Department of Neurobiology and Center for Neuronal Computation, Institute of Life Sciences, Hebrew University, Jerusalem 91904, Israel
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ABSTRACT |
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Matzner, Henry,
Yoram Gutfreund, and
Binyamin Hochner.
Neuromuscular System of the Flexible Arm of the Octopus:
Physiological Characterization.
J. Neurophysiol. 83: 1315-1328, 2000.
The octopus arm is an outstanding example of
an efficient boneless and highly flexible appendage. We have begun
characterizing the neuromuscular system of the octopus arm in both
innervated muscle preparations and dissociated muscle cells.
Functionally antagonistic longitudinal and transverse muscle fibers
showed no differences in membrane properties and mode of innervation. The muscle cells are excitable but have a broad range of linear membrane properties. They are electrotonically very compact so that
localized synaptic inputs can control the membrane potential of the
entire muscle cell. Three distinct excitatory neuronal inputs to each
arm muscle cell were identified; their reversal potentials were
extrapolated to be about 10 mV. These appear to be cholinergic as
they are blocked by hexamethonium, D-tubocurarine, and
atropine. Two inputs have low quantal amplitude (1-7 mV) and slow rise
times (4-15 ms), whereas the third has a large size (5-25 mV) and
fast rise time (2-4 ms). This large synaptic input is most likely due
to exceptionally large quantal events. The probability of release is
rather low, suggesting a stochastic activation of muscle cells. All
inputs demonstrated a modest activity-dependent plasticity typical of
fast neuromuscular systems. The pre- and postsynaptic properties
suggest a rather direct relation between neuronal activity and muscle
action. The lack of significant electrical coupling between muscle
fibers and the indications for the small size of the motor units
suggest that the neuromuscular system of the octopus arm has evolved to
ensure a high level of precise localization in the neural control of
arm function.
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INTRODUCTION |
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This report is part of a comprehensive study of
the octopus arm as a model system for motor control and biomechanical
functions of flexible arms (Gutfreund et al. 1996,
1998
). The octopus arm, like other cephalopod tentacles,
vertebrate tongues, and the elephant trunk, lacks any form of rigid
skeleton. In contrast to articulated appendages, the muscles in these
structures supply skeletal support as well as generating movements.
Because these structures are composed mainly of incompressible muscle
tissue, Kier and Smith (1985)
have termed them muscular hydrostats.
Apart from the specialized neuromuscular system of the chromatophors
(Bone et al. 1995; Florey et al. 1985
;
Packard 1995
), little is known about the physiology of
cephalopod neuromuscular systems. Electromyographic recordings of
muscle activity have provided some insights into the neuromuscular
system of the muscular hydrostats comprising the mantle and fin of
cephalopods (Gosline et al. 1983
; Kier et al.
1989
; Wilson 1960
; for review, see Bone et al. 1995
). Synaptic transmission first was investigated in these neuromuscular systems by Stockbridge and Stockbridge
(1988)
, who showed that spontaneous synaptic activity consists
of very large, presumably quantal, events and that the nerve-evoked
release is very unsynchronized. However, because of technical
limitations they were unable to address several important issues such
as the pattern and mode of innervation, the postsynaptic integrative properties of the muscle cells, and the characteristics of transmitter release properties. Further characterization of such systems is needed
to determine whether cephalopod muscular hydrostats have evolved unique
neuromuscular mechanisms.
We show here that the neuromuscular system of the octopus arm has several features not found in other neuromuscular systems. The small, electrically compact and excitable muscle cells are innervated by three distinct cholinergic excitatory synaptic inputs. One of these inputs has an exceptionally large unitary size, and all showed a modest activity-dependent plasticity. Longitudinal and transverse muscle groups are composed of uniform neuromuscular units with rather simple integrative and transformational properties. These results demonstrate several neuromuscular features that may have evolved as an adaptive solution to the complex problem of motor control in flexible arms.
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METHODS |
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Specimens of Octopus vulgaris were collected by local
fishermen in the Mediterranean Sea or imported from the Stazione
Zoologica, Naples, Italy. The octopuses were kept individually in
aquaria of artificial seawater, which circulated through a closed
system of biological filters. Aquaria were regulated to 17°C, 12 h light/dark cycle, and the octopuses were fed fish meat once a day.
These conditions enabled us to keep the octopuses for 6 mo, during which they gained weight at what seemed to be a normal rate.
