Download Ultrastructure and Function of Cephalopod Chromatophores

AM. ZOOLOGIST, 9:429-442 (1969).
Ultrastructure and Function of Cephalopod Chromatophores
ERNST FLOREY
Department of Zoology, University of Washington,
Seattle, Washington 98105
SYNOPSIS. Each chromatophore organ consists of a pigment cell and of several radial
muscle fibers that represent separate cells. The pigment granules are contained within
an elastic sacculus within the pigment cell. The sacculus is attached around the
equator of the chromatophore to the cell membrane by zonal haptosomes. In turn, the
cell membrane is attached to the radial muscle fibers by a dense basal lamina. The
cell membrane of the retracted chromatophore is highly folded. Contraction of the
radial muscle fibers is initiated by (a) excitatory junction potentials, (b) miniature potentials, or (c) spike potentials. The latter arise spontaneously in the muscle
fibers when these have undergone some internal (metabolic?) change. The contraction
of the muscle fibers causes expansion of the pigment-containing sacculus. Relaxation of the muscle fibers permits the sacculus to assume its original lenticular or
near-spherical shape; the energy for this is stored within the expanded elastic
components of the sacculus. In normal skin the chromatophore organs are entirely
under the control of the central nervous system, the muscle fibers being activated only
by local, excitatory postsynaptic potentials initiated by motor nerve impulses. That
postsynaptic potentials are non-propagating insures that individual motor fibers can
be activated individually, thus permitting a delicate control of skin color by recruitment as well as by frequency. Tonic contractions and pulsations, involving spontaneous
release of transmitter from nerve terminals and spike generation within the muscle
fibers, respectively, are the result of altered, abnormal conditions within the skin.
The rapid changes in color exhibited by
cephalopod molluscs have fascinated naturalists since antiquity but no serious
study of the underlying physiological
mechanisms was undertaken until Sangiovanni (1819) published the first paper
on cephalopod chromatophores. He described "chromophore organs" in the skin
of cephalopods (we must remember that
the cell concept had not yet been formulated). In a later paper Sangiovanni
(1829) described conditions of "systole"
and "diastole" which were controlled by
the nervous system. Another Neapolitan
naturalist, Delle Chiaje (1829) supplemented and expanded Sangiovanni's findings. He discovered that the expansion
of the chromophores was accomplished by
muscle fibers that were radially arranged
The work reported in this paper was supported
by USI'HS Grant No. NB-04145 from the National
Institute of Neurological Diseases and Blindness,
and by a Training Grant, GM-1194, from the National Institute of General Medical Sciences.
around the periphery of each chromophore. Thus, the chief morphological and
physiological features of cephalopod color
change were described even before the existence of chromatophores in other animal
groups had been discovered. Today we regard the chromatophores of cephalopods
as unique: in all the other chromatophores
the pigment moves within highly branched cells, the movement being elicited by
the action of hormones or nerve impulses.
As we see it now, the cephalopod chromatophores are deformed by radially attached
muscle fibers (Fig. 1). The change in color
of a squid or octopus is controlled by a
motor innervation that activates the chromatophore muscles; thus the coloration of
a cephalopod obeys the laws that govern
the coordination of muscular movement:
there are colored twitches, red, yellow, and
brown tetani, and there is paling relaxation.
Although Sangiovanni and Delle Chiaje
had recognized the basic elements of
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430
ERNST FLOREY
CEPHALOPOD CHROMATOPHORES
431
FIG. 1. Interference-contrast photomicrograph of
chromatophoric organs in the skin of the squid,
Loligo opalescens (unstained fixed in 2.5% glutaraldehyde in sea water). All the larger chromatophores are of the brown variety. The yellow chromatophores are marked (y). The small nerve bun-
dle (N) supplies a group of chromatophoric organs
not shown in this picture. The chromatophore
at the center of the left margin shows particularly
well the bridge-like junction (b) between adjacent
muscle fibers.
cephalopod chromatophore function, their
discoveries were not generally accepted.
Throughout the nineteenth century a
heated debate was carried on between two
major camps: those who believed and supported the passive role of the chromatophore and the muscular nature of the radial fibers, and the others who were convinced that the radial fibers were only elastic connective tissue strands and that the
chromatophores themselves were actively
expanding and retracting. Numerous authors, from Holland, Belgium, France, Italy, Germany and Austria participated in
the argument. When van Rynberk reviewed the topic in 1906 he cited over 90
publications.
