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The Journal of Neuroscience, September 1, 2000, 20(17):6529-6539
Structural and Functional Alterations of Neuromuscular Junctions
in NCAM-Deficient Mice
Victor F.
Rafuse,
Luis
Polo-Parada, and
Lynn T.
Landmesser
Department of Neurosciences, Case Western Reserve University,
School of Medicine, Cleveland, Ohio 44106-4975
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ABSTRACT |
The role of neural cell adhesion molecule (NCAM) in the
development and maturation of the neuromuscular junction (NMJ) was explored by characterizing structurally and functionally NMJs from
postnatal day 11 (P11) to P30 +/+, +/ , and / NCAM null mutant mice. Differences in NCAM levels resulted in alterations in the
size and shape of NMJs, with / NMJs being smaller. Additionally both the withdrawal of polyneuronal innervation and the selective accumulation of synaptic vesicle protein in the presynaptic terminal were delayed. These observations suggest that the bidirectional signaling responsible for these events is impaired at / NMJs. Functionally, miniature end plate potential size, end
plate potential size, and quantal content did not differ from that of
wild type under either normal or low release conditions. However at
normal release conditions, / NMJs, unlike +/+ NMJs, lacked
paired-pulse facilitation. The most striking abnormality was the
inability of NCAM null junctions to maintain transmitter output with
repetitive stimuli. Combined electrophysiological and
FM1-43-labeling studies suggest that NCAM null junctions are
unable either to dock or to mobilize a sufficient number of vesicles at
high but physiological rates of transmitter release. Taken together our
observations show that many aspects of transmission are normal and,
thus, that many presynaptic and postsynaptic molecules have assembled
properly in the absence of NCAM. However, the fact that NCAM was
required for specific aspects of transmission, including paired-pulse
facilitation and reliable transmission with repetitive stimuli,
suggests that NCAM either is directly involved in these processes or is
required for the proper organization and/or function of other molecules underlying these processes.
Key words:
NCAM; synaptic depression; FM1-43; synapse elimination; neuromuscular transmission; synapse maturation
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INTRODUCTION |
For proper nervous system function,
developing neurons must grow to, and form connections with, their
appropriate targets. Subsequent interactions transform these initial
connections into highly specialized synapses (for review, see
Sanes and Lichtman, 1999 ). During development of the neuromuscular
junction (NMJ), synaptic vesicles that are initially distributed along
the length of the motor axon become clustered at presynaptic active
zones (Kelly and Zacks, 1969; Lupa and Hall, 1989; Dahm and Landmesser, 1991). Postsynaptically, the density of acetylcholine receptors (AChRs)
increases dramatically both by the clustering of diffusely distributed
AChRs (Anderson and Cohen, 1977; Frank and Fischbach, 1979; Nitkin et
al., 1987) and by transcriptional activation of AChR genes in
subsynaptic myonuclei (Usdin and Fischbach, 1986; Fischbach and Rosen,
1997). Postnatally, as the muscle fibers grow, the shape and complexity
of the NMJ change, with small uniform plaques of AChRs expanding into
pretzel-shaped structures as the nerve terminal grows (Balice-Gordon
and Lichtman, 1990). Additionally, there is a withdrawal of all but one
motor axon from the initially multiply innervated NMJ (Redfern, 1970;
Brown et al., 1976; Balice-Gordon and Lichtman, 1993: Balice-Gordon et
al., 1993).
These processes involve the translocation of numerous molecules and
presumably alterations in adhesive interactions between prejunctional
and postjunctional membranes. Cell adhesion molecules play critical
roles in the development and maturation of the Drosophila NMJ, as well as other vertebrate synapses (for review, see Fields and
Itoh, 1996; Landmesser, 1997, 1998). Using the power of
Drosophila genetics, Goodman and colleagues showed that the
stability and size of the NMJ were controlled by the relative level of
fasciclin II (FasII) on developing muscle and nerve (Schuster et al.,
1996a,b). Although synapses initially formed in mutants lacking FasII,
they later regressed (Schuster et al., 1996a). Surprisingly, there was
enhanced synaptic growth in mutants that expressed only 50% of the
FasII protein. This observation becomes understandable by the
demonstration that activity-dependent downregulation of FasII was both
sufficient and necessary for the presynaptic sprouting that normally
accompanies growth of the NMJ (Schuster et al., 1996b).
Does neural cell adhesion molecule (NCAM), the vertebrate homolog of
FasII (Lin and Goodman, 1984), play a similar role at vertebrate NMJs?
NCAM, specifically its polysialic acid (PSA) moiety, is required for
the activity-dependent induction of long-term potentiation (LTP) and
long-term depression (LTD) in the hippocampus (Muller et al., 1996).
Sanes and colleagues showed recently that NMJs from NCAM null mice were
smaller and that postsynaptic junctional fold development was delayed
(Moscoso et al., 1998); however no functional studies were performed.
To investigate possible roles of NCAM in the formation and maturation
of the NMJ, we compared the structure and function of postnatal NMJs in
wild-type and NCAM null mice. We found that NCAM-deficient end plates
were smaller, synaptic efficacy was compromised, and transmitter
release could not be sustained with repetitive stimuli, supporting a
role for NCAM in the proper development, function, and plasticity of
the synapse.
Parts of this paper have been published previously in abstract
form (Rafuse et al., 1998).
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MATERIALS AND METHODS |
Mice
The NCAM-deficient mice used were originally generated by Cremer
et al. (1994) in the C57B/6 strain and were subsequently raised and
bred locally. For each experiment wild-type (+/+), heterozygote (+/),
and homozygote (/) mice were identified by PCR using primers that
distinguish wild-type from mutant alleles (Cremer et al., 1994).
Immunostaining
Mice were killed in a CO2 chamber, and the
semitendinosus muscles were quickly dissected and frozen without
fixation in ornithine carbamyl transferase (OCT; Miles, Elkhart, IN) in
isopentane that was cooled in dry ice. Rhodamine-conjugated
 -bungarotoxin (-BTX; Molecular Probes, Eugene, OR) and SV2 (a
gift of K. Buckley, Harvard University) were used to visualize AChRs
and synaptic vesicles, respectively. Briefly, 50 µm longitudinal
sections were mounted on previously subbed glass slides, fixed in 3.7%
formaldehyde for 10 min, rinsed, incubated overnight in
rhodamine-conjugated -BTX and SV2, washed several times in PBS,
incubated for 2 hr in fluorescein-conjugated IgG secondary antibody
(Sigma, St. Louis, MO), washed in PBS, and, finally, coverslipped with
50% glycerin-PBS containing 0.03 mg/ml p-phenylenediamine
to prevent fading. Images were digitally photographed (40×) with an
upright Nikon Diaphot 300 microscope equipped with a Javelin Ultrachip
CCD camera (Javelin Electronics, Los Angeles, CA). Images were acquired
and analyzed using an Argus Hamamatsu Image Processor (Hamamatsu
Photonics K.K.) in series with the Metamorph Imaging System (Universal
Imaging Corporation, West Chester, PA). The size of the NMJs was
quantified by measuring both the area occupied by the AChRs and the
circumference of the total end plate region. Acquired images were
cropped in Corel Draw to generate the figures. Any modifications to the
acquired digital figures were limited to changes in color brightness
and contrast.
