesicles (Katz, 1969 ), (2) the
diffusion of transmitter in the synaptic cleft after fusion has
occurred, (3) the transient opening of postsynaptic channels after
binding of transmitter to its cognate receptors (del Castillo and Katz,
1957), and (4) clearance of transmitter. Some of these steps have been
elucidated in the mammalian brain for the glutamatergic giant synapse
in the medial nucleus of the trapezoid body (MNTB) in the acoustic
pathway. The large somatic terminals of bushy cells in the ventral
cochlear nucleus (VCN), known as calyces of Held, form synapses with
cell bodies of the principal neurons of the MNTB (Held, 1898; Lenn and
Reese, 1966; Nakajima, 1971; Petelina, 1975; Casey and Feldman, 1985; Kandler and Friauf, 1993; Forsythe, 1994; Rowland et al., 2000). The
trains of APs in the bushy cells of the VCN are converted to an
equivalent inhibitory signal transmitted to the lateral superior olive
(LSO) (Smith et al., 1998).
In young rats [postnatal day 9 (P9)] both the presynaptic and
postsynaptic elements of the synapse can be patch clamped
simultaneously (Borst et al., 1995; Takahashi et al., 1996; Borst and
Sakmann, 1998) to measure transmitter release under defined internal
and external ionic and membrane potential conditions. The size and time
course of AP-evoked calyceal Ca2+ influx
(Borst and Sakmann, 1996, 1998), occupancy of the putative Ca2+ sensor driving vesicle fusion
(Bollmann et al., 2000; Schneggenburger and Neher, 2000), and the
equilibration of intracellular Ca2+ with
the endogenous Ca2+ buffer and the
eventual Ca2+ clearance (Helmchen et al.,
1997) can be measured accurately as well as the latency, size, and time
course of evoked quantal EPSCs and multiquantal EPSCs (Borst and
Sakmann, 1996; Sahara and Takahashi, 2001).
A quantitative knowledge of the geometry of the presynaptic and
postsynaptic structures is required for a more detailed understanding of the mechanisms underlying synaptic transmission and provides important constraints for numerical simulations. On the presynaptic side, time- and space-dependent build-up and collapse of
[Ca2+]i domains
around the internal pore of Ca2+ channels
at an active zone (AZ), the buffered diffusion, and the subsequent
interaction of free Ca2+ with the
Ca2+ sensor can, at present, be simulated
only (Yamada and Zucker, 1992; Bertram et al., 1999; Smith, 2001;
Meinrenken et al., 2002). Realistic values of the number, distribution,
and geometry of synaptic contacts (SCs) including the distribution of
synaptic vesicles are essential for constraining such simulations. On
the postsynaptic side the time course and amplitude of
spontaneous and evoked EPSCs are used to infer the rate of quantal
release (del Castillo and Katz, 1954). This inference requires
simulations of the transient increase of glutamate concentration in the
synaptic cleft, reversible binding of glutamate to glutamate
receptor (GluR) channels, and eventual uptake and diffusion of
glutamate out of the cleft. To a large extent these processes are
governed by the geometry of the synaptic cleft and the postsynaptic
densities (PSDs).
Here we report the three-dimensional reconstruction of a giant synaptic
structure formed between the calyx of Held and the soma of a principal
neuron in the MNTB, made from contours of serial ultrathin sections.
The present study provides a quantitative analysis of the number,
density, and area of SCs and their associated synaptic vesicles and the
volume and geometry of the synaptic cleft.
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MATERIALS AND METHODS |
Fixation and tissue processing. All experiments were
performed in accordance with the animal welfare guidelines of the
University of Freiburg and the Max Planck Society.
For the complete reconstruction of the calyx and the principal neuron,
the brain of one Wistar rat was used (selected from four perfused
animals, P9, body weight ~15 gm). The animals were anesthetized
deeply with sodium pentobarbital (Nembutal, 60 mg/kg body weight) and
perfused transcardially with physiological saline, followed by a
phosphate-buffered solution (PB; 0.1 M solution, pH 7.4)
containing 2.5% glutaraldehyde (Polyscience Europe GmbH, Eppelheim,
Germany) for 15-20 min at room temperature. After 1 hr of postfixation
the brains were removed from the skull and stored overnight in fresh
fixative at 4°C. Serial 100-µm-thick vibratome sections (Leica
VT1000S, Wetzlar, Germany) were cut in the frontal plane through the
MNTB complex. After a thorough rinsing in 0.1 M PB (3-5×,
10 min for each step), the sections were postfixed for 1 hr in
PB-buffered 1% osmium tetroxide, pH 7.4 (containing 6.86 gm
sucrose/100 ml), at room temperature in the dark. After several rinses
(3-5×, 15 min for each step) in 0.1 M PB the sections
were stained en bloc with aqueous 1% uranyl acetate (1 hr in the
dark). After several rinses they were dehydrated in an ascending series
of ethanol (30 min for each step) and propylene oxide (2×, 10 min for
each step) and then flat-embedded in Durcopan (Fluka, Neu-Ulm,
Germany). Finally, the sections were polymerized at 60°C for 2-3 d.
From one flat-embedded vibratome section through the middle portion of
the MNTB, serial ultrathin sections (~70 nm in thickness, silver to
light gray interference colors) were cut through the MNTB complex on a
Reichardt Ultracut S (Leica) and collected on Formvar-coated slot
copper grids. Of each of the 270 serial ultrathin sections, four to six
electron micrographs of the calyx and the principal cell body were
photographed at a primary magnification of 4500×. The micrographs were
scanned at high resolution (1800 dpi) on a Zeiss SCAI scanner
(Oberkochen, Germany). These images provided the basis for the
subsequent three-dimensional morphological reconstruction of the
presynaptic and postsynaptic structures.