Hofmann (1907-1910) published exhaustive histological and physiological
studies, followed 20 years later by Bozler
(1928—1930) whose papers remained the
definitive statement of cephalopod chromatophoric physiology. TenCate (1928)
and Hill and Solandt (1954) did not add
anything fundamentally new.
By the turn of the century nobody
doubted the muscular nature of the radial
fibers that are attached to the chromatophores; the histological and physiological
evidence was overwhelming. It was also
clear that the chromatophoric muscles
were controlled by nerves. Other problems
did not have an easy answer. If muscles
were responsible for the expansion of the
chromatophores, what caused the retraction? Suggested answers were: (1) the
chromatophore itself contracts actively,
(2) the cell membrane is elastic and provides the force for retraction, and (3) the
chromatophore is surrounded at its equator by an elastic cellular ring. At the time
of van Rynberg's review the first alternative could be dismissed, because the thorough histological studies of several authors
had disproved the earlier claims of a di-
rect innervation of the chromatophore itself or of circular myofibrils within the
chromatophore. Hofmann (1907, p. 418)
noted that pieces of Sepia skin exposed to
ammonia vapor showed complete retraction of all chromatophores and concluded
"that neither muscle contraction nor any
other 'vital' force is necessary for retraction
and that purely physical forces such as the
elasticity of the cell membrane, and perhaps surface tension, are sufficient to
achieve the retraction from the shape of a
flat disc to that of a sphere." Earlier authors, notably Boll (1869), Klemensiewicz
(1878), and Phisalix (1892) had described
a cellular sheath that surrounds each
chromatophore like a wreath or collar. It
was most prominent in retracted chromatophores, but flattened out and eventually
disappeared when the chromatophores expanded. This ring of cells was interpreted
by many as the seat of the elastic forces
that aided or caused the retraction of the
chromatophore proper. Bozler avoided
commitment to one or the other view by
simply stating that retraction of the chromatophore is caused by the elasticity of the
"cell sheath."
In view of what is now known about
plasma membranes it is clear that the cell
membrane of the chromatophore cannot
provide the elastic force required to stretch
the relaxing muscle fibers and to transform
the expanded pigment cell into a small
sphere that is often less than one-tenth
the diameter of the expanded cell. Cell
membranes are invisible in the light
microscope, although Rabl (1900) pleaded that he had indeed seen a cell membrane separating the chromatophore proper from its muscle fibers, despite the claims
of other histologists that they could not
see it. Van Rynberk (1906) favored the
view that the entire chromatophore together with its complement of muscle
432
ERNST FLOREY
fibers formed a syncytium. The position
adopted by Hofmann (1907, p. 385) was
one of extreme caution: he felt that the
broad connecting bridges between adjacent
muscle fibers where they attached to the
body of the* chromatophore proper, forced
one .tp.the conclusion that the chromatophores must be considered a functioning
syncytium. But he also stated later that
when the radial muscle fibers relax, "the
pigment body retracts, essentially due to
the elasticity of a cell membrane that surrounds it" (1910, p. 44), and he pointed
out (p.- 45) that to- understand the observed behavior of the musculature of the
ehromatophores, the chromatophores must
be considered multinucleate single cells
(Chun, 1901) so that it is possible "to
observe even the behavior of single parts
(the different radial fibers) of a cell after
stimulation."
I emphasize Hofmann's position intentionally because his statements represented
the last word- on the structure of
cephalopod chromatophores when Richard
Gloney and I began to study their ultrastructure two years ago!
How important it is to know.whether
the chromatophore or its. muscle fibers represent a syncytium or not, becomes evident
when one considers the functional behavior of the chromatophores. Bozler (1928,
1930) showed clearly that individual muscle fibers could.be activated independently
through select stimulation of their nerve
supply. Bozler concluded, therefore, that
the muscle fibers of a chromatophore do
not fqrm a syncytium. On the other hand
it-had already been shown by earlier au?
thors, in -particular by Hofmann (1907),
that chromatophores sometimes pulsate,
even in the absence of nervous stimulation,
l a these cases all muscle fibers seem to con-!
tract synchronously (evidence for Hofmann
of. the syncytial nature of the chromatophoric musculature). Bozler (1928) felt that
such pulsations occurred only as a transient
phenomenon and that they were due to excitation of the peripheral nerve branches.