Muscle fiber cross-sectional area measurements
Mice were killed in a CO2 chamber, and the
semitendinosus muscles were quickly removed, pinned at proximal and
distal ends to a cork after being extended to their approximate
in vivo length, which was measured when the leg was in a
position midway between flexion and full extension, mounted in OCT
(Miles), and immediately frozen in isopentane that was cooled in dry
ice. Fourteen micrometer sections were taken from the midregion of the
muscle and dried on a previously subbed glass slide. Sections were
incubated overnight (4°C) with a monoclonal antibody (1:100 dilution)
that exclusively recognizes slow skeletal muscle myosin (Sigma), fixed
in 3.7% formaldehyde for 10 min, rinsed, washed several times in PBS, incubated for 2 hr (room temperature) with a goat anti-mouse
fluorescein-conjugated secondary antibody (Sigma), washed in PBS, and,
finally, mounted in 50% glycerin-PBS containing 0.03 mg/ml
p-phenylenediamine to prevent fading. Images were digitally
photographed (20× magnification) with a Nikon Diaphot 300 microscope
as described above. Background fluorescence permitted visualization and
cross-sectional area measurement of both nonlabeled fast skeletal
muscle fibers and labeled slow fibers. The cross-sectional areas of
individual muscle fibers in the midbelly region of the semitendinosus
muscles were measured using the Metamorph Imaging system (Universal
Imaging Corporation).
In vitro isometric tension and
electromyogram measurements
Mice were killed in a CO2 chamber, and the
semitendinosus muscles, along with the nerve supply and contributing
spinal roots (L4-L6), were isolated and immediately placed into well
oxygenated (95% O2 and 5%
CO2) Tyrode's solution (125 mM NaCl,
24 mM NaHCO3, 5.37 mM
KCl, 1 mM MgCl2, 1.8 mM
CaCl2, and 5% dextrose). Extra care was taken to
ensure that the spinal roots and nerve supply were left intact and not
damaged. The proximal end of the muscle was carefully pinned in a
Sylgard (Dow Corning, Midland, MI)-coated recording chamber containing
well oxygenated Tyrode's solution maintained at 27-29°C. The distal
muscle tendons were securely fastened with silk thread (no. 1) and tied
to a force transducer (model 373; Harvard Apparatus, Holliston, MA) to
measure total muscle force. In all cases a small piece of femur was
left in continuity with the tendon to prevent slippage of the thread
during maximal contractions. Two fine-tipped polyethylene suction
stimulating electrodes [pulled from polyethylene tubing (PE-190; Clay
Adams, Parsippany, NJ)] were used to stimulate each of the spinal
nerves separately, or together, using a Grass S88 stimulator (Grass, Quincy, MA). Tight electrode seals made it possible to stimulate each
spinal nerve separately without the spread of any electric current to
the unstimulated spinal nerve (Landmesser and O'Donovan, 1984).
Electromyogram (EMGs) were recorded from the midbelly of the
semitendinosus muscles using a third polyethylene suction electrode,
amplified with a bandwidth between 3 Hz and 10 kHz, and displayed on an
oscilloscope (R5030; Tecktronix, Beaverton, OR). Short (0.5-1 msec),
monophasic electrical stimuli, isolated from ground with a Grass
PISU6P stimulus isolation unit, were used to evoke EMG and force
responses that were displayed on a Gould chart recorder (Gould,
Cleveland, OH) and stored on analog tape (Vetter, Rebersburg, PA) for
later analysis. The signal was digitized in parallel using a Cygnus
FLA-01 8 Pole Bessel Filter, connected to a analog-to-digital converter
system (DigiData 1200; Axon Instruments, Foster City, CA), on a pentium
Dell V-350 computer. Axoscope 8.0 (Axon Instruments) software was used
to digitize (50 MHz) and perform measurements. In some cases,
10 7 to
105
M D-tubocurarine (D-TC;
Sigma) was added to the Tyrode's solution to reduce the safety
margin of transmission (Wernig and Herrera, 1986). At least 30 min
was allowed after changing from one perfusion medium to another
before EMG and force measurements were taken.
Electrophysiological determination of polyneuronal innervation
The force occlusion technique was used to estimate the presence
or absence of polyneuronal innervation in the neonatal mice (Wernig and
Herrera, 1986). Briefly, twitch and tetanus force responses were
recorded after stimulation of each of the two contributing spinal
nerves separately and then together. If each fiber is only innervated
by a single motoneuron (i.e., not polyneuronally innervated), then the
forces recorded by stimulating each spinal nerve separately should
mathematically sum to equal the force recorded after stimulating both
spinal nerves simultaneously. If each muscle fiber is innervated by
more than one motoneuron (i.e., polyneuronally innervated), then the
force evoked by stimulating both nerves simultaneously will be less
than the mathematical summation of the forces recorded by stimulating
each spinal nerve separately (see Results for further details).
Intracellular recording
Semitendinosus muscles, with the nerve supply intact, were
isolated as described above and immediately placed into well oxygenated Tyrode's solution. The muscles were gently extended and pinned flat in
a Sylgard (Dow Corning)-coated recording chamber. The nerves were
sucked tight into polyethylene stimulating electrodes pulled from
polyethylene tubing (PE-190; Clay Adams). Standard electrophysiological
techniques were used to record miniature end plate potentials (mepps)
and evoked end plate potentials (Epps) in a
high-Mg2+,
low-Ca2+ Tyrode's solution (125 mM NaCl, 5.37 mM KCl, 24 mM
NaHCO3, 12 mM
MgCl2, 1 mM
CaCl2, and 5% dextrose). Alternatively, evoked Epps alone were recorded in normal Tyrode's solution (1 mM
Mg2+ and 1.8 mM
Ca2+) containing between 0.5 and 10 µM D-TC (Sigma) to block muscle contraction.
Briefly, sharp glass electrodes were pulled (20-40 M
resistance) and filled with 3 M KCl, and single muscle
fibers were impaled near the motor end plate. The initial resting
potentials were between 70 to 85 mV and usually remained stable
throughout the duration of the experiments. Electrophysiological
measurements were not recorded if the resting potential decreased by
>15% of its original value. Potentials were recorded via an
intracellular amplifier (World Precision Instruments) using Axotape or
Axoscope software (10 kHz sampling rate; Axon Instruments) and stored
simultaneously on a ZIP drive (Iomega, Roy, UT) for later
analysis. mepps (100-200) were recorded from each muscle fiber in
high-Mg2+,
low-Ca2+ Tyrode's solution over a 2-4
min recording period. Single Epps (recorded at 0.5 Hz for 3-4 min),
paired-pulse Epps, or trains of Epps (5-200 Hz) were recorded in
high-Mg2+,
low-Ca2+ Tyrode's solution or in normal
Tyrode's solution containing 0.5-10 µM
D-TC, using short (0.5-1 msec), monophasic electrical
stimuli isolated from ground with a Grass stimulus isolation unit.
