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The Journal of Neuroscience, February 15, 1998, 18(4):1374-1382
Synaptic Structure and Transmitter Release in Crustacean Phasic
and Tonic Motor Neurons
M.
Msghina1,
C. K.
Govind2, and
H. L.
Atwood1
1 Departments of Physiology and Zoology, University of
Toronto, Toronto, Ontario, Canada M5S 1A8, and 2 Life
Sciences Division, Scarborough Campus, University of Toronto, West
Hill, Ontario, Canada M1C 1A4
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ABSTRACT |
The paired phasic and tonic motor neurons supplying the extensor
muscle in the crayfish leg were investigated to establish whether
differences in synaptic structure could account for large differences
in transmitter release at the neuromuscular junctions. Nerve terminals
with transmitter release that had been assessed from recordings made
with a focal "macro-patch" electrode were subsequently labeled,
processed for electron microscopy, and reconstructed from serial
sections.
At a frequency of 1 Hz, quantal contents of phasic terminals were
90-1300 times greater than those of tonic terminals when both were
recorded at the same location. At higher frequencies, facilitation was
pronounced at tonic, but not phasic, terminals.
Reconstructions of recording sites showed that both phasic and tonic
terminals possessed many small synapses, usually with one or more
structurally defined active zones. Mean synaptic contact area was
larger for tonic terminals, and the number of individual synapses per
length of nerve terminal was also larger. Active zones were not
different in size for the two terminals.
At low frequencies, quantal emission per synapse is much greater for
phasic terminals. The higher quantal content of phasic terminals and
their synapses cannot reasonably be accounted for by more or larger
synapses or active zones at the recording sites. Because structural
features alone are not likely to produce the very large differences in
quantal content of phasic and tonic terminals observed at low
stimulation frequencies, it is likely that other properties of the
nerve terminal are largely responsible for these differences.
Key words:
crayfish; quantal; synapse; ultrastructure; neuromuscular
junction; active zone
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INTRODUCTION |
Crustacean motor neurons are diverse
in their physiology and morphology. A broad distinction between
"tonic" and "phasic" motor neurons has been drawn (Kennedy and
Takeda, 1965a ,b; Atwood and Wojtowicz, 1986): tonic neurons
continuously produce impulses during locomotion, whereas phasic neurons
fire in brief bursts for occasional rapid movements. Synaptic
physiology differs correspondingly: phasic motor neurons are
specialized for rapid initial release of transmitter and generate large
EPSPs in the innervated muscle fibers, whereas tonic motor neurons
release less transmitter initially and generate smaller EPSPs that
exhibit marked facilitation as impulse frequency increases (Hoyle and
Wiersma, 1958; Bittner, 1968a; Bradacs et al., 1997).
Several muscles in the limbs of crustaceans receive a pair of
excitatory neurons, one phasic and one tonic, which ramify together throughout the muscle and innervate the same muscle fibers (Wiersma, 1961; Lnenicka et al., 1986; Bradacs et al., 1997). In such cases, synapses made on the same postsynaptic element can be compared, physiologically and structurally. Observations on two of these muscles
in the crayfish, the claw closer muscle and the extensor of the
carpopodite in the leg, have provided structural comparisons of phasic
and tonic nerve terminals. Paradoxically, phasic terminals are much
thinner and contain fewer mitochondria, a smaller vesicle population,
and a lower concentration of the neurotransmitter glutamate than tonic
nerve terminals on the same muscle fiber (Lnenicka et al., 1986;
Shupliakov et al., 1995; King et al., 1996).
These observations raise an interesting question: how do thin phasic
nerve terminals, less well endowed with neurotransmitter, produce much
larger EPSPs than thicker, more transmitter-rich tonic nerve terminals?
Possibilities to be considered include numerical or structural
differences in synapses or their active zones, and molecular or
biophysical differences in the neurons. In the present study, we
addressed the structural possibility by reconstructing nerve terminals
from electron micrographs after recordings had been made from them to
assess their neurotransmitter output (Wojtowicz et al., 1994; Cooper et
al., 1995a). The reconstructions allowed us to count and measure all
the individual synapses on a defined region of the nerve terminal, and
thus to compare structural features associated with known transmitter
output for the two types of nerve terminal.