The animals were anesthetized in cold seawater containing 2% ethanol. A short segment, 1-3 cm long, was dissected from the middle of the arm and kept in artificial seawater (ASW) at ~10°C. ASW composition was (in mM) 460 NaCl, 10 KCl, 55 MgCl2, 11 CaCl2 10 glucose, and 10 HEPES, pH 7.6. The animal displayed normal behavior after the operation, and the amputated arm readily regenerated as in the natural environment.
Dissociated muscle cells
Dissociated muscle cells were prepared according to the method
of Brezina et al. (1994). A small piece of arm muscle
was taken from a designated area of the intrinsic musculature of the
arm under microscopic control. The tissue was incubated at 25°C for 2-4 h in 0.2% collagenase (Sigma Type I) dissolved in Leibovitz L15
culture medium (Biological Industries, Bet Haemek, Israel), adjusted to
the concentration of salts in the seawater. Rinsing with L15 terminated
the enzymatic treatment. The tissue then was triturated manually until
an appreciable concentration of dissociated cells could be detected in
the supernatant. These cells were kept at 4°C for
4 days; their
physiological properties did not appear to deteriorate during this
period. For electrophysiological experiments, an aliquot of the cells
was transferred to a plastic petri dish mounted on an inverted
microscope. The cells settled on the bottom of the dish after a few minutes.
The electrical properties of the isolated muscle cell were investigated in the whole cell current-clamp mode, using the Axoclamp 2B (Axon Instruments). The patch pipettes were filled with the following internal solution (in mM): 465 K-gluconate, 2 MgCl2, 1 CaCl2, 10 K-EGTA, 5 Na2ATP, 50 HEPES, buffered to pH 7.2 with KOH. In several experiments, EGTA concentration was reduced to 10 µM to avoid major disturbance to the cell's buffering system while still chelating excess Ca2+ in the internal pipette solution.
Innervated muscle preparation
A small muscle strand of ~10 mm2 was
dissected out in the planes shown in Fig.
1 together with three to six ganglia of
the arm axial nervous system (a nerve cord length of ~6-12 mm). We concentrated on the longitudinal (Fig. 1C) and transverse
(Fig. 1B) muscle fibers in the lateral and dorsal muscle
groups (Fig. 1, top inset) (Graziadei 1971;
Kier 1988
). These are built of closely packed, obliquely
striated muscle fibers typical of cephalopods (Bone et al.
1995
; Kier 1985
). These groups play a
significant role in arm bending (Kier and Smith 1985
)
and are thus important in the generation of the arm extension and
reaching movements being investigated in our laboratory
(Gutfreund et al. 1996
, 1998
). The lateral nerve roots
innervating the muscle of interest (Graziadi 1971
;
Martoja and May 1956
) were left intact for stimulation
with bipolar Ag/AgCl electrodes (Fig. 1, A-C). The
preparation was pinned down on a silicone elastomer (Sylgard)-coated
dish and perfused with aerated artificial seawater at room temperature. Stimulating the nerve in fresh preparations frequently caused weak
muscle twitches. These disappeared as the preparations equilibrated in
the experimental bath. Intracellular recordings were made with sharp
glass microelectrodes (25-40 M
when filled with 3 M K-acetate plus
0.1 M KCl) using the Axoclamp 2B in bridge mode. The results were
stored on video recorder (Neuro-Corder, NeuroData) for later analysis.
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The small size of the cells (Bone et al. 1995;
unpublished results) made it difficult to obtain stable long-term
recordings. We therefore constructed a superfusion system that allowed
fast changes of solution at the recording site (see Fig.
1C). Briefly, the recording site was superfused continuously
via a polyethylene tip drawn to a diameter of ~50 µm and mounted on
a micromanipulator. Hydrostatic pressure was used to drive four
different solutions (only 2 are shown in Fig. 1C) through
the polyethylene tubing (1.14 mm ID) right into the tip. The free space
at the tip was rather small (~20 µl), enabling exchange between the
different solutions within a few seconds. Dye added to the solutions
helped aim the stream at a desired location and control its flow.
However, as recordings were made from cells in deeper tissue layers,
the latencies of the effects of solution changes were variable, and the
effective concentration of the drugs could not be precisely determined.
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RESULTS |
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Passive and active membrane properties of the muscle fibers
The electrical properties of the muscle fibers were examined using whole cell recordings in enzymatically dissociated cells and in the innervated muscle preparation using sharp microelectrodes in a bridge mode (see METHODS).