For the reconstruction of the 52 individual SCs and associated synaptic
vesicles, two Wistar rats (P9) were used. One rat brain was perfused
transcardially, fixed, and processed according to the protocol
described above; the other brain was perfused with a PB-buffered
solution containing 1% paraformaldehyde, 2.5% glutaraldehyde, and
0.1% picric acid, pH 7.4. Vibratome sections of the MNTB complex were
processed further according to a modified protocol originally developed
by Phend et al. (1992).
Serial ultrathin sections (~60 nm in thickness) through individual
synapses were taken from different animals to check for inter-individual differences. Electron microscopic (EM) images were
photographed at a primary magnification of 20,400×, and SCs were
reconstructed from ultrathin sections taken from both osmium-treated (n = 32; 187 sections) and osmium-free (Phend et al.,
1992) material (n = 20; 136 sections). No structural
differences were found between the two EM preparations, and data sets
were pooled. SCs were selected on the basis of their completeness and
optimal angle of sectioning, i.e., perpendicular to the synaptic cleft.
Distinction between SCs and puncta adherentia. For the
reconstruction of individual SCs (n = 52) only those
membrane specializations were used that met the following three
criteria: (1) presence of synaptic vesicles in close proximity to the
presynaptic density, (2) an asymmetry between presynaptic and
postsynaptic density, and (3) a widening of the synaptic cleft. For the
complete reconstruction all membrane specializations that showed
vesicle accumulations and met criterion 1 were classified as SCs.
Because of the often nonideal orientation of the synaptic cleft to the
plane of sectioning, criteria 2 and 3 applied in only 27% of all SCs.
All other membrane specializations were classified as puncta adherentia
(PA). A membrane specialization classified as SC had, on average,
10.6 ± 8.9 synaptic vesicles (mean ± SD) located within a
distance of 50 nm to the AZ membrane. In a randomly chosen subset of
n = 50 PA, we found, on average, 0.6 ± 1.1 vesicles within 50 nm from the membrane specialization. The
classification of PA versus SCs agreed among three observers in >98%
of the cases.
Three-dimensional reconstruction. Three-dimensional
reconstructions were made by alignment of serial EM images of the
synaptic structure between a single calyx and its principal neuron with the use of a software tool named CAR (Contour Alignment Reconstruction; contact kurt.saetzler{at}iwr.uni-heidelberg.de) running on a Silicon Graphics computer, as described in detail previously (Sätzler, 2000, 2001). In brief, two successive sections were aligned via rotation and translation such that corresponding structures like mitochondria and membrane specializations in the two sections superimposed. To compensate for distortions introduced by the sectioning, we had to transform the images anisotropically and linearly. Alignment was followed by a contouring of membranes by
visually defining markers. The subsequent linear interpolation between
these markers resulted in a polygonal membrane "contour." The
three-dimensional geometry was reconstructed from the stacks of all
contoured sections. By filling these stacks with tetrahedra (Boissonnat, 1988; Boissonnat and Geiger, 1992; Eils and Sätzler, 1999), we calculated volumes and surface areas of the presynaptic and
postsynaptic structures of interest.
Data analysis. Surface area values were obtained by summing
those triangular areas that formed the boundary of the reconstructed object. Accordingly, the volume of the object was determined as the sum
of the volumes of all tetrahedra inside the object (Eils and
Sätzler, 1999), which are directly proportional to the estimated section thickness. Varying the average section thickness in the reconstruction by 14% (assuming a mean thickness of 60 or 80 nm instead of the 70 nm used for the reconstruction) leads to the same
variation in the reconstructed volume estimate. This represents also an
upper bound of the error in surface area measurements introduced by
variation of section thickness, although surface measurements are
dependent on the real shape and the actual orientation of the
reconstructed structure within the slices. For the structure of the
reconstructed calyx, varying the section thickness by 14% results in a
variation of the calyx surface area by 5%.
To estimate the number and size of SCs (or PA), we first marked all
surface points on the three-dimensional reconstruction of the
presynaptic cell membrane that were part of a SC (or PA). In a second
step all those triangles of the surface of the calyx were labeled if at
least two of the corner points were found to be part of a SC (or PA).
In a final step the connected surface patches were defined as follows.
Starting from a single, labeled triangle, all those triangles were
taken to be connected that met the following criteria: (1) the triangle
itself was labeled, and (2) the triangle had at least one side in
common with a triangle of the surface patch. Performing this final step
iteratively provided individual and connected surface patches defining
single SCs (or PA). These surface patches were regrouped manually in
part by inspecting the original EM images to compensate for
"linking" errors introduced by locally misaligned sections. For
each individual patch the surface area and the center of gravity were
calculated. We compared this surface area estimate to the product of
the summed contour length of a complete AZ and section thickness
(two-dimensional estimate) in a subset of 12 perpendicular cut SCs. The
two-dimensional estimate was 17% lower.
For simulations of nearest-neighbor distances between SCs or between PA
we used the triangulated mesh of the contact area (see below),
attributing contacts randomly to triangles with a probability
proportional to their area. Each triangle could contribute to a contact
only once. For the simulations we constrained the nearest-neighbor
distances to be at least 100 nm for SCs and PA (similar to the measured
values of the reconstructed calyx).
