The role of muscarinic receptors and intracellular Ca2+ in the spectral reflectivity changes of squid iridophores
1 The Marine Biological Association of the United Kingdom, The Laboratory,
Citadel Hill, Plymouth PL1 2PB, UK
2 Department of Animal and Plant Sciences, University of Sheffield,
Sheffield S10 2TN, UK
* Author for correspondence at present address: Vision, Touch and Hearing Research Centre, University of Queensland, Brisbane, Queensland 4072, Australia (e-mail: l.mathger{at}uq.edu.au)
Accepted 24 February 2004
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Summary |
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Key words: squid, Alloteuthis subulata, invertebrate, multilayer reflector, reflectivity, iridophore, calcium, acetylcholine, muscarine receptor
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Introduction |
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Ca2+ serves in many second messenger systems in a variety of
cell types and triggers numerous physiological phenomena (for reviews, see
Berridge, 1998;
Bootman et al., 2001
). To date
it has, however, not been shown directly whether Ca2+ is involved
in the functioning of animal reflectors. In this study we measured
intracellular Ca2+ concentrations and we present data to suggest
that reflectivity changes of squid iridophores are mediated by cytoplasmic
Ca2+. Hanlon et al.
(1990
) suggested that
reflectivity changes in squid may be mediated by Ca2+, but were
unable to substantiate these claims, as no direct measurements of
Ca2+ were made.
Mäthger and Denton
(2001) described the
organisation and possible functions of the iridophores of the small squid
Alloteuthis subulata, found off the coast of Northern Europe. They
found that the reflective properties of squid iridophores resembled those of
ideal quarter-wavelength multilayer reflectors. Light reflected from a
reflector of this kind is almost always coloured. Multilayer reflectors are
characterised by the fact that they contain thin plates of a higher refractive
index than the spaces separating them. In an ideal multilayer reflector the
plates and spaces both have an optical thickness (actual thickness multiplied
by refractive index) of a quarter of the wavelength reflected by the stack at
normal incidence. `Ideal' here means that such a stack has the highest
reflectivity in comparison with `non-ideal' reflectors, for which the plates
and spaces differ in optical thickness. It therefore becomes obvious that a
change in the thicknesses of the plates and/or spaces will change the
wavelengths reflected from the stack
(Huxley, 1968
;
Land, 1972
).
Fig. 1B shows the location of
the various reflective stripes found in A. subulata and the
orientation of the iridophores with respect to the skin surface. Mäthger
and Denton (2001
) described
only one reflective state of the iridophore stripes of this squid. However,
the iridophores of these stripes can often be observed in varying reflective
states. They can be highly reflective as well as non-reflective and they can
undergo changes in spectral reflectivity. The present paper describes in
detail the reflectivity changes of iridophores of the squid A.
subulata. The aim was to study the ultrastructure of iridophores in a
whole-skin preparation as well as at the cellular level, and to study the
pharmacology and the Ca2+ dynamics of these cells.
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Materials and methods |
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Transmission electron microscopy (TEM)
After decapitation the mantles of 5 squid were fixed in 2.5% glutaraldehyde
in artificial sea water (ASW, in mmol l-1: NaCl 470, KCl 10,
CaCl2 10, MgCl2 60, Hepes 10, pH 7.8). Fixing the entire
mantle ensured that the iridophores maintained their original orientations
when tissue was processed for TEM. Small samples were cut out from various
skin areas containing the different reflective stripes and dehydrated in a
graded series of acetone. Specimens were embedded in Spurr's resin (TAAB,
Aldermaston, UK). Sections were cut on a ReichertJung Ultracut
microtome using glass knives (TAAB), stained with uranyl acetate and lead
citrate and examined on a transmission electron microscope (JEOL JEM 200 CX,
Welwyn Garden City, UK). Following dissociation, isolated cells were treated
in the same way. However, a 15 min period was given between each dehydration
step to allow cells to settle.
Whole skin preparation
The effects of different agonists and antagonists on the various iridophore
stripes were studied. The shape of the mantle was maintained by inserting a
cylinder made of black plastic foil into the mantle cavity. This was crucial
to determine the angle of incidence at which the reflective stripes were
observed. The cylinder was firmly attached inside a 100 ml perfusion chamber.
Rotation of the cylinder allowed all reflective stripes to be observed. All
drugs were superfused across the preparation. The responses of iridophores
were observed through a dissecting microscope. Unless specified otherwise, all
observations were made at near-normal incidence (approximately 20°) to the
individual reflective stripes (see Fig.
2A for orientation of normal incidence). Photographs were taken on
a camera (Nikon UFX-II, Kingston Upon Thames, UK) attached to the microscope.
A video camera (Panasonic VHS, Osaka, Japan) was used to record changes in
spectral reflectivity in living squid.
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Electrical stimulation was carried out using a bipolar silver wire field electrode. The squid mantle was cut open ventrally and pinned onto the Sylgard base of a Petri dish containing ASW. While observing the spectral reflectivities of iridophores, areas, including the iridophore and chromatophore layers and the stellate ganglion, were stimulated using a Grass SD 5 Stimulator (Rhode Island, USA). Iridophores were stimulated for up to several seconds with voltages between 0.8 and 80 V, applied in pulses lasting 0.220 ms at frequencies of 220 Hz.
