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THE INITIATION AND SPREAD OF ACTIVATION OE
THE IRIDOPHORE CELLS IN THE SOUID
LOLIGO OPALESCENS
Annette C. Cholon
Hopkins Marine Station
Spring Class 198
ABSTRACT
The squid Loligo opalescens exhibits iridescent colors in its
third der mal layer through the action of a specific cell type, the
iridophore. Although much is known about the neural control of the
squids pigmented chromatophore organs, little is known about the
action of iridophore cells, other than the neurotransmitter
acetylcholine is responsible for their activation. This paper sets out to
examine the possible mechanism of ACh delivery to the iridophore
cells, through an analysis of the activation and spread of iridescence
in response to electrical stimulation.
INTRODUCTION
The skin of the common market squid, Loligo opalescens is
capable of rapid changes in both color and pattern. These changes are
effected through the action of two cell types, chromatophores and
iridophores. Together, chromatophores and iridophores allow the
squid to camouflage themselves from predators and to signal to othe
members of the species. The most spectacular displays of color are
produced by the iridescent iridophore cells, which may reflect light in
metallic hues of pink, green, blue, orange and yellow.
The organization of color producing cells into units and patterns
has been well described. (Hanlon,1982). Iridophores appear in two
basic arrangements, dorsal splotches and ventral sheets. The dorsal
splotches are arranged as rings, which our lab has fondly nicknamed
doughnuts", and contain a number of iridophore cells with a
chromatophore in the center. The ventral sheets are a more ordered
array of iridophore cells, arranged as continuous sheets of color.
Iridescent cells known as reflector cells cover the optic lobes, ink sacs
and various internal organs of the squid.
Both reflector cells and iridophore cells are characterized by
non-pigmented arrays of thin membrane bound platelets. In both
cell types, these platelets are of a higher refractive indexs than the
material around them, and reflect light to produce structural colors.
The basic difference between reflector cells and iridophores is in the
orientation of the platelets. In iridophores the platlets are oriented on
edge relative ot the skin surface, while in reflector cells they are flat
with a broad surface facing out (Brocco & Cloney, 1980). It is thought
that iridophores produce structural colors by acting either as a thin
film interference device or as a diffraction grating. (Brocco & Cloney,
1980; Land,1972). The basic difference between iridophores that
are in a non-iridescent and those in an iridescent state is in the
ultrastructure of the platelets, which coalesce from a non uniform to
highly structured material of uniform refractive index (Cooper &
Hanlon, 1986).
It has been reported that the pigmented chromatophore organs
are under neural control. (Cloney & Florey, 1968; Mirow,1972; Cloney
& Brocco, 1983). However, little is known about the control of
iridophore cells. Previous investigation has shown the
neurotransmitter, acetylcholine (ACh) to be a key factor in the
activation of structural changes in iridophore cells. Although ACh has
been found in the dorsal mantle iridophore der mal layer of L. brevis
at levels of 1 nmol Ach mg * protein, nothing has yet been deduced
about the delivery of the ACh to the cells, as the presence of nerves
has not yet been anatomically established (Cooper & Hanlon, 1986).
Several possible mechanisms for ACh delivery do exist. One is that the
ACh diffuses across a distance, originating in the chromatophore
layer. Another is that the ACh is delivered through the circulatory
system, acting almost as a hormone. A third possibility, also the
most likely, is that the iridophores are innervated by a very fine
nerve structure which releases ACh at its terminals. This paper sets
out to examine the possible mechanism of activation of the iridophore
cells, how the ACh is delivered, and to quantify the spread and
intensity of iridescence produced as iridophores are activated.
METHODS AND MATERIALS
Schooling L. opalescens caught locally in Monterey Bay were
kept in running sea water tanks. Both male and female squid were
used, with males being used more often because of their greater size.