FM1-43 optical imaging
FM1-43 dye uptake during a 10 min depolarization with high
K+. In an initial series of
experiments the dye FM1-43 (Betz and Bewick, 1993; Reid et al., 1999)
obtained from Molecular Probes was loaded into presynaptic vesicles by
high-K+ depolarization, in a muscle in
which end plates have been labeled previously by exposure to 20 µM rhodamine -bungarotoxin for 1 hr. The extracellular
solution bathing a muscle was then switched from normal saline to one
containing 60 mM K+ and 12 mM FM1-43 for 7 min. The preparation was then washed with saline containing 12 mM Mg+2
and 0.5 mM Ca+2 for 15 min to
wash the dye from the bath and to reduce spontaneous release of
vesicles during this process. End plates were then visualized with a
40× water immersion objective on a Nikon Microphot fluorescence
microscope equipped with the appropriate filter cubes for rhodamine and
FM1-43. Different concentrations of FM1-43 and different loading times
were tried, with the procedure described above producing the most
intense staining of end plates with minimal nonspecific labeling of
other structures. Pictures were taken with a Nikon camera at the same
exposure and filter settings so that the pattern and intensity of
staining could be compared between wild-type and NCAM null junctions.
Dynamic imaging of vesicle release with FM1-43. In a
subsequent series of experiments, muscles in which end plates had been made visible by labeling with 20 µM -bungarotoxin for
30 min had FM1-43 loaded into synaptic vesicles by stimulating the
muscle nerve for 10 min at 10 Hz, while the muscle was bathed in normal saline containing 12 µM FM1-43. These parameters produced
optimal loading of the dye. After loading, the preparation was washed with high Mg+2 (1.75 mM) and
low Ca+2 (0.5 mM) for 10 min
to remove the dye from the solution. Images were then captured with a
40× water immersion objective on a Nikon Microphot microscope by means
of an SIT camera (Hamamatsu C2400) connected to a frame grabber (Matrox
IM-LC) on a computer (Dell pentium V350) in parallel with a
videocassette recorder (Toshiba KV-6200A). A neutral density filter was
used to reduce light damage and to ensure that the camera was within
its linear range. At the same time a second image was captured to show
rhodamine -bungarotoxin labeling to demonstrate overall end plate
morphology. The preparation was then returned to normal saline (1.8 mM Ca+2 and 1 mM
Mg+2) for 5 min, and images were captured
every 10 sec. To evaluate changes in the intensity of labeling, the
videotape was played back, and the selected frames were captured and
digitized using the same computer system. Images were stored digitally
and later color-coded for pixel intensity using the Metamorph Image
Analysis System. The montage and labeling of the pseudocolored images
were created using Corel Draw 7.0. Loss of dye with time after trains of stimuli at different frequencies allowed us to monitor synaptic vesicle exocytosis via the loss of FM1-43 fluorescence.
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RESULTS |
Alterations in the size and shape of end plates in NCAM null and
heterozygous mice
Mammalian neuromuscular junctions are initially formed during
embryonic development, and at birth in mice and rats all end plates are
polyneuronally innervated (Redfern, 1970; Brown et al., 1976;
Balice-Gordon et al., 1993). Fibers become singly innervated during the
first 2 postnatal weeks, and in the semitendinosus muscle that was the
focus of the present study, we found this process to be complete by
postnatal day 11 (P11) in +/+ mice. Thus P11 was chosen as the first
time point to assess the effect of the absence of NCAM on the size and
shape of end plates.
By P11, AChRs had become clustered at NCAM null end plates at a
density comparable with that in +/+ end plates as judged by the
intensity of rhodamine -BTX labeling (Fig.
1A,B). Because end
plates are complex structures including regions of high AChR density
interspersed with regions lacking AChRs, we quantified end plate size
in two ways: (1) the total area exhibiting high AChR density
(Figs. 1, 2, BTX-binding area
histograms) and (2) the total area enclosed by a line encircling
the end plate and thus containing areas of both high and low AChR
density (Figs. 1, 2, total end plate area histograms). By
either measure, end plates from P11 / mice were ~20% smaller
than were those from +/+ littermates. Although there was considerable
overlap in the end plate size histograms (Fig. 1), those
from the NCAM null mice (Fig. 1D) were clearly
skewed toward smaller values than were those from +/+ mice (Fig.
1C), this reduction being statistically significant
(p < 0.05, one-way ANOVA).
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Figure 1.
Size and shape of end plates in P11 NCAM-deficient
and wild-type neuromuscular junctions. A, Three examples
of P11 wild-type end plates visualized with rhodamine -bungarotoxin
staining show regions of high AChR density. C, The total
area of high AChR density was quantified (BTX-binding area) for 42 end
plates, and the values are expressed in the left
histogram as the proportion of end plates occurring within each
bin size. The right histogram illustrates the total end
plate area and includes the area contained within a line
encircling the end plate (i.e., including regions of high AChR density
as well as the interspersed areas that lack AChRs). B,
Three examples of similarly stained P11 NCAM null end plates are shown.
D, In general the NCAM null synapses are smaller in
size, as is apparent from the histograms showing the
distribution of the BTX-binding area (left) and the
total end plate area (right) from 46 NCAM null end
plates.
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Figure 2.
Size and shape of end plates from P30 wild-type,
heterozygous, and NCAM null mice. A,
Left, Two examples of P30 wild-type end plates stained
with rhodamine -bungarotoxin are shown. Right,
Histograms show the size distribution for the
BTX-binding area (left histogram) and the total end
plate area [area enclosed by a line encircling the end plate
and containing regions of both high and low ACh receptor density
(right histogram)] from 65 wild-type end plates.
B, Both the examples of BTX-stained NCAM null end plates
(left) and the histograms
(right) generated from 64 such end plates show that both
the BTX-binding areas (left histogram) and total end
plate areas (right histogram) are skewed toward smaller
sizes, compared with wild type. C, Left,
Two examples of similarly stained end plates from a heterozygous mouse
illustrate that such end plates were often less compact.
Right, Although the area of membrane containing a high
density of ACh receptors was similar to that of wild type (left
histogram; 65 junctions analyzed), many junctions had total end
plate areas (right histogram) that were larger than that
of wild type, because of the less compact shape of the end
plates.
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As muscle fiber diameter increased during the next several weeks, end
plates in both +/+ and / mice increased in size. However as shown
by the histograms for both BTX-binding area and total end
plate area, NCAM null end plates at P30 (Fig. 2B)
remained smaller in size than did wild-type end plates (Fig.
2A); the histograms were clearly skewed
toward smaller sizes, and the mean size was reduced by ~12%. At P30
we also analyzed +/ mice, which would be expected to have NCAM levels
of ~50% of wild type. The size of these end plates, as measured by
the area of the membrane with high AChR density, was not different from
control (Fig. 2A,C, left histograms). Thus
the apparent reduction in NCAM levels to 50% of wild type did not
result in an increase in the junctional area as occurred in
Drosophila when FasII was reduced by 50% (Schuster et al.,
1996a). We noticed, however, that end plates in +/ mice were much
less compact than were those of either +/+ or / mice. Although not
all end plates were as open in shape as those shown in Figure
2C, there was a strong tendency for +/ end plates to be
less compact and thus to occupy more total end plate area (Fig. 2,
compare A,C, right histograms; the means were
statistically different, p < 0.05, one-way ANOVA).
