AMER. ZOOL., 13:337-355 (1973).
Crustacean Neuromuscular Mechanisms: Functional Morphology
of Nerve Terminals and the Mechanism of
Facilitation
FRED LANG
Boston University Marine Program, Marine Biological Laboratory,
Woods Hole, Massachusetts 02543
AND
HAROLD L. ATWOOD
Department of Zoology, University of Toronto, Toronto, Ontario, Canada
SYNOPSIS. Two aspects of crustacean neuromuscular physiology are discussed: (1) the
ultrastrucUiral identification of the excitatory and inhibitory nerve terminals, and (2)
the characteristics of, and the possible mechanisms for, facilitation.
The first problem was studied in crayfish opener muscle which has one excitatory
and one inhibitory axon. One of the nerves was stimulated in the presence of DNP
until synaptic transmission failed; the preparations were then fixed for electron microscopy. Whenever the excitatory nerve was stimulated, the terminals with round
synaptic vesicles were depleted while nearby terminals with smaller elongate vesicles
were normal. When the inhibitory nerve was stimulated, the converse was true.
The possible reasons for the diversity in crustacean neuromuscular properties are
discussed. Large EPSP's with a high quantal content (in) , appear to be produced by
terminals which are invaded by a propagated spike. Small EPSP's (small m) appear
to be produced by terminals which don't spike and which are depolarized by a decrementally conducted potential. There is an inverse relationship between m and the
amount of facilitation. The physiological basis for facilitation is discussed; previous
hypotheses are found wanting and a new one is proposed, that of slow depolarization.
One of the characteristics of invertebrate axons, but inhibitory axons as well (Fig.
nervous systems is that they have evolved 1).
in a parsimonious fashion and nowhere is
The synaptic transmitter substances of
this as evident as in the innervation of these axons have been shown to be differcrustacean skeletal muscle. Here, as has ent. The work of Kravitz and his colleagues
been pointed out so often, there are only (Kravitz, 1967, 1968) and of Takeuchi and
a few axons serving each muscle, and in- Takeiichi (1967) led to the conclusion that
deed, some axons are shared by two or the inhibitory transmitter is y-aminobutymore muscles. In contrast to the vertebrate ric acid (GABA). Other work has supportsystem, where hundreds of axons serve each ed the view that the excitatory transmitter
ntajor skeletal muscle, the crustacean sys- in crustacean leg muscles may be glutatem seems very simple. However, as the mate (Takeuchi and Takeuchi, 1964; Taearly investigators showed, upon close in- raskevich, 1971). However, a recent study
spection, the simplicity breaks down (for suggests that this may not be the case for
review, see Wiersma, 1961). The crustacean all crustacean skeletal muscles (Futamachi,
limb muscles, as well as those of other ar- 1972).
thropods (Pearson, 1973; Fourtner and
The inhibitory influence on the muscle
Sherman, 1973) receive not only excitatory fibers can be of two sorts, direct and indirect. In the former, termed postsynaptic inhibitio
" ' * e " ^ ^ i t o r y nerve terminals
grant A-2352 (H. L. A.). Contribution No. 556
from the Bermuda Biological station.
synapse directly on the muscle fiber. In the
latter, termed presynaptic inhibition, the
337
338
FRED LANG AND HAROLD L. ATWOOD
NERVE
TERMINALS
EXCITATORY
FIG. 1. Diagrammatic representation o£ the excitatory and inhibitory innervation of a crustacean
muscle fiber. Excitatory nerve terminals form neuromuscular synapses. Inhibitory terminals form
neuromuscular synapses
(postsynaptic inhibition) and axo-axonal synapses upon excitatory terminals (presynaptic inhibition) .
inhibitory nerve terminals synapse upon
the nerve terminals o£ the excitatory nerve
which in turn synapses on a muscle fiber
(Fig. 1). The latter type of inhibition is
more restricted in its distribution than the
former (Atwood, 1968). Thus, there are
two very different kinds of nerves, which
are physiologically and pharmacologically
distinct, serving most crustacean muscles.
The ultrastructural identification of the
nerve terminals of these axons is the first
problem to which this paper will be addressed.
In addition, the excitatory and inhibitory nerve terminals themselves each exhibit heterogeneity. The physiological variations for both are similar (Atwood and
Bittner, 1971), so the discussion will center upon the excitatory terminals which
are more easily studied. If one looks at the
excitatory postsynaptic potentials (EPSP's)
in a crustacean muscle with a single excitatory axon, a wide range of variation is evident. Some muscle fibers have EPSP's
which are initially large (10-30 mV) and
which, with repetitive stimulation show
little facilitation (Fig. 2). Other fibers in
the same muscle have small EPSP's (1.0-5
mV) which show considerable growth or
facilitation with repetitive stimulation.
The differences have been shown to be pre-
synaptic in origin and attributable to differences in the quantal content of released
transmitter (Dudel and Kuffler, 19616; Atwood, 1967; Bittner and Kennedy, 1970).
These types of terminals have been known
or suspected for some time (Hoyle and
Wiersma, 1958), but it is only within the
last, few years that their importance for
control of muscle contraction has been understood (Atwood, 1965; Bittner, 1968).
However, the physiological basis for the
different kinds of terminal, that is, the
poorly facilitating and the strongly facilitating, has received little attention. It is
this problem which will be the subject of
the second part of the paper.
ULTRASTRUCTURAL IDENTIFICATION
OF NERVE TERMINALS
Early attempts to characterize differences
between crustacean excitatory and inhibitory nerve terminals were not successful
(Peterson and Pepe, 1961). Later studies
proved more rewarding; it was shown that,
on the average, the synaptic vesicles in putative excitatory nerve terminals were circular while those in putative inhibitory
terminals were smaller and ellipsoid or irregular in shape (Uchizono, 1967; Atwood
and Jones, 1967). These studies were concerned with inhibitory nerve terminals on
the crayfish stretch receptor (Uchizono,
1967) and with crayfish axo-axonal synEXCITATORY NERVE
I .,
FIG. 2. Excitatory synapses on two muscle fibers
of a crustacean muscle with a single motor axon.
The long sarcomere fiber (left) receives terminals
which form large synapses and which give large,
poorly facilitating EPSP's (low F c ). The short sarcomere fiber receives terminals which form small
synapses and which give initially small but strongly facilitating EPSP's (high F o ). Characteristic
EPSP's are shown below each fiber.
339
CRUSTACEAN NEURO>TUSCULAR MECHANISMS
apses (Atwood and Jones, 1967). The observations, although suggestive, were still
based on inference, and a more direct
method of identification, particularly of
neuromuscular terminals was desirable.
A method which can be used to differentiate between the two types of terminals is
to selectively deplete the synaptic vesicles
from the terminals of one axon so that
they can be easily identified ultrastructurally (Atwood et al., 1972). For this purpose,
the crayfish opener muscle was used because it is innervated by a single excitatory and a single inhibitory axon, which
provide a relatively simple situation for
investigation.
Several characteristics of crustacean systems render some of the conventional methods for identifying terminals useless. Severing an axon to cause degeneration cannot
be used because crustacean nerve terminals
remain viable for several months after they
have been separated from the soma by cutting the axon (Hoy, 1969). Likewise, continuous stimulation of one axon at relatively high frequencies (10-20 Hz) for several hours doesn't lead to failure of transmission and depletion of transmitter. On
the contrary, in addition to the initial rapid facilitation of the EPSP, stimulation
over a long period leads to a steady increase in EPSP size. This phenomenon,
called "long term facilitation," leads to a
slow increase in quantal output and is
probably due to accumulation of sodium
ions in the terminals (Sherman and Atwood, 1971).
Our approach to the problem was to
repetitively stimulate one of the axons to
the muscle while attempting to interfere
with replenishment of the transmitter
stores by using a metabolic inhibitor. For
this purpose we used 0.2 to 0.7 mM 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation (Loomis and Lipman,
1948) which has been shown to penetrate
nerve cells (Caldwell, 1960). If DNP is added to the physiological solution while the
excitatory axon is stimulated at 10-15 Hz,
the EPSP exhibits a very rapid increase in
size (10-12X) within 10 minutes (Fig. 3).
Control
2 min
,r
30 ••
40
•
10 ms
FIG. 3. Effect of DNP on crayfish opener muscle.
EPSP's in control and DNP treated preparations after 2, 20, 30, and 40 min. Continuous
stimulation at 10 Hz.