The observations of Hofmann and of
Bozler can be summarized as follows: in
freshly excised skin the chromatophoric
muscles are completely relaxed and the
chromatophores are retracted. Stimulation
of the skin nerves with single pulses causes
twitch-contractions; repetitive stimulation
above 6-10 pulses/sec gives rise to a tetanus. Not. all the muscle fibers of a given
chromatophore are activated by the same
nerve, but a given nerve branch activates
muscles of many chromatophores. Previously denervated skin, or skin that had
been excised for several hours, exhibits
chromatophoric responses that have been
described and discussed in several of the
older papers: chromatophores show varying degrees of steady or fluctuating expansion, and some pulsate. The spontaneous
expansions have been ascribed to a tonic
contraction of the muscle fibers, considered
not to be a tetanus for the following reasons (Bozler, 1928, 1929, 1930):. muscle
fibers often show intermediate states of
contraction that cannot be duplicated by
nervous stimulation since this always gives
rise to maximal contraction; partially contracted .muscle. fibers can be brought to
maximal contraction through tetanic stimulation of their nerve supply; and, lastly,
the tonic contractions can- persist for
hours, and can thus be assumed to .persist
without great expenditure of energy as
would be the case if the contraction were
a tetanus. When maximal tonic contractions are -present,, nervous stimulation
causes relaxation: single stimuli produce
only a short-lasting relaxation; repetitive
stimuli (referred to as faradic stimulation)
lead to relaxations of longer duration that
are more complete. Bozler interpreted this
as evidence for the presence of: inhibitory
nerve fibers. He assumed that the nervous
stimulation caused inhibition when the
motor neurones had already died and only
the more resistant inhibitory neurones :remained. A similar opinion was expressed
by TenCate (1928). The concept of inhibitory innervation was not new: Hofmann
(1907, p. 419) seriously considered it but
decided "that there is no definite evidence
for the existence of an inhibitory innervation of the chromatophores-"
433
CEPHALOPOD CHROMATOPHORES
5/sec
FIG. 2. Tracings of contractions of chromatophoric
muscle fibers resulting from stimulation of
their motor axons. The upper channel records
contraction, the lower channel the stimuli. The
numbers indicate the frequency of stimulation in
pulses per second.
The notion of inhibitory innervation received new impetus when Kahr (1959) discovered that 5-hydroxytryptamine (5-HT)
caused retraction of chromatophores in the
skin of Octopus, an action which he found
was antagonized by acetylcholine (ACh).
Kahr ignored the function of the radial
muscle fibers and spoke of a "cell-contracting" action of 5-HT. The analogy to the
pharmacological behavior of the anterior
byssus-retractor muscle of the mussel, Mytilus, is striking: this muscle has been studied in great detail as a representative example of molluscan "catch" muscle. As
Twarog (1954) discovered, ACh causes tonic contraction in these muscles, while 5-HT
effectively abolishes tonic contraction ( =
"catch"). Twarog's interpretation that 5HT may be the transmitter substance of
relaxing nerve fibers, has found wide acceptance. Interpreting Kahr's observations
in the light of the known function of the
radial muscle fibers, one is led to speculate
that ACh might be the transmitter of the
motor neurones and that the relaxing action of 5-HT might be evidence for a tryptaminergic inhibitory innervation of the
chromatophoric muscle fibers. Bozler (1928)
had already emphasized the similarity of
the tonic behavior of the chromatophoric
muscles of squid with that of molluscan
catch muscle.
It became clear that a thorough investigation of the structure and function of
cephalopod chromatophores, employing
modern'techniques of electron microscopy
and electrophysiology was necessary to
resolve the many puzzling problems posed
by these intriguing organs. What is the
nature of the tonic muscle contractions? If
they are not initiated by nerve impulses,
what causes them? What is the nature of
the innervation; is there evidence for more
than one kind of nerve fiber—and where
are the neuro-muscular junctions? What
kinds of postsynaptic potentials are generated by stimulation of the nerve supply,
and what are the effects of ACh and 5-HT
on the muscle membrane? It was also
necessary to find out if the chromatophores, or at least their muscle fibers, form
a syncytium. Finally, an explanation had
to be found' for the fact that nervous stimulation could independently activate individual muscle fibers, while the spontaneously arising contractions occurred simultaneously in all the fibers of a chromatophore during pulsations.