To estimate the cleft volume, we first extracted all those triangles
from the reconstructed surface of the calyx and the cell body that were
in close apposition to each other (<70 nm), which we named the contact
area. We then estimated the mean cleft width by measuring the mean
distance between presynaptic and postsynaptic membranes from the
reconstruction (28 ± 9 nm, measured at n = 184,087 points), which was in good agreement with the cleft width measured directly in sections perpendicularly cut to the synaptic cleft
(27 ± 4 nm; n = 1673). The product of mean
distance and apposition area provided the estimate of the cleft volume.
The distribution of synaptic vesicles at AZs was determined in
individual SCs (n = 52). In these contacts we traced
the AZ and measured vesicle diameters at the middle of the membrane
bilayer. All vesicle-to-AZ distances were determined by subtracting the radius of the vesicle from the distance between the center of gravity
of each vesicle and the membrane trace of the AZ. This distance denotes
a membrane-to-membrane distance.
When the number of docked or clustered vesicles is estimated, the
correction factor for possible vesicle double counts according to
Abercrombie (1946) would be ~0.56. Because, however, we counted only
those vesicles with a clear ring-like structure, this would cause an
underestimate of the true number; therefore, the numbers reported in
this study remain uncorrected.
The number of synaptic vesicles clustered at an AZ was determined by
counting all vesicles within a 200 nm distance in sections in which the
AZ could be seen. To correct for not including the vesicles located in
sections directly adjacent to an AZ, we applied a correction factor of
1.3. This factor represents the ratio between the total intracellular
volume within a 200 nm distance from a (idealized) planar circular AZ
(radius, 178 nm) and the part of that volume contained in the analyzed sections.
Glutaraldehyde fixation and release. To estimate the amount
of vesicular release that might occur during tissue fixation (Smith and
Reese, 1980), we measured the presynaptic APs, EPSCs, and kainate-evoked synaptic currents during fixation of an acute MNTB slice
preparation. Whole-cell recordings of principal cells and stimulation
of afferent fibers were performed as described in detail previously
(Borst and Sakmann, 1996). During fixation the afferently evoked EPSCs
were abolished (Fig.
1A, left)
before the presynaptic action potentials or the sensitivity to kainate (Fig. 1A, right) was affected. In
addition, the fixation was not accompanied by an increase in the
frequency of spontaneous or delayed EPSCs (Fig. 1B).
These results suggest that it is unlikely that the fixative induced a
substantial change in the distribution of synaptic vesicles near the
presynaptic membrane. The view that negligible vesicular release occurs
during fixation also is supported by the fact that we did not observe
large numbers of omega-shaped profiles at SCs in the EM images.
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Figure 1.
Transmitter release at a MNTB synapse during
chemical fixation. A, Left, Afferently evoked prespikes
(presynaptic AP recorded in postsynaptic cell) and EPSCs at three
different time points during bath perfusion of the experimental chamber
with fixatives (1% paraformaldehyde/2.5% glutaraldehyde, dissolved in
Ringer's solution, pH 7.4). At bottom, the control is
shown before fixation. The middle traces show ~20 sec
after the start of fixation and the top traces at ~2
min after the start of fixation. A, Right, The same
traces are shown at a lower time resolution, illustrating postsynaptic
response to kainate (KA; 1 mM, dissolved in
Ringer's), which was applied by pressure ejection from a nearby patch
pipette. EPSCs were blocked before prespikes or before the sensitivity
to KA was clearly changed (middle traces).
B, Chemical fixation was not accompanied by an increase
in spontaneous or delayed EPSCs. In this experiment fixatives were
applied by pressure ejection from a nearby pipette. In the
bottom trace before fixation the afferently evoked EPSC
(amplitude, ~1.5 nA at  80 mV) was followed by delayed and
spontaneous EPSCs. In the middle trace early during the
fixation both evoked EPSCs and spontaneous EPSCs were reduced. In the
top trace late during the fixation both evoked and
spontaneous EPSCs were blocked. In A and
B the traces have been offset vertically for display
purposes.
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RESULTS |
General description and structures of interest
Figure 2A shows an
EM cross section through the synapse between a calyx of Held and an
MNTB principal neuron. The image represents a patchwork of five EM
micrographs comprising the outlines of the calyx highlighted in yellow
and the cell body of the principal neuron in blue, with its nucleus
shown in brown. The calyx is a complex nerve terminal forming a cap
with finger-like stalks that envelope the principal neuron. These
presynaptic stalks could be followed over several micrometers along the
surface of the cell body, sometimes approaching the opposite pole of
the principal neuron where they terminate abruptly. The cup-like
structure covered ~40% of the ovoid soma of the principal neuron.
The remaining surface area of the principal neuron was not studied in
detail; however, some of the finger-like stalks appeared to be
surrounded by glial processes. The thickness of the calyx was variable
and mostly <1 µm. The diameter of the principal cell body was ~22 µm in the longer and 18 µm in the shorter axis. The nucleus had an
eccentric position, being located closer to the synaptic membrane. Figure 2B shows the two membrane specializations in
the apposition zone between the calyx and postsynaptic membrane (marked
region of Fig. 2A at higher magnification), which
were classified as SCs (Fig 2, arrow) and PA (Fig. 2,
arrowhead), as described in Materials and Methods.
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Figure 2.
Cross section through a calyx of Held and a
principal neuron in the MNTB. A, EM image of the nerve
terminal and the cell body of the principal neuron in the MNTB. The
image was obtained by a computer-aided montage of five electron
micrographs of a single ultrathin section (imaged at a magnification of
3000×). The structures of interest were outlined and laid
transparently over the montage; the presynaptic calyx is shown in
yellow, the postsynaptic principal neuron in
blue, and the nucleus in brown. Scale
bar, 5 µm. B, The boxed region in
A at higher magnification (imaged at 20,400×). The SC
is marked by an arrow, and a neighboring PA is marked by
an arrowhead. Scale bar, 0.3 µm.