This increase is sustained for several minutes, after which the EPSP amplitude declines rapidly until it is no longer visible
30-40 minutes after introduction of the
DNP (Fig. 4, continuous line). If stimulation is stopped for a few minutes, a small
but transient recovery of the EPSP is obtained; if stimulation is resumed at this
point, the EPSP rapidly disappears. Stimulating the axon intermittently (for a 20 second period once every 4 minutes) in the
presence of DNP, results in an increase in
EPSP amplitude, but the increase is not as
rapid or striking as with continuous stimulation (Fig. 4, dashed line). In addition,
>
E
E
<
4
-10
/A \
3DN P
6
/
A
o
1
10
Time
20
30
40
SO
M
(minutes)
FIG. 4. Effects of continuous and intermittent stimulation on DNP treated crayfish opener muscle.
Stimulation at 10 Hz continuously (solid line) and
intermittently, for 20 sec every 4 min (dashed
line).
340
FRED LANG AND HAROLD L. ATWOOD
resulting from ouabain treatment, this compound is ineffective for depletion studies.
Shortly after the maximal EPSP size was
obtained, the EPSP amplitudes exhibited
large fluctuations, and transmission failures were common. Complete blockage of
transmission followed soon after, probably
as a result of depolarization blockade in
the fine nerve branches of the stimulated
axon (Sherman and Atwood, 1971). An alternative explanation for the action of
DNP on transmitter release is suggested by
the work of Lehninger et al. (1967) and
Godfraind et al. (1971). They suggest that
DNP might be effective in releasing Ca2+
from mitochondria. This increase in intracellular calcium might be effective in increasing transmitter release (Llinas et al.,
1972).
Electron microscopic examination of
these preparations confirmed our deductions regarding the presence of synaptic
vesicles. In ouabain treated preparations,
nerve terminals invariably contained a full
complement of synaptic vesicles (Sherman
and Atwood, 1971). Examination of stimulated, DNP-treated preparations on the
other hand, revealed that the nerve terminals of the stimulated axon were selectively depleted of their synaptic vesicles. When
the excitatory axon was stimulated in the
presence of DNP, nerve terminals which
contained round vesicles were found to
contain few, if any, synaptic vesicles; nearby terminals with less regular vesicles appeared completely normal (Fig. 6). The
converse was true if the inhibitory axon
was stimulated.
In addition to loss of synaptic vesicles, a
common feature in the DNP-treated preparation was vacuolation and apparent swelling of the terminals of the stimulated axon
(Fig. 6). Similar features have been described in nerve terminals of vertebrate
sympathetic ganglia and neuromuscular
junctions treated for 3 hours with cardiac
glycosides (Birks, 1962). In this case, the
FIG. 5. Effects of ouabain on EPSP's in crayfish loss of synaptic vesicles and the vacuolation
opener muscle. Continuous stimulation (10 Hz) in occurred without stimulation and was atthe presence of ouabain (\0~* M) results in a strik- tributed to swelling of terminals due to acing increase in EPSP size (solid line) as compared
cumulation of Na+.
to a control (dashed line) .
transmission failure does not occur, the increase in EPSP size is sustained for at least
1-5 hours.
The increase in size of the EPSP was
shown to be due to a presynaptic mechanism. Recording from single synaptic sites
with an external niicroelectrode (see Dudel
and Kuffler, 1961a) showed that the changes
in EPSP size reflected changes in the quantal content of released transmitter from the
nerve terminals.
The effects of DNP were complex, but
the use of other techniques which mimicked some of its actions shed light on some
of the mechanisms involved. DNP is a powerful blocker of oxidative phosphorylation
and would cause a rapid depletion of the
energy reserves of a metabolically active
cell. This, in turn, would affect all energy
dependent processes, including active transport. More specifically, interference with
the sodium pump would result in an accumulation of sodium in the nerve terminals which might cause the increase in
transmitter release (Sherman and Atwood,
1971). This hypothesis is sustained by experiments in which stimulated preparations were bathed in physiological solution
containing ouabain. In this case, EPSP's increased six to eight times their initial
height (Fig. 5, continuous line) in contrast
to preparations stimulated without ouabain, in which the EPSP increased two to
three times (Fig. 5, dashed line). In spite
of the large increase in transmitter output
CRUSTACEAN NEUROMUSCULAR MECHANISMS
341
FIG. 6. Effects of DNP on the presence o£ synaptic vesicles in nerve terminals from crayfish opener
muscle. The excitatory nerve was stimulated for 1
hi" at 10 Hz in the presence (A,C,D) and absence
(li) of 0.5 mM DNP. The synapses are indicated
by arrows in these electron micrographs. In A and
B, both excitatory (e) and inhibitory (i) terminals
are present; the former contain round synaptic
vesicles (sv) while the latter contain smaller,
elongate vesicles. A, adjacent e- and i-terminals
are located just under the sarcolemma. The e-ter-
minal contains only a few synaptic vesicles while
the i-termiual contains many. In B, a control, the
i-terminal forms an axoaxonal synapse with e,
which in turn forms neuromuscular synapses with
the muscle fiber (m) . Vesicles are present even
after 1 hr of stimulation at 10 Hz. C, synapse of
a depleted e-lerminal, and D, an adjacent i-terminal in a different preparation from A. Note
dense-cored vesicle (d) and microtubule (t) in C.
Scale: A, 0.5 ^m; B-D, 0.3 ^m. (From Atwood
et al., 1972.)
Thus, the DNP experiments confirm, by
a direct method, the previous identification
of nerve terminals with spherical and lessregular synaptic vesicles as excitatory and
inhibitory respectively. It also provides a
technique for identifying the terminals of
any specific neuron, even in a complex system, provided that the neuron can be selectively stimulated.
The results also lend support to the vesicle hypothesis of synaptic transmission;
transmission was shown to depend upon
the presence of synaptic vesicles. In addi-
tion, the presence and replenishment of
synaptic vesicles in these terminals appears
to depend on a rapid energy-dependent
process which occurs in the terminals themselves. Indeed, stimulation of the nerves,
even for several hours at 15 Hz, failed to
impede synaptic transmission unless DNP
was present. This suggested that replenishment of vesicles is not primarily via axoplasmic flow from the soma, if it occurs at
all. This last contention is supported by
two previous observations. First, crustacean
nerve terminals remain viable even several
342
FRED LANG AND HAROLD L. ATWOOD
months after being severed from the rest
of the neuron (Hoy, 1969). Secondly, there
is some evidence which suggests that either
synaptic vesicles are formed directly or indirectly from the nerve terminal membrane
or that they merely empty their contents
into the synaptic cleft by exocytosis and
are reused (Bittner and Kennedy, 1970;
Holtzman et al., 1971).
A POSSIBLE BASIS FOR FACILITATION AT
CRUSTACEAN NEUROMUSCULAR
JUNCTIONS
Differences in nerve terminals
Facilitation, in the present context, is
the process whereby repetitive stimulation
causes a greater probability of transmitter
release at a synapse. Thus, each EPSP in a
train is successfully larger than the previous one until a final maximal level is
reached. A convenient measure of facilitation is F e (Atwood and Bittner, 1971)
where:
EPSP size at 10 Hz
EPSP size at 1 Hz
Both the amount of transmitter release
(quantal content) at low frequencies of
stimulation (< 1 Hz) and the amount of
facilitation at higher frequencies (> 2-4
Hz) varies widely in crustacean neuromuscular systems, even from synapse to synapse
among the terminals of a single axon. For
example, the stretcher muscle of Grapsus
is innervated by one excitatory axon. The
EPSP's evoked in the muscle fibers by
stimulation of this axon exhibit a variety
of characteristics which run the gamut between two extremes: (1) large EPSP's (1030 mV) with a large quantal content at low
frequencies and which facilitate poorly at
high frequencies (low Fe), and (2) small
EPSP's ( < 1 mV-5 mV) with a low quantal content at low frequencies which facilitate strongly at high frequencies (high Fe).
Examples of the extreme types as well as
an intermediate type are shown in Fig. 7.
The physiological basis for these differences in transmitter release and facilitation
FIG. 7. Diversity of electrical responses in three
muscle fibers of Grapsus stretcher muscle. A, the
EPSP's are large at low frequency and do not facilitate at higher frequency (low F6 fiber) . B, the
EPSP's are very small initially, but exhibit strong
facilitation (high Fe) ; at high frequency, an active membrane response is present (Bs) . C, an intermediate fiber, has fairly large EPSP's which
facilitate; large graded membrane responses occur
at relatively low frequency. Stimulation frequencies: A,, 1 Hz; A,, 2 Hz; As, 5 Hz; At, 10 Hz; B,,
20 Hz; Bt, 40 Hz; B,, 60 Hz; C, 10 Hz. Calibration:
vertical, 20 mV; horizontal, A-B, 1 sec; C, 0.5 sec.