Improving a technique' already employed by Bozler (1930), I used a photocell to monitor the contractions of chromatophoric muscles as they were reflected in
the moving boundary' of the lightabsorbing pigment body of chromatophores as this was projected through the
lenses of a microscope. With the aid of
suitable amplifiers these were transcribed
on a pen-writing chart-recorder. Whereas
Bozler stimulated the nerve elements by
passing a current across the entire piece of
isolated skin, I made use of suction electrodes to stimulate small bundles, usually
consisting of less than 10 axons. Figure 2
shows examples of records obtained when
supramaximal stimuli were applied at different frequencies. When the applied voltage was reduced gradually, there was a
stepwise reduction in the strength of the
muscle contraction even when the pulsefrequency remained constant. The image
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ERNST FLOREY
•
•
•
•
•
•
•
•
•
•
•
0
•
m
*
1,2,3
^
V
—— = motor unit 1
motor unit 2
motor unit 3
FIG. 3. Stepwise expansion of chromatophores due
to recruitment of additional motor units. The upper set of four diagrams shows the appearance of
the same group of brown chromatophores in the
absence of stimulation (O), and when 1, 2 ( =
1+2) and 3 ( = 1+2+3) motor axons of the
appropriate nerve bundle are stimulated at the
same frequency of 20 pulses/sec. The lower set
of diagrams illustrates the distribution of the motor units. The diagrams are based on a series of
photomicrographs taken during an experiment.
Only three of the observed six motor units are
shown here.
of the responding chromatophores showed
that this was due to a reduction in the
number of activated muscle fibers. The
nerve bundle supplied several chromato-
phoric organs, each motor axon activating
several muscle fibers of each. Thus, using
the same impulse frequency, the nerve, in
the intact animal, can produce increasing
CEPHAI.OPOD CHROMATOPHORES
shades of brown by a stepwise expansion of
each of the brown chromatophores due to
recruitment of more muscle fibers (Fig.
3). It also became clear that differently
colored chromatophores were independently innervated.
Bozler's observation that the muscle
fibers of a given chromatophore could be
individually and independently activated
was easily confirmed. But during the same
experiments there invariably occurred pulsating chromatophores as the preparations
aged. When all nerve-mediated responses
were stopped by applying tetrodotoxin
(pufferfish toxin, known to abolish spike
potentials involving sodium-activation)
the pulsations, as well as slow, tonic contractions, persisted. Clearly, neither tonic
contractions nor pulsations could be due to
spontaneously arising nerve impulses. Yet
during the pulsations the entire complement of muscle fibers of the pulsating
chromatophores contracted in synchrony!
This could no longer be explained by nervous coordination. How then does the excitation arise and how can excitation
spread from fiber to fiber, when nerveactivated muscle fibers do not influence
their neighbors?
The surprise solution of the paradox
came through electrophysiological studies
(Kriebel and Florey, 1968; Florey and
Kriebel, 1969). Using intracellular microelectrodes we found that the muscle fibers
responded to nervous stimulation only
with local excitatory postsynaptic potentials. These potentials showed no facilitation, nor was there appreciable summation.
It was impossible to elicit spike potentials.
Even when the muscle cells were depolarized with the aid of a second, intracellular,
current-passing electrode no spikes could
be produced. The cell membrane proved
to be electrically inexcitable. The local potentials, on the other hand, could be recorded anywhere along the entire length of
the muscle fiber with similar amplitude.
This indicated some form of polyterminal
innervation.
Changing the stimulating voltage we
found that there was a stepwise change in
435
FIG. 4. Excitatory postsynaptic potentials recorded
with an intracellular microelectrode from a chromatophoric muscle fiber. The motor nerve, containing several axons, was stimulated with single
pulses of increasing current. The records are superimposed to show the stepwise increase in the amplitude o£ the postsynaptic potentials. Note the
difference in rise-time (data of Florey and Kriebel).
the amplitude of the excitatory postsynaptic potentials, (Fig. 4). Up to six or seven
steps occurred, clearly suggesting polyneuronal innervation. It was puzzling, however, that the smaller, low threshold potentials usually had a longer rise-time, and
often a longer latency. If motor nerves elicited only small, local potentials then it
was easy to understand that the muscle
fibers could be individually and independently activated: even if they formed a
syncytium, the non-conducted postsynaptic
potentials would insure independent activation.