Voltage responses to injection of long current pulses in dissociated
cells are shown in Figs. 2, A
and B, and 3, A
and B. Responses in innervated muscle fibers are given in
Figs. 2C and 3, C and D. The time
constants and input resistances obtained in the whole cell
configuration are much larger than those recorded intracellularly in
the innervated muscles. In dissociated fibers, Rin = 480 ± 414 M (mean ± SD; n = 16) and
m= 102 ± 55 ms (n = 15), but these only average 32.2 ± 12.8 M
(n = 15) and 23.8 ± 16.3 ms
(n = 14) in the innervated muscle preparations. These differences are usually attributed to damage caused by the sharp microelectrodes (see Marty and Neher 1995
).
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The current-voltage relationship of both dissociated and innervated
muscle cells shows a wide range of linear membrane properties. As shown
in Fig. 2, B and C, this linearity exists in the
range of membrane potential from about 100 to about
40 mV; the
resting potential is
74 to
63 mV in both experimental conditions.
Outward rectification starts at about
40 mV, which is close to the
level of initiation of active currents (see following text). These
findings indicate that the octopus muscle fibers serve as linear
voltage integrators at a broad range of membrane potentials. (Note that fibers can contract at membrane potentials below the threshold of the
regenerative potentials.)
The muscle cells can generate several types of regenerative responses
that are activated at a relatively high membrane potential. Figure 3,
A and B, illustrates the two main classes of
regenerative activity detected in dissociated cells. We termed the cell
in Fig. 3B a "spiky" cell because it responds to a
long-lasting depolarization with a train of overshooting spikes. The
threshold for spike initiation was about 30 mV, and spike frequency
was related to current intensity. The cell in Fig. 3A showed
a more complex behavior, which we term "oscillatory." At around
45 mV (bottom trace), slow fluctuations in membrane
potential appear during the current injection. At around
40 mV (Fig.
3A,
), a slow regenerative response is initiated, followed by gradually increasing oscillations. These oscillations build
up into a train of low-amplitude spikes. Some cells showed both
patterns of behavior; after massive activation, the cell in Fig.
3B changed from spiky to oscillatory. Despite the large differences in the apparent passive membrane properties, similar types
of cells showing either accelerating oscillations (Fig. 3C)
or overshooting spikes (Fig. 3D) were observed in the
innervated muscle preparation. This suggests that the excitable
membrane properties of the cells are not drastically altered by the
dissociation or recording procedures.
An important functional issue is whether or not the longitudinal and transverse muscle fibers differ in their electrical properties. We found no significant differences in either the excitable or passive properties in the innervated muscle preparation (Table 1).
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Muscle cells are electrotonically compact
Double whole cell recordings were performed on three dissociated
muscle fibers to assess the spatial integrative properties of the
muscle fibers (see inset in Fig.
4). The voltage responses recorded by the
two micropipettes were identical over the whole range of voltage
waveforms as well as during active spike generation (Fig. 4). Thus the
muscle fibers appear to be isopotential cells and can serve as linear
spatial integrators of synaptic inputs. On the basis of the input
resistance, time constant and the dimensions of the cell (900 × 12 µm), we calculated a specific membrane resistance of 93 K
· cm2 and a specific capacity of 1.55 µF/cm2. Assuming cytoplasmic resistivity of 70
· cm (Laurienti and Blankenship 1996
), the
space constant of such fiber is 7.9 mm.
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Muscle cells are innervated by three distinct types of excitatory synaptic inputs
Spontaneous postsynaptic potentials (sPSPs) are shown in Fig.
5A. Some of these are
exceptionally large, as also found in squid fin and mantle muscle cells
(Stockbridge and Stockbridge 1988). In contrast to these
squid muscles, almost every recording from octopus arm muscle cells
showed distinct classes of sPSPs that could be distinguished by their
rise times and amplitudes (Fig. 5A). The two-dimensional
distributions of the amplitudes and rise times (time from onset to
peak) of the sPSPs are shown in Fig. 5C1. One group is
formed by sPSPs with fast rise times (2-4 ms) and large amplitudes
(
20 mV, Fig. 5C1, *). This group is clearly distinct from
a large class of sPSPs with broadly skewed rise times (4-16 ms) and
relatively low amplitudes (<10 mV). The distributions of rise times
and amplitudes of the slow sPSPs are not uniform and can be viewed as
combinations of two groups (Fig. 5C1, ** and ***).