For some experiments a Ca2+ free solution was used (EGTA Ca2+ free ASW, in mmol l-1: NaCl 470, KCl 10, MgCl2 10, Hepes 10, EGTA 10, pH 7.8).
Isolation of iridophores
The iridophore layers were dissected from 1.52 cm2 pieces
of skin, taken from the dorsal, lateral and ventral regions of the mantle. The
iridophore layers were incubated in centrifuge tubes (1 ml volume) for
1520 min in ASW containing 1 mg ml-1 trypsin (type III,
Sigma Aldrich, Poole, UK) at room temperature. After washing in ASW the cells
were transferred to a solution of ASW containing 3 mg ml-1 papain
(Sigma) and 3 mg ml-1 collagenase P [Boehringer Mannheim (Roche
Diagnostics), East Sussex, UK]) (filtered through a 2 µm filter) for
2030 min at room temperature. The tissue was then washed thoroughly in
ASW and strongly agitated in a Petri dish containing glass coverslips in ASW.
The cells were allowed to settle for 10 min and were kept viable in the
refrigerator for up to 4 h. In order to establish whether the dissociated
cells were iridophores, some isolated material was processed for TEM.
Dye filling using whole cell patch-clamp electrodes
The coverslips holding the isolated iridophores were transferred to the
chamber of a pre-cooled stage (1113°C) of an inverted microscope
(Diaphot, Nikon, Kingston Upon Thames, UK). Cells were filled with Lucifer
Yellow after establishing whole-cell configuration, by dialysing the pipette
internal solution (0.3% of the dye). The bath solution used was ASW. The
pipette solution contained (in mmol l-1):
caesium-D-aspartate, 450; MgCl2, 15; EGTA-Cs, 15;
MOPS-Cs, 30 and TEA-Cl, 6 (pH 7.2). Patch electrode resistance ranged from
24 M. In this study, we used a laboratory-made amplifier with a
headstage currentvoltage converter, with a feedback resistance of 1
G
.
Ratiometric Ca2+ measurements
Isolated iridophore cells were ester loaded with the Ca2+
sensitive dye Fura-2 AM (Molecular Probes, Oregon, USA) in ASW for 30 min
(final loading concentration of dye was 5 µmol l-1).
Fluorescence from Fura-2 loaded iridophores was measured on a pre-cooled stage
(1113°C) of a microscope (Optiphot-2, Nikon), which was equipped
with a photometric analysis system (Cairn, Kent, UK). A spinning wheel with
filters of 340, 360 and 380 nm was used to provide the excitation wavelengths
needed to record the Ca2+ dynamics of Fura-2 loaded cells. The
light source was a Xenon lamp (75W; AMKO, Tornesch, Germany). Fluorescence
emission was measured at 450520 nm. During the measurements,
iridophores were visualised on a monitor, using an infra-red video camera.
Some experiments were done in EGTA Ca2+ free ASW. The solution was the same as mentioned above, only the concentration of EGTA was reduced to 3 mmol l-1.
Calculations of `resting' Ca2+ concentrations
The dual-wavelength fluorescence data (ratio 340 nm/380 nm) was calibrated
using an adaptation of the method given by Thomas and Delaville
(1991), in which cells,
permeabilised by ionomycin (1 µmol l-1), are bathed in EGTA
Ca2+ free ASW to obtain minimal Ca2+ concentrations.
Saturating Ca2+ concentrations are obtained by bathing cells in
ASW. At the end of the calibration, 2 mmol l-1 MnCl2 is
used to determine auto/background fluorescence. This procedure is fully
described and illustrated in Benech et al.
(2000
).
Drugs
The following drugs were prediluted in the bathing solutions (ASW and EGTA
Ca2+ free ASW) and superfused across preparations: acetylcholine
(ACh), carbachol, L-glutamate (L-glu), serotonin
(5-hydroxytryptamine; 5-HT), atropine, muscarine, nicotine, caffeine and
potassium chloride (KCl). All chemicals were obtained from Sigma (UK).
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Results |
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The reflective plates of the `red' stripe were oriented approximately parallel to the skin surface. Plates were on average 103±2.38 nm thick (mean ± S.E.M., N=13).
The iridophore plates of the `blue' stripe lay at angles of 5070° with the skin surface. This corresponds to an angle between the normals of the iridophores and the horizontal of between 50 and 70°. The plates were on average 190±2.01 nm thick (mean ± S.E.M., N=13).
The iridophore plates of the ventral iridophores lay at angles ranging from 60 to 70° with the skin surface. For specimens taken from the area halfway between the ventral midline and the blue stripe this corresponds to an angle between the normals of the iridophores and the horizontal of 1020°. Plates were on average 102±3.06 nm thick (mean ± S.E.M., N=13). No nerve terminals were found in the iridophore layer of the mantle.