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The SCs are spread over the apposition zone between the cell body and
the terminal, including the finger-like stalks. They are always
characterized by a cluster of synaptic vesicles located in the
proximity of the presynaptic density, asymmetric presynaptic and
postsynaptic densities, and a broadening of the synaptic cleft (Fig.
3). In their apposition zone the
presynaptic and postsynaptic membranes show another membrane structure,
which we refer to as PA. These have been described in the adult cat
MNTB to be part of an organelle assembly named mitochondria-associated
adherens complex (MAC) (Rowland et al., 2000). Figure
4 shows a serial section through a
PA.
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Figure 3.
Serial section through a synaptic contact.
A-H, Selected serial sections of a SC
(arrow) between a presynaptic calyx and the postsynaptic
principal neuron. The open arrowhead in D
points to a vesicle fused with the presynaptic membrane. Scale bar: (in
H) A-H, 0.25 µm.
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Figure 4.
Serial section through a PA. A-H,
Serial sections through a PA are shown. In A, two
adjacent SCs are visible for a comparison of the two different types of
membrane apposition. The PA is labeled by arrowheads,
and SCs are labeled by arrows. In contrast to the SCs,
the PA shows symmetric presynaptic and postsynaptic densities and
lacks a synaptic vesicle accumulation and a widening of the cleft. The
whole PA appears to be perforated, as seen in B-D, G.
Scale bar: (in H) A-H, 0.25 µm.
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Because the structure of the calyx changes during development from a
cup- to a multifinger-like shape, we measured the mean thickness of the
calyx at membrane specializations (MS), compared this value with the
mean thickness measured at randomly chosen points, and found that the
mean thickness of the calyx at MS is larger than the mean thickness at
randomly chosen points. The difference was small but significant (MS,
790 ± 550 nm; random, 690 ± 550 nm; Student's t
test; p < 0.001).
Membrane specializations
Figures 3 and 4 show serial sections through
specialized membrane contacts at high magnification. These were
referred to as synaptic contacts (SCs) when synaptic vesicles were
accumulated close to the presynaptic dense region (Fig.
3A-H). The presynaptic and postsynaptic densities
formed bands of electron-dense fuzzy material, and both densities were
interrupted frequently, appearing as perforated structures. Regularly,
dense material connecting the presynaptic and postsynaptic membranes
was seen in the synaptic cleft. The SCs shown in Figure
3A-H also exhibited a characteristic widening of the
synaptic cleft and an asymmetry in the presynaptic and postsynaptic
densities. At some SCs coated vesicles or endocytotic half-vesicles
were located at the borders of AZs. Approximately one-third of the
membrane specializations in the apposition zone were identified as SCs.
The more frequently occurring type of membrane specialization were PA
(Rose et al., 1995). These were characterized by two parallel bands of
electron-dense material of approximately the same width at the
presynaptic and postsynaptic membranes (Fig. 4A-H) but lacking synaptic vesicles and the
wide broadening of the cleft typical for SCs.
In the entire calyx a total of 29 "spine-like"
protrusions of the postsynaptic membrane was observed (Fig.
5A,B). They resembled the
somatic appendages or somatic spicules described by Tolbert and Morest
(1982). In all, 32 SCs were located on these appendages. The
protrusions were variable in size, and some of them lacked a spine neck
whereas others were comparable with the "stubby" spines on
dendrites of neocortical and hippocampal pyramidal neurons, although we
never observed a spine apparatus.
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Figure 5.
Spine-like protrusion at the soma of the principal
neuron. A, B, Two successive EM images of sections
through two typical spine-like membrane protrusions of the soma of the
postsynaptic cell (labeled s). The protrusion on the
left has a narrow "spine neck." Note the presence of
SCs identified by the dense accumulation of synaptic vesicles
(black arrows) and numerous PA. Only the SCs on
spine-like protrusions are labeled. Scale bar, 0.5 µm.
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Figure 6 shows a three-dimensional
reconstruction of a spine-like protrusion with two AZs that are located
opposite to each other at the neck. They are relatively large-sized and
not perforated. The reconstruction of the volume and surfaces of such
structures was made from stacks of contoured sections (Fig.
6A) by filling the stacks with tetrahedra (Fig.
6B).
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Figure 6.
A three-dimensional reconstruction from contoured
sections. A, Superposition of contoured sections to form
a "stack of contours" from serial EM images of a spine-like
protrusion. The postsynaptic membrane is marked in blue,
the AZs in red, and PSDs in orange. The
two SCs are 0.5 µm apart. B, The stack of contours
shown in A was filled by tetrahedra, which generates a
triangulated surface (outlines of
triangles marked in gray).
Section thickness, 60 nm.
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Geometry of the synapse
Surfaces of calyx and cell body
Figure 7 shows
three-dimensional surface views of the calyx and principal cell body
obtained by surface reconstruction from a stack of 270 serial ultrathin
sections with a thickness of ~70 nm. The calyx is shown in yellow and
the principal neuron in blue. The surface of the calyx and cell body is
viewed from two sides by rotating the reconstruction around the
horizontal axis (Fig. 7A,B). The calyx rests like a cup on
the soma, covering ~40% of the surface of the principal neuron. From
the surface of the calyx numerous "finger-like" stalks enclose the
cell body. In Figure 7C the postsynaptic membrane was made
transparent to illustrate the eccentric location of the nucleus,
positioned closer to the innervated membrane of the cell body.