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Postsynaptic potentials were evoked by stimulating the lateral nerve roots (see METHODS and Fig. 1). Suprathreshold stimulation, at ~50% above PSP threshold intensity, ensured repeated activation of the same population of motor neurons. Nerve stimulation also revealed three classes of synaptic inputs. These were especially clear under conditions of low probability of release, which allow monitoring of single release events. These conditions were created by lowering the Ca2+ concentration in the bathing solution to 2 mM (Fig. 5B).
The traces in Fig. 5B show PSPs evoked by double pulse stimulation. These nerve-evoked PSPs are very similar to the sPSPs recorded in the same cell (Fig. 5A). The amplitude and rise-time distributions of the evoked (Fig. 5C, 2 and 3) and spontaneous PSPs (Fig. 5C1) clearly show three clusters with the three peaks similarly located on the rise time axis. The upward amplitude expansion of the three clusters of the evoked PSPs (Fig. 5C, 2 and 3), in comparison with the spontaneous PSPs (Fig. 5C1), can be explained by the summation of multiple releases, which would be predicted to be ~20% at this probability of release (45% failures). A further difference is that the population of small amplitude spontaneous PSPs is evoked very rarely by stimulation.
There was no significant difference in the amplitude versus rise-time
distributions of the sPSPs in the transverse and longitudinal muscle
fibers (cf. Fig. 6, A and
B), suggesting that both muscle groups are similarly
innervated. The sPSPs' frequency, which was variable among different
muscle cells, could be elevated by increasing the tonicity of the ASW
with sucrose. This treatment has been found to increase the frequency
of miniature PSPs by a mechanism independent of external
Ca2+ concentration (Fatt and Katz
1952; Manabe et al. 1992
; Mochida et al.
1998
; Shimoni et al. 1977
). Figure 6,
A1 and B, 1 and 2, are derived from
experiments with ASW to which 0.5 M sucrose was added. The sPSP
distributions obtained under these conditions were not qualitatively
different from those obtained under normal conditions (cf. Fig.
6A, 2 and 3, and B3). Sucrose
therefore was added to increase the sPSP sample in various experiments.
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Several findings suggest that the sPSPs are equivalent to "miniature
postsynaptic potentials (mPSPs)" and therefore represent the random
release of synaptic vesicles ("quanta"). First, the probability of
occurrence of the spontaneous sPSPs appears to be random. Second, sPSP
frequency and amplitude did not change significantly after superfusion
with TTX, which abruptly inhibited evoked release. It is less likely
that sPSPs are due to spontaneous Ca2+ spikes as
lowering Ca2+ concentration affected neither the
amplitude nor the frequency of sPSPs. Third, under conditions of low
release probability, the size of the single unit of the different
classes of evoked PSPs matched the amplitude and shape of the
spontaneous PSPs (Fig. 5) and, in some cases (e.g., Fig. 9), it is
possible to discern discrete large units that comprise nerve-evoked
release. And finally, as described in the preceding text, increasing
the osmolarity of the ASW by adding sucrose elevated the frequency
without affecting the amplitude distribution of the sPSPs. However, we
cannot exclude the possibility that the sPSPs, especially the large and
fast sPSPs, are composed of multiple vesicular releases. Indeed, the presence of a group of fast but low amplitude sPSPs (e.g., Figs. 5C1 and 6B1) may represent a class of such
subvesicles or, alternatively, input from another unidentified source.
Subminiatures and giant miniature PSP have been described in several
studies (see for review, Van der Kloot 1991). Our
electron microscopic studies, however, show conventional small (30-50
nm) clear core vesicles at areas of dense pre- and postsynaptic
membrane junctions (unpublished observations).
Properties of the different classes of synaptic inputs
We next examined the pharmacology, ionic mechanisms, and plastic properties of the different neuromuscular connections to evaluate possible differences in the functions of the various synaptic inputs.
DECAY TIME COURSE OF THE PSPS.
To further characterize the different synaptic inputs and to assess the
electrical dimensions of the muscle cells in innervated muscle, the
decay of the synaptic and passive potentials were analyzed. A slow PSP,
fast PSP and the response to current injection are shown in Fig.
7A after normalization and
alignment of the peaks. The decay of the passive potential and fast PSP
demonstrated a good overlap, whereas the slow PSP decayed more slowly.