(From Atwood and Bittner, 1971.)
have yet to be fully elucidated and, in fact,
have received little attention. Sherman and
Atwood (1972) have shown that large EP
SP's are associated with relatively large synaptic release areas (of opposed synaptic
membrane) while small EPSP's are associated with much smaller release areas.
However, the number of terminals and
the total amount of release area is not
known for each type of fiber. Physiological
evidence, to be presented below, suggests
that the total amount of release area may
not differ greatly between the low and high
Fe fibers.
Another possible basis for differences
among nerve terminals could involve quantitative differences in Ca2+ mobilization,
since a residuum of Ca2+ within the terminal is thought to be the basis of facilitation
at frog and squid synapses (Katz and Miledi, 19656., 1968a). However, the calcium
residue hypothesis is less able to account
for facilitation at crustacean neuromuscular synapses since the relationship between
external Ca2+ concentration and transmitter release is linear in a crayfish muscle
(Bracho and Orkand, 1970). This relationship would not generate the observed
amounts of facilitation in this system. Furthermore, it has been found that facilita-
CRUSTACEAN NEUROMUSCULAR MECHANISMS
tion at crab neuromuscular junctions is not
significantly affected by a wide range of external Ca2+ concentrations (T. Linder, personal communication).
A third possibility for differences among
nerve terminals could be differential invasion of the action potential in different
nerve terminals (Atwood, 1967). This hypothesis stems from the work of Dudel and
Kuffler (1961a>) and Dudel (1965a) who
recorded directly from single synaptic sites
using focal, extracellular electrodes. In this
manner, it is possible to visualize not only
externally recorded synaptic potentials (ER
SP's) from a single synaptic area, but also
a nerve terminal potential (NTP). The
NTP precedes the ERSP by one half to one
milliseconds and is a monopolar recording of current flow from the nerve terminal. Using this technique on the strongly
facilitating synapses of the crayfish opener
muscle, Dudel and Kuffler (1961a) showed
that NTP's recorded near the synapses of
nerve terminals were monophasic and positive, suggesting decremental conduction.
More proximal recordings from the nerve
terminal resulted in diphasic or triphasic
waveforms with a prominent negative
phase, suggesting that an action potential
propagated through this region. Atwood
(1967), on the other hand, suggested that
poorly facilitating synapses were located on
large nerve branches which support a
propagated action potential. Thus, it is
possible that some terminals are more completely invaded by the action potentials
than others, thereby giving different
amounts of transmitter release.
Another interpretation for the development of monophasic positive NTP's has
been made by Katz and Miledi (1965a).
Recording from frog neuromuscular junctions, which are believed to be fully invaded by an action potential, these workers
have found that monophasic positive
NTP's can be obtained when the electrode
is over a closed end of the nerve terminal.
However, Dudel (19656) found that during presynaptic inhibition in the crayfish
preparation, the biphasic NTP can be converted into a monophasic positive NTP.
343
This observation suggests that the nerve
terminals have a low safety factor for transmission, a property which is utilized during presynaptic inhibition. In addition,
Dudel's (19656) results suggest that monophasic positive waveforms can be recorded
at other than closed endings of nerve terminals. Thus, the fine nerve branches
which have been shown to be present at
these terminals (Lang et al., 1972) may conduct only decremental potentials rather
than propagated spikes.
Our experiments have tended to support
the view of Dudel (1965a) regarding the
shape of the NTP. In preparations where
it had proved possible to follow fine nerve
branches visibly, we were able to record
from several locations along the length of
the nerve terminal regions. On poorly facilitating muscle fibers the recorded NTP's
were invariably biphasic or monophasic
with a prominent negative phase (Fig. 8A).
We were unable to record monophasic
positive NTP's from such fibers, although
theoretically, this should be possible (e.g.,
Katz and Miledi 1965a). In strongly facilitating fibers, on the other hand, we were
able to record NTP's similar to those in
the poorly facilitating fibers as well as
monophasic positive NTP's (Fig. 8B). Using the amplitude of the ERSP as a criterion for proximity to the synapse, the
largest ERSP's were associated with monophasic positive NTP's in the strongly facilitating fibers. An example of two records
from a nerve branch are illustrated in Fig.
8B. The diphasic NTP was associated with
a smaller synaptic potential and a longer
average latency than the monophasic potential, which was seen when the recording
electrode was moved 20-30/;. distally, along
the same nerve branch. Both of these criteria, the size of the ERSP and the latency,
suggest that the biphasic NTP was recorded further from the actual site of transmitter release than the monophasic NTP.
However, the known complexity of synaptic areas does not permit an unequivocal
interpretation (Lang et al., 1972).
These observations suggest an hypothesis for the differences in transmitter release
344
FRED LANG AND HAROLD L. ATWOOD
B,
L
FIG. 8. Recording o£ NTP's and ERSP's along a
nerve branch. Fine nerve branches were visualized
on Pachygrapsus stretcher muscle fibers and focally applied, extracellular microelectrodes were used
to record from the neuromuscular junctions. Intracellular EPSP's were monitored with a second
microelectrode (records not shown) . A, a low Fe
fiber, four successive records were made at 20-30
^m steps along the nerve. The order of the records
is (1) Au top; (2) Au bottom; (3) Alt top; (4)
A,, bottom. The NTP's are all diphasic, suggesting
active conduction of a spike. The ERSP's are relatively constant in size, suggesting a large release
area. B, a high Fo fiber, two successive records at
about 25 iim separation. B, shows a diphasic NTP;
Bi, made near the point where the nerve branch
was no longer distinguishable, has a monophasic
positive NTP, suggesting decremental conduction.
Bi has a larger ERSP than B, suggesting proximity
to the synaptic release site. All records are 128
sweeps, averaged. Stimulation frequency: A, 1 Hz;
B, 4 Hz. Calibration: vertical, A, 0.2 mV; B, 0.1
mV; horizontal, A, 2 msec; B, 1 msec.
among terminals: The poorly facilitating
synapses produce large EPSP's which
change little in size, even at high frequencies, because the terminals are fully depolarized, or nearly so, as a result of each
axon spike. Thus, they release a relatively
large fraction of the "immediately available store" of transmitter. Some of the
strongly facilitating synapses, on the other
hand, may be located on terminals which
are partially depolarized with each axon
spike due to decremental conduction into
the fine nerve branches. Successive spikes
would either depolarize these terminals
more and more by some mechanism which
would then result in an increasing amount
of transmitter release, or they would operate to recruit more Ca24~ and thereby release successively larger amounts of the
"immediately available" transmitter.
An alternate hypothesis would be that
both the strongly and poorly facilitating
terminals support a spike for all or most
of the length of the terminals and that facilitation is due to some subsequent step
in what has been termed excitation-secretion (E-S) coupling (Douglas and Rubin,
1961). If the alternate hypothesis is correct, one would expect that an increase in
the depolarization of the nerve terminals
would not have a striking effect on the
amount of transmitter release, or if it did
increase transmitter release, one would not
expect a large differential effect on the two
types of terminals.
Experiments with cesium ion
To test the hypothesis, we used Cs+ to
replace the K.+ in the physiological solution. Cs+ has been shown to increase transmitter release at both crustacean (Gainer
et al., 1967) and frog (Ginsborg and Hamilton, 1968) neuromuscular junctions. In
both of these studies, the action of Cs +
was shown to be presynaptic; its effect
could not be explained by removal of K +
from the solution, changes in sensitivity of
the postsynaptic receptors to the transmitter, changes in postsynaptic membrane resistance, or changes in the reversal potential for EPSP's (Gainer et al., 1967).
Although Cs+ is impermeant in squid
axon membrane (Pickard et al., 1964),
when introduced intracellularly it increases the duration of the action potential in both squid giant axon (Baker et
al., 1962; Sjodin, 1967; Adelman and
Senft, 1967) and cat spinal motoneurons
(Araki et al., 1962). For this reason both
Gainer et al. (1967) and Hamilton and
Ginsborg (1968) suggested that Cs+ might
prolong the presynaptic action potential
and thereby increase transmitter release in
this manner. Our studies have sustained
tin's hypothesis and have also shown that
over a period of several hours, Cs+ has
little effect on crab motor axons in the
leg nerve but can apparently be taken up
by synapse-bearing nerve terminals (Atwood and Lang, 1973).