Unless otherwise noted, skin preperations were made by first
decapitating the squid and then removing all of the internal organs,
including the gladius. The mantle was then pinned out, and the first
two dermal layers were peeled off. The remaining third layer,
containing the iridophores, was left on the mantle to prevent any
damage to possibly existing nerves. The preperation was periodically
run under natural seawater to retain its viability.
Circulatory experiments were run with the internal organs of the
squid intact. Glass microelectrodes were used to cannulate various
major arteries and viens, including the celiac arteries and cephasic
viens.
All experiments relating to neurophysiology were perfor med on
the common mantle preperation. Electrical stimulation was performed
through a fine glass suction electrode. Stimulation was carried out at
differing parameters: voltage ranged between 3V to 30V, frequency
of 1 to10Oper second, with duration 6 to 6ms However, the
parameters of 3V, 100 shocks per second, and 6 ms duration were
shown to be most effective and used for the majority of the
experiments. Results were observed through a Wild dissecting
microscope, and recorded on a VCR using a Hitachi Video Camera.
Mantle preperations were immersed in 50 ml bathes of various agents
to test for the activation of iridophores by electrical stimulation. The
solutions were in concentrations as follows: TTX - 1x10*° in natural
sea water; high Ca' - natural sea water + 50 mM Cacl»; high Zn'-
natural sea water + 10mM ZnCl; 0 Na’- Tris replaced Na’ in
artificial sea water; 0 Ca’- 0 Ca' artificial sea water; high Mg' -
60mM MgCl, in artifiial sea water; Scopolamine - 1x10 4 in natural
sea water. Quantitative analysis of video taped data was performed
on a Mega Vision Image Processor.
RESULTS
Injecting ACh into the circulatory system of the squid resulted in
no change in the status of the iridophores, or even of the
chromatophores. Varying concentrations of 1x10-7, 1x10-6, 1x10-4
were all tried. Neither injections into the major arteried nor into the
major viens proved effective in activating the iridophores to produce
iridescence. Bath applied ACh at a concentration of 1x10-6 after
negative injection results did activate the iridophores, thus the skin of
the preperations was still viable.
Direct electrical stimulation of the third dermal layer of skin
proved to be much more fruitful. A single shock of 7.5V or greater
produced iridescence in a single, or a few iridophores. Multiple shocks
of 3V at a frequency of 100 per second for 1 to 5 seconds activated
large numbers of iridophores in a single doughnut, with a ring size of
about 2.5mm, and often caused the activation of iridescence to
spread to adjacent rings up to almost Smm away. Once it was
established that electrical stimulation was possible in activating
iridophores, various solutions were applied to the mantle
preperations, with results illustrated in Table 1.
Video tapes taken of the stimulation of iridophores in different
solutions were then analysed on the Image analyser. Images of the
iridophores at differents times during the activation process were
projected upon the screen. The image was mapped, and the
percentages of white pixels in each picture represented the area of
iridophore activation. The results from such analysis of both contross
(in natural sea water) and of experimental preperations in high Mg-
and Scopolamine can be seen in figure 1 and figure2.
The actual time of activation through electrical stimulation of the
iridophores ranged between 10 and 60 seconds, with a peak intensity
appearing between 30 and50 seconds. The deactivation of the
iridophores was much longer and more varied, ranging between 5
and 30 minutes while under running sea water.
It was observed that during activation the iridophores passed
through various stages of color, intensity and density. Upon initial
stimulation the iridophores often began iridescing yellow and blue,
later turning to the more common pink and red stage. The spread of
iridescence did not appear to occur in concentric circles about the
electrode tip. Rather, it occured randomly within a ring, with a few
iridophores in various locations activating, to later be joined by others
between them, thus increasing the density of activated within a
doughnut. (see figures 3 and 4).
DISCUSSION
The negative results of the circulatiory injection experiments,
coupled with the activation of iridiphores through electrical
stimulation lead one to believe that ACh is not carried through the
circulatory system to activate iridophore cells. This, in any event,
seemed unlikely from the start, as Ach is easily degraded in the
curculation, and if carried in the blood, would affect many other
organs besides the iridophores.