These observations suggest that differing levels of NCAM can affect the
process by which end plates grow in size during postnatal maturation.
Because end plate size is usually well matched to the diameter of the
postsynaptic muscle fiber to maintain effective neuromuscular transmission (Kuno et al., 1971; Harris and Ribchester, 1979), end
plates in NCAM null mice might be smaller simply because muscle fibers
were smaller. This was in fact suggested in a previous study
(Moscoso et al., 1998) that found that end plates in the sternomastoid muscle of NCAM null mice were ~12% smaller than those
of wild type, a reduction similar to what we report in the present
study. We therefore measured muscle fiber diameter in P30 +/+ and /
semitendinosus muscles from frozen cross sections taken through the
middle portion of the distal head of the muscle. This head, from which
all end plate morphology and most electrophysiology were obtained, is
composed primarily of fast-twitch muscle fibers but also contains a
small number of slow-twitch fibers that are confined to the internal
portion of the muscle (these appear in Fig.
3A-C as dark
profiles because they were stained with a slow myosin-specific
antibody). The process of primary and secondary myogenesis, which
produces this pattern of fiber type distribution, appeared to have
occurred normally in the NCAM null mice, because both the number and
distribution of slow fibers (Fig. 3A,C) were similar to that
of wild type (Fig. 3B). Furthermore the mean fiber cross-sectional area did not differ between +/+ and /, as indicated by the histograms in Figure 3, D and
E, respectively (p > 0.05, one-way
ANOVA). The histogram of muscle fiber cross-sectional areas
was clearly not skewed toward smaller sizes as was the end plate area
histograms (see Fig. 2) in these mice, as would be expected
if the smaller end plate areas were a result of smaller muscle fiber
diameters. The slow-twitch fibers (Fig. 3D,E, filled bars) were smaller than the fast-twitch fibers (open
bars) in both +/+ and /. Thus, overall, the absence of NCAM
and PSA does not affect the process of myogenesis or alter the size of
the muscle fibers. Although we did not quantify total muscle fiber or
motoneuron number, as judged from the size of the muscles and the
diameters of the muscle nerves, there did not appear to be a reduction
in these parameters in the NCAM null mice.
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Figure 3.
Size and spatial distribution of fast- and
slow-twitch muscle fibers in the semitendinosus muscle from wild-type
and NCAM null P30 mice. A, Low-magnification view of a
frozen cross section from a wild-type muscle stained with an antibody
specific for slow myosin. The slow-twitch muscle fibers (stained
darkly) are sparsely distributed throughout the interior of the
muscle, interspersed with larger fast-twitch muscle fibers that
comprise the majority of the muscle. B, C, Higher
magnification views of portions of wild-type (B)
and NCAM null (C) muscles showing that the
distribution and size of both slow- and fast-twitch muscle fibers are
similar. D, E, Histograms showing the
cross-sectional area distributions of all fibers within a square region
taken from the center of a wild-type (D) and NCAM
null (E) muscle and extending from the top to the
bottom surface. The slow-twitch fibers are illustrated as filled
bars; fast-twitch fibers are open bars in the
histograms. It is clear that the
histogram of muscle fiber cross-sectional areas from the
NCAM-deficient muscle (E) is not highly
skewed toward smaller sizes as were the end plate area
histograms from the (/) mice shown in Figure 2.
Scale bar: A, 82 µm; B, C, 50 µm.
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NCAM null mice exhibit a delay in the withdrawal of
polyneuronal innervation
We estimated the extent of polyneuronal innervation from P0 to P30
functionally by the method of tension occlusion. If all muscle fibers
were singly innervated, then the tension obtained by stimulating those
fibers innervated by spinal nerve L4 alone and the tension obtained by
stimulating those innervated by L5 alone should sum linearly when L4
and L5 are stimulated simultaneously. We found that by this criteria
both +/+ and +/ semitendinosus muscles were no longer polyneuronally
innervated by P11 (see, for example, Fig.
4, top). In contrast, P11
/ muscles did not exhibit any summation of tension and appeared to
be completely polyneuronally innervated (Fig. 4, middle). It
is also apparent that the P11 / muscle exhibited extensive fatigue
during the 1 sec, 50 Hz train. The reason for this fatigue, which was
not observed under similar conditions in +/+ or +/ muscles, will be
considered below. In this figure, muscles were activated by a 1 sec
train at 50 Hz. Comparable data were also obtained when twitch tension
was analyzed (data not shown). By P17 (Fig. 4, bottom),
withdrawal of polyneuronal innervation also had taken place in the
/ mice. Thus the absence of NCAM delays, but does not prevent, the
process of neonatal synapse elimination. Similar observations have been
made morphologically for NCAM null sternomastoid muscles
(Moscoso et al., 1998).
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Figure 4.
Tension produced by semitendinosus muscles in
response to a 50 Hz, 1 sec train of stimuli applied to spinal nerves
L4, L5, or L4 + L5. Top, Traces from a
P11 heterozygote show that the tension produced by stimulating both
nerves simultaneously is equivalent to the sum of the tensions produced
by stimulating L4 and L5 separately, indicating that the majority of
the muscle fibers are no longer polyneuronally innervated.
Middle, In contrast, a P11 homozygous muscle does not
exhibit any summation of tension, indicating that most muscle fibers
are still polyneuronally innervated. In addition the NCAM null muscle
is unable to maintain tension for 1 sec when stimulated at the 50 Hz
frequency. Bottom, By P17, the NCAM null muscle exhibits
summation of tension, indicating that synapse elimination is primarily
complete. By P17 the NCAM null muscle is also able to maintain tension
when stimulated at 50 Hz for 1 sec.
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NCAM null mice exhibit a delay in the selective localization of the
synaptic vesicle antigen SV2 to the end plate
After the growing tip of an axon stops and forms a synapse, there
is a rapid redistribution of synaptic vesicle proteins from an initial
diffuse localization along the nerve to the presynaptic terminal where
they become highly concentrated (Lupa and Hall, 1989; Dahm and
Landmesser, 1991). Thus by P11 in +/+ end plates, SV2 immunolabeling
(Fig. 5A) was confined to the
region immediately overlaying the end plate as visualized with BTX
staining (Fig. 5B). In contrast, in NCAM null end plates,
SV2 immunolabeling remained high within the nerve, as well as at the
end plate. This allowed us to confirm by morphological means that these
NCAM null junctions were polyneuronally innervated (Fig.
5C,E, arrows indicate separate axons). Synaptic
vesicle proteins gradually became cleared from the intramuscular axons
in NCAM null mice, but even at P30, pale labeling of the preterminal
axon was often visible in NCAM null but not wild-type synapses. These
observations suggest that the process by which synaptic vesicle
proteins (and presumably synaptic vesicles) become selectively
targeted to the presynaptic terminal occurs less effectively in
NCAM-deficient mice.