The effect of Cs + was studied in three
species of crab, Hyas, Grapsus, and
CRUSTACEAN NEUROMUSCULAR MECHANISMS
Pachygrapsus, and the results were consistent among the animals. When the K+
(8 imi) in the physiological solution was
replaced by a like amount of. Cs+, an increase in EPSP size was obvious after 3060 minutes. The increase in EPSP size occurred in conjunction with an increase in
duration of the externally recorded nerve
terminal potentials (NTP's) of both low F c
and high Fo fibers (Fig. 9). The amplitude
of the NTP's did not appear to change significantly, but one cannot be completely
confident of changes in this parameter
since the height of the NTP is so critically dependent on the electrode placement.
In addition, experiments in which the
solution was changed to introduce Cs+
usually lasted nearly an hour. Small
changes in the relative position of the
preparation and the recording electrode
may have occurred during this time,
not only from disturbance of the solution, but also from twitches which often
occur in Cs+-treated, high Fe fibers in response to stimulation at 1 Hz. The duration of the NTP, on the other hand, is
probably not as greatly influenced by electrode placement as is the amplitude. It
Control
Cs+
L
FIG. 9. Effect on Cs+ on ERSP's and NTP's at
Crapsus (A) and Hyas (B) neuromuscular junctions. A,, A2, top trace, EPSP's; bottom trace,
NTP's and ERSP's from a low Fc fiber,
B,, B,, NTP's and ERSP's of high F e fiber obtained with a signal averager. A,-B,, control; ArB»
40 min after Cs+ treatment, all 1 Hz stimulation.
Note increase in duration of NTP's and larger amplitude of ERSP's. Calibration: vertical, A, top,
10 mV; A, top, 20 mV; A,-A3 bottom, 0.4 mV;
BrB2, 0.1 mV; horizontal, A, 4 msec; B, 3 msec.
(Atwood and Lang, 1973.)
345
Bi
FIG. 10. Effects of Cs* on EPSP's (top traces) and
ERSP's (bottom traces) , in Grapsus stretcher
muscle. Each series (A,B,C) is from a single fiber
at 1 Hz stimulation, with two-to-four sweeps
superimposed. A,, Bv Cu control; At, B,, C,, 10
min after Cs+; AB, Bs, 20 min after Cs+; At, Bt,
Cj, 35 min after Cs+. Note spontaneous ERSP
(arrow) in Bt. Calibration: horizontal, A-B 4 msec;
C, 20 msec. Vertical, upper traces: A,, 4 mV; A,.t,
B,.t, C,, 10 mV; C^s, 20 mV. Lower traces:
A,-%, B,, 0.4 mV; B2.t, C,.,, 1 mV. ERSP in B, has
been retouched. (Atwood and Lang, 1973.)
seems likely that Cs+ increases transmitter output through its action in prolonging the nerve terminal potential.
Although an increase in EPSP size was
observed in all impaled muscle fibers, the
effect was far more striking in those that
originally had small, strongly facilitating
(high FP) EPSP's than in those with large,
poorly facilitating (low Fe) EPSP's. The
latter showed an increase of about 2.5
times their initial size while the former
increased 30-80 times their initial size (Fig.
10). In addition, after the effect was maximal (60-90 minutes) the initially high Fo
fibers were statistically indistinguishable
from the low Fe fibers (Fig. 11) on the
basis of EPSP size and facilitation; the
differences in their postsynaptic membrane
properties persisted, however (Atwood and
Lang, 1973).
The increase in EPSP size in the presence of Cs+ was shown to be due to an
increase in quantal content of transmission at individual synaptic areas. Using
intracellular and focally applied extracel-
346
FRED LANG AND HAROLD L. ATWOOD
FIG. II. Effect of Cs+ on El'SP amplitude and Fc
in Grapsus stretcher muscle fibers. Dashed line indicates best fit for a large sample of EPSP's measured without Cs+. Open circles show records
from single fibers before Cs+ and closed circles,
joined by solid line, from same fiber after 1 hr
of Cs+ treatment. (Atwood and Lang, 1973.)
lular microelectrodes, EPSP's and ERSP's
(externally recorded synaptic potentials)
were recorded from synapses on muscle
fibers having different Fe values. The
ERSP's of the low F,, fibers, which were initially large, increased severalfold in Cs+,
paralleling the increase in EPSP size in
these fibers (Fig. IOC). The ERSP's of the
high Fp fibers, which were initially small
and often had many failures, increased
strikingly in Cs+, and there were few
failures (Fig. 10/4). After prolonged treatment with Cs+, EPSP's which were strongly facilitating to start with were converted
to dcjacilitating EPSP's. Defacilitation
was shown at single synaptic foci on these
fibers by external microelectrode recordings. Apparently, the drain on the immediately available transmitter store is increased to such an extent by Cs+ treatment that facilitation no longer occurs,
and the synapse comes to resemble the
mammalian neuromuscular junction in its
behavior (for review, see Hubbard et al.,
1969).
What can account for the differential
effect of Cs on the low Fe and high Fe
terminals? We believe that high Fe terminals show a greater response to Cs+ because initially they are less completely depolarized by the axon spike. After Cs+
treatment, they become more completely
depolarized and are able to release a much
larger fraction of their "immediately
available" transmitter store for each nerve
impulse.
It is possible that Cs+ has some other
effect, perhaps on a step subsequent to depolarization in E-S coupling. For instance,
Cs+ may increase the immediately available store in high Fe fibers to a much
greater extent than in low Fe fibers. This
seems unlikely, however. In spite of the
fact that high Fe fibers have very small
EPSP's at 1 Hz, the immediately available
store of transmitter must be large in these
terminals. When they are stimulated at
high frequencies, EPSP's facilitate to relatively large amplitudes. In addition,
EPSP's evoked by post-tetanic potentiation
(PTP) are very large, even after a tetanic
train of several seconds duration. This
suggests that the immediately available
store of transmitter is very large in these
high Fe fibers or that mobilization of transmitter is unusually rapid. The fact that
Cs+ increased the depolarization of the
terminals, and thereby obscured the differences between the low Fe and high Fe
synapses, suggests that the level of depolarization may be the critical difference between them.
In spite of the fact that the EPSP's of
the high Te fibers in Cs+ were as large as,
or larger than those of low Tv fibers, the absolute size and quantal content of ERSP's
remained smaller. At single synapses of
Grapsus, the quantal content of low F c
ERSP's increased three to eight times after
Cs+ treatment to final m values of 10-20,
whereas the quantal content of high Fe
ERSP's increased 10-100 times after Cs+
treatment, to final m values of 1-5. This
finding supports the ultrastructural finding that the high FP synapses are smaller
than the low F(. synapses (Sherman and
Atwood, 1972) and suggests that they are
capable of releasing smaller amounts of
transmitter even with optimal E-S cou-
347
CRUSTACEAN NEUROMUSCULAR MECHANISMS
pling. It further suggests that there may be
more terminals on high Fe fibers and that
the total amount of transmitter release
areas may not differ greatly between the
two types of fibers, since after Cs+ treatment the EPSP's are of similar size in all
fibers at low frequencies of stimulation.
A crude comparative estimate of the
number of synapses on crab stretcher
muscle fibers is given in Table 1, where
the relative number of terminals is computed for different types of fiber. The calculations depend on a number of assumptions: (1) individual quanta from all terminals are equal in size, (2) ERSP's of a
given fiber are similar at all synaptic foci,
and (3) quantal effectiveness is constant
(i.e., a single quantum produces a constant
amount of depolarization for all fibers).
The first assumption is reasonable since all
terminals belong to a single axon. The
work of Sherman and Atwood (1972) suggests that the second assumption is a good
approximation for small crab fibers, although further testing is necessary, especially for large fibers (Lang et al., 1970) in
which there are indications of different
types of synapse along the length of the
fibers. The third assumption is not strictly a good one since the low Fe fibers have
a higher input resistance, thus making each
quantum more effective (Sherman and Atwood, 1972). This is at least partially offset by the fact that the low Fe fibers also
exhibit more pronounced membrane rectification with depolarization than high Fe
fibers (Sherman and Atwood, 1972). However, for small EPSP amplitudes, where
rectification is minimal, the difference in
quantal effectiveness must be taken into
account by correcting for differences in
input resistance.