It was not easy to successfully penetrate
pulsating muscle fibers with recording microelectrodes, but we were successful in
several preparations. The results were startling: these muscle cells generated spikesl
Each spike potential was preceded by a
slowly rising generator potential (pacemaker potential) (Fig. 5). Not all spikes
led to complete depolarization, and often
there was a sequence of several spikes of
increasing amplitude. Such episodes sometimes occurred at regular intervals. Visual
observations showed that the large spikes
were accompanied by synchronous contractions of all muscle fibers of the particular
chromatophore, giving rise to a typical
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ERNST FLOREY
FIG. 5. Spike potential recorded from a pulsating
muscle fiber of a brown chromatophore (Loligo
opalescens). Note the slowly rising generator depolarization. The small potential during the hyperpolarizing after-potential has been seen in several
instances; it is possibly a movement-artifact. The
upper trace represents zero membrane potential,
(data of Florey and Kriebel).
pulsation. Here then was the solution to
the problem of synchronization: conducted
spikes could be assumed to spread from
FIG. 6. Diagram of the ultrastructure of a retracted chromatophoric organ of Loligo opalescens. A,
axon; C, contractile cortex of muscle fiber; F, folds
of cell membrane of chromatophore; G, glia cell
that coveTS the axon and its terminal structures;
H, haptosomes attaching folds of the cell membrane to the elastic sacculus (S) that encloses the
one muscle fiber to the next. A further
question remained: Is there a connection
between them that would permit HQW of
current from one fiber to the next? .-•
At this point, only an analysis of the
ultrastructure of the chromatophores could
provide the answer. The results have already been published (Cloney and Florey,
1968), and I shall refer here only to the
highlights (Fig. 6). So many problems required an answer. There was not only the
question whether the muscle fibers formed
a syncytium, but it was not even known
how the muscle fibers were constructed.
Bozler reported that he had seen internal longitudinal fibrils ("Tonusfibrillen"),
while the earlier histologists had simply
described the core of the fibers as granular.
According to Bozler, the fibrils were responsible for the tonic contraction, while
the outer, contractile cortex of the fibers
pigment granules, m, mitochondria in the core of
the muscle fiber; N, nerve terminals; n, nucleus of
muscle cell; M, muscle fibers; J, junction between
adjacent muscle fibers. The sheath cells that cover
the chromatophore and the muscle fibers are not
shown. (After Cloney and Florey, 1968).
CEPHALOPOD CHROMATOPHORES
gave rise to the phasic contractions initiated by nervous impulses. Considering the
similarity of contractile behavior and
pharmacology 6f chromatophoric muscles
and of molluscan catch muscle it seemed
quite possible that the ultrastructure of the
two types of muscles would show similarities. In particular, do chromatophoric
muscles contain the thick paramyosin filaments that are so characteristic of the byssus retractor muscle and other . lamellibranch "holding" muscles? And, of course,
there was the question of the innervation:
how many motor fibers were involved in
the innervation of a given- muscle fiber,
and what .was the structure of the neuromuscular junction? Was there any evidence for inhibitory neurons, perhaps a
difference in the kind of synaptic vesicles
of different terminals?..
In addition, there was the old problem
of the elastic elements responsible for the
retraction of the chromatophore. Was
there an elastic cell membrane? Or "Was
there an elastic cellular ring surrounding
each chromatophore?
.
Nothingis more amazing than the truth.
We found no tonus fibrils in the interior
of the muscle fibers; instead the entire core
of each fiber consisted of tightly packed
mitochondria. The outer, contractile cortex contained no paramyosin filaments but
staggered arrays of thick and thin filaments of dimensions similar to those of
typical myosin and actin filaments of vertebrate striated muscle. Each set of filaments,
was. separated from the neighboring sets
by obh'quely oriented sheaths of sarcoplasmic reticulum. This arrangement gives the
muscle the appearance of an oblique striation—hence the term "obliquely striated
muscle."
A nerve strand runs over the entire
length of the muscle fiber in a zig-zag pattern that obviously allows for the great
change in length of the contracting muscle
(Fig. 6). One to four axon profiles were
seen in cross-section. Synaptic vesicles were
prominent in all of them. The axon (s)
lay in a groove and made intimate contact
437
with the sarcolemma; the glial cells of the
sheath were displaced so that they covered
the groove (Fig. 7). The glial cells and
muscle were covered by a common external lamina which', in turn, was surrounded
by several layers of sheath cells. The electron micrographs indicate that die synaptic
junction between axon (s) and muscle extends over the entire length of the contrac1
tile portion of the muscle cell. This explains why the local postsynarptic' potentials can be recorded anywhere along the
entire muscle fiber.
Where they join the pigment cell, the
individual muscle fibers 'make contact with
each other: everywhere else they are.surrounded by a basal lamina, but here this
lamina is absent. The membranes of adjacent muscle fibers. are closely apposed,
the distance between them narrowing for
wide regions to about 30 A (osmium staining) to form "close junctions" (Trelstad,
Hay, and Revel, 1967), suggesting a pathway of low resistance between adjacent
cells that permits' passage of action cur.rent.