(2) Changes in spectral reflectivity
In order to describe the changes in spectral reflectivity that occurred
during iridophore activity a `resting' state of reflectivity had to be
recognised. This resting state was found by observing the iridophore
reflections of a freshly decapitated squid at angles around normal incidence
until no further changes in spectral reflectivity took place. This occurred
after approximately 1520 min of killing the squid. These resting
reflective states could also be observed in living squid.
When in their resting state, the `red' stripe iridophores are non-reflective. The `blue' stripe iridophores have reflectivities in the blue and the ventral iridophores reflect weakly in the red parts of the spectrum. All the experiments described in section 3 were done using iridophores in their resting state.
(3) Pharmacology of iridophores using whole skin preparations
The `red' stripe
When `red' stripe iridophores in their resting state (i.e. non-reflective)
were superfused with ACh or carbachol in ASW they `switched on', that is, they
reflected red light (Fig. 2A).
This response was observed within 1015 s of ACh perfusion (2050
µmol l-1) (N=10). Maximum reflectivity was reached
within 11.5 min. Washing for approximately 15 min in ASW reversed the
effects of ACh. Assuming that the changes in reflectivity are elicited by
changes in the spacing between adjacent iridophore plates, this would suggest
that, when non-reflective, best reflectivity would be in the infra-red, while
ACh causes the plates to become closer together, resulting in reflections of
red light. There are other ways in which this colour change can be produced
(see Discussion). Perfusion of muscarine (3050 µmol l-1)
(N=5) resulted in the same spectral shifts as caused by ACh and
carbachol. The effects of ACh and muscarine could be blocked by 510
µmol l-1 atropine in ASW (data not shown) (N=3).
Superfusions of nicotine (1050 mmol l-1), caffeine (30
µmol l-1 to 60 mmol l-1), KCl (20100 mmol
l-1), L-glu and 5-HT (each 1050 mmol
l-1) had no effects on spectral reflectivities (for each experiment
N=5).
The effect of the absence of external Ca2+ was also investigated. The preparation was kept in EGTA Ca2+ free ASW for 15 min. This had no effect on the reflective properties of the iridophores. The preparation was then superfused with 50 µmol l-1 ACh in EGTA Ca2+ free ASW, which caused the iridophores to `switch on' (N=5). This suggests that ACh acts via mechanisms independent of the presence of external Ca2+.
We also investigated whether the spectral changes could be based on osmotic changes within the iridophore. This was done by perfusing the preparations with hyperosmotic ASW (from 30 to 50 mmol l-1 sucrose in ASW, with osmolarities of 10991158 mOsm kg-1) and hyposmotic ASW (5075% ASW, with osmolarities of 536769 mOsm kg-1). Even after 40 min, neither solution produced any change in spectral reflectivity (N=5). However, the iridophores remained viable, as they still responded to perfusions of ACh after these experiments.
When in a reflective state, the iridophores of the `red' stripe reflect
green light at angles of incidence around 45°. Iridophores that are
observed at 45° during the process of `switching off' reflect first yellow
and then red light (Fig. 2B),
before they `switch off' completely. During the stage of red reflection at
45°, measurements of reflectivity and polarisation, using the methods
described by Mäthger and Denton
(2001), showed that the
reflected red light is polarised (data not shown).
Fig. 2C shows images taken from
a video. At the beginning of filming, the `red' stripe reflected strongly in
the bluegreen (Fig. 2Ca)
(note that the angle of incidence to the `red' stripe is approximately
4550°). After approximately 30 min of filming, the iridophores
began to reflect red and yelloworange light at that angle, showing that
the reflective shifts occur in a living squid.
The `blue' stripe
The `blue' stripe iridophores responded to perfusions of 2050
µmol l-1 ACh or carbachol by `switching off', that is the
reflections became invisible to the human eye (N=10). Assuming that
the activity pattern is based on a platelet spacing change, this would suggest
that the reflections would shift from blue into the UV. Reflectivity changes
of the `blue' stripe took longer than those of the `red' stripe iridophores.
They set in after approximately 30 s of drug perfusion and were complete
within 25 min (Fig. 2D).
These spectral changes were also observed when superfused with 3050
µmol l-1 muscarine (N=5). Atropine (510 µmol
l-1) blocked the effects of ACh and muscarine (N=3).
Nicotine (1050 mmol l-1), caffeine (30 µmol
l-1 to 60 mmol l-1), KCl (20100 mmol
l-1), L-glu and 5-HT (each 1050 µmol
l-1) had no effects on spectral reflectivity (for each experiment,
N=5). Neither the absence of external Ca2+ (N=5)
nor hyperosmotic or hyposmotic ASW (N=5) had any visible effect on
spectral reflectivity.