Table 1 includes EPSP sizes from a
variety of fiber types and calculations of
m, the average quantal size, from single
terminals on the same fibers at 1 Hz stimulation. Assuming a quantal effectiveness,
x, of 0.1 mV for low Fe synapses (as observed from measurements of spontaneous
miniature potentials), the quantal effectiveness, x, for other fibers can be estimated by correcting for differences in input resistance (Sherman and Atwood,
1972). The relative number of synapses (S)
can be calculated by:
EPSP (mV)
rn -x
c
Although the calculation cannot be construed as being accurate in absolute terms,
it is obvious that high Fe fibers have 4 to
20 times as many synapses as low F e fibers,
depending on the assumptions used to
make the calculations. An intermediate
fiber (# 3) falls between the extremes.
It may be that the actual number of foci
for the high Fe fibers falls somewhere between the corrected and uncorrected
values.
In the crayfish opener muscle, Bittner
and Kennedy (1970) estimated 45 synaptic
foci per muscle fiber for both high and
low Fe fibers. In crabs, the differences in
synaptic properties among the various
muscle fibers are greater than for crayfish, and one might expect more variation
in the numbers of terminals.
TABLE 1. Estimate of the number of synaptic foci on G-rapsus stretcher muscle fibers.
Corrected*
Un&orreeted
Fiber
1
2
3
4
5
6.1
4.3
3.3
1.6
1.5
(mV) 1 Hz
m
X (mV)
S
X (mV)
S
0.10
0.35
0.01
0.07
3.5
6.0
7.2
1.4
4.1
3.2
0.1
0.1
0.1
0.1
0.1
100
50
25
15
22
0.025
0.035
0.05
400
150
50
15
22
X = Quantal effectiveness.
S = Number of synaptie foci, calculated by S = EPSP (mV)/ro • X.
+ z= Corrected for relative membrane resistance.
(Raw data from Atwood and Lang, 1973).
0.1
0.1
348
FRED LANG AND HAROLD L. ATWOOD
Factors which may be involved in facilitation
From the above discussion, it can be
seen that several factors may be involved
in facilitation at crustacean neuromuscular
junctions. These include:
(1) Residuum of "active" calcium taken
up by nerve terminals during an action
potential, as per the hypothesis of Katz
and Miledi (1965&; 1968a). Although some
of the short-term facilitation could result
from this mechanism, data in the literature (Bracho and Orkand, 1970; Ortiz and
Bracho, 1972) suggest that transmitter release is linearly related to external Ca2+
concentration at a crayfish neuromuscular
junction and not to the fourth or fifth
power of external Ca2+ as at the frog
neuromuscular synapse (Dodge and Rahamimoff, 1967; Werman, 1971). The observed facilitation at the crayfish synapse
could not readily be generated by a calcium residue mechanism with a first-power
relationship between external Ca2+ and
transmitter release. Furthermore, several
workers (e.g., Norman and Maynard, unpublished results) have observed that a
single exponentially decaying variable
(e.g., "active" calcium) cannot mathematically account for facilitation at certain
crustacean synapses. This contrasts with results for the frog neuromuscular synapse
where most of the facilitation can be
explained by a calcium residue hypothesis
(Mallart and Martin, 1967; Rahamimoff,
1968). It appears that more than one factor may be involved in facilitation and
that factors other than "active" Ca2+ may
be very important in the crustacean system.
(2) Sodium accumulation. Sherman and
Atwood (1971) have shown that long-term
facilitation at crustacean neuromuscular
synapses is probably related to sodium
accumulation by active nerve terminals.
Increased intracellular sodium could act
either to increase the level of available
intracellular Ca2+ (Birks and Cohen,
1968), thereby facilitating transmitter release, or to alter electrical conditions in
the nerve terminal. It is not yet certain
whether sodium accumulation can play a
role in short-term facilitation in small crustacean nerve terminals.
(3) The "immediately available" store
of transmitter and mobilization of transmitter. The observations with Cs+ suggest that facilitation occurs most strongly
in terminals where a small fraction of the
immediately available transmitter store is
released by low frequency stimulation.
When the fraction is increased (as with
Cs+) facilitation decreases. The relative
size of the immediately available store and
the rate of mobilization probably act as
limiting factors in facilitation.
(4) Progressive electrical changes in the
nerve terminal. These will be discussed
in more detail below, since electrical
changes have been previously proposed to
account for facilitation at crayfish neuromuscular junctions.
Although it is not presently possible to
draw up a complete model of facilitation
which will account for observed effects at
different crustacean nerve terminals, or
even at a single nerve terminal, it seems
likely that several of the above factors may
be involved and would have to be taken
into account in drawing up a complete
model.
Electrical behavior of nerve terminals
The question of the electrical behavior
of nerve terminals has been studied in several systems with variable results. From the
work of Katz and Miledi (1965a, 19686)
it is clear that in frog neuromuscular junctions most or all of the length of the nerve
terminal will support a propagated action
potential. The same is true of mammalian
neuromuscular terminals (Hubbard and
Schmidt, 1963; for a review, see Hubbard
et al., 1969). For this reason and for several others, including the large size of the
vertebrate nerve terminals, the amount of
facilitation, and the action of calcium,
comparisons with the crustacean system
must be made with caution.
Two other systems that have received
attention in the present context are the
squid giant synapse (for review, see Gage,
CRUSTACEAN NEUROMUSCULAR MECHANISMS
1967) and the chick ciliary ganglion (Martin and Pilar, 1964). In both of these
preparations the presynaptic nerve terminals have been shown to support a
propagated action potential, but again,
comparisons with the crustacean system
must be made with caution. The nerve terminals in both of these preparations are
quite large, large enough, in fact, to permit their penetration with microelectrodes.
Crustacean terminals are diminutive by
comparison (Atwood and Morin, 1970;
Lang et al., 1972). In addition, the amount
of facilitation at the frog, chick, and squid
synapses does not appear to approach the
values obtainable in the crustacean high
Fe fibers; this is somewhat uncertain, however, because stimulus train parameters
differed widely in these investigations and
comparable values are not available for
the squid preparation.
Other studies on the squid (reviewed by
Gage, 1967) further suggested that a propagated action potential, per se was not necessary to evoke transmitter release. In fact,
neither the Na+ or K+ current nor a
change in internal Na+ was necessary for
transmitter release; the critical factor appeared to be the level of membrane depolarization (Gage, 1967). Thus, a decrementally conducted potential could be sufficient to trigger transmitter release. Such
a mechanism would also permit a large
amount of facilitation since the amount of
transmitter release has been shown to be
critically dependent on the amount of presynaptic depolarization in the squid (Hagiwara and Tasaki, 1958; Kusano et al., 1967)
and crayfish (Dudel, 1971) preparations.
The notion that decremental conduction can cause transmitter release at a
synapse is supported by several studies.
Werblin and Dowling (1968) showed that,
in addition to the receptors, both types of
neurons in the outer plexiform layer of
mudpuppy retina do not spike. Rather,
they conduct decremental signals along
their relatively short length. More recently, Shaw (1972) showed that the photoreceptors of barnacle lateral eye also transmit only graded potentials. Similarly, Ripley et al. (1968) and Paul (1972) have found
349
a giant stretch receptor in crabs which conducts only graded potentials. Presumably,
all of these cells excite postsynaptic neurons
via synapses which release transmitter in
response to the decrementally conducted
potentials.
Observations on presynaptic inhibition
in crayfish (Dudel, 19656) indicate that the
action potential can become decremental
during the inhibitory action in places
where it is initially fully propagated. Furthermore, there is a relationship between
decreased spike amplitude in the terminals
and reduced transmitter output, which
suggests that NTP size governs transmitter
output.
Thus, the available evidence, although
not conclusive, strongly suggests that decremental conduction may occur in some
crustacean nerve terminals. Variation in
extent of conduction of the nerve impulse
in different terminals could be involved
in the physiological differences they exhibit, but this can only explain why m
differs greatly between high and low Fo
terminals at low frequencies of stimulation. It fails to explain why the amount of
facilitation also differs greatly at higher
frequencies. In this regard, several hypotheses have been suggested which attempt to account for facilitation at crustacean nerve terminals in terms of progressive changes in the electrical responses
in the terminals during stimulation. Briefly, the available hypotheses are: (1) recruitment or progressive invasion of unactivated terminals, (2) progressive increase
in NTP amplitude, (3) hyperpolarization,
and (4) slow depolarization. The last hypothesis has not, to our knowledge, been
previously proposed for crustaceans, but
merits consideration.