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MATERIALS AND METHODS |
Animals. Freshwater crayfish, Procambarus
clarkii, obtained from the Atchafalaya Biological Supply Company,
were maintained in tanks of dechlorinated tap water at 12-15°C.
Walking legs were removed at the point of autotomy to make the
preparations. Dissection of the extensor muscle in the meropodite
followed procedures described previously (Bradacs et al., 1997). The
preparations were superfused with standard crayfish solution (modified
van Harreveld's solution) cooled to 11-13°C (Wojtowicz and Atwood,
1986).
Stimulation and recording. The exposed inner side of the
extensor muscle was superfused for 2-3 min with crayfish solution containing 2 µM of 4-Di-2-Asp
(4-(4-(diethylamino)styryl)-N-methylpyridinium iodide) to
make the nerve terminals visible with fluorescence microscopy
(Harrington and Atwood, 1995). The nerve terminals remain functional if
the exciting illumination is used for brief periods (Cooper et al.,
1995a). A "macro-patch" electrode (~15 µm inner diameter) was
positioned under visual control over selected nerve terminals.
Locations were chosen such that synaptic currents generated by both the
tonic and phasic terminals could be recorded without moving the
electrode. The nerve bundle containing both the tonic and phasic axons
was dissected out in the ischiopodite and drawn into a tightly fitting
suction electrode for stimulation, using chlorided silver wires inside
and outside the stimulation electrode. First the tonic axon, which has
a larger diameter (Bradacs et al., 1997) and a lower stimulus
threshold, was excited selectively by adjusting the amplitude or
duration of the stimulus, and 500-600 sweeps were recorded at
frequencies of 1, 2, 5, 10, and 20 Hz. After this the stimulus
intensity was increased to recruit also the phasic axon, and 500-600
pulses were recorded at 1 Hz. At low frequencies, the quantal output
from tonic terminals was generally very low (see Fig. 1), so that its
contribution to the phasic excitatory synaptic currents (ESCs) was
negligible. To avoid muscle contractions during the recordings, muscle
fibers were prestretched. Excitatory synaptic currents were recorded on
tape and computer for subsequent analysis.
Electron microscopy. The macro-patch electrode was dipped
into a solution containing spherical polystyrene beads (0.5 µm
diameter) (Duke Scientific Company) and then allowed to dry before
being filled with the crayfish solution for recording. During the
recording session, beads were deposited at the recording site on the
surface of the muscles in a ring corresponding to the rim of the
electrode (Cooper et al., 1995a). The beads marked the recording site,
which could be identified during all stages of specimen preparation and
also in electron micrographs (see Fig. 3) (Wojtowicz et al., 1994).
Fixation, embedding, and serial sectioning of specimens containing
recording sites followed standard procedures (King et al., 1996).
Micrographs were taken from serial sections and enlarged for
measurements. The surface contact area of each synapse was calculated
by measuring its linear dimension in each section in which it appeared,
multiplying this by the thickness of the section, and summing the
results for each section. Synaptic active zones (dense bodies) were
counted, and their lengths were measured.
Data analysis. For the tonic axon, synaptic currents could
usually be resolved into quantal components (see Fig. 2), and the number of quanta released by each impulse at the recording site could
be determined with a high degree of certainty. Quantal contents at
several frequencies were determined, as described by Cooper et al.
(1995b). Quantal counts were analyzed by statistical procedures to
determine whether they could be fitted by Poisson or binomial distributions, as described previously (Cooper et al., 1995a,b). For
the phasic axon, the ESCs were too large and contained too many quantal
units to be resolved accurately in this way. Quantal content was
estimated by averaging the evoked ESCs and dividing this value by the
average of the asynchronous "late" quantal units, which were
resolved in the traces. No attempt was made to determine binomial
parameters for phasic synapses.
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RESULTS |
Properties of transmitter release
Recordings of synaptic currents were made with a macro-patch
electrode at selected sites where phasic and tonic nerve endings were
closely apposed (Figs. 1,
2). Separate stimulation of the two axons
in the motor nerve produced synaptic currents of very different
properties. Synaptic currents generated by the phasic axon were many
times larger than those of the tonic axon; responses of the latter were
small at low frequencies, but facilitated greatly at higher frequencies
(Bradacs et al., 1997).