It is also clear that ACh is not only delivered as a secondary
response to chromatophore activity. Direct observation of the squid
showed that the iridophores did not activate each time the
chromatophores expanded, and that their activation was selective to
the animals particular behavior.
There are several possible explanations for the fact that electrical
stimulation activates iridophores. One possivility is that voltage gated
channels are being opened due to the voltage change caused by the
shock. Although this may account for the activation of iridiphores
directly beneath the electrode, it in no way explains the iridescence
produced in peripheral iridophores, far from the area of direct
stimulation.
Another possibility is that the iridophores are electrically
coupled. That is, that all of the iridophores in a single doughnut are
directly connected by gap junctions. Because an assay for gap proteins
was not performed, this possibility can not be discounted. However,
the activation of peripheral iridophores, as far away as in an adjacent
ring, seems to weaken the possibility of the existance of gap junctions.
A third and more likely explanation for the activation of
iridophores through electrical stimulation is that the iridophores are
all connected through a fine network of nerves. The fact that a higher
frequency of stimulation increases the spread of iridophore activation
suggests that some kind of facilitated activation is talking place. The
high frequency of stimulation may build upon action potentials, thus
allowing them to traverse several synapses to activate a large number
of iridophore cells within the network.
The possible innervation of iridophore cells is also supported
through the preliminary results of stimulation experiments in several
different bath applied solutions (see results Table 1). Sodium is
needed to carry action potentials along nerves, thus the failure of the
spread of iridescence in zero sodium, as well as a partial blockage of
spread in TTX, implies that nerve function may be hindered. This
result is strengthened by iridophore failure in zero calcium solution,
as calcuim is also quite necessary for nor mal nerve transmission.
Magnesium blocks calcium channels at neural synapses, thus a high
magnesium solution would block the efficacy of synaptic transmission.
Scopolamine, working as an ACh antagonist also blocked the spread of
iridophore activation. Therefore, the results of these preliminary
experiments suggest that nerves of some kind may possibly be
involved in the activation and spread of iridophore iridescence.
Examination of the time courses analysed on the image processor
show clearly that iridophore activation does not occur in concentric
circles, spreading away from the electrode. Rather, the iridophores
light up randomly with their density increasing as more and more are
activated. The image processed data also shows that most of the
iridophores show a similar time course of activity, with a peak
intensity of brightness reached about 30 to 50 seconds after
stimulation. It is interesting to note that those iridophores which are
brightest at first also seem to fade first.
In summary, the preliminary evidence presented here indicates
that the iridophore cells of squid L. opalescens could possibly be
under neural control, with a fine network of nerves present in the
third dermal layer. Thus, ACh could be delivered at these nerve
terminals, and not from a distance or through the circulation.
However, many experiments remain to be performed before any solid
conclusions about the presence of nerves in the third layer can be
made, the most obvious of which is a thorough anatomical search. A
great deal of analytical potential also lies in the use of the Mega Vision
Image Processor, which presents a good method of quantifying the
spread and intensity of iridescence. Overall, the study of the
activation of iridophore cells may lead to a better understanding not
only of squid behavioral color patterning, but also of the nervous
control of other ACh based systems.
O
ACKNOWLEDGEMENTS
I am grateful to Dr. Stuart Thomson for his advice, patience, and
encouragement, to Dr. William Gilly for his help, and to Natasha
Fraley for being kind enough to let me use her equipment and office.
O
TABLE 1.
IRIDIPHORE RESPONSE IN VARIOUS SOLUTIONS
Bolution
Iridescence
TTX I 10-6
+ -
SCOPOLAMINE 110-41
HIGH MG¬
+ -
- - - -
oca¬
- - - -
O Na¬
+ -
O Na-//w/Li-
+- - partial blockage of iridescence
--- - - full blockage of iridescence
C
cte dere
3
0 00
160
o
G-
a
2
0
20
30
TIME(SEC)
40
CONTROL
SCOPOLAMINE +
50
60
FIGURE 4
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