The ventral iridophores
It was previously reported that the iridophores of the ventral mantle of
Lolliguncula brevis were `inactive'
(Hanlon et al., 1990;
Cooper et al., 1990
). This is
not true of A. subulata. Perfusions with 2050 µmol
l-1 ACh, carbachol (N=10) and muscarine (N=5)
resulted in the weakly red reflective ventral iridophores
(Fig. 2Ea), reflecting
orange-yellow light within approximately 1.52 min
(Fig. 2Ed). These effects were
blocked by 510 µmol l-1 atropine (N=3). Nicotine
(1050 mmol l-1), caffeine (30 µmol l-1 to 60
mmol l-1), KCl (20100 mmol l-1), L-glu
and 5-HT (each 1050 µmol l-1) had no effects on their
spectral reflectivity (for each experiment, N=5). Neither the absence
of external Ca2+ (N=5) nor hyperosmotic or hyposmotic ASW
(N=5) had any visible effect on spectral reflectivity.
(4) Electrical stimulation
Electrical stimulation of the stellate ganglion and localised stimulation
of the chromatophore and iridophore layers in the skin led to contractions of
the mantle and expansion of the chromatophores, but no changes were seen in
spectral reflectivity of the iridophores. This supports the data reported by
Hanlon et al. (1990), who
stimulated the stellate ganglion, the stellar nerves and the iridophore layers
of the squid Lolliguncula brevis.
(5) Isolated iridophore cells
Morphology of isolated iridophore cells
Fig. 3A,B shows electron
micrographs of enzymatically isolated iridophore cells processed for TEM. The
iridophores in these images are 50300 µm long. Similar measurements
were made in iridophores that had been enzymatically isolated (for example,
see Fig. 3C). The iridosomes
containing the iridophore plates can clearly be distinguished. The structures
look similar to those shown for squid iridophores by, for example, Mirow
(1972) and Hanlon et al.
(1990
; see also
Fig. 1B).
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Having injected a cell and filled it with Lucifer Yellow, it was clear that
the dye, seen fluorescing in Fig.
3C, appeared to be taken up only within the iridosomes. Iridosomes
have been shown to contain the plates and spaces that produce iridescence
(Mirow, 1972;
Cloney and Brocco, 1983
). This
was found for all iridophores filled with Lucifer Yellow (N=5). We
filled iridophores from the dorsal (N=3), lateral (N=1) and
ventral (N=1) regions of the skin and found no difference in the way
the dye distributed within the iridosomes. It seems likely, therefore, that
the iridosomes are one continuous structure and that they function as one unit
during iridophore activity. The cells filled uniformly within 35 min,
suggesting the absence of gap junctions.
Ratiometric Ca2+ measurements
The Ca2+ concentration of isolated iridophore cells under
resting conditions was on average 66.16±18.71 nmol l-1 (mean
± S.E.M., N=4). In ASW, addition of KCl
(1050 mmol l-1) evoked a transient increase in cytoplasmic
Ca2+ (N=10) (Fig.
4A). In ASW, caffeine (2.510 mmol l-1) also
evoked an increase in Ca2+ (N=9)
(Fig. 4B). This internal
Ca2+ flux was observed irrespective of the Ca2+
concentration in the external medium (see
Fig. 4E). Addition of
50100 µmol l-1 ACh resulted in a transient increase in
cytoplasmic Ca2+ (N=12)
(Fig. 4C). This effect was also
observed using similar concentrations of carbachol (N=6) (data not
shown). Addition of 25 mmol l-1 nicotine had no effect on
cytoplasmic Ca2+ concentrations (N=8) in cells for which
the response to ACh was still evident (see
Fig. 4C). In contrast to the
nicotine response, muscarine (2550 µmol l-1) evoked a
strong Ca2+ response (N=7), which could be blocked by
pre-treating cells with 3570 µmol l-1 atropine for
510 min (N=4). The blockage could be reversed by washing the
cell with ASW for at least 1015 min
(Fig. 4D). These results
suggest that the iridophore cells are activated by muscarinic acetylcholine
receptors, which is further supported by the finding that neither
L-glutamate (50100 µmol l-1) (N=4),
nor 5-HT (50100 µmol l-1) (N=7) caused an
increase in cytoplasmic Ca2+ (data not shown).
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Experiments were conducted to investigate whether Ca2+ in the external medium is important to evoke the cytoplasmic increases in Ca2+ concentration. Cells were superfused for 10 min with EGTA Ca2+ free ASW before application of drugs. We found that intracellular Ca2+ concentration increased in response to 50 µmol l-1 ACh and 2 mmol l-1 caffeine even in the absence of external Ca2+ (N=4) (Fig. 4E).
Although the results using fresh tissue suggest that the reflective changes are not based on osmotic changes, experiments were conducted to investigate the effects of osmotic changes on the cellular level. Fura-2 loaded iridophore cells were superfused with ASW containing 50 mmol l-1 sorbitol (1112 mOsm kg-1; s-ASW) to increase the osmolarity of the extracellular solution and 75% ASW (769 mOsm kg-1) to decrease the osmolarity of the extracellular solution. Neither of these solutions had any effect on the Ca2+ concentration of the iridophore cells (N=5) (Fig. 4F). The Ca2+ levels remained low and subsequent addition of 50 µmol l-1 ACh evoked a strong Ca2+ response, showing that the cells were still viable.