"
" _ .,-.\v-'. • '
Kriebel and I have measured the electrical coupling between neighboring muscle
cells by using two. intracellular electrodes,
one for current-passing, the other for recording. Keeping the inter-electrode distance constant it is possible to measure the
coupling coefficient by .determining the
voltage change produced in the same and
in the neighboring muscle cell, The values
ranged from 0.1 to 0.16. From this and
from measurements of input and transfer
resistances, the specific membrane resistance has been calculated, using electronmicrographs for determining the various
membrane areas. The specific membrane
resistance was found to be 1.0 to 1.3 X 103
Ohm.cm2 for the regular surface membrane
of the muscle fibers, "and 12.8 to 22.6
Ohm.cm2 for the close junctions. Assuming
the resistivity of the sarcoplasm to be twice
that of sea water (23 Ohm-cm), the length
constant is about 1.5 mm.
The ultrastructure of the pigment cell
itself proved full of surprises; what could
438
ERNST FLOREY
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CEPHALOPOD CHROMATOPHORES
FIG. 7. Electron micrograph of neuromuscular
junction (Loligo opalescens). The same axon (Ax)
is sectioned twice as it weaves in and out of
the plane of section. On its outer side (facing away
from the muscle fiber) it is covered by a glia
cell (G) that contains granulated vacuoles (V).
Muscle fiber and glia cell are covered by a common
external lamina (EL) that separates them
from several layers of sheath cells (S). Myofila-
ments (MF) can be seen in the outer cortex of the
muscle fiber; the thick filaments have diameters of 100—300 A, the thin filaments a diameter
of 60—80 A. The muscle fiber is sectioned
near its surface. Near the right margin can be seen
several surface folds (F) of the sarcolemma. Mitochondria (M) are near the layer that contains the
myofibrils. (Cloney and Florey, 1968).
easily be mistaken for a ring of cells
around the chromatophore turned out to
be a set of large folds of the chromatophore's cell membane. The membrane of
retracted chromatophores is incredibly
folded whereas the pigment body is contained in a smooth-cont,ured sac composed
of ultrafine filamentous material (Fig. 8).
We call it the cyto-elastic sacculus and
ascribe to it the elastic force responsible
for the retraction of the chromatophore.
In the expanded condition this sacculus is
thinly stretched over the flattened disc-like
pigment body. The cell membrane under
these conditions is unfolded and smoothly
covers the pigment body (Figure 8).
Around the equator of the pigment cell
the elastic sacculus is attached to the cell
membrane by a continuous dense structure which we named "zonal haptosome."
The cell membrane in turn is attached to
the surrounding muscle fibers by a dense
basal lamina. The upper and lower surface
of the pigment sacculus shows spot-
attachments of the cell membrane. These
"focal haptosomes" (Fig. 6) may be assumed to serve in the maintenance of order and organization of the complex folding pattern which the chromatophoric
membrane assumes during retraction.
Nothing has yet been said about the
mechanism underlying the tonic contractions. Our electrophysiological investigation (Kriebel and Florey, 1968; Florey and
Kriebel, 1969) brought the surprising answer: these tonic muscle contractions are
caused by miniature potentials, that is by
spontaneous, quantal release of transmitter
substance from motor nerve terminals.
Examples of miniature potentials are
shown in Figure 9. At times the frequency
of these potentials increased enormously;
at the same time the muscle fibers could be
seen to go into tonic contraction. The
miniature potentials reach considerable
amplitudes, the largest ones almost equaling the postsynaptic junction potentials resulting from nervous stimulation. We con-
retraction
expansion
FIG. 8. Diagram of cross sections, perpendicular to
the plane of expansion, through a chromatophore
and its muscle fibers during retraction and expan-
contraction
sion. The pigment granules are contained in an
elastic sacculus. For details of the ultrastructure,
see Cloney and Florey (1968).
440
ERNST FLOREY
FIG. 9. Miniature potentials recorded from chromatophoric muscle fibers (Loligo opalescens) during tonic contraction. Manual adjustment displaced
the- sweep in a Vertical direction in order .to
show a large number of traces on the same image.
The two photographs represent two muscle fibers.
That of A had been- treated with tetrodotoxin
(10"6g/ml)' and showed no response to nervous
stimulation. Calibration: 10 mV; 0.1 sec. (data of
Florey and Kriebel).
elude, therefore, that the tonic contractions
represent a tetanus—a miniature tetanus, so
to speak.