The idea of recruitment of unactivated
nerve terminals is not a new one (see Dudel and Kuffler, 1961b). This hypothesis
suggests that not all of the nerve terminals
in a facilitating system are invaded by impulses and that larger numbers are recruited during facilitation. Dudel and Kuffler, 19616) successfully showed that this
was not applicable to crayfish neuromuscular synapses. At low frequencies of stim-
350
FRED LANG AND HAROLD L. ATWOOD
ulation, when there was little facilitation,
the number of failures of transmission approximated the number predicted by Poisson's theorem. In addition, an EPSP after
a failure showed just as much facilitation
as when no failure occurred; thus, the terminal must have been invaded.
Bittner and Harrison (1970) provided
evidence to support the idea that not all
branches of the axon are invaded by each
action potential, but they did not show a
progressive recruitment of terminals or a
progressive invasion which could account
for facilitation. In addition, in his exhaustive study, Dudel (1965«,6) never observed
monophasic positive NTP's changing to
biphasic NTP's during facilitation, and he
suggested that this probably wasn't occurring.
The second alternative, a progressive
increase in size of the monophasic positive
NTP, was put forward as a mechanism of
facilitation by Dudel (1965a). He reported
that the monophasic positive NTP recorded from crayfish nerve terminals grew
linearly with increases in frequency of
nerve stimulation. This growth of the NTP
paralleled increases in both the externally
recorded synaptic current (ERSP) and the
EPSP. Our experience along with others
(Ortiz, personal communication) suggests
that Dudel was recording an increase in a
"nonspecific" potential (Katz and Miledi,
1965«; Atwood and Johnston, 1968) which
accompanies and just precedes the ERSP.
This potential does grow rapidly and linearly with stimulation frequency at facilitating terminals and is probably a result
of recording "source" current from nearby
activated synaptic "sinks." This potential
can be easily eliminated by placing a second microelectrode within 50-100 ^ of the
monitoring electrode and recording differentially. In this case, the monophasic positive NTP does not exhibit any striking
change in size with increasing frequency of
stimulation (Fig. 12). A similar conclusion
was reached by Ortiz (personal communication) using the crayfish opener muscle.
He employed the same preparation and essentially the same technique as Dudel
(196ja), but also cooled the preparation
FIG. 12. Effect of stimulation on the size of the
NTP's (arrow, A,) from high Fo muscle fibers of
Pachygrapsus {A) and Hyas (B) . All responses are
averaged; the NTP in A, is slightly larger and
has a longer duration than At; this is probably
not significant, as records taken before and after
this one showed no growth or a slight decline in
amplitude as compared to the NTP in A,. Stimulation frequency: A,, B,, 1 Hz; A2, 5 Hz; Bt, 4 Hz.
Calibration: horizontal, A, 3 msec; B, 1 msec; vertical, 0.1 mV.
to greatly increase the synaptic delay. In
this case, the nonspecific potential still
preceded the ERSP, suggesting a common
origin. The NTP which greatly preceded
the ERSP was then uncontaminated by the
nonspecific potential and did not exhibit
an increase in size with increasing frequency of stimulation.
The third possibility, hyperpolarization
of the nerve terminals during a train of
impulses, was recently supported by Dudel
(1971). Hyperpolarization of nerve terminals has been shown to increase the
transmitter output resulting from a subsequent impulse in mammalian (Hubbard
and Willis, 1962, 1968), squid (Gage, 1967),
and crayfish (Dudel, 1971) synapses. Based
on their work, Hubbard and Schmidt
(1963) and Dudel (1971) came to the conclusion that hyperpolarization might be
the mechanism which controls facilitation
at mammalian and crayfish terminals, respectively. Gage and Hubbard (1966a,ft)
later determined that post-tetanic hyperpolarization of mammalian nerve terminals is probably not the mechanism for posttetanic potentiation of transmitter release
since the two phenomena can be uncoupled in many different ways. Dudel
(1971) found that hyperpolarization ap-
CRUSTACEAN NEUROMUSCULAR MECHANISMS
plied to crayfish nerve terminals increases
the duration and amplitude of the action
potential recorded near the terminal and
also increases transmitter output (probably
as a result of changes in the action potential). Hyperpolarization of the nerve terminals could be induced normally by a
train of impulses either through activation
of membrane potassium channels (e.g.,
Junge, 1972) or through activation of an
electrogenic sodium pump (e.g., Kuno et
al., 1970).
There are two reasons which suggest
that the hyperpolarization mechanism may
not apply under physiological conditions.
First, as yet, it has not been shown that
crayfish nerve terminals undergo a lasting
hyperpolarization during repetitive stimulation; on the contrary, they probably slowly depolarize (see below). A second and
more compelling reason is that the effect
of hyperpolarization was seen to develop
on the order of seconds after the application of the current in both squid (Gage,
1967) and crayfish (Dudel, 1971). Facilitation resulting from nerve stimulation is
apparent on the order of milliseconds after
each impulse. Thus, while the mechanism
of hyperpolarization cannot yet be ruled
out with direct evidence, the available data
suggest that it is not the likely mechanism
which underlies facilitation.
The fourth hypothesis, that of slow depolarization, suggests that facilitation may
be linked to a progressive decrease in the
membrane potential of the non-spiking
nerve terminal. If the decrementally conducted potentials resulting from the spike
are all about the same size, when superimposed upon the slow depolarization,
they might progressively attain higher and
higher absolute levels of depolarization.
One of the prerequisites for the operability of this mechanism is that the facilitating terminals do not spike. In the squid
giant synapse, background depolarization
decreases the amount of transmitter release evoked by a depolarizing pulse (Gage,
1967V Thus, the postsynaptic EPSP resulting from a presynaptic current pulse is
smaller if the pulse is superimposed upon
a small depolarizing conditioning pulse.
351
Presumably, the decrease in transmitter
output is due to rectification resulting
from the depolarizing activation of K+
channels; in the presence of tetraethylammonium (TEA), which blocks K+ channels, the effect of the conditioning pulse
is abolished or even reversed in sign (Katz
and Miledi, 1971). Thus, if the facilitating
crustacean nerve terminals are electrically
inexcitable, it is a plausible assumption
that there are no electrically activated K+
channels which can cause rectification. The
consequences of these phenomena are clear;
the slow depolarization would be sufficient
to poise the E-S coupling mechanism at a
higher and higher level for the arrival of
each successive impulse, thus resulting in
facilitation.
There are several lines of evidence which
suggest that, in fact, there may be a slow
depolarization of nerve terminals resulting
from repetitive stimulation. First, depolarization of nerve terminals was shown to increase the frequency of spontaneous miniature potentials in frog (del Castillo and
Katz, 1954), rat (Hubbard and Willis,
1968), and crayfish (Dudel, 1971). After repetitive stimulation, at certain frequencies,
the frequency of spontaneous miniature
potentials increased at crayfish terminals,
suggesting maintained depolarization (Dudel andKuffler, 1961 b). Secondly, if the
nerve terminals do become depolarized during repetitive stimulation, one would expect to see a decrease in the size of the action potential at the spiking portion of the
membrane. This has been observed in crayfish (Dudel, 1965a) nerve terminals.
From the available evidence, it seems
likely that there is a slow depolarization of
nerve terminals as a result of repetitive
stimulation. Such a phenomenon has been
shown for many types of unmyelinated
axons, and it appears that it may be the
result of a transient increase in extracellular K+ concentration near the membrane
(Shanes, 1951; Frankenhaeuser and Hodgkin, 1956). Frankenhaeuser and Hodgkin
(1956) suggested that the change in K+
concentration around the axolemma of the
squid giant axon would be considerable
even for a single impulse, because the K+
352
FRED LANG AND HAROLD L. ATWOOD
becomes trapped in the narrow space between the axolemma and the enveloping
Schwann cells. The increase is in excess of
1 ITIM per spike. At high frequencies of
stimulation the increase in K+ accumulation is great enough (15 mM at 125 Hz)
to result in a depolarization in excess of
13 mV. In crab leg nerve fibers Shanes
(1949) found a depolarization of 2 mV
with a frequency of stimulation of 1-6 Hz.
In the case of crustacean motor nerve terminals, the effect might be even more striking (e.g., Eccles et al., 1969) since the terminals have a small diameter (ca. 1 ^). In
addition, they are embedded in clefts beneath the sarcolemma surrounded by extremely narrow (100-200 A) extracellular
spaces (Atwood and Morin, 1970; Lang et
al., 1972) which would permit accumulation of K+ in a confined region around
the terminals.