The semilogarithmic plot in Fig. 7B shows that the main part
of the decay phase of the passive and fast PSP, i.e., after ~10 ms,
can be fitted with a single exponent, indicating a finite and
relatively short electrotonic length of the cell (Rall
1969).
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REVERSAL POTENTIALS.
The reversal potential of the different synaptic inputs reveals
whether they function as excitatory or inhibitory inputs. Figure
8A shows the
amplitude-rise-time distribution of sucrose-induced sPSPs at five
different membrane potentials imposed by DC current injection. To
estimate the reversal potential of each class of sPSP, the potentials
first were separated into three rise-time groups, fast, moderate, or
slow. The rise-time windows marked in Fig. 8A, left, were
chosen to minimize overlap between the three groups. The dependence of
average sPSP amplitude on membrane potential was evaluated by linear
regression for each rise-time window (Fig. 8B), and the
reversal potential was estimated by extrapolating to zero amplitude.
The error was evaluated by resampling the data using the bootstrap
method (Manly 1997) to estimate the SD of the intercept.
The reversal potential (±SD) of the fast synapse was
4.2 ± 20.8 mV, the moderate PSP reversed at
0.1 ± 13.8 mV, and the
slow PSP at +8.7 ± 24.1 mV.
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TWIN PULSE MODULATION OF TRANSMITTER RELEASE.
Twin pulse facilitation can be used to characterize the
activity-dependent modulation of transmitter release and also allows analysis of mechanisms of transmitter release (for reviews, see Parnas and Parnas 1994; Zucker 1989
).
Because we could not reliably stimulate each motor neuron separately,
we studied twin pulse modulation of release using suprathreshold stimulation.
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PHARMACOLOGICAL ANALYSIS.
Acetylcholine (ACh) has been identified as the neuromuscular
transmitter in most studies in mollusks (Bone et al.
1982, 1995
; Cohen et al. 1978
; Kozak et
al. 1996
; McPherson and Blankenship 1991
),
although some reports suggest glutamate as a potential neuromuscular
transmitter (Bone et al. 1982
; Florey et al.
1985
; Fox and Lloyd 1999
). ACh receptor
antagonists, hexamethonium, D-tubocurarine (dTC), and
atropine were tested here to identify the neuromuscular transmitter in
the octopus arm. The pharmacological experiments on the innervated
muscle preparation required a relatively high range of drug
concentrations (0.2-10 mM), possibly due to the fast local superfusion
method of application (see METHODS); in addition, the
muscle cells are packed densely (Bone et al. 1995
;
unpublished observations), and this may impede penetration of the drugs
into deep muscle layers where the recorded cells most likely lie.
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DISCUSSION |
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The results presented here provide the first analysis of the neuromuscular system of the octopus arm. They suggest that some of the unique properties of this neuromuscular system reflect evolutionary adaptation for the efficient control of a muscular hydrostat. These include the small morphological and electrical dimensions of the muscle cells, the innervation of each muscle cell by three classes of excitatory motor neurons, the lack of profound activity-dependent synaptic plasticity and the similarity in the properties of antagonistic longitudinal and transverse muscle cells.
Passive properties of the muscle cells
A most significant finding is the compact electrical
dimensions of the muscle cells of both longitudinal and transverse
muscle groups. The cells are virtually isopotential with very
high-input resistance (Fig. 4). Indirect evidence suggests that this
may be a general characteristic of cephalopod muscles (Bone et
al. 1995; Gilly et al. 1996
; Rogers et
al. 1997
; Stockbridge and Stockbridge 1988
); as
well, compact electrical dimensions are shown by the dissociated muscle
fibers of the parapodial swim muscle (Laurienti and Blankenship
1996
) and the buccal mass (Brezina et al. 1994
) of the mollusk Aplysia, although the latter cells are
electrically coupled in the intact muscle (Cohen et al.
1978
). The electrical compactness has functional implications
for the control of the muscle activation as the entire muscle fiber is
activated simultaneously. Unlike vertebrate striated muscle (e.g.,
Fatt and Katz 1951
), the isopotentiality of the octopus
muscle cell indicates that the regenerative membrane properties are not
likely to serve the transmission of electrical signals along the muscle fibers.