The action of acetylcholine was also explained: this substance did not affect the
muscle membrane directly but enormously
increased the frequency of miniature potentials. The action of acetylcholine is presynaptic and causes increased quantal release of transmitter.
It now remained to discover the
mechanism of action of 5-hydroxytryptamine.' This compound effectively abolished
tonic contraction and antagonized the
effect of acetylcholine. As with acetylcholine, we were unable to detect any
alteration in the electrical characteristics
of the muscle* fiber membrane. We abo
found that'5-HT does hot interfere with
neuro*muscular transmission;. On the other
hand there was a reduction in .the. frequency of miniature potentials, indicating
presynaptic action, and there were signs of
a postsynaptic action: the velocity of shortening and of relaxation of the muscle
fibers was increased after application of
5-HT (Florey, 1966). The latter effect implied an intracellular action within the
muscle cells. •
The effect of applied 5-HT is reminiscent of that of nervous stimulation: both
cause relaxation of tonically contracted
muscle fibers. In view of the" fact that
5-HT does not affect the electrical responses of the postsynaptic membrane, this substance cannot be regarded as a transmitter
substance. Selective stimulation of single
axons (these can be seen and picked up
under the microscope for stimulation)
caused -relaxation of tonically contracted
muscle fibers, but when the latter were
relaxed, stimulation of the same axon was
always followed by contraction. In either
case the micro-electrode picked up only an
excitatory junction potential. In our
studies on miniature potentials we have
never seen any evidence for miniature potentials of differing polarity nor did we
ever observe postsynaptic potentials with
different reversal potentials in experiments
in which we altered the membrane potential by passing current through a second
intracellular microelectrode. In the absence of any evidence for inhibitory (or
relaxing) innervation, die relaxing effect
of 5-HT and of motor nerve stimulation
required a new interpretation.
The diminishing of the frequency of
miniature potentials by 5-HT did not explain the effect on the speed of contraction
and relaxation. The key to this problem
lies in the results of close visual observation of muscle fibers in their normal phasic,
and in the. tonic, condition. When miniature potentials occur in freshly dissected
preparations, the muscle fibers fibrillate.
441
CEPHALOPOD CHROMATOPHORES
Similar jittery movements can be observed
in older preparations after these have been
subjected -to 5-HT. In the tonic condition, muscle fibers often fail to relax after
stimulation of motor nerves,- and-after episodes of miniature potential activity have
subsided. We have often noted that muscle
fibers in steady tonic contracture -showed
no miniature potentials, even though they
exhibited- frequent miniature potentials at
the time they went into tonic contraction.
I should like to summarize our hypothesis as follows: The tonic condition that
develops in aging preparations represents a
failure to relax due to failure of intracelliilar calcium to return to its binding sites
within the sarcoplasmic reticulum. Repetitive nervous stimulation or 5-HT restore
intracellular conditions that permit activation of calcium transport into sarcoplasmic
reticulum. Calcium is an important link
not only in excitation-contraction coupling
of muscle, but also in excitation-secretion
coupling in nerve terminals. If excess free
calcium is responsible for spontaneous
transmitter release (and miniature potentials), then calcium binding initiated by
5-HT can be expected to reduce the spontaneous transmitter release. There , is a
great deal of circumstantial evidence in
support of this interpretation (Florey and
Kriebel, 1969).
REFERENCES
Boll, F. 1869. Beitrage zur vergleichenden Histologie des Molluskentypus. Arch. Mikr. Anat. 5
Bozler, E. 1928. tJber die Tatigkeit der einzelnen
glatten Muskelfaser bei der Kontraktion. II.
Mitt. Die Chromatophorenmuskeln der Cephalopoden. Z. Vergl. Physiol. 7:379-406.
Bozler, E. 1929. Weitere Untersuchungen zur Frage
des Tonussubstrates. Z. Vergl. Physiol. 8:371-390.
Bozler, E. 1930/31. Ober die Tatigkeit der
einzelnen glatten Muskelfaser beider Kontraktion. III. Mitt. Registrierung der Kontraktionen
der Chromatophorenmuskelzellen von Cephalopoden. Z. Vergl. Physiol. 13:762-772.
Chun, C. 1901. Ober die Natur und die Entwicklung der Chromatophoren bei den Cephalopoden. Verh. Deut. Zool. Ges. 11:162-182.
Cloney, R. A., and E. Florey. 1968. infrastructure
of cephalopod chromatophore organs. Z. Zellforsch. 89:250-280.
• ,:.L6iigo- opateti&Tis.' Gomp. Biochent. Physiol.