Accumulation of sodium ions in nerve
terminals during prolonged repetitive
stimulation could affect either the level of
membrane potential in the terminal or
the shape of the NTP, and it is conceivable that these effects may be involved in
long term facilitation (Sherman and Atwood, 1971) or even in short term facilitation in small terminals. It is suggestive
that marked facilitation can occur under
conditions in which the nerve terminals
are probably partially depolarized—such as
after soaking in ouabain or DNP (Figs. 35). Apparently, prolonged partial depolarization of nerve terminals does not diminish transmitter release through Ca 2+ inactivation (e.g., see Katz and Miledi, 1971)
or reduction of spike amplitude (Dudel,
1965b). On the contrary, transmitter release is greatly augmented until nerve
branches are blocked (as with ouabain) or
vesicles are depleted (as with DNP).
Whether the augmentation of transmitter
release is due to depolarization per se, and
its effects on the E-S coupling mechanisms,
or to sodium-calcium competition, is not
yet certain. However, the above evidence
makes the "slow depolarization" hypothesis at least as good an alternative as the
"hyperpolari/ation" hypothesis.
CONCLUSION
In conclusion, it can be unequivocally
stated that crustacean neuromuscular junctions exhibit a wide range in their facilitation properties, even among the nerve
terminals of a single motor axon. The advantages for this system and for nervous
systems in general are obvious; the mechanism, permits integrative activity such that
the postsynaptic response will vary both
spatially and temporally depending not
only on the average frequency but also on
the pattern of presynaptic activity. If all
the nerve terminals were functionally allor-none, they would act only as relay systems; postsynaptic responses would vary
only temporally, their size being constant.
This latter system has developed in vertebrate skeletal muscle, where control of contraction resides at the level of the spinal
cord and is relayed via the final common
pathway of motor units. The former system has evolved in invertebrate C.N.S.
(Maynard, 1966) and probably in vertebrate C.N.S. as well (for review, see Hubbard et al., 1969). The physiological value
of a heterogeneous population of synapses
to the invertebrate system and some speculation as to their development are covered
by Atwood (1973).
Note added in proof: Since this paper was submitted for publication, Krnjevic and Morris (1972)
(Can. J. Physiol. Pharm. 50:1214) have reported
that stimulation of primary afferent fibers in cat
spinal cord results in an increase in extracellular
K+ activity (as measured with a K+ selective microelectrode) at the nerve terminals of these
fibers. This increase in K+ was associated with a
depolarization in the fibers. In addition, Atwood
(unpublished observations) has found that in
crustacean neuromuscular preparations, the nerve
terminal potential (NTP) exhibits a prolongation following treatment with ouabain or DNP.
REFERENCES
Adelman, W. J., and J. P. Senft. 1967. Voltage
clamp studies on the effect of internal cesium
ion on sodium and potassium currents in the
squid giant axon. J. Gen. Physiol. 50:279-293.
Araki, T., M. Ito, P. G. Kostyuk, O. Oscarsson,
and T. Oshima. 1962. Injection of alkaline cations into cat spinal motoncurons. Nature (London) 196:1319-1320.
Atwood, II. L. 1965. Excitation and inhibition in
CRUSTACEAN NEUROMUSCULAR MECHANISMS
crab muscle fibers. Comp. Biochcm. Physiol. 16:
409-426.
Atwood, H. L. 1967. Variation in Lhe physiological
properties of crustacean motor synapses. Nature
(London) 215:57-58.
Atwood, H. L. 1968. Peripheral inhibition in crustacean muscle. Experientia 24:753-763.
Atwood, H. L. 1973. An attempt to account for the
diversity of crustacean muscles. Amer. Zool. 13:
357-378.
Atwood, H. L., and G. D. Bittner. 1971. Matching
of excitatory and inhibitory inputs to crustacean
muscle fibers. J. Xeurophysiol. 34:157-170.
Atwood, H. L., and H. S. Johnston. 1968. Neuromuscular synapses of a crab motor axon. J.
Exp. Zool. 167:457-470.
Atwood, H. L., and A. Jones. 1967. Presynaptic
inhibition in crustacean muscle: axo-axonal synapse. Experientia 23:1036-1038.
Atwood, H. L., and F. Lang. 1973. Differential
responses of crab neuromuscular synapses to
cesium ion. J. Gen. Physiol. (In press)
Atwood, H. L., F. Lang, and W. A. Morin. 1972.
Synaptic vesicles: selective depletion in crayfish
excitatory and inhibitory axons. Science 176:13531355.
Atwood, H. L., and W. A. Morin. 1970. Neuromuscular and axoaxonal synapses of the crayfish
opener muscle. J. Ultrastruct. Res. 32:351-369.
Baker, P. F., A. L. Hodgkin, and T. I. Shaw. 1962The effects of changes in internal ionic concentrations on the electrical properties of perfused giant axons. J. Physiol. 164:355-374.
Birks, R. I. 1962. The effects of a cardiac glycoside on subcellular structures within nerve cells
and their processes in sympathetic ganglia and
skeletal muscle. Can. J. Biochem. Physiol. 40:
303-315.
Birks, R. I., and M. W. Cohen. 1968. The influence
of internal sodium on the behaviour of motor
nerve endings. Proc. Roy. Soc. London B 170:401421.
Bitlner, G. D. 1968. Differentiation of nerve terminals in the crayfish muscle and its functional
significance. J. Gen. Physiol. 51:731-758.
Bittner, G. D., and J. Harrison. 1970. A reconsideration of the Poisson hypothesis for transmitter release at the crayfish neuromuscular junction. J. Physiol. 206:1-23.
Bittner, G. D., and D. Kennedy. 1970. Quantitative
aspects of transmitter release. J. Cell Biol. 47:
585-592.
Bracho, H., and R. K. Orkand. 1970. Effect of
calcium on excitatory neuromuscular transmission in the crayfish. J. Physiol. 206:61-71.
Caldwell, P. C. 1960. The phosphorus metabolism
of squid axons and its relationship to the active
transport of sodium. J. Physiol. 152:545-560.
del Castillo, J., and B. Katz. 1954. Changes in endplate activity produced by presynaptic polarization. J. Physiol. 124:586-604.
Dodge, F. A., and R. Rahamimoff. 1967. Cooperative action of calcium ions in transmitter release
at the neuromuscular junction. J. Physiol. 193:
353
419-432.
Douglas, W. W., and R. P. Rubin. 1961. The role
of calcium in the secretory response of the adrenal medulla to acetylcholine. J. Physiol. 159:
40-57.
Dudel, J. 1965a. Potential changes in the crayfish
motor nerve terminal during repetitive stimulation. Pflugers Arch. 282:323-337.
Dudel, J. 1965b. The mechanism of presynaptic
inhibition at the crayfish neuromuscular junction. Pflugers Arch. 284:66-80.
Dudel, J. 1971. The effect of polarizing current on
action potential and transmitter release in crayfish motor nerve terminals. Pflugers Arch. 324:
227-248.
Dudel, J., and S. W. Kuffler. 1961«. The quantal
nature of transmission and spontaneous miniature potentials at the crayfish neuromuscular
junction. J. Physiol. 155:514-529.
Dudel, J., and S. W. Kuffler. 1961b. Mechanism of
facilitation at the crayfish neuromuscular junction. J. Physiol. 155:530-542.
Eccles, J. C, H. Horn, G. Taborikova, and N.
Tsukahara. 1969. Slow potential fields generated
in cerebellar cortex by mossy fiber volleys. Brain
Res. 15:276-280.
Fourtner, C. R., and R. G. Sherman. 1973. Chelicerale skeletal neuromuscular systems. Amer. Zool.
13:271-289.
Frankenhaeuser, B., and A. L. Hodgkin. 1956. The
after-effects of impulses in the giant nerve fibres
of Loligo. J. Physiol. 131:341-376.
Futamachi, K. J. 1972. Acetylcholine: possible neuromuscular transmitter in Crustacea. Science 172:
1373-1375.
Gage, P. W. 1967. Depolarization and excitationsecretion coupling in presynaptic terminals. Fed.
Proc. 26:1627-1632.
Cage, P. W., and J. I. Hubbard. 1966a. The origin
of post-tetanic hyperpolarization in mammalian
motor nerve terminals. J. Physiol. 184:335-352.
Cage, P. W., and J. I. Hubbard. 1966b. An investigation of the post-tetanic potentiation o£ the
end-plate potentials at a mammalian neuromuscular junction. J. Physiol. 184:353-375.
Gainer, H., J. P. Reuben, and H. Grundfest. 1967.
The augmentation of postsynaptic potentials in
crustacean muscle fibers by cesium. A presynaptic mechanism. Comp. Biochem. Physiol. 20:877900.
Ginsborg, B. L., and J. T. Hamilton. 1968. The
effect of caesium ions on neuromuscular transmission in the frog. Quart. J. Exp. Physiol. 53:
162-169.