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Figure 7.
Surface views of the three-dimensional
reconstruction of a calyx of Held and the principal neuron.
A, Top view of the calyx and the cell body of the MNTB
principal neuron. The calyx (left) is shown in
yellow and the principal neuron (right)
in blue. The nerve terminal forms a cup-like structure
covering ~40% of the surface area of the principal neuron. The final
part of the preterminal axon that gives rise to the giant terminal is
seen on the left. B, Side view after
rotating the reconstruction by 90° around the x-axis.
At the edges of the cap numerous finger-like stalks can be identified.
C, Surface view of the reconstruction in which part of
the surface membrane was made transparent to illustrate the eccentric
location of the nucleus (brown) next to the calyx. The
membrane specializations can be seen as small spots in
red (SCs) and magenta (PA), respectively.
The postsynaptic axon (bottom) and dendritic processes
(right) were not reconstructed fully.
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Volume and surface area of calyx and soma
The area of the plasma membrane surrounding the calyx, including
both the apposition zone with the principal neuron and the noninnervating part of the membrane, was ~2500
µm2. The calyceal membrane surrounded a
volume of ~480 µm3 or 0.48 pl. The
area of the surface membrane of the principal cell body was 2400 µm2 of which ~40% (1000 µm2) was apposed to the calyx. The
volume of the cell body was ~3400 µm3
or 3.4 pl, more than sevenfold larger than the volume of the calyx
(Table 1).
Volume of the synaptic cleft
The volume estimate of the cleft separating the two
apposed surface membranes of calyx and soma depends on the degree of
"smoothing" of the rugged surface created by triangulation of the
contours between successive sections. Distortions of the surface were
caused by variations in section thickness (we assumed an average
thickness of 70 nm) and the sectioning procedure. The distribution of
distances measured between the presynaptic and postsynaptic membrane
shows that the mean distance was 28 ± 9 nm. This value, in
combination with the area of the apposition zone, yields an
extrapolated volume of 28.0 µm3 or 0.028 pl for the synaptic cleft separating presynaptic and postsynaptic cell,
~20-fold smaller than the volume of the calyx.
Number and size of specialized contacts
Figure 8 illustrates a view onto the
synaptic surface of the calyx. The locations of SCs (Fig.
8A,C, red) and PA (Fig.
8B,C, magenta) are indicated. The number
of PA (n = 1010) was nearly twofold larger than that of
SCs (n = 554). The specialized contact zones of the
calyx and cell body membrane, not differentiating between SCs and PA,
comprised an area of ~100 µm2, which
is ~10% of the apposition area. Approximately one-half (55 µm2) of the specialized contact area was
occupied by SCs and the other one-half (46 µm2) by PA, indicating that the SCs were
larger, on average.
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Figure 8.
Distribution of membrane specializations in the
apposition zone between the calyx and the principal neuron.
A, En face view on the synaptic surface
of the calyx (yellow) showing the distribution of
SCs highlighted in red. B, Reconstruction
of the same calyx surface as shown in A with the
distribution of PA highlighted in magenta.
C, Same view as in A and B
showing an overlay of the distributions of SCs and PA. Same color code
is used as in A and B.
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The mean surface area of a SC was 0.100 µm2, and its size distribution was
characterized by a large variability [coefficient of variation
(CV) = 0.9] (Fig. 9A).
The surface area of a PA had a mean of 0.046 µm2, approximately one-half of the size
of the area of a SC. Their size distribution shows a similar degree of
variability (CV = 0.9) (Fig. 9A).
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Figure 9.
Geometry of membrane specializations in the
apposition zones between the calyx and the principal neuron.
A, Histogram (left) of the distribution
of surface areas of SCs (gray; mean 0.100 ± 0.086 µm2) and PA (black; mean
0.046 ± 0.042 µm2) and the corresponding
cumulative histogram (right). Bin width, 0.01 µm2. B, Histogram of the
distribution of nearest-neighbor distances between SCs marked in
gray (mean, 0.589 ± 0.296 µm) and PA marked in
black (mean, 0.374 ± 0.179 µm). The right
panel shows the corresponding cumulative histograms (same color
code). Bin width, 0.02 µm. The dashed lines are
nearest-neighbor distances taken from a simulated representation of SCs
(dashed gray line) and PA (dashed black
line), which were spread randomly across the apposition area of
calyx and principal neuron.
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Distance between specialized contacts
The average distance between nearest-neighbor SCs, measured from
their center of gravity, varied widely between 0.15 and 1.3 µm,
with a mean of 0.59 µm (CV = 0.5). The distribution of distances between a PA and its nearest neighbor indicates that PA were, as
expected, more closely spaced, the mean nearest-neighbor distance between PA being 0.37 µm (CV = 0.5) (Fig. 9B).
To check whether SCs and PA were distributed homogeneously on the
apposition zone, we simulated a distribution of points on the calyx
membrane that represented the centers of gravity of SCs and PA,
respectively. SCs and PA (numbers taken from measurements) were spread
randomly across the apposition area. We then compared means and SDs of
the distances between nearest-neighbor SCs and PA for the following
combinations: distance from SC to closest SC (SC-SC) and closest PA
(SC-PA), distance between PA and closest PA (PA-PA) and closest SC
(PA-SC), and distance between membrane specializations irrespective of
whether they are a SC or a PA (MS-MS). We compared these random
distributions with the measured distributions by using a
Kolmogorov-Smirnov (K-S) test to establish whether the simulated and
measured distributions of nearest-neighbor distances were different
(Press et al., 1992).