-18:305:3?*.
••
'
•••'•
'".:••-':•
Flprey, E.,-.and M. ^ Kriebel'. :4969. EleC6»cal and
^••'hjBch^nfeal;. respojnises 0f dhrpmatoph'oje;' njuscle
. Vfibeis' of ^je iquid), Loligo qpalesi^nSi-,Ur:nerve
stimulation and "drags. Z. y.ergF.* Physiol. (In
j h " A. V., and D. Y. Solajldi: 1954, MtySgramis
from the chromatophores of; S'epiif.- J. iPhysioK
""•' '
126:13P-14P.
Hplmanrt,, F. -B., 1907. ^[istologische Unters,uchun,: gen,tiber die Inrtervation der-glatten;.und der, (Hr
. veriy&ndten Muskolatitr- der-. \Virbeltiere ;un^
der Molliusken. Axchi" Mikf. Anat. '70;36l-413.
Hofmann, F. B,ir 1907. Gibt es in- der Muskulat'ur
der Mollu$ken .periphere, fcotitinuierlich leiterfde
Nervennetze bei Abwesenheit von Ganglienzellen?
I. Untersirchungen an Cephalopoden.(. Pfliigers
Arcli. Ges."PKysiql. 118:375-412. '• -VHofmann, .F:" B'.,. 19O7-. Ober einen peripherep
Tdnus dpr, Cephaloppden-Ghromatophoren un<3
uber ihre Beefriflussung -<lurch Gifte.. Pfliigers
Arch. Ges. Physiol. 118:413^451.
Hofmann, F. B., 1910;. Chemische. Reizung und
Lahmung markloser Nerven und glatter Muskelti
wirbelloser Tiere. Untersuchungen an den
Chromatophoren • der Kephalopoden. Pfliigers
Arch. Ges. Physiol. 132:82430.
Hofmann, F. B., 1910. Gibt es in der Muskulatur
der. Mollusken periphere, kontinuierlich leitende
Nervennetze bei Abwesenheit von Ganglienzellen?
II. Weitere Untersuchungen an den Chromatophoren der Kephalopoden. Innervation' der
Mantellappen von Aplysia. Pflugers Arch. Ges.
Physiol. 132:43-81.
Kahr, H. 1959. Zur endokrinen Steuerung der Melanophoren-Reaktion bei Octopus irulgaris. Z.
Vergl. Physiol. 41:435-448.
Klemensiewicz, R., 1878. Beitrage' zur Kenntnis des
Farbwechsels der Cephalopoden. Sitz. Ber. Akad.
Wiss. Wien, Math.-natwiss. Kl. (3) 78:7-50.
Kriebel, M. E., and E. Florey. 1968. Electrical and
mechanical responses of obliquely striated muscle
fibers of squid to ACh, 5-hydroxytryptamine
and nerve stimulation. Fed. Proc. 27 (2):236.
Phisalix, C. 1892 Recherches physiologiques sur les
chromatophores des cephalopodes. Arch. Physiol.
Norm. Pathol. 4:209-224.
Rabl, H. 1900. Ueber Bau und Entwicklung der
Chromatophoren der Cephalopoden nebst allgemeinen Bemerkungen uber die Haut dieser Tiere.
Sitzungsber. K. Akad. Wiss. Wien, Math. Naturwiss. Cl. 109:341-404.
Sangiovanni, G. 1819. Descrizione d'un particolare
sistema di organi cromophoro espansivodermoideo e dei fenomeni ch'esso produce, sco-
4 42
ERNST FI.OREY
perto nei molluschi cefalopodi. Giorn. Endclopedico di Napoli 13, nr. 9 .
Sangiovanni, G. 1829. Description d'un systeme particulier d'organes chromophores chez plusieurs
mollusques cephalopodes. Ann. Sci. Naturelles
Zoologie 16:308-315.
TenCate, J. 1928. Contribution a la question de
1'innervation des chromatophores chez Octopus
vulgaris. Arch Neerl. Physiol. 12:568-599.
Trelstad, R. L., E. D. Hay, and J. P. Revel. 1967.
Cell contact during early morphogenesis in the
chick embryo. Develop. Biol. 16:78-106.
Twarog, B. M., 1954. Effects of acetylcholine and
5-hydroxytryptamine on the contraction of a
molluscan smooth muscle. J. Physiol. 152:236-242.
Van Rynberk, G., 1906. Der Farbenwechsel der
Weichtiere und besonders der Cephalopoden.
Ergeb. Physiol. 5:353-394.