Godfraind, J. M., H. Kawamura, K. Krnjevic, and
R. Pumain. 1971. Actions of dinilrophenol and
some other metabolic inhibitors on cortical
neurones. J. Physiol. 215:199-222.
Hagiwara, S., and I. Tasaki. 1958. A study on the
mechanism of impulse transmission across the
giant synapse of the squid. J. Physiol. 143:114128.
Holtzman, E., A. R. Freeman, and L. A. Kashner.
354
FRED LANG AND HAROLD L. ATWOOD
1971. Stimulation-dependent alterations in peroxidase uptake at lobster neurouiuscular junctions.
Science 173:733-736.
Hoy, R. R. 1969. Degeneration and regeneration
in abdominal flexor motor neurons in the crayfish. J. Exp. Zool. 172:219-232.
Hoyle, G., and C. A. Wiersma. 1958. Excitation at
neuromuscular junctions in Crustacea. J. Physiol.
143:403-425.
Hubbard, J. I., R. Llinas, and D. M. Quastel.
1969. Electrophysiological analysis of synaptic
transmission. Edward Arnold Ltd., London.
Hubbard, J. I., and R. F. Schmidt. 1963. An electrophysiological investigation of mammalian
motor nerve terminals. J. Physiol. 166:145-167.
Hubbard, J. I., and W. D. Willis. 1962. Hyperpolarization of mammalian motor nerve terminals. J. Physiol. 163:115-137.
Hubbard, J. I., and W. D. Willis. 1968. The effects
of depolarization of motor nerve terminals upon
the release of transmitter by nerve terminals. J.
Physiol. 194:381-405.
Junge, D. 1972. Increased K-conductance as promimate cause of post-stimulus hyperpolarization in
Tritonia neurones. Coinp. Biochem. Physiol. 42:
975-981.
Katz, B., and R. Miledi. 1965a. Propagation of
electric activity in motor nerve terminals. Proc.
Roy. Soc. London B 161:453-482.
Katz, B., and R. Miledi. 19656. The effect of calcium on acetylcholine release from motor nerve
terminals. Proc. Roy. Soc. London B 161:496503.
Katz, B., and R. Miledi. 1968a. The role of calcium in neuromuscular facilitation. J. Physiol.
195:481-492.
Katz, B., and R. Miledi. 1968b. The effect of local
blockage of motor nerve terminals. J. Physiol.
199:729-741.
Katz, B., and R. Miledi. 1971. The effect of prolonged depolarization on synaptic transfer in the
stellate ganglion of the squid. J. Physiol. 216:
503-512.
Kravitz, E. A. 1967. Acetylcholine, -y-aminobutyric
acid, and glutamic acid: physiological and chemical studies related to their roles as neurotransmitter agents, p. 433-444. In G. C. Quarton, T.
Mclncchuk, and F. O. Schmitt [cd.], The neurosciences. Rockefeller Univ. Press, Xew York.
Kravitz, E. A. 1968. A study of synaptic chemistry
in single neurons, p. 67-72. In F. D. Carlson
[ed.], Physiological and biochemical aspects of
nervous integration. Prentice-Hall, Englewood
Cliffs, N. J.
Kuno, M., J. T. Miyahara, and J. N. Weakly. 1970.
Post-tetanic hyperpolarization produced by an
electrogenic pump in dorsal spinocerebellar tract
neurones of the cat. J. Physiol. 210:839-855.
Kusano, K., D. R. Livengood, and R. Werman.
1967. Correlation of transmitter release with
membrane properties of the presynaptic fiber of
the squid giant synapse. J. Gen. Physiol. 50:
2579 2601.
Lang, F., H. L. Atwood, and \\*. A. Morin. 1972.
Innervation and vascular supply of the crayfish
opener muscle: scanning and transmission electron microscopy. Z. Zellforsch. 127:189-200.
Lang, F., A. S. Sutterlin, and C. L. Prosser. 1970.
Electrical and mechanical properties of the
closer muscle of the Alaskan King Crab, Paralithodes camtschatica. Comp. Biochem. Physiol.
32:615-628.
Lehninger, A. L., E. Carafolk, and C. S. Rossi.
1967. Energy-linked ion movements in mitochondrial systems. Advan. Enzymol. 29:259-320.
Llinas, R., J. R. Blinks, and C. Nicholson. 1972.
Calcium transient in presynaptic terminal of
squid giant synapse: detection with aequorin.
Science 176:1127-1129.
Loomis, W. F., and F. Lipman. 1948. Reversible inhibition of the coupling between phosphorylation and oxidation. J. Biol. Chem. 173:807-808.
Mallart, A., and A. R. Martin. 1967. An analysis
of facilitation of transmitter release at the neuromuscular junction of the frog. J. Physiol. 193:
679-694.
Martin, A. R., and G. Pilar. 1964. Presynaptic
and post-synaptic events during post-tetanic potentiation and facilitation in the avian ciliary
ganglion. J. Physiol. 175:17-30.
Maynard, D. M. 1966. Integration in crustacean
ganglia. In G. M. Hughes [ed.], Nervous and
hormonal mechanisms of integration. Symp. Soc.
Exp. Biol. 20:111-149.
Ortiz, C. L., and H. Bracho. 1972. Effect of reduced calcium on excitatory transmitter release
at the crayfish neuromuscular junction. Comp.
Biochem. Physiol. 41:805-812.
Paul, D. H. 1972. Decremental conduction over
"giant" afferent processes in an arthropod. Science 176:680-682.
Pearson, K. G. 1973. Function of peripheral inhibitory axons in insects. Amcr. Zool. 13:321-330.
Peterson, R. P., and F. A. Pepe. 1961. The fine
structure of inhibitory synapses in the crayfish.
J. Biophys. Biochem. Cytol. 11:157-169.
Pickard, W. F., J. Y. Letvin, J. W. Moore, M.
Takata, J. Pooler, and T. Bernstein. 1964. Cesium
ions do not pass the membrane of the squid
axon. Proc. Nat. Acad. Sci. U.S.A. 52:1177-1183.
Rahamimoff, R. 1968. A dual effect of calcium ions
on neuromuscular facilitation. J. Physiol. 195:
471-480.
Ripley, S. H., B. M. Bush, and A. Roberts. 1968.
Crab muscle receptor which responds without
impulses. Nature (London) 218:1170-1171.
Shanes, A. M. 1949. Electrical phenomena in nerve.
II. Crab nerve. J. Gen. Physiol. 33:75-102.
Shanes, A. M. 1951. Potassium movements in relation to nerve activity. J. Gen. Physiol. 34:795807.
Shaw, S. R. 1972. Decremental conduction of the
visual signal in barnacle lateral eye. J. Physiol.
220:145-175.
Sherman, R. G., and H. L. Atwood. 1971. Synaptic facilitation: long-term neuromuscular facilitation in crustaceans. Science 171:1248-1250.
Sherman, R. G., and H. L. Atwood. 1972. Cor-
CRUSTACEAN NEUROMUSCULAR MECHANISMS
related electrophysiological and ultrastructural
studies of a crustacean motor unit. J. Gen.
Physiol. 59:586-615.
Sjodin, R. A. 1967. Long duration responses in
squid giant axons injected with "'Cesium sulfate
solutions. J. Gen. Physiol. 50:269-278.
Takeuchi, A., and N. Takeuchi. 1964. The effect
on crayfish muscle of iontophoretically applied
glutamate. J. Physiol. 170:296-317.
Takeuchi, A., and N. Takeuchi. 1967. Electrophysiological studies of the action of GABA on
the synaptic membrane. Fed. Proc. 26:1633-1638.
Taraskevich, P. S. 1971. Reversal potentials of Lglutamate and the excitatory transmitter at the
neuromuscular junction of the crayfish. Biochem.
355
Biophys. Acta 241:700-704.
Uchizono, K. 1967. Inhibitory synapses on the
stretch receptor neurone of the crayfish. Nature
(London) 214:833-834.
Werblin, F. S., and J. E. Dowling. 1969. Organization of the retina of the mudpuppy, Xecturus
maculosus. II. Intracellular recording. J. Neurophysiol. 32:339-355.
Werman, R. 1971. The number of receptors for
calcium ions at the nerve terminals of one endplate. Comp. Gen. Pharm. 2:129-137.
Wiersma, C. A. G. 1961. The neuromuscular system, p. 191-240. In T. H. Waterman fed.], The
physiology of Crustacea. Vol. II. Academic Press,
New York.