The results of the K-S test showed that the measured and simulated
distributions of nearest-neighbor distances were significantly different for SC-SC distances (p = 0.01) and
for all other combinations (p < 0.001) (Fig.
9B, Table 2). This suggests
that membrane specializations are not distributed randomly across the
apposition area.
Presynaptic active zones
We attempted to identify subsets of synaptic vesicles as
anatomical correlates for different pools of releasable quanta of transmitter as postulated from measurements of release rates (for review, see Schneggenburger et al., 2002). The total number of synaptic
vesicles in the calyx and the average number of clustered synaptic
vesicles at an AZ were estimated by measuring the number of vesicles
located from the AZ within a specified distance of ~500 and 200 nm,
respectively. Furthermore, the number of "anatomically docked"
vesicles was estimated by counting those vesicles in which no
cytoplasmic space was visible between the vesicle and the AZ membranes
(Fig. 10A-C). A total of 52 SCs was reconstructed, including the vesicles clustered at the AZs.
Figure 10C shows an EM image of one representative section
through a SC with a cluster of synaptic vesicles located in close
proximity of the presynaptic density. The geometric quantities relating
to the location and size of synaptic vesicles at an AZ in a section are
indicated schematically in Figure 10D.
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Figure 10.
Quantification of synaptic vesicle cluster
geometry at SCs. A, B, Two different SCs at high
magnification with anatomically docked vesicles (marked by
asterisks). Scale bar, 0.1 µm. C,
Electron micrograph of another single SC showing the dense accumulation
of synaptic vesicles at the AZ. Scale bars: C, D, 0.25 µm. D, Same micrograph as in C.
Schematic representation of geometric measurements is shown for a subset of synaptic vesicles: AZ membrane (red
line) and shortest distance (green straight
lines) between the membrane of the synaptic vesicles
(green circles) and the AZ membrane. Anatomically
docked vesicles are marked by asterisks. All parameters
were measured in two dimensions.
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Clusters of synaptic vesicles
First we determined the number of synaptic vesicles that were
found within a certain distance from the membrane of a given SC in all
consecutive sections in which the SC could be identified. In the calyx
cup-like compartment this distance was ~500 nm, somewhat smaller than
the mean thickness of the terminal in this region. Typically, four to
five ultrathin sections were needed to reconstruct a SC and to
determine the number of associated synaptic vesicles. Figure
11A illustrates a
three-dimensional reconstruction of a single SC in which the number of
synaptic vesicles around an AZ could be determined unequivocally.
Figure 11B shows two SCs located opposite to each
other at a spine-like protrusion with the associated synaptic vesicles,
illustrating that, in some cases, distinction between vesicles
associated with different SCs is ambiguous. Extrapolating from the
sample of 52 SCs, with on average 125 ± 82 synaptic vesicles, to
the total number of AZs (n = 554), the calyx would
contain at least 70,000 synaptic vesicles (Table
3). More likely, the total number of
synaptic vesicles in a calyx is somewhat higher because those located
in sections adjacent to SCs and those not associated with SCs were not
counted.
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Figure 11.
A three-dimensional reconstruction of SCs and
associated vesicle clusters. A, Cluster of synaptic
vesicles near a SC. The surface membrane of the principal neuron is
shown in blue, the AZ in red, and
synaptic vesicles in green. Note the large size of the
pool of synaptic vesicles at this SC (n = 304).
B, Two spine-like protrusions, one with two SCs (on the
right). The SCs are located opposite to each other on
the neck of the protrusion. Same color code is used as in
A. Here the individual clusters of synaptic vesicles are
not clearly discernible at these two SCs (n = 358).
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Approximately 50% of the synaptic vesicles were located within a
distance of 200 nm from an AZ (data not shown). Because this distance
is approximately one-half of the average distance between nearest-neighbor SCs, synaptic vesicles found within this distance were
considered to form a "cluster" associated with the SC, as shown in
one example (Fig.
12A). Here, 28 synaptic vesicles of those shown (green) formed a cluster
according to our 200 nm criterion (Fig. 12B), with
the average nearest distance from the AZ membrane being 84 nm. The
distribution of the number of synaptic vesicles (within a 200 nm
distance cluster) measured for 52 SCs shows that each cluster comprises
a mean of 63 ± 29 synaptic vesicles (Fig. 12C), which
are located at an average distance of 87 ± 15 nm from an AZ,
corresponding to approximately two vesicle diameters (see below).
Extrapolation to 554 SCs and accounting for vesicles located in
sections adjacent to the AZ (see Materials and Methods) suggest that in
the calyx ~45,000 synaptic vesicles were clustered around the
AZs.
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Figure 12.
Geometry of synaptic vesicle clusters.
A, Three-dimensional view of a single SC showing the
distribution of distances of synaptic vesicles
(green) from the AZ membrane
(red). Distances are shown as lines
connecting a synaptic vesicle and the nearest point on the AZ. For
clarity, a relatively small SC was chosen for display.
B, Histogram of the distribution of distances of
synaptic vesicles from the presynaptic membrane at the SC shown in
A. The total number of synaptic vesicles was 37. The
number of synaptic vesicles clustered within 200 nm was 28. C, Histogram showing the number of SCs with 0-4, 5-9,
etc. synaptic vesicles within 200 nm from the AZ. D,
Number of SCs with 0, 1, 2, etc. anatomically docked vesicles (i.e., no
visible cytoplasmic space between synaptic vesicle and AZ membrane).
The number of SCs lacking docked vesicles is represented by the
gray column. E, Diagram showing the
number of synaptic vesicles per SC (mean ± SD) located within a
given distance from the AZ membrane. F, Histogram of the
distribution of vesicle diameters (mean, 46 ± 7 nm;
n = 7799).
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Docked synaptic vesicles and vesicle size
Synaptic vesicles that are docked anatomically (Fig.
10A,B,D, asterisks) to an AZ may represent
a morphological correlate of the readily releasable pool (RRP) of
quanta as estimated from EPSCs (Schneggenburger et al., 1999; Wu and
Borst, 1999) and capacitance measurements (Sun and Wu, 2001). Figure
12D illustrates the distribution of the number of
anatomically docked vesicles. The number of docked vesicles increased
with AZ area (rPearson = 0.62; data
not shown). On average, 2.0 ± 2.0 synaptic vesicles were docked
per SC (n = 104 for 52 SCs). We also analyzed the
average number of synaptic vesicles located within a given distance
from an AZ (10-50 nm) (Fig. 12E). The number of
synaptic vesicles located within 10 nm from the AZ membrane (1.9 ± 2.0) was in good agreement with the number of anatomically docked
vesicles. At larger distances from the AZ membrane this number
increased from 4.8 ± 3.8 (within 20 nm) up to 18.1 ± 8.3 (within 50 nm).
The diameter of all analyzed synaptic vesicles was 46 ± 7 nm
(Fig. 12F). We did not observe a significant
difference in diameters of synaptic vesicles depending on their
location or their distance from the AZ (data not shown).
| |
DISCUSSION |
Three-dimensional reconstruction of a calyx of Held revealed a
structure composed of a cup with several fingers enveloping its
postsynaptic target, as previously observed by light microscopy (Kandler and Friauf, 1993). The membrane area (2500 µm2) and volume (0.48 pl) of the
reconstructed calyx corresponded well with estimates derived from
measurements of capacitance (2400 µm2,
which includes <30 µm of axon and assumes 10 fF/µm2) (Borst and Sakmann, 1998) and
fluorescence (0.4 pl; Helmchen et al., 1997) of unfixed tissue from
animals of the same age (P9). This similarity suggests that tissue
fixation and sectioning did not distort overall morphology substantially.
The average surface area of an AZ was 0.100 µm2, which lies between the average size
of AZs in the end bulb of Held of the juvenile rat (0.066 µm2; Nicol and Walmsley, 2002) and of
the adult cat (0.14 µm2; Ryugo et al.,
1997) and is somewhat larger than that in the adult cat calyx of Held
(0.07 µm2; Rowland et al., 2000). In
other mammalian CNS synapses smaller and larger values are observed
(range, 0.03-0.14 µm2; Pierce and
Mendell, 1993; Harris and Sultan, 1995; Schikorski and Stevens, 1999;
Xu-Friedman et al., 2001). In most of these studies a preponderance of
small AZs also was observed, suggesting that AZ size distribution is
independent of the general morphology of the nerve terminal.
PA are not distributed randomly and may form focal adhesion complexes
that stabilize the apposition of presynaptic and postsynaptic membranes. PA also have been reported in the mossy fiber CA3
pyramidal cell synapse (Chicurel and Harris, 1992) and in the CA1 area
of the hippocampus (Spacek and Harris, 1998).
Geometry of the calyx of Held as a determinant of release
A comparison of the number of Ca2+
channels estimated to be open during an AP (~12,000; Borst and
Sakmann, 1996) and the number of SCs in the reconstructed calyx (554)
suggests that, for each AZ, ~20 channels open during the peak of the
Ca2+ influx. This value is comparable with
the numbers estimated for synaptic boutons in layer 2/3 pyramidal
neurons (Koester and Sakmann, 2000). However, not all calcium channels
are concentrated at AZs (Wu et al., 1999). A lower estimate is obtained
if calcium channels are assumed to be distributed evenly over the
entire calyx membrane. In this case only ~0.5 channels would open per
AZ, because the AZ area constitutes 2% of the total surface area
(Table 3). The nearest-neighbor distance between open channels would be
>400 nm. Such low estimates are not in agreement with the apparent low
variance in the calcium signals controlling release (Borst and Sakmann,
1999; Meinrenken et al., 2002).
Quantal content of evoked EPSCs and number of SCs
The estimate of 554 SCs agrees well with the number of functional
SCs estimated from fluctuation analysis of evoked EPSCs (Meyer et al.,
2001). A single AP releases ~200 quanta (Borst and Sakmann, 1996;
Schneggenburger et al., 1999; Bollmann et al., 2000), suggesting that
only ~35% of all AZs release a packet of glutamate. Estimates of the
size of the readily releasable pool (RRP) of vesicles vary between
~800 (Schneggenburger et al., 1999; Wu and Borst, 1999; Bollmann et
al., 2000) and up to ~5000 (Sun and Wu, 2001). The differences are
probably attributable to differences in the exact definition of the
pool and to the methods used for estimating its size.
The morphological correlate for the RRP of quanta may be the number of
anatomically docked vesicles, i.e., those vesicles being in direct
contact with the AZ membrane (Schikorski and Stevens, 2001) (but see
Xu-Friedman et al., 2001). In the present study the calyx had <1200
docked vesicles, in agreement with the lower estimates of the RRP size.
If we assume, however, that synaptic vesicles located within 20 nm from
the AZ can be released rapidly, the total number of available synaptic
vesicles would be ~3000, comparable with the midrange estimates of
the RRP size (Schneggenburger and Neher, 2000; Sakaba and Neher, 2001).