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Figure 1.
Comparison of synaptic currents during stimulation
of phasic and tonic axons. A1, Individual records of
phasic (P) and tonic (T) responses at the same recording site
during 1 Hz stimulation. A2, Averaged currents (500 stimuli) for phasic and tonic axons during 1 Hz stimulation.
B1, Individual measurements (current-time integrals) of
phasic responses (open circles) and tonic responses (filled circles) for a series of 500 stimuli
delivered at 1 Hz. The regression line shows a characteristic slow
decline in amplitude with time for the phasic responses. Most of the
tonic responses were failures of release at this frequency.
B2-4, Individual responses for 500 stimuli recorded
during stimulation of the tonic axon at 2 Hz (B2), 5 Hz
(B3), and 10 Hz (B4). Most of the
responses at 2 Hz are failures or single quantal events; single quantal events are frequent at 5 Hz, whereas a significant number of
multi-quantal events appear at 10 Hz.
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Figure 2.
Synaptic currents recorded at a single site with
stimulation of the tonic axon. Nerve terminal potentials are indicated
by single asterisks, asynchronously released quantal
units by double asterisks, and near-synchronously
released quantal units by small arrows. Cases of zero,
one, two, three, and four quanta released by nerve impulses during 10 Hz stimulation are illustrated. Individual quantal components are well
resolved.
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When the tonic axon was activated at low frequencies, many impulses
failed to produce a response, although the nerve terminal potential was
always recorded. When synaptic currents did occur, they appeared to be
single quantal units (Figs. 1, 2). As the frequency of stimulation was
increased, the percentage of failures diminished, and a few
multi-quantal responses occurred. Almost always it was possible to
count the number of quantal units in each response by observing the
inflections on the rising phase of the recorded current (Fig. 2).
Variation in quantal unit size occurred, as in previously studied
synapses (Wojtowicz and Atwood, 1986; Wojtowicz et al., 1994).
Therefore, quantal content was determined by counting all the quanta
released by a series of impulses and calculating the mean number of
quanta released per impulse (Cooper et al., 1995b). As expected (Dudel
and Kuffler, 1961), quantal content increased with frequency (Fig. 1,
Table 1).
From counts of the number of impulses releasing different numbers of
quanta, statistical calculations were made to determine whether the
observed distributions could be best fitted by Poisson, uniform
binomial, or nonuniform binomial distributions (Wojtowicz et al., 1991,
1994; Cooper et al., 1995a,b). Results from three sites that were
subsequently reconstructed from electron micrographs are given in Table
1. Quantal contents for these sites (and others not included) were
always <1 at frequencies below 10 Hz. At some sites, quantal content
became >1 at 10 Hz, but at most, quantal contents of 1 or more were
attained at frequencies of 20-40 Hz. At low frequencies (0-5 Hz), the
observed distributions were best fitted by Poisson distributions. At
frequencies of 5 Hz and above, some of the sites became binomial, with
estimates of 1-10 for n in the binomial distributions
(Table 1). Site 3 remained best fitted by Poisson distributions even at
20 Hz. Higher frequencies were not investigated because of the risk of
nonstationarity attributable to movement.
Activation of the phasic axon at low frequencies generated much larger
synaptic currents at the same sites, with no failures of transmission
(Fig. 1). The responses were multi-quantal, and it was not possible to
count reliably the number of quanta in each response. Maintained
stimulation at low frequency generally produced a slowly developing
decrease in the evoked current (Fig. 1); such decline did not occur at
the terminals of the tonic axon. Estimates of quantal content for
phasic terminals were obtained by taking the ratio of the averaged
evoked response to the averaged "late quantal events" seen in the
tail of emission of unitary events after evoked release (Erulkar and
Rahamimoff, 1978; Kita et al., 1981; Ravin et al., 1997). These
estimates of quantal content ranged from 5.2 to 18.0 at the three sites
selected for ultrastructural analysis (Table
2). At other sites not included in the
present analysis, values of quantal content ranged from 8.3 to 15.2. The ratios of phasic to tonic quantal content at the selected sites
ranged from 94 to 1300 (Table 2). There is clearly a large difference
in quantal content at the same recording site in these examples.