We attribute the significant differences between the input resistances
and time constants of the dissociated muscle cells, and those of
innervated muscle fibers, to damage caused by the intracellular
recording electrodes (Marty and Neher 1995). Although we
have not found any morphological indications for gap junctions (unpublished observation), electrical coupling between muscle cells, as
found in other mollusk muscles (Cohen et al. 1978
), could be an alternative explanation. According to this explanation, the
PSPs with low amplitude and slow time course may originate from fast
PSPs in neighboring cells. Theoretically the degree of the DC coupling
coefficient between coupled RC compartments can be estimated from the
ratio of the areas under the PSPs (Rinzel and Rall
1974
). The ratios of the areas under slow and fast PSPs suggest
coupling coefficients between 1:3 for the moderate PSPs and 1:5 for the
slow PSPs. This may indicate a relatively strong coupling between the
muscle cells that could account for the large difference in input resistance.
On the other hand, several findings are incompatible with the mediation
of the two slow groups of PSPs by electrical synapses. 1)
The exponential decay of the slow PSPs is significantly slower than the
membrane time constant calculated from the exponential decay of the
passive potentials or from the fast PSPs (Fig. 7). This suggests that
an active current participates in the decay phase of the slow PSPs
(Rall 1964). Contrasting with the slow PSPs, the
striking similarity in the decay phases of the passive and fast PSPs
strongly suggests a passive decay for the fast PSP, and it therefore
cannot serve as a current source for the active decay of the slow PSPs.
This suggests differences in synaptic mechanisms that cannot be
attributed to electrical coupling. 2) The pharmacological
results, especially the fact that each drug caused a specific and
different combination of effects on PSP rise times, indicate the
presence of synaptic inputs with pharmacologically distinct
postsynaptic ACh receptors for the slow and fast PSPs (Fig. 11, Table
2). 3) The estimated reversal potentials of the different
classes of PSPs lie in the same range; with electrical coupling, the
reversal potential of the synapse originating in the neighboring cells
would be higher due to voltage and current attenuation. For example,
the DC coupling, estimated in the preceding text, suggests a three to
five times more positive reversal potential for a PSP originating in
the coupled cell (+110 mV for moderate PSPs to +300 mV for the slow
PSPs), which clearly exceeds the range of variability of our results.
And 4) the fact that the slow and fast PSPs show different
twin pulse facilitation properties at low Ca2+
concentration (Fig. 10, E and F) also supports
their uniqueness. We therefore suggest that the muscle cells in the
octopus arm are unlikely to function as a syncytium of highly coupled
muscle cells. However, because our analysis centers on the properties of the different PSPs, we cannot exclude the possibility of a low
coupling, too weak for transmission of a significant synaptic potential, that could serve to coordinate activity in an ensemble of
muscle cells.
Active membrane properties of the muscle cells
The presence of active membrane properties in octopus arm muscle fibers suggests that the mechanical output of the muscle is not a simple function of the synaptic activity at all voltage ranges. We found that octopus muscle cells are capable of generating a repertoire of active membrane potentials, which include overshooting action potentials, oscillations, and slow regenerative potentials (Fig. 3). Because the electrical compactness of the muscle fibers implies that the electroresponsiveness of the cells is not required for spreading excitability along the cell, these properties are more likely involved in the activation of the contractile machinery.
The threshold for activation of the regenerative potentials is
relatively high (approximately 40 mV with resting potential close to
70 mV). Muscle contractions can be generated at membrane potentials
below this threshold (unpublished observation), ensuring a wide
potential range where synaptic inputs can directly control muscle
contraction. The regenerative processes, therefore may come into play
when vigorous contractions are required. The nature of the active
currents are still unknown; in cephalopods, fast sodium currents
(Gilly et al. 1996
) and an L-type
Ca2+ current have been shown to mediate muscle
contraction (Rogers et al. 1997
). If slow
Ca2+ currents mediate some of the regenerative
responses observed here, for example, the slow regenerative potentials,
then these currents, together with oscillations or trains of spikes,
may serve as a very powerful mechanism for introducing
Ca2+. This may be an effective solution for
muscle cells that lack a specialized system for transferring the signal
into the cell, such as the T-system found in other muscular systems
(Bone et al. 1995
).
Neural control of muscle activation
On the basis of morphological studies, Young (1965)
estimated that 3.8 × 105 motor neurons
innervate the intrinsic muscles of each arm via the numerous nerve
roots projecting from the axial nerve cord which runs along the arm
(Graziadei 1971
; Martoja and May 1956
). Our physiological findings support a high level of localization in the
neural control of these muscles; stimulation of the lateral nerves
(Fig. 1) evokes PSPs only in muscle cells close to the stimulated
nerves (unpublished observations). Further morphological investigation
is needed to characterize number, types, size, and innervation pattern
of the neurons in these nerve roots.