There was no apparent difference between iridophores from the dorsal, lateral or ventral side in their intracellular Ca2+ response to the various drugs.
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Discussion |
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The plate thicknesses of the `red' stripes and the ventral iridophores
suggest that they act as ideal quarter-wavelength (/4) stacks. A
reflective plate of chitin with a thickness of approximately 100 nm would have
best reflectivity at 624 nm (
=4nd;
Huxley, 1968
;
Land, 1972
), where
n=1.56, the refractive index for chitin, and d is the actual
thickness of the plate). The plates of the `blue' stripe are too thick to act
as ideal
/4 stacks. However, this could be the thickness required for
the plates to act as a
wavelength stack, for which
max=4/3nd. For a plate with a thickness of 200 nm,
maximum reflectance would be at 416 nm.
The space thicknesses were very irregular. Assuming that the spaces between
the iridophore plates are filled with cytoplasm, the dehydration of the tissue
would have caused the spaces to shrink substantially. Therefore, from this
study, we cannot be certain about the thicknesses of the spaces and
consequently whether squid iridophores act as ideal or non-ideal multilayer
reflectors. Optically, however, both ideal and non-ideal multilayer reflectors
have very similar reflective properties
(Land, 1972).
Changes in spectral reflectivity of iridophores
Active iridophores are found in a number of aquatic animals and are often
associated with different behavioural states of the individual. Although the
anatomy and physiology underlying fish iridophores differ greatly from those
of cephalopods, it is worth mentioning the blue damselfish Chrysiptera
cyanea, whose body colouration is mainly attributed to multilayer
reflectors, which can change colour. The wavelength changes are produced by a
change in distance between the adjoining plates of the multilayer reflectors
and it has been shown that the iridophores are under the control of the
sympathetic (adrenergic) nervous system (Oshima et al.,
1985a,b
;
Kasukawa et al., 1986
,
1987
). The colour change in
the paradise whiptail Pentapodus paradiseus has also been shown to be
based on this system (Mäthger et al.,
2003
). Colour changes mediated by active iridophores have also
been reported in amphibians (e.g. Butman et
al., 1979
) and reptiles (e.g.
Morrison et al., 1991
).
When observing squid, the chromatophores are the most obvious colour
changing media. The reflections from the iridophores are often inconspicuous,
or even absent. If the stripes are fully reflective, however, they produce
very clear reflective patterns that contribute substantially to the overall
appearance of the animal. The `red' stripes, for example, are often visible
when the animal is threatened (e.g. by a hand net) or during intraspecific
encounters. Similar observations were reported on Loligo plei by
Hanlon (1982).
In the squid used in this study, the changes in spectral reflectivity are
consistent with the hypothesis that these iridophores act as multilayer
reflectors. The patterns of spectral changes were consistent in all stripes:
ACh, muscarine and carbachol shifted the reflected wavelengths towards the
shorter end of the spectrum with respect to the wavelength that they reflected
in their resting state, i.e. the `red' stripe iridophores changed from
(presumably) infrared to red, the `blue' stripe changed from blue to
(presumably) UV and the ventral iridophores changed from red to yellow. Hanlon
et al. (1990) also reported
that in the dorsal iridophores of L. brevis the spectral reflections
underwent a shift towards shorter wavelengths in response to topical
applications of ACh. It therefore seems very likely that the distances between
the plates or the thicknesses of plates and/or spaces are altered to produce
these spectral changes. This hypothesis finds further support when observing
the reflective changes of the `red' stripe at 45° incidence during the
process of `switching off'. We found that the reflected light is polarised at
45° incidence, independently of which wavelength is reflected. Another
hypothesis has been proposed by Cooper et al.
(1990
). They suggest that a
change in the refractive index of the plates may also change the reflected
wavelengths.
The spectral changes were also elicited using muscarine and blocked by
atropine, suggesting the existence of muscarinic ACh receptors in the
iridophore system. Cooper and Hanlon
(1986) and Hanlon et al.
(1990
), who used atropine to
block the effects of ACh, also suggested that a muscarinic ACh receptor type
may be involved. Here we confirm their findings and also found no evidence for
the presence of nicotinic receptors.
It was surprising to discover that changing the osmolarity of the external
medium had no effect on spectral reflectivity of iridophores. In fish scales
and in lizard skin, changes in the osmotic pressure of the external solution
results in clear shifts of the reflected wavelengths
(Foster, 1933;
Denton and Land, 1971
;
Morrison et al., 1991
). The
most likely explanation, that the squid iridophores were protected by
connective tissue and that the solutions had no access to the iridophores, was
disputed after the same experiments were repeated on isolated squid iridophore
cells and, clearly, the access problem in these preparations was minimal. The
failure of the iridophores to respond to changes in osmolarity may also be
because the iridophore spaces do not contain cytoplasm, a possibility that has
already been proposed by Cooper et al.
(1990
).
In contrast to the results of the study of Cooper et al.
(1990) and Hanlon et al.
(1990
), the results of the
present study show that the iridophores of all stripes are active, that is,
all respond to certain drugs with changes in the spectrum of the light they
reflect. These changes can also be observed in living squid.