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Figure 5.
The distribution of the synaptic vesicle protein
SV2 at P11 wild-type and NCAM null end plates. A, In the
wild-type muscle, the synaptic vesicle antigen SV2 selectively
localized to the presynaptic nerve terminals. B, The
same wild-type terminals stained with rhodamine -bungarotoxin,
showing good colocalization with the SV2 staining shown in
A. C, E, Two examples of SV2 staining
from a P11 NCAM null muscle, showing extensive staining for SV2 all
along the intramuscular axons, in addition to the presynaptic terminal
overlying the end plate. This extensive axonal staining also allows
morphological confirmation that NCAM null junctions are polyneuronally
innervated at this stage of development. Arrows indicate
separate axons that innervate the same end plate. D, F,
-Bungarotoxin labeling of the same NCAM null end plates shown in
C and E, respectively. Scale bar, 50 µm.
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Synaptic efficacy is reduced in NCAM-deficient muscles during
repetitive stimulation
While making tension recordings to assess the degree of
polyneuronal innervation, we observed that NCAM null muscles often exhibited a falloff in tension during the 1 sec stimulation trains especially at higher stimulus frequencies (100 Hz or higher), whereas
wild-type muscles were able to maintain tension effectively (Fig. 4,
middle). Because NCAM null muscles could maintain tension when directly stimulated (data not shown), this suggested that the
decline in tension was synaptic in origin.
To address this possibility more directly, we recorded compound action
potentials (EMGs) with suction electrodes from +/+ and / muscles in
Tyrode's solution containing normal Ca2+
and Mg2+. As shown in the example in
Figure 6A, the
wild-type junction (top) maintained the original level of
transmission for the first five stimuli during a 200 Hz train. Even by
the end of the 1 sec train, the compound action potentials were ~50%
of the original amplitude. In contrast, the NCAM null junction (Fig.
6A, bottom) showed a decrement in
transmission even during the first five stimuli, and transmission had
failed completely by the end of the train. Although there was some
variation in the degree of transmission failure from muscle to muscle,
in general all of the NCAM null muscles showed a severe decrement in
transmission at higher stimulus frequencies (i.e., 100 and 200 Hz; Fig.
6B), indicating that in most fibers transmission had
fallen below threshold for action potential generation. This appears to
be a result of defective synaptic transmission, because direct
recording of compound action potentials from the muscle nerve as it
enters the muscle indicated that NCAM-deficient axons were able to
conduct action potentials without failure even at repetition rates in
excess of 200 Hz (data not shown). This decline in effective
transmission was accentuated in preparations in which
D-TC had partially blocked the NMJ (data not
shown). This inability of NCAM null junctions to maintain transmission
at high repetition rates does not appear to reflect simply a delay in
synaptic maturation, because P65 junctions exhibited the same defect
(Fig. 6B).
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Figure 6.
EMG recordings from P30 wild-type and NCAM null
muscles reveal differences in the efficacy of NCAM null end plates when
activated repetitively. All data were obtained from muscles with
physiological levels of Ca2+ (1.8 mM)
and Mg2+ (1 mM). A,
Examples of EMGs from a wild-type (top) and NCAM null
(bottom) muscle stimulated at 200 Hz. B,
Bar graphs showing the ratios of the amplitude of the
fifth over the first response (left) and the last over
the first response (right) at P30 (open
bars, wild type; black filled bars, NCAM null)
and at P65 (gray filled bars, NCAM
null). The bars represent the mean ± SE from 28 recordings from
six muscles from three different wild-type mice, 22 recordings from
four muscles from three different NCAM null mice at P30, and 14 recordings from two different muscles from one NCAM null mouse at P65.
Each recording was performed twice with 1 sec trains of 10, 20, 50, 100, and 200 Hz with 8-10 sec between each train.
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In summary these observations show that NCAM null junctions are more
prone to failure especially with repetitive stimulation. The
retardation of junctional fold development that has been described (Moscoso et al., 1998) could reduce the safety factor for
transmission (Wood and Slater, 1997). Additionally a reduction in
the amount of transmitter released could contribute to the
observed fatigue. To define better which presynaptic or postsynaptic
changes might contribute to this reduction in synaptic efficacy, we
characterized different parameters of transmission at P30 junctions
using intracellular recordings, limiting our studies to +/+ and / mice.
Properties of synaptic transmission at P30 junctions under
conditions of low transmitter release
To prevent contraction and to allow for quantification of both
spontaneous mepps and evoked Epps, we reduced the probability of
release by raising Mg2+ to 12 mM and lowering Ca2+ to 1 mM, resulting in the release of only several quanta per stimulus. Under these conditions, mepp amplitudes were unimodally distributed, and for all the cells that were analyzed, the entire distribution was clearly above noise level. The mean mepp amplitude (Fig. 7A) did not
differ between +/+ and / junctions, indicating that a single
quantum of transmitter produced the same postsynaptic depolarization in
both cases. Because muscle fiber diameters did not differ on average
between +/+ and / (see Fig. 3), the input resistance of the fibers
would be expected to be similar. This suggests that both the amount of
transmitter contained per vesicle and its effect, which would be
dependent on the density of ACh receptors, are essentially normal at
junctions lacking NCAM. There was however a slight decrease in the
frequency of mepps compared with that at wild-type junctions (Fig.
7B).
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Figure 7.
Parameters of synaptic transmission at P30
wild-type and NCAM null junctions under conditions of low transmitter
release (extracellular Ca+2, 1 mM;
Mg+2, 12 mM). A,
Bar graph showing mean mepp amplitude
(Amp.) ± SD. There was no statistical difference
between +/+ and / end plates (p > 0.05, one-way ANOVA). B, Bar graph showing mepp
frequency, which was slightly reduced at the / junctions
(p < 0.05, one-way ANOVA).
C, Bar graph showing Epp amplitude in
response to a single suprathreshold stimulus to the nerve. The /
and +/+ junctions were not statistically different
(p > 0.05, one-way ANOVA).
D, Bar graph showing mean quantal
content ± SD for Epps evoked by a single stimulus and calculated
from the ratio of mean Epp amplitude/mean mepp amplitude. The quantal
contents were not statistically different between +/+ and /
junctions. E, Responses to pairs of pulses to determine
paired-pulse facilitation. Top, Trace
from +/+ junctions showing facilitation at 10, 6, and 4 msec intervals
(left to right).
Bottom, Trace from a / junction
showing similar facilitation at the three intervals. F,
Bar graph showing the mean amplitude ± SD of the
second Epp of the pair as a percent of the amplitude of the first Epp
in the pair at 10, 6, and 4 msec intervals. Both +/+ and /
junctions showed a similar amount of facilitation at each interval.
Data were obtained from 10 +/+ and 10 / junctions.
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When release was evoked by single suprathreshold stimuli to the nerve,
the Epp amplitude also did not differ from that of wild type (Fig.