In contrast to the muscle cells of other invertebrates (Bullock
and Horridge 1965; Hoyle 1977
), the cells in the
octopus arm are isopotential, and thus each synaptic input can control
the membrane potential of the entire cell via a single localized
synaptic junction. This proposition is supported by the similarity in
membrane time constant calculated from the decay of fast PSP and
passive voltage decay (Fig. 7). Indeed, our unpublished results show
that the number of synaptic junctions is comparable with the number of
nuclei in cross-sections of the arm muscles. This morphological finding
suggests a low density of innervation of each muscle cell.
Each muscle fiber, in both longitudinal and transverse muscles,
receives three types of synaptic inputs. In polyneuronal neuromuscular systems, the synaptic inputs to one muscle cell may be excitatory or
inhibitory; they may be "fast" (phasic), i.e., large amplitude and
nonfacilitating, or "slow" (tonic), i.e., low-amplitude and facilitating (Parnas and Atwood 1966). The situation in
the octopus arm appears simpler. First, the estimated PSP reversal
potentials indicate that the innervation is exclusively excitatory.
Second, despite the difference in amplitude, all the synaptic inputs
are phasic synapses showing only modest twin pulse modulation. Thus simple postsynaptic summation serves as the main mechanism for transforming the presynaptic activity into muscle action.
The fast PSPs are composed of very large, probably quantal units, and
thus few are required to activate the muscle; the slow PSPs are
composed of smaller unitary responses, and temporal summation of such
PSPs may be effective in activating the muscle. The low-amplitude PSPs
have a slower time course; this increases their capacity for
postsynaptic summation. Thus from a functional point of view, one may
hypothesize that the fast PSPs participate in phasic and vigorous
responses that require active currents generated at relatively high
membrane potentials (40 to
30 mV). On the other hand, trains of
slow PSPs, with their slow time course and low amplitude, may mediate
sustained muscle contraction by regulating the membrane potentials
below the threshold for active currents (between
70 and
40 mV).
Interestingly, our electromyogram (EMG) and modeling studies
(Aharonov et al. 1997
; Gutfreund et al.
1998
) suggest an important role for tonic regulation of muscle
stiffening in the generation of arm movements.
A unique feature of the fast motor neuron input to the octopus arm
muscle is the unusually large unitary postsynaptic response. Stockbridge and Stockbridge (1988) obtained similar
results in the neuromuscular system of the fin and mantle of the squid,
and so this may be a general property of cephalopod neuromuscular systems. These large PSPs cannot be attributed solely to high-input resistance of the muscle fibers because we found a second class of slow
PSPs with lower amplitude. The functional role of this large unitary
response needs to be explored further; nevertheless, large quantal
size, together with the low probability of release (Fig. 9), suggest a
stochastic mechanism for recruitment of muscle fibers. This is possibly
an adaptive mechanism for the control of a system composed of many
small muscle cells. In a system composed of many small cells with the
same function, the force generated can be determined by the number of
cells activated, and not only by the level of activation.
Acetylcholine is the probable neuromuscular transmitter in the octopus arm muscles
Acetylcholine and L-glutamate have been suggested as
excitatory neuromuscular transmitters in cephalopods (Bone et
al. 1982, 1995
; Florey 1985
) and other mollusks
(Cohen et al. 1978
; Fox and Lloyd 1999
;
Kozak et al. 1996
; McPherson and Blankenship
1991
). Indirect experiments led Bone et al.
(1982)
to suggest ACh as the putative transmitter in the
octopus arm. Our results directly support the involvement of ACh in the
neuromuscular synapses in the octopus arm. The ACh antagonists,
hexamethonium, curare, and atropine, block both spontaneous and
nerve-evoked fast and slow PSPs. In addition, nerve-evoked contractions
were inhibited by ACh, but L-glutamate had no effect. The
postsynaptic receptors of the octopus arm muscles resemble those of the
cationic ACh receptor channels in Aplysia ARC muscle
(Cohen et al. 1978
; Kozak et al. 1996
;
Laurienti and Blankenship 1999
), which, unlike other invertebrate neuromuscular junctions, are blocked by hexamethonium (Colquhoun et al. 1991
). As in other invertebrate
neuromuscular ACh receptors (see Colquhoun et al. 1991
),
the muscarinic antagonist, atropine, and the nicotinic blocker, curare,
were effective in blocking the neuromuscular junctions (but see
following text).