Response amplitude for the phasic axon initially grew slightly at
higher frequencies (cf. Bradacs et al., 1997), but the rate of
depression also increased with continuing stimulation, and the
responses were not analyzed further in the present study.
Ultrastructure of nerve terminals
Recording sites were labeled with polystyrene microspheres
deposited around the perimeter of the site by the recording electrode (Fig. 3). The nerve terminals usually
retained their integrity, and individual synapses could be identified
and measured. Electron-dense, uniformly separated pre- and postsynaptic
membranes identified individual synapses, whereas electron-dense
presynaptic projections (dense bodies) marked the active zones (Jahromi
and Atwood, 1974). However, the recording and preparative procedures
caused some degradation in the fine structure of the terminals, as was
evident from comparisons with the same terminals adjacent to the
recording site. In particular, the phasic terminal, which in many
locations was very thin, was sometimes damaged within the recording
site. Three successful reconstructions of recording sites were
completed (out of six attempted), but in one of these the structural
data from the phasic terminal were not complete because of damage, and
data were collected from the same terminal just outside the recording
site so that comparison with the tonic axon in this region could be
completed.
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Figure 3.
Electron micrographs of recorded specimens.
A, Recording site labeled with polystyrene microspheres
(b) left by the recording microelectrode on the
muscle fiber surface. Phasic (p) and tonic (t) nerve terminals populated with clear
spherical vesicles are surrounded by subsynaptic reticulum, and each
shows a synaptic contact (between arrows). Myofibrils
(f) are located below the terminal
region. B, Terminals from the recorded area showing a phasic (p) terminal that is much smaller in size
than its tonic (t) counterpart and has fewer
synapses (between arrows). Note abundant mitochondria in
tonic terminal. s, Subsynaptic reticulum; v, synaptic vesicles. C, Surface view of
an active zone denoted by a dense bar (arrowhead) with
several docked synaptic vesicles (v) in a tonic
(t) nerve terminal. s, Subsynaptic
reticulum. Magnification: A, 21,000×; B,
35,000×; C, 115,000×. Scale bars: A, 2 µm; B, 1 µm; C, 0.2 µm.
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Reconstructions illustrate the general morphological differences
between phasic and tonic terminals (Fig.
4). Larger varicosities and more numerous
synapses per unit length distinguished the tonic terminals. These
features have also been documented in previous observations with light
and electron microscopy (King et al., 1996; Bradacs et al., 1997).
General comparison of the size and complexity of the individual
synapses indicated no major differences between the two terminals (Fig.
4A,B). Most synapses of both terminals had a single
active zone (defined by electron-dense material and associated docked
vesicles at the presynaptic membrane). Synapses with no active zone, or
with two or more, also occurred on both terminals, but generally were
in the minority. At one of the sites (Table
3, Site 2A), the tonic axon in the
recorded area had a higher percentage of "complex" synapses (two
active zones) than observed elsewhere; this site also had the highest
quantal content (Table 1). However, the percentage of "blank"
synapses (no active zones) was also high at this site.
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Figure 4.
A, Three-dimensional
reconstructions of terminals at a recording site, illustrating terminal
morphology of tonic (t) and phasic (p) axons, and individual synapses of varying
complexity (no active zones, red; one active zone,
yellow; two active zones, green). Scale
bar, 2.5 µm. B, Two-dimensional representation of
individual synapses at the recording site, illustrating relative size
and synaptic complexity for the two axons. (Color coding is the same as
in A). There are more individual synapses on the
terminal of the tonic axon, but no obvious superiority in synaptic size
or complexity for either axon at this site. Scale bar, 2.5 µm.
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Morphometric data for the three reconstructed sites (Table 3)
illustrate the major features of the two terminal types. The number of
synapses per unit length of terminal was consistently higher for the
tonic axon. At one site (Site 3), the number of synapses in the
recording site was larger for the phasic axon, but this was
attributable to the presence of two branches of the phasic axon in this
region. The number of synapses per unit length of terminal was larger
for the tonic axon, here as elsewhere. The number of active zones per
synapse was quite similar for the two axons, although the phasic axon
had overall slightly more complex synapses with two or more active
zones. This observation was made also in a previous morphometric study
(King et al., 1996). Synaptic contact area was variable (Fig.