7C). Furthermore quantal content calculated by the ratio of
the Epp/mepp amplitude was ~2 for both +/+ and / junctions (Fig.
7D). Finally, NCAM-deficient junctions exhibited paired-pulse facilitation to the same level as wild-type junctions at
10, 6, and 4 msec intervals (Fig. 7E,F). Taken
together these observations suggest that most of the presynaptic and
postsynaptic machinery required for normal synaptic transmission has
become properly assembled at NMJs in the absence of NCAM.
Properties of synaptic transmission at P30 junctions under normal
levels of transmitter release
To study synaptic transmission at levels of transmitter release
that would be expected in vivo, we recorded from junctions in normal Ca2+ and
Mg2+, by using D-TC
(5 µM) to reduce transmission to below
threshold for muscle action potential generation, thereby preventing
contraction. Under these conditions Epp amplitude did not differ
between +/+ and / junctions (Fig.
8A). Because this
concentration of D-TC reduced mepp amplitude to
within the noise level, we estimated quantal content from the
coefficient of variation of Epp amplitude (Miyamoto, 1975; Clements and
Silver, 2000). The number of quanta released by single stimuli did not
differ significantly between wild-type and NCAM null junctions and was
~60 at this stage of development (Fig. 8B).
However, unlike wild-type synapses that continued to show paired-pulse
facilitation at normal levels of transmitter release, the
NCAM-deficient synapses failed to show paired-pulse facilitation at
intervals between 6 and 50 msec. Examples of traces at the 6 and 10 msec intervals are shown in Figure 8, C and
D. At the 4 msec interval, the NCAM-deficient synapses in
fact exhibited a moderate degree of depression (Fig. 8C,D).
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Figure 8.
Parameters of synaptic transmission at P30
wild-type and NCAM null junctions at normal levels of transmitter
release. All data were collected in a physiological saline containing
in vivo levels of Ca+2 (1.8 mM) and Mg+2 (1 mM) and 5 µM D-TC to partially block end plates to
avoid muscle contraction. A, Mean Epp amplitudes ± SD for P30 +/+ and / junctions were not statistically different.
B, Quantal content, calculated from the coefficient of
variation of Epp amplitude (see Materials and Methods for additional
details), also did not differ between +/+ and / junctions
(p > 0.05, one-way ANOVA for both A,
B). C, Epps to pairs of pulses separated by 10, 6, or 4 msec (left to right) to
assay paired-pulse facilitation are shown. Top,
Trace from a +/+ junction shows that the response to the
second stimulus is facilitated at all intervals. Bottom,
Trace from an NCAM null junction that did not exhibit
facilitation at any of the intervals is shown. D,
Bar graphs show the mean amplitude ± SD of the
second Epp as a percent of the first. Although the wild-type junctions
(open bars) exhibited a modest degree of facilitation at
10, 6, and 4 msec intervals, the NCAM null junctions
(filled bars) did not exhibit facilitation at the
10 and 6 msec intervals and were in fact depressed at the 4 msec
interval.
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NCAM-deficient synapses are unable to sustain transmitter output
with repetitive stimuli
P30 wild-type and NCAM-deficient junctions were next stimulated in
5 µM D-TC and normal
Ca2+ and Mg2+
with 1 sec trains of stimuli at 10, 20, 50, 100, and 200 Hz. In
agreement with the extracellular recordings of compound action potentials, we observed that NCAM-deficient junctions were unable to
maintain effective transmission when challenged with multiple stimuli.
Such a deficiency was most pronounced at high repetition rates as shown
by the 200 Hz, 1 sec train illustrated in Figure 9A (the trace
on the left shows Epps occurring at the beginning of
the train; the trace on the right occurs at the
end of the train). The wild-type junction shows some facilitation of
Epp amplitude during the first few stimuli and even at this high
stimulation rate was able to maintain Epp amplitude at ~50% of the
initial value at the end of the 1 sec train (see also Fig.
9C,D, bar graphs). In contrast, the NCAM null
synapse did not show facilitation, and even by the ninth stimulus, Epp
amplitude was already reduced to 25% of the initial value (Fig.
9B). Transmission at this junction had essentially failed by
the end of the 1 sec train. Although most extreme at high stimulus
rates, as illustrated by the bar graph in Figure
9D, the NCAM-deficient synapses showed some falloff in
efficacy of transmission even at moderate repetition rates (i.e., 10 and 20 Hz; Fig. 9C).
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Figure 9.
Inability of NCAM null junctions to maintain
transmitter output when stimulated repetitively. All data were obtained
from intracellular recordings from muscles in saline with physiological
levels of Ca+2 (1.8 mM) and
Mg+2 (1 mM) and 5 µM
D-TC to block contraction. A, Epps from a
wild-type junction during a 1 sec, 200 Hz train are shown. The Epps
exhibited facilitation during the beginning of the train
(left) and even by the end of the train
(right) were ~50% of the initial Epp amplitude.
B, A similar trace from an NCAM null
junction is shown. There is no facilitation at the beginning of the
train (left), and even by the ninth stimulus, Epp
amplitude was reduced to less than one-half of the initial Epp. By the
end of the train (right), transmission had almost
completely failed. C, D, Bar graphs show
the mean amplitude ± SD of the fifth and the last
(lst) Epp in the train, respectively, as a percent of
the first Epp for a number of junctions at a variety of different
stimulus repetition rates. C, Compared with wild type,
the NCAM null junctions show a rapid depression in transmission to
~60% by the fifth pulse at all frequencies tested. D,
By the end of the 1 sec train, the NCAM null junctions are more
depressed than wild type at all intervals except for 50 Hz. The
depression with respect to wild type is most extreme at 200 Hz where
the wild-type junctions maintain transmission at ~50% of the initial
value, whereas the NCAM null junctions are almost completely
blocked.
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Thus one of the most striking defects in the NCAM-deficient NMJ is an
inability to sustain transmission at an optimal level over time. This
would most likely represent a presynaptic defect in the ability either
to release synaptic vesicles or to mobilize vesicles from the reserve
pool, after those in the readily releasable pool (Kuromi and Kidikoro,
1998; Stevens and Sullivan, 1998; Wang and Kaczmarek, 1998) have been
exhausted. To confirm that the reduction in Epp amplitude does in fact
represent a reduction in the number of quanta (vesicles) released per
stimulus, we calculated quantal content from the coefficient of
variation of Epp amplitude (Miyamoto, 1975) at several wild-type and
NCAM-deficient junctions after a series of 1 sec trains separated by
sufficient time for full recovery. We chose the 10th stimulus of the
200 Hz train to compare because in NCAM null synapses, Epp amplitude
was considerably depressed but not completely blocked as at the end of
the train. As an example, the quantal content in one junction was
reduced from 94 to 18 by the 10th stimulus. We quantified this
reduction for eight NCAM null junctions and found a mean reduction of
68.8% (range, 41-82%).
The data showing a difference in synaptic depression between wild-type
and NCAM null junctions were obtained in the presence of 5 µM D-TC to prevent muscle contraction.