In mollusks, ACh receptors have been found to mediate both excitatory
(nonspecific cationic currents) and inhibitory (chloride) currents
(Gardner and Kandel 1977; Kehoe and McIntosh
1998
; Kozak et al. 1996
; Laurienti and
Blankenship 1999
), but here only functionally excitatory
connections were found. The reversal potential of these synaptic
potentials (about
10 mV) may fit with two mechanisms described in
ACh-mediated neuromuscular transmission. One involves chloride and
cationic (Na+) currents (ARC muscle of
Aplysia) (Kozak et al. 1996
), whereas the
second involves a nonspecific cationic current
(K+ and Na+), as in
vertebrate neuromuscular junctions (Takeuchi and Takeuchi 1960
), neuronal nicotinic receptors in mollusks (Ascher
et al. 1978
), and ACh-mediated depolarizing currents in the
parapodial muscle of Aplysia brasiliana (Laurienti
and Blankenship 1999
).
The specific drug actions on the time course of the different classes
of PSPs suggest the existence of several types of postsynaptic ACh
receptors. dTC did not affect the rise time of the fast and slow PSPs,
whereas hexamethonium caused a shortening of the fast PSP only and
atropine shortened all PSPs. No significant changes in passive membrane
properties were observed, and thus the faster rise of the PSP is most
likely due to shortening of the synaptic currents. This shortening
effect is most probably due to blocking agents binding to the open
channel. Such behavior is characteristic for the interaction of several
ACh receptor antagonists with other molluscan neuronal cationic
channels, especially dTC and hexamethonium (Kehoe and McIntosh
1998). In contrast to the pharmacological differences between
the fast and slow PSPs, none of the antagonists used so far
demonstrated any significant differences between the two classes of
slow PSPs that were identified physiologically.
Implications of neuromuscular organization for the function of the octopus arm
The main feature of the octopus arm is that it does not contain a
rigid skeleton, forcing the muscle tissue to serve both as a dynamic
skeletal support and for movement generation. This is achieved by
keeping the volume of the arm constant (Kier and Smith
1985). Mechanically this structure allows the arm unlimited degrees of freedom because the arm can elongate, shorten, twist, and
bend at any point along it. Several of the results reported here may
represent adaptive mechanisms for the control of flexible muscular hydrostats.
The octopus arm does not show several features that endow the
neuromuscular systems of other invertebrates with nonlinear transformation properties (Bullock and Horridge 1965;
Hoyle 1977
). First, the synaptic control of membrane
potential does not include postsynaptic inhibition. Second, the
integrative properties of the muscle cells are linear due to the linear
membrane properties and the isopotentiality of the cells. Third, the
identified synaptic inputs do not possess profound activity-dependent
plastic properties. Therefore it is temping to speculate that this
design leads to rather simple and more direct transformation of neural
activity (spike frequencies) into muscle action.
The similarities between the functional units of the longitudinal and
transverse muscle groups include the morphological structure of the
muscle cells, their passive and active membrane properties, their
pattern of innervation, and the properties of their synaptic connections. Kier (1985) similarly has found that squid
arms, which are involved mainly in bending, are built of muscle cells of similar ultrastructural characteristics. In contrast, in the elongating tentacles, the transverse muscle cells show morphological adaptation consistent with the major role of the transverse muscle group in tentacle elongation. The findings presented in this study extend this organizational principle to the neuronal and physiological levels.
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ACKNOWLEDGMENTS |
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We thank N. Feinstein for the morphological data, Dr. Graziano Fiorito, from the Stazione Zoologica di Napoli, for collaboration and contributions to various aspects of this research, Dr. Jenny Kien for critical readings of this manuscript, Prof. Yosef Yarom for valuable comments and discussions during the course of this research, and Prof. Idan Segev for advice on analyzing the electrotonic properties.
This work was supported by the Office of Naval Research (N00014-94-1-0480), by the Israel Academy of Sciences and Humanities (190/95-1), and by the United States-Israel Binational Science Foundation (95-00170).
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FOOTNOTES |
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Address reprint requests to B. Hochner.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9 July 1999; accepted in final form 28 October 1999.
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REFERENCES |
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