The properties of isolated iridophore cells
Lucifer Yellow
A striking result of the Lucifer Yellow injections was that the dye
diffused into the iridosomes. It has so far been assumed that the iridosomes,
which contain the plates and spaces (210 per iridosome), are separate
units within an iridophore cell (Mirow,
1972; Cloney and Brocco,
1983
). The Lucifer Yellow injections presented here show that the
iridosomes are interconnected and that during iridophore activity they most
probably function as one unit. Furthermore the dye diffused uniformly into the
iridophore cell. It therefore seems certain that the iridosomes are not
electrically coupled, but that they are one unit.
Cytoplasmic Ca2+ measurements
The resting Ca2+ concentrations calculated during this study are
within the physiological range, which reflects the healthy conditions of the
cells. Similar concentrations have been obtained for isolated squid
synaptosomes from the optic lobe (Benech et
al., 2000) and isolated squid chromatophore muscles
(Lima et al., 2003
). It can
therefore be concluded that the process of enzymatic isolation and Fura-2
loading did not impair the healthy condition of the iridophore cells.
Cytoplasmic Ca2+ was found to increase transiently in response
to ACh, carbachol and muscarine, but not to nicotine. This study suggests that
ACh acts locally, on a post-synaptic level, via muscarinic
acetylcholine receptor. This is an interesting finding, as electron microscopy
has revealed no nerve terminals in the vicinity of iridophores
(Cooper et al., 1990) and
electrical stimulation of fresh tissue had no effect on reflectivity (see also
Cooper and Hanlon, 1986
;
Cooper et al., 1990
). How ACh
is released remains to be established. Hanlon et al.
(1990
) suggest that it may act
as a hormone. Certainly, ACh is abundant in the brain and optic lobes of
several cephalopod species (Tansey,
1979
).
Addition of KCl resulted in an increase in cytoplasmic Ca2+,
which shows clearly that isolated iridophores are excitable cells that respond
to changes in the electrical properties of their membrane, revealing the
existence of voltage-activated channels. It is, however, interesting to note
that KCl had no effect on whole skin preparations of iridophores, in contrast
to some tropical fish, where increasing the external KCl concentration changed
reflectivity (Oshima et al.,
1985a; Fujii et al.,
1989
; Nagaishi and Oshima,
1989
; Goda and Fujii,
1998
). The lacking KCl response in whole skin preparations may be
due to an accessibility problem, as other tissue surrounds the iridophores.
Also, the K+ concentrations inside the iridophore cells (as well as
outside) when in intact tissue are not known. Therefore, it is impossible to
predict the magnitude of the induced depolarisation (by increasing the
external K+ concentration to 50 mmol l-1), as the
potassium equilibrium was not determined.
The response to caffeine and the Ca2+ response in the absence of
external Ca2+ show the existence of Ca2+ stores inside
the cells. The experiments described here strongly suggest the existence of
ryanodine receptors on the Ca2+ stores in iridophore cells, as
caffeine has been used as a specific agonist of ryanodine receptors
(Shmigol et al., 1995;
Verkhratsky and Shmigol, 1996
;
Koizumi et al., 1999
;
Huang 1998
). Hanlon et al.
(1990
) suggested the existence
of Ca2+ stores, although no exact localisation could be inferred
from their study. The results presented here, on both fresh tissue and
isolated cells, suggest that Ca2+ is released from the stores
independently of extracellular Ca2+. This is an interesting
finding, as all muscular systems of invertebrates need external
Ca2+ for contraction, a feature shared with vertebrate cardiac
muscles (Fabiato, 1983
;
Inoue et al., 1994
). In
isolated iridophores both KCl and muscarine evoked vigorous Ca2+
transients, suggesting that calcium-induced-calcium-release may not be the
only mechanism for triggering Ca2+ release. Further research is
required to investigate this point.
It is unclear to date what the role of Ca2+ is during iridophore
activity. Ca2+ is a trigger for many physiological phenomena. For
example, fluctuations in intracellular Ca2+ concentration can
change the structure of microtubules
(Weisenberg, 1972;
Zhang et al., 1992
;
Barton and Goldstein, 1996
;
Jones et al., 1980
).
Ca2+ is also required for the movement of flagella and cilia
(Holwill and McGregor, 1975
;
Walter and Satir, 1979
). Both
microtubules and microfilaments have been observed in fish and lizard
iridophores (Harris and Hunt,
1973
; Rohrlich,
1974
; Nagaishi and Oshima,
1992
; Oshima and Fujii,
1987
). Neither microfilaments nor microtubules have been detected
in squid iridophores, but the possibility that they provide the mechanism of
iridophore colour change in squid should be further investigated.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
Present address: Department of Pharmacology, University Walk, University of
Bristol, Bristol BS8 1TD, UK
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References |
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---|
Barton, N. R. and Goldstein, L. S. B. (1996).
Going mobile: microtubule motors and chromosome segregation. Proc.
Natl. Acad. Sci. USA 93,1735
-1742.