4B), but the mean value was larger for the tonic
terminals (Table 3). Active zone lengths were essentially the same for
both terminals. Thus, for the recording sites sampled, there were no
qualitative differences that stood out in comparisons of the two
terminal types; rather, the differences were quantitative and
relatively minor.
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DISCUSSION |
The present study compared, at the same recording sites on
identified postsynaptic targets, the physiological and morphological parameters of two identifiable neurons known from previous studies to
be physiologically different (Bradacs et al., 1997). An earlier morphological study of these neurons (King et al., 1996) had suggested that ultrastructural differences may not be able to account for the
physiological ones. The present study confirms this view and indicates
clearly that additional factors must be considered in attempting to
explain physiological differences among synapses of different
neurons.
For crustacean motor neurons, many studies have shown that there are
differences in the rate of quantal release among endings of a single
tonic-type neuron (crab opener-stretcher neuron: Atwood, 1967; Atwood
and Bittner, 1971; Sherman and Atwood, 1972; crayfish opener motor
neuron: Bittner, 1968a,b; Cooper et al., 1995a; lobster accessory
flexor motor neuron: Frank, 1973; Walrond et al., 1993). Ultrastructural work on these neurons has consistently yielded structural correlates for synaptic strength. Crustacean synapses are
readily distinguished in electron micrographs on the basis of their
heavily stained, uniformly spaced pre- and postsynaptic membranes, and
the dense bodies of the active zones are also clearly identifiable.
Synapses of terminals releasing relatively large amounts of transmitter
show more numerous active zones and more complex synapses with multiple
active zones (Atwood and Marin, 1983; Cooper et al., 1996) and longer
active zones (Atwood and Marin, 1983; Walrond et al., 1993). More
numerous active zones at a single synapse could provide the conditions
for lateral interaction among them (Cooper et al., 1996), whereas
larger active zones may provide more calcium channels at or near sites
of vesicle docking (Walrond and Reese, 1985; Walrond et al., 1993).
Structural influences on transmitter release can be readily envisioned
from these results.
The present study shows that such arguments, developed from comparisons
of different terminals of the same neuron, cannot be easily
extended to comparisons between different neurons. Synaptic size, active zone size, synaptic complexity, and the number of individual synapses at a recording site are not sufficiently different to account for a 50- to 1500-fold difference in quantal content at the
recording sites. In fact, two of these measures (synapse size and
number of synapses per length of terminal) were inversely related to
quantal content for phasic and tonic terminals in the present
study.
We can also rule out several other possible explanations for the
differences in quantal content at low frequencies. Transmitter availability, judged from quantitative assessments of intraterminal glutamate, is less at phasic than at tonic terminals (Shupliakov et
al., 1995). Vesicles are not markedly more plentiful at phasic synapses
(King et al., 1996); some studies have shown them to be less plentiful
(Atwood and Johnston, 1968; Atwood and Jahromi, 1978). Nerve terminal
action potentials can be consistently recorded extracellularly at both
phasic and tonic terminals (Figs. 1, 2), although some previous studies
have suggested that some of the terminals of the motor axon of the
crayfish opener muscle, a tonic-type neuron, are not fully excitable
(Dudel, 1981). Intracellular recording of action potentials from paired
phasic and tonic motor axons in a crab (Pachygrapsus) have
shown that the action potentials are similar in amplitude and duration
(Stephens et al., 1983; Stephens, 1992). Although it is possible that
the action potentials differ in the fine terminals (where they have not
been recorded with intracellular electrodes), they do not appear to be
different in duration in extracellular recordings at these
locations.