Although this is an accepted procedure, it is known that
D-TC can enhance synaptic depression at NMJs by acting on
presynaptic ACh receptors (Magelby et al., 1981; Tian et al., 1994).
However we observed a similar difference between +/+ and /
junctions in synaptic depression in the absence of D-TC,
while recording EMGs during repetitive stimulation as described
previously (Fig. 6). Thus it does not seem that differences in the
sensitivity of wild-type and NCAM null junctions to this action of
D-TC can account for our observations.
One explanation for the excessive depression at NCAM null junctions
would be that the pool of releasable vesicles is so small that all have
been exhausted by the end of the 200 Hz, 1 sec train (Schneggenburger
et al., 1999). Alternatively, the NCAM-deficient synapses may have
difficulty in mobilizing or docking vesicles at these high rates
(Pozzo-Miller et al., 1999; Wu and Borst, 1999). To distinguish better
between these possibilities, we have made preliminary observations of
transmitter release properties using the dye FM1-43 (Betz and Bewick,
1993; Reid et al., 1999).
Aspects of synaptic vesicle pool size and release dynamics as
revealed by FM1-43 labeling
The fluorescent dye FM1-43 is taken up into synaptic vesicles
during the process of exocytosis/endocytosis and can be used in
conjunction with electrophysiology to estimate synaptic vesicle pool
size and to monitor both temporally and spatially synaptic vesicle
dynamics (Betz and Bewick, 1993; Reid et al., 1999). After determining
optimum conditions for loading with FM1-43 (see Materials and Methods
for details), we found that with low-frequency stimulation both
wild-type and NCAM null synapses could be loaded with dye to
approximately the same level. Although there was some variation from
junction to junction, as shown by the photos in Figure
10, junctions from both types of
animals exhibited similar intensities of staining, which was unevenly
distributed, with intense hot spots of staining interspersed with lower
intensity, somewhat more diffuse staining. These patterns are similar
to those described by others at both mouse and rat NMJs (Ribchester et
al., 1994; Reid et al., 1999). When comparing FM1-43 labeling with
rhodamine BTX staining of the same junctions to delineate the
end plate area, all regions of the NCAM-deficient end plates appeared
capable of being loaded.
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Figure 10.
Wild-type and NCAM null junctions labeled
by FM1-43 dye uptake to indicate synaptic vesicles participating in the
exocytotic/endocytotic cycle. A, B, Two wild-type
junctions visualized with rhodamine -bungarotoxin to label
postsynaptic ACh receptors. C, D, The same junctions in
A and B, respectively, visualized by
FM1-43 taken up during a 7 min depolarization in 60 mM KCl
containing 12 mM FM1-43. E, F, Two NCAM null
junctions visualized with rhodamine -bungarotoxin. G,
H, The same junctions in E and F,
respectively, visualized by FM1-43 after depolarization as described
above. Scale bar, 30 µm.
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To visualize FM1-43 labeling dynamically and to compare the loss of dye
with the amount of transmitter release detected electrophysiologically, we acquired FM1-43 images with a digital camera after complete loading
and at various intervals during stimulation at 200 Hz. In Figure
11 the first pair of images
(left) shows a wild-type (top) and NCAM-deficient
(bottom) junction at the end of FM1-43 loading, when the
intensity of staining has been color-coded. At the end of a 1 sec, 200 Hz train, dye has been lost from both junctions, but a large amount of
labeled vesicles remains in both. After an additional 30 sec of
stimulation, much of the remaining dye can be released from the
NCAM-deficient junction. Thus the failure in transmission cannot be
caused simply by a very small pool of releasable vesicles, which is
completely exhausted by the end of the 1 sec train. The observations
rather suggest some defect in the NCAM-deficient junctions either in
docking vesicles at the presynaptic active zones at these high stimulus
rates or in mobilizing vesicles from the reserve to the readily
releasable pool (Stevens and Sullivan, 1998; Wang and Kaczmarek, 1998).
Nevertheless, as Figure 11 shows, a consistent finding, which at least
superficially seems at odds with this suggestion, was that
NCAM-deficient junctions lost FM1-43 labeling much more rapidly than
did wild-type junctions (L. Polo-Parada and L. T. Landmesser,
unpublished observations). Furthermore this rate of destaining was
inconsistent with the much smaller number of quanta detected
electrophysiologically. One possibility is that many of the vesicles
loaded with FM1-43 in the / junctions do not contain transmitter.
Another possibility is that at the NCAM-deficient junctions, vesicles
are also released at sites that are not directly apposed to the
postsynaptic muscle membrane and therefore are not detected by
intracellular recordings. Ongoing experiments are being performed to
distinguish among these possibilities and to determine the cellular
mechanism underlying the depression we have observed.
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Figure 11.
Loss of FM1-43 label from a wild-type (top
row) and NCAM null (bottom row) junction by
electrical stimulation of the nerve. The two junctions were loaded by
stimulating the nerve for 10 min at 10 Hz in normal saline.
Left, After washout of FM1-43 from the bath in
high-Mg+2, low-Ca+2 saline for 10 min, the muscle was perfused with physiological saline, and an image
was captured. Middle, An image was captured at the end
of a 1 sec, 200 Hz train. Right, Dye remaining after
another 30 sec of stimulation at 200 Hz is shown. All images were
captured with a digital camera, and pixel intensity was later
color-coded by means of the Metamorph imaging program, with
red indicating the highest intensity.
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DISCUSSION |
Although many aspects of neuromuscular maturation occurred
normally in NCAM null mice (see also Moscoso et al., 1998), we detected a number of differences including the size and shape of end
plates and delays in the withdrawal of polyneuronal innervation and the
selective targeting of synaptic vesicle proteins to the presynaptic
terminal. Most striking were specific functional defects in synaptic
transmission: an absence of paired-pulse facilitation and an inability
to maintain effective transmission with repetitive stimuli. Each of
these differences will be discussed in turn.
NCAM can act as a homophilic adhesion molecule (Rutishauser and
Jessell, 1988) and could contribute to initial NMJ formation by
promoting interactions between the presynaptic and postsynaptic membrane. However the large width of the synaptic cleft and the interposition of a basal lamina would seem to preclude such homophilic interactions across postnatal NMJs. It seems more likely that NCAM,
which postnatally becomes concentrated both prejunctionally and
postjunctionally (Covault and Sanes, 1986), would interact heterophilically with other molecules in the synaptic basal lamina, such as neural agrin (Ferns et al., 1993), to which it can bind (Burg
et al., 1995). NCAM might also interact with other molecules enriched
in synaptic regions including synaptic laminins (Sanes et al., 1990;
Patton et al., 1997; Sanes and Lichtman, 1999). Such
heterophilic interactions, acting via adhesion or signaling in either a
cis or trans manner, could contribute to the
structural and functional alterations that we documented. PSA can also
modulate NCAM function (Rutishauser and Landmesser, 1996), and a role
for it cannot be excluded until effects of its enzymatic removal on junctional properties of +/+ mice are determined. However we found that
junctional PSA levels were quite low during the postnatal period we investigated.