Benech, J. C., Lima, P. A., Sotelo, J. R. and Brown, E. R. (2000). Ca2+ dynamics in synaptosomes isolated from the squid optic lobe. J. Neurosci. Res. 62,840 -846.[CrossRef][Medline]
Berridge, M. J. (1998). Neuronal calcium signaling. Neuron 21,13 -26.[Medline]
Bootman, M. D., Lipp, P. and Berridge, M. J. (2001). The organisation and functions of local Ca2+ signals. J. Cell Sci. 114,2213 -2222.[Medline]
Butman, B. T., Obika, M., Tchen, T. T. and Taylor, J. D. (1979). Hormone-induced pigment translocation in amphibian dermal iridophores, in vitro: changes in cell shape. J. Exp. Zool. 208,17 -34.[Medline]
Cloney, R. A. and Brocco, S. L. (1983). Chromatophore organs, reflector cells, iridocytes and leucophores in cephalopods. Amer. Zool. 23,581 -592.
Cooper, K. M. and Hanlon, R. T. (1986). Correlation of iridescence with changes in iridophore platelet ultrastructure in the squid Lolliguncula brevis. J. Exp. Biol. 121,451 -455.[Medline]
Cooper, K. M., Hanlon, R. T. and Budelmann, B. U. (1990). Physiological color-change in squid iridophores. II. Ultrastructural mechanisms in Lolliguncula brevis. Cell Tissue Res. 259,15 -24.[Medline]
Cornwell, C. J., Messenger, J. B. and Hanlon, R. T. (1997). Chromatophores and body patterning in the squid Alloteuthis subulata. J. Mar. Biol. Assn. UK 77,1243 -1246.
Crookes, W. J., Ding, L., Huang, Q. L., Kimbell, J. R., Horwitz,
J., McFall-Ngai, M. J. (2004). Reflectins: The unusual
proteins of squid reflective tissues. Science
303,235
-238.
Denton, E. J. and Land, M. F. (1971). Mechanism of reflexion in silvery layers of fish and cephalopods. Proc. R. Soc. Lond. A 178,43 -61.
Fabiato, A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol. 245,C1 -C14.[Medline]
Foster, K. W. (1933). Color changes in Fundulus with special reference to the color changes of the iridosomes. Proc. Natl. Acad. Sci. USA 19,535 -540.
Fujii, R., Kasukawa, H. and Miyaji, K. (1989). Mechanisms of skin coloration and its changes in the blue-green damselfish, Chromis viridis. Zool. Sci. 6, 477-486.
Goda, M. and Fujii, R. (1998). The blue coloration of the common surgeonfish, Paracanthurus hepatus II. Color revelation and color changes. Zool. Sci. 15,323 -333.
Hanlon, R. T. (1982). The functional organization of chromatophores and iridescent cells in the body patterning of Loligo plei (Cephalopoda: Myopsida). Malacologia 23,89 -119.
Hanlon, R. T. (1988). Behavioral and body patterning characters useful in taxonomy of field identification of cephalopods. Malacologia 29,247 -264.
Hanlon, R. T. and Messenger, J. B. (1996). Cephalopod Behaviour. Cambridge University Press.
Hanlon, R. T., Cooper, K. M., Budelmann, B. U. and Pappas, T. C. (1990). Physiological color-change in squid iridophores. I. Behavior, morphology and pharmacology in Lolliguncula brevis.Cell Tissue Res. 259,3 -14.[Medline]
Hanlon, R. T., Maxwell, M. R., Shashar, N., Loew, E. R. and
Boyle, K. L. (1999). An ethogram of body patterning behavior
in the biomedically and commercially valuable squid Loligo pealei off
Cape Cod, Massachusetts. Biol. Bull.
197, 49-62.
Hanlon, R. T., Smale, M. J. and Sauer, W. H. H.
(1994). An ethogram of body patterning behaviour in the squid
Loligo vulgaris reynaudii on spawning grounds in South Africa.
Biol. Bull. 187,363
-372.
Harris, J. E. and Hunt, S. (1973). The fine structure of iridophores in the skin of the atlantic salmon (Salmo salar L.). Tissue and Cell 5, 479-488.[Medline]
Holwill, M. E. J. and McGregor, J. L. (1975). Control of flagellar wave movement in Crithidia oncopelti.Nature 255,157 -158.[Medline]
Huang, C. L. (1998). The influence of caffeine
on intracellular charge movements in intact frog striated muscle.
J. Physiol. 512,707
-721.
Huxley, A. F. (1968). A theoretical treatment of the reflexion of light by multi-layer structures. J. Exp. Biol. 48,227 -245.
Inoue, I., Tsutsui, I., Bone, Q. and Brown, E. R. (1994). Evolution of skeletal muscle excitation contraction coupling and the appearance of dihydropyridine sensitive charge movement. Proc. R. Soc. B 225,181 -187.