Although more direct experiments are needed to ascertain that vesicles
located at >10 nm from the membrane can be released within 10 msec,
the distance from the membrane could contribute to the heterogeneity in
their apparent Ca2+ sensitivity (Sakaba
and Neher, 2001).
For the largest estimate of the RRP (~5000 releasable vesicles
suggested by capacitance measurements) >8 vesicles per SC would be
released within a few milliseconds. The observed capacitance increase
of ~0.4 pF (Sun and Wu, 2001) corresponds to an increase in the
active zone size of 40 µm2, almost
doubling the AZ area. Under such conditions the diffusional distance
between synaptic vesicles and open calcium channels could be increased
substantially, which would contribute to the lower release probability
of releasable synaptic vesicles during recovery from synaptic
depression (Wu and Borst, 1999; Meinrenken et al., 2002). On the other
hand, a single AP will lead to an increase in the AZ area of
<10%.
Properties of SCs at other CNS synapses
Synaptic contacts had, on average, 2.0 docked vesicles, whereas
SCs in cerebellum, hippocampus, or cortex contain, on average, at
least 10 docked vesicles (Harris and Sultan, 1995; Schikorski and Stevens, 1999; Xu-Friedman et al., 2001). The relatively high sensitivity of the release process to the fixatives, i.e., the rapid
block of release during superfusion with fixative, argues against a
substantial influence of the fixatives on synaptic vesicle distribution
in the calyx, in agreement with previous results obtained in cultured
neurons (Rosenmund and Stevens, 1997). We cannot exclude that the
number of docked vesicles may increase during development, but in the
calyx of Held in the adult cat or the end bulb of Held in the juvenile
rat these numbers were also comparatively low (Rowland et al., 2000;
Nicol and Walmsley, 2002). In most studies, including the present one,
the number of docked vesicles varied greatly, with CVs typically
ranging between 0.5 and 1.0. This variability may be explained in part by the large variability in AZ sizes, because large AZs generally have
more docked vesicles (Schikorski and Stevens, 1999).
Geometry of PSDs and GluR channels
The estimated mean number of quanta released per AP (~200; Borst
and Sakmann, 1996) indicates that the distance between PSDs activated
by released glutamate is 0.9 µm, on average, assuming random
activation of SCs across the apposition zone. Therefore, cross-activation of GluR channels between different SCs is unlikely to
shape the rising phase and peak of the EPSC during a single AP-evoked
transmission. However, AMPA receptors (AMPARs) desensitize rapidly
(Geiger et al., 1995), and simulations of the cleft diffusion of
glutamate (A. Roth, unpublished data) indicate that during repetitive
stimulation the distance could be short enough to cause GluR activation
and/or desensitization by spillover from neighboring release sites
(Otis et al., 1996). At experimentally induced maximal release rates
this could cause an underestimate of the RRP measured from the EPSC
amplitude (Sakaba and Neher, 2001; Sun and Wu, 2001).
Density of GluR channels
Approximately 20 AMPAR channels open during the peak of a quantal
EPSC (Sahara and Takahashi, 2001). Assuming a peak open probability of
0.5 for AMPAR channels at saturating glutamate concentrations (Geiger
et al., 1995; Koh et al., 1995), the number of functional channels per
PSD would be ~40. This is a lower limit considering that AMPARs may
not be saturated during the release of a single quantum (Silver et al.,
1996; Ishikawa et al., 2002). When distributed evenly across the PSD
area (0.100 µm2), the density of
functional AMPAR channels would be ~400
µm 2 with a spacing of >40 nm. This
estimate is comparable with an estimate of the functional AMPAR channel
density of PSDs in CA1 and CA3 pyramidal neurons in rat hippocampus
(Nusser et al., 1998).
NMDA receptor (NMDAR)-mediated peak current during an evoked EPSC is
~4 nA at -80 mV in the absence of external magnesium (J. H. Bollmann, unpublished data). If we assume a channel conductance of
~50 pS (Spruston et al., 1995; Clark et al., 1997), then ~1000 NMDAR channels are open during the peak of the EPSC, suggesting that
approximately five NMDAR channels open at each of the 200 PSDs. Because
NMDAR occupancy and open probability depend on the subunit composition
(Wyllie et al., 1998), which is not known, these estimates are speculative.
Thus at those PSDs exposed to a transmitter packet, ~20-30 GluR
channels are opened during the peak of the EPSC. The variability (CV = 0.9) of PSD area suggests that the variability in
miniature EPSC amplitude (CV of ~0.5) (Borst and Sakmann,
1996; Sahara and Takahashi, 2001) is attributable mostly to the
variability in the number of AMPARs present at different SCs.
| |
FOOTNOTES |
Received Nov. 12, 2001; revised Sept. 23, 2002; accepted Sept. 27, 2002.
*
K.S. and L.F.S. contributed equally to this work.
We thank S. Nestel for her extraordinary skills in producing large
series of ultrathin sections. We thank A. Roth for performing simulations of glutamate diffusion in the synaptic cleft and of AMPAR-mediated EPSCs. We also thank him, together with Drs. T. Kuner,
C. Meinrenken, R. Schneggenburger, and J. Waters and Professor E. Neher, for critically reading a previous version of this manuscript. In
addition, we thank M. Winter and I. Dehof for excellent technical assistance. Finally, we thank Professor W. Jäger and Drs. R. Eils
and K. Saracoglu from the Interdisciplinary Center of Scientific Computing in Heidelberg for technical support and fruitful discussions.
Correspondence should be addressed to Bert Sakmann, Department of Cell
Physiology, Max Planck Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany.
| |
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TOP
ABSTRACT
INTRODUCTION