Alterations in size and shape of NMJs that lack NCAM
The usual tight relationship between end plate size and muscle
fiber diameter (Kuno et al., 1971; Harris and Ribchester, 1979) ensures
effective transmission. Such size matching was impaired in NCAM null
mutants because end plates were smaller although fiber diameter was
normal. We also observed differences in the shape and compactness of
end plates in both / and +/ compared with +/+ mice. Furthermore,
the +/ and / mice differed from each other. The diverse end plate
shapes result from the loss of AChRs under portions of the
initially plaque-shaped endings during the first few postnatal weeks
(Balice-Gordon and Lichtman, 1993), a process that appears to involve
bidirectional signaling. Our results indicate that this process is
somehow affected by differing NCAM levels. Subsequent growth without
shape change has been proposed to be mediated by adhesion between
terminal and muscle fibers, resulting in intercalary terminal growth as the muscle fiber increases in size (Balice-Gordon and Lichtman, 1990).
This could also be affected by alterations in NCAM-mediated adhesion or
signaling. Ectopic expression of muscle NCAM in mice was shown recently
to result in terminal sprouting and increased NMJ area (Walsh et al.,
2000). In Aplysia (Mayford et al., 1992; Zhu et
al., 1995) and Drosophila (Schuster et al., 1996a,b)
alterations in levels of NCAM-like molecules also affect synaptic
growth and plasticity. Additional insight into how differing levels of
NCAM affect the size and shape of mouse NMJs, and how this might relate to the observations in invertebrates, will probably require dynamic observations of junctions over time (Balice-Gordon and Lichtman, 1990).
Presynaptic structural maturation is delayed in the absence
of NCAM
Immature motor axons have synaptic vesicle proteins
diffusely distributed (Lupa and Hall, 1989; Dahm and Landmesser, 1991) and are able to release ACh (Chow and Poo, 1985) along their length. Contact with a muscle fiber results in specialization of the
presynaptic terminal, with synaptic vesicle proteins becoming
concentrated there and cleared from the rest of the axon (Lupa and
Hall, 1989; Dahm and Landmesser, 1991). Although the synaptic vesicle
protein SV2 became concentrated at NCAM null junctions, this was
delayed and incomplete even by P30. This observation also suggests some impairment in the process of bidirectional signaling at the / NMJ.
Neonatal synapse elimination is delayed in NCAM null junctions
Neonatal synapse elimination is a competitive (Betz et al., 1989;
Balice-Gordon and Lichtman, 1994) and activity-dependent (O'Brien et
al., 1978; Thompson, 1985; Busetto et al., 2000) process by which all
but one of the multiple nerve terminals are eliminated from the NMJ
(for review, see Sanes and Lichtman, 1999). We found that synapse
elimination was significantly delayed in the NCAM null semitendinosus
muscle, as also observed for the sternomastoid muscle (Moscoso
et al., 1998). Why this should result from lack of NCAM is not
immediately apparent, although a general retardation in development
seems unlikely. Nerve ingrowth and initial synapse formation occurred
between embryonic day 12 (E12) and E14 as in +/+ mice (S. Banerjee and
L. T. Landmesser, unpublished observations), and other aspects of
neuromuscular development occurred on schedule including secondary
myogenesis and growth of muscle fibers.
Because synapse elimination is activity dependent, the fact that NCAM
null junctions were less effective, especially with repetitive
stimulation, might have contributed to this delay. Alternatively, the
competitive process by which one terminal becomes functionally stronger
at the expense of the other (Balice-Gordon et al., 1993; Colman
et al., 1997) might be affected more directly by the absence of NCAM.
Kopp and Balice-Gordon (1999) found recently that differences in the
probability of transmitter release between competing terminals precede
subsequent structural alterations and synapse elimination. The absence
of paired-pulse facilitation under normal release conditions at NCAM
null junctions suggests that the release probability may already be
maximal. This in turn could impede early events in the divergence of
synaptic strength that contribute to synapse elimination.
Alterations in the function of NCAM null junctions
At P30, many properties of transmission appeared normal in NCAM
null homozygotes. Mepp amplitude was normal, as were Epp amplitude and
quantal content at both low and normal levels of transmitter release.
That both Epp amplitude and quantal content were normal, although the
junctional areas in NCAM / mice were skewed toward smaller sizes,
suggests that some functional compensation (see Sandrock et al., 1997)
may have occurred. Paired-pulse facilitation was also normal under low
transmitter release conditions. Taken together these observations
indicate that the large number of molecules required for both
presynaptic and postsynaptic function (Sanes and Lichtman, 1999) become
appropriately organized without NCAM.
Nevertheless we observed two striking differences in transmission.
First, at normal levels of extracellular
Ca+2 and
Mg+2, NCAM null junctions
did not exhibit paired-pulse facilitation, suggesting that some aspect
of the release process had become saturated. This might occur if
intracellular Ca+2 levels were elevated or
if the release machinery was more sensitive to
Ca+2 in / terminals, resulting in a
maximal probability of release even to the first stimulus.
Alternatively, the entire readily releasable pool of vesicles (Kuromi
and Kidikoro, 1998; Stevens and Sullivan, 1998; Wang and Kaczmarek,
1998; Schneggenburger et al., 1999) might be released by each
stimulus, leaving no additional vesicles or docking sites for enhanced
release to the second stimulus (for review, see Zucker, 1999). Ongoing
studies to measure presynaptic Ca+2, to estimate the
binomial release parameters n and p, and to measure vesicle pool sizes with FM1-43 imaging and EM should help distinguish among these possibilities.
The second major defect was a profound inability of NCAM null junctions
to maintain transmitter output with repetitive stimuli. Because quantal
content to single stimuli did not differ from +/+, this synaptic
fatigue cannot result from an increase in the number of released
vesicles to compensate for a postsynaptic reduction in efficacy. This
process occurred in neuregulin heterozygotes (Sandrock et al., 1997)
and resulted in synaptic fatigue. Total vesicle pool size appeared
approximately normal on the basis of the amount of FM1-43 that could be
loaded, and all of this dye was releasable by stimuli to the nerve.
Thus NCAM null junctions appear to have some defect in docking and
releasing the appropriate number of vesicles at high stimulus
repetition rates. Whether this is caused by some defect in the
machinery involved in docking and release or in mobilizing vesicles to
replenish those lost (see Pozzo-Miller et al., 1999) remains to be
determined. As noted previously there may be additional alterations in
vesicle cycling and release at the NCAM-deficient junctions that may
contribute to the observed depression.
Although NCAM null mice lack hippocampal LTP and LTD, this appears to
be caused by a lack of PSA (Muller et al., 1996). It will be
interesting to see whether the alterations we have found at the NMJ
also occur in central synapses. A more complete characterization of the
dynamics of synaptic vesicle cycling should help to define the
mechanisms underlying the transmission defects and thus the potential
role of NCAM. It is possible that presynaptic NCAM, acting in a
cis manner, helps to organize other molecules involved in
transmitter release (see Thomas et al., 1997). If true, this would |
TOP
ABSTRACT
INTRODUCTION