Jones, H. P., Lenz, R. W., Palevitz, B. A. and Cormier, M. J. (1980). Calmodulin localization in mammalian spermatozoa. Proc. Natl. Acad. Sci. USA 77,2772 -2776.[Abstract]
Kasukawa, H., Oshima, N. and Fujii, R. (1986). Control of chromatophore movements in dermal chromatic units of blue damselfish II. The motile iridophore. Comp. Biochem. Physiol. 83C,1 -7.
Kasukawa, H., Oshima, N. and Fujii, R. (1987). Mechanism of light reflection in blue damselfish motile iridophore. Zool. Sci. 4,243 -257.
Koizumi, S., Lipp, P., Berridge, M. J. and Bootman, M. D.
(1999). Regulation of ryanodine receptor opening by lumenal
Ca2+ underlies quantal Ca2+ release in PC12 cells.
J. Biol. Chem. 274,33327
-33333.
Land, M. F. (1972). The physics and biology of animal reflectors. Progr. Biophys. Mol. Biol. 24, 75-106.[CrossRef][Medline]
Lima, P. A., Nardi, G. and Brown, E. R. (2003). AMPA-like and NMDA-like glutamate receptors at the chromatophore neuromuscular junction of the squid: role in synaptic transmission and skin patterning. Eur. J. Neurosci. 17,1 -10.[CrossRef][Medline]
Mäthger, L. M. and Denton, E. J. (2001). Reflective properties of iridophores and fluorescent `eyespots' in the loliginid squid Alloteuthis subulata and Loligo vulgaris.J. Exp. Biol. 204,2103 -2118.[Medline]
Mäthger, L. M., Land, M. F., Siebeck, U. E. and Marshall,
N. J. (2003). Rapid colour changes in multilayer reflecting
stripes in the Paradise Whiptail, Pentapodus paradiseus. J. Exp.
Biol. 206,3607
-3613.
Mirow, S. (1972). Skin color in the squids Loligo pealii and Loligo opalescens. II. Iridophores. Z. Zellforsch. 125,176 -190.[Medline]
Morrison, R. L., Sherbrooke, W. C. and Frostmason, S. K. (1991). A temperature mediated mechanism of iridophore-based structural color-change in a lizard. Am. Zool. 31, A83.
Nagaishi, H. and Oshima, N. (1989). Neural control of motile activity of light-sensitive iridophores in the neon tetra. Pigment Cell. Res. 2,485 -492.[Medline]
Nagaishi, H. and Oshima, N. (1992). Ultrastructure of the motile iridophores of the neon tetra. Zool. Sci. 9,65 -75.
Oshima, N. and Fujii, R. (1987). Motile mechanisms of blue damselfish (Chrysiptera cyanea) iridophores. Cell Motil. Cytoskel. 8,85 -90.
Oshima, N., Kasukawa, H. and Fujii, R. (1985a). Effects of potassium ions on motile iridophores of blue damselfish. Zool. Sci. 2,463 -467.
Oshima, N., Sato, M., Kumazawa, T., Okeda, N., Kasukawa, H. and Fijii, R. (1985b). Motile iridophores play the leading role in damselfish coloration. In Pigment Cell 1985: Biological, Molecular and Clinical Aspects of Pigmentation (ed. J. Bagnara, S. N. Klaus, E. Paul and M. Schartl), pp. 241-246. Tokyo: University of Tokyo Press.
Parker, G. H. (1948). Animal Colour Changes and their Neurohumours. Cambridge University Press.
Rohrlich, S. T. (1974). Fine structural
demonstration of ordered arrays of cytoplasmic filaments in vertebrate
iridophores. J. Cell Biol.
62,295
-304.
Schäfer, W. (1937). Bau, Entwicklung und Farbentstehung bei den Flitterzellen von Sepia officinalis. Z. Zellforsch. 27,222 -245.
Shmigol, A., Verkhratsky, A. and Isenberg, G. (1995). Calcium-induced calcium release in rat sensory neurons. J. Physiol. Lond. 489,624 -636.
Tansey, E. M. (1979). Neurotransmitters in the cephalopod brain. Comp. Biochem. Physiol. 64C,173 -182.[CrossRef]
Thomas, A. P. and Delaville, F. (1991). The use of fluorescent indicators for measurements of cytosolic-free calcium concentration in cell populations and single cells. In Cellular Calcium (ed. J. G. McCormack and P. H. Cobbold), pp.1 -54. Oxford: Oxford University Press.
Verkhratsky, A. and Shmigol, A. (1996). Calcium-induced calcium release in neurones. Cell. Calcium 19,1 -14.[Medline]
Walter, M. F. and Satir, P. (1979). Calcium does not inhibit active sliding of microtubules from mussel gill cilia. Nature 278,69 -70.[Medline]
Weisenberg, R. C. (1972). Microtubule formation in vitro in solutions containing low calcium concentrations. Science 177,1104 -1105.[Medline]
Williams, L. (1909). The Anatomy of the Common Squid, Loligo pealii. Leiden, Holland: E. J. Brill.
Zhang, D. H., Wadsworth, P. and Hepler, P. K. (1992). Modulation of anaphase spindle microtubule structure in stamen hair-cells of Tradescantia by calcium and related agents. J. Cell Sci. 102,79 -89.