Because in all likelihood the above factors cannot account for the
large difference in low-frequency transmitter release, an explanation
must be sought in other aspects of synaptic transmission. Calcium
channels, and the factors that regulate them, may differ in the two
terminal types. Preliminary evidence for more calcium entry in phasic
terminals has been obtained (Msghina et al., 1995), which suggests a
higher probability for calcium channel opening, or more available
calcium channels, at phasic synapses. Preferential location of more
calcium channels near docked vesicles (Walrond and Reese, 1985) is
another interesting possibility. In addition, there is evidence that at
least one synaptically active regulatory molecule may differ in its
expression in phasic and tonic terminals (Atwood et al., 1995). Other
molecules of the vesicle docking and release complex (Bennett and
Scheller, 1994; Südhof, 1995) have not been assessed
quantitatively. Additional indirect evidence of a role for calcium
channels comes from studies in which calcium currents of crustacean
neurons are altered by neuronal activity in a way that correlates with
activity-induced changes in synaptic transmission: evoked transmitter
release (Lnenicka and Atwood, 1985) and one type of calcium current are
both adaptively downregulated in phasic neurons subjected to more than
the normal amount of electrical activity (Hong and Lnenicka, 1995,
1997).
The fact that terminals of such different properties occur together on
the same postsynaptic target strongly indicates that presynaptic
influences are important in establishing and regulating synaptic
phenotype. Neuronal activity level is one presynaptic influence that is
thought to determine both synaptic phenotype (Lnenicka and Atwood,
1988) and terminal morphology (Lnenicka et al., 1986) in phasic and
tonic crustacean neurons. In contrast, when different endings of the
same motor neuron innervate separate postsynaptic targets, retrograde
influences from the latter are likely to play a major role in
determining synaptic phenotype (Frank, 1973; Davis and Murphey,
1994).
The tonic axon of the leg extensor muscle is unusual among crustacean
motor neurons in its very low quantal content at frequencies below 20 Hz (Bradacs et al., 1997). This leads to typical Poisson distributions
at low frequencies (Table 1). At higher frequencies, binomial
distributions become the best fits for some sites, suggesting the
emergence of a few synapses that transmit more reliably. Values for
binomial n remain much smaller than the total number of
available synapses, but n increases with frequency in this
neuron, as in the crayfish opener motor neuron (Wojtowicz et al.,
1994). These observations are in accordance with the hypothesis that
binomial n represents the number of active synapses or
performing active zones (Zucker, 1973; Schikorski and Stevens, 1997),
and that in the crayfish tonic neurons more synapses are recruited to
observable activity as the frequency increases. By contrast, in the
phasic neuron, a greater percentage of the available synapses are
active at low frequencies, contributing to the observed (often large) values for quantal content. Optical studies of active synapses with the
fluorescent dye FM1-43 lend support this general hypothesis (Msghina
et al., 1995; Quigley et al., 1996).
The observations on synaptic diversity and its morphological
correlates, which have a long history in crustacean (Atwood, 1967;
Jahromi and Atwood, 1974) and amphibian (Nudell and Grinnell, 1983;
Robitaille and Tremblay, 1987) motor neurons, is now paralleled by an
increasing number of studies on mammalian central neurons (Pierce and
Mendel, 1993; Harris and Sultan, 1995; Murthy et al., 1997; Schikorski
and Stevens, 1997; Turner et al., 1997). Some of the same general
structure-function relationships are being found, including
heterogeneity of release properties and correlation between active zone
structural features and release properties. The present observations,
together with previous ones on phasic and tonic crustacean neurons,
emphasize that the structure-function relationships can be altered
from one neuron to another, even when two neurons innervate the same
postsynaptic targets.
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FOOTNOTES |
Received Oct. 15, 1997; accepted Nov. 24, 1997.
This work was supported by grants from the Medical Research Council of
Canada (H.L.A.) and Natural Sciences and Engineering Research Council
of Canada (C.K.G.) and the Faculty of Medicine, University of Toronto
(M.M.). Contributions of Dr. Leo Marin and Joanne Pearce to electron
microscopy, and Marianne Hegström-Wojtowicz to manuscript
preparations were important for completion of this work.
Correspondence should be addressed to Dr. Harold L. Atwood, Department
of Physiology, Medical Sciences Building, University of Toronto,
Toronto, Ontario, Canada M5S 1A8.
Dr. Msghina's present address: Department of Physiology and
Pharmacology, Karolinska Institutet, S-171 77, Stockholm,
Sweden.
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Top
Abstract
